Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Bacterial infectious disease requires the knowledge of the etiologic agent which can be investigated in the following ways:
- Isolating and identifying the organism in culture
- Serologic tests that demonstrate antibodies in the patient’s blood against an organism or specific bacterial antigens
- Detection of pathogen specific antigen
- Detection of pathogen specific DNA/RNA
- Immune function assays (e.g., γ-interferon-release assays).
Isolating and identifying the organism in culture is the gold standard in microbiological diagnostic testing for numerous infectious diseases. As a rule, the culture of non fastidious, fast growing bacteria is simple, safe, fast, economic and, relevant to therapy. After isolating and identifying the organism in culture testing the susceptibility of the organism to antibiotics is important. Depending on the type of pathogen, its requirements for growth and longer generation times, the investigation interval required until a result is available may be days or even several weeks (e.g., for mycobacteria).
Alternatively, microorganisms that are difficult to culture, such as atypical pneumonia organisms, can be detected by direct serological tests based on pathogen specific antigens using specific polyclonal or monoclonal antibodies, by direct immunofluorescence, enzyme linked immunosorbent assay or immunochromatographic assay (e.g., the presence of legionella antigen in urine).
Molecular biological techniques are the methods of choice if results should be available immediately such as PCR of MRSA, or if the in vitro reproduction of a fastidious pathogen is difficult or very time consuming (e.g., pertussis, mycobacteriosis) /, , , /.
A target sequence is a genetically stable, clearly defined, specific genomic structure of a species or genus in microbiology. Samples obtained from primarily non sterile specimens should be used to search for pathogen specific targets. By contrast, primarily sterile specimens are suited for the more universal search and detection of pathogens based on consensus sequences .
The analytical specificity of molecular biological method is determined by the mutual exclusivity for a target or a specific pathogen, thus avoiding false positive results due to cross reacting molecular structures in closely related pathogens .
The analytical sensitivity represents the probability to detect a target sequence in a nucleic acid preparation from a typical clinical specimen (e.g., genome equivalents per milliliter or per volume of PCR mixture) .
Diagnostic sensitivity and specificity of nucleic acid amplification techniques
Contrary to the analytical sensitivity and specificity of molecular biological methods, the diagnostic sensitivity relates to true positive samples in patients and the diagnostic specificity relates to true negative samples in a reference population. The test identifies individuals with clinically unequivocal characteristics of the relevant disease and/or reference populations without the relevant infectious disease or with closely related bacterial infections .
A DNA probe is a short, labeled, single stranded sequence of a nucleic acid that is complementary to the DNA or RNA segment to be detected. The length of the probes (DNA or RNA fragments) can vary (usually 15–50 nucleotides).The DNA probe analysis technique is based on the property of nucleic acids to reversibly bind to complementary nucleic acid sequences by forming specific base pairs (hybridization) at suitable experimental conditions. Common test formats are liquid phase, solid phase and in situ hybridization.
Liquid phase hybridization is most widely used. Probe tests using pathogen specific target sequences are highly specific but their analytical sensitivity is low. For direct pathogen detection in clinical samples, probe tests have, therefore, become inferior to NAA techniques and are mainly used for the identification of microorganisms and the detection of resistance genes from culture isolates.
Gene probe testing is characterized by four procedural steps:
- Denaturation (physical separation of the two strands of DNA double helix)
- Hybridization of pathogen DNA or RNA and the complementary gene probe
- Removal of excess (non hybridized) gene probes (washing)
- Detection of hybridization products by means of a suitable test system.
To increase the detection limit of DNA target molecules in the sample classical enrichment methods or molecular biological amplification, such as PCR, are performed prior to hybridization.
Signal amplification methods are special types of the gene probe analysis which do not primarily increase the concentration of pathogen specific target sequences, but amplify the detection signal after the binding of gene probes to specific target DNA, thus improving analytical sensitivity. This includes, for instance, the hybrid capture method or the branched DNA method, which are, however, better suited for high sample pathogen concentrations and are primarily used in virological diagnostics.
Nucleic acid amplification (NAA) techniques
NAA techniques are laboratory tests that involve the in vitro synthesis of many copies of DNA or RNA from one original target sequence. NAA techniques such as the polymerase chain reaction (PCR) or ligase chain reaction (LCR) are largely automated.
Principle of PCR assay
The amplification of pathogen specific DNA presupposes the knowledge of the DNA target sequence at the 5’ and 3’ ends to be able to hybridize two primer oligonucleotides to start the synthesis reaction. PCR is characterized by three procedural steps:
- DNA denaturation: physical separation of the two strands of DNA double helix at a temperature of 94–98° C for 20–30 seconds
- Annealing: at a temperature of 50–65 °C for 20–40 seconds the primers that are complementary to the target DNA region anneal to each separated single stranded DNA as the target sequence
- Extension: a heat stable DNA polymerase (Taq polymerase) binds to the primer template hybrid and synthesizes a new DNA strand complementary to the DNA template in the 5’ to 3’ direction.
This technique increases the amplification efficiency and reduces the concentration of nonspecific PCR products due to the amplification of untargeted primer binding sites. Nested PCR integrates two different pairs of primer sets used in two successive PCR steps. After the first PCR of the target sequence a second step of primer pairs amplifies a secondary target within the fist step PCR product; this is the source of the term nested. As a result, the nested PCR analysis has an improved detection limit because a sufficient amount of target DNA through a two step amplification procedure. Techniques of this type are susceptible to contamination and should be integrated in the same reaction vessel (single tube assay), thus avoiding to open the vessel and minimizing the risk of contamination.
The multiplex PCR combines different primer pairs to ideally detect several pathogens in the same sample at the same time. Such PCR is used, for example, to synchronously detect typical respiratory pathogens in the diagnosis of pneumonia. Multiplex PCR is usually not equally sensitive to all pathogens and, in most cases, is less sensitive than the corresponding monoplex PCR .
Ligase chain reaction (LCR)
The LCR is a rarely used technique and similar to the PCR, with the difference that, after thermal denaturing of the nucleic acid sequences, target complementary oligonucleotides are attached in the second step of the cycle. In the third step of the cycle, thermostable ligase is added and will covalently join only those adjacent hybridized oligonucleotides that are not separated by gaps.
The real-time PCR is a variation of the PCR technique intended to amplify and simultaneously quantify a target molecule. The key feature is that the amplified DNA fragment of classical PCR is detected as the reaction proceeds in real time. Two methods are common for detection of real time PCR products:
- Non specific fluorescent dyes (e.g., SYBR Green) that intercalate with double stranded DNA
- Sequence specific oligonucleotide probes (e.g., TaqMan) probe that are labeled with a fluorescence reporter dye. These dyes permit detection only after hybridization of the probe with its complementary target sequence.
Identification of PCR products
A simple way to detect PCR amplification is to use intercalating dyes (e.g. SYBR green) that preferentially bind to double stranded DNA molecules, thereby increasing their fluorescence.
Another possibility of measuring and identifying PCR products is to employ specifically labeled DNA probes and molecular beacons, consisting of dye labeled, short fragments of nucleic acids that bind to their specific targets and, via conformational change, lead to a differentiated interaction of attached donor and acceptor dyes. The resulting change in the fluorescence behavior of these dyes compared to the initial situation can be measured in terms of quality and quantity using special optics .
DNA micro arrays
They consist of immobilized oligonucleotides bound to a variety of reaction fields of miniaturized microchips, in some cases using special micro formats. The oligonucleotides function as solid phase coupled hybridization probes. After successful binding of the targets, the complexes are visualized by coupled color and fluorescence detection. These tests are used to directly detect specific target molecules in the specimen and to detect and identify PCR products at high sensitivity, for instance for the molecular detection of resistance mechanisms.
The culture independent nucleic acid amplification assay with is relatively high analytical sensitivity and specificity is a valuable alternative to minimize the time for the detection and identification of specific and especially fastidious pathogens between sample collection and result /, , , /. This refers to microorganisms, in particular, which are difficult to culture, or to cases, where no specific immune response is to be expected, for example, in immune compromised patients. These advantages are opposed by the following potential disadvantages:
- Molecular biological techniques can for methodological reasons not distinguish between vital and non vital pathogens
- While their detection limit is high, they imply the risk of false positive results caused by environmental or laboratory related contamination /, , , /
- From an economic point of view, novel molecular biological detection methods are in many cases much more expensive than conventional methods
- The low degree of standardization in many fields regarding targets, method of investigation and sample treatment (DNA and RNA isolation) is another disadvantage to be considered
- The evaluation of advanced molecular biological methods in the literature and the results of external quality assurance for a reason reveal significant variance in the performance of in-house tests and commercially available analyzers. This especially applies to the poorly standardized NAA methods in medical bacteriology, mycology and parasitology compared to molecular methods in virology
- Whenever possible, the clinician and the laboratory physician should only interpret molecular biological results in the context with other microbiological findings and available clinical information.
Specimens for nuclear amplification assays (NAA) can be refrigerated at 2–8 °C for up to 3 days. For longer storage, samples should be stored at –20 °C for DNA analyses and at –70 °C for RNA analyses. It must be kept in mind when storing liquid samples that freezing and thawing may lead to the lysis of bacteria and cellular components .
- Prevent contamination
- Perform the pre amplification, amplification and post amplification steps in separate, self contained chambers, if required
- Have the laboratory personnel adhere to a one way sample flow, if necessary
- Check nucleic acid isolation procedures at regular intervals
- Ensure nucleic acid decontamination.
- Negative controls: run parallel controls in all processing steps
- Positive controls: perform defined sensitivity checks near the detection limit
- Use control samples with determined pathogen concentrations in quantitative methods
- Use quantitative control specimens that comply with international standard preparations
- Perform inhibition control for each patient sample.
- Hybridization, sequencing or restriction enzyme analysis
- The mere sizing in gel or melting curve analysis with SYBR Green is not sufficient.
External quality assurance
Within the scope of external quality control, inter laboratory proficiency testing has been established in respect of bacterial genome detection and is to be performed every half year (e.g., in Germany) :
- Bordetella pertussis
- Borrelia burgdorferi
- C. trachomatis, C. pneumoniae
- Helicobacter pylori
- Legionella pneumophila
- Listeria monocytogenes
- Mycobacterium tuberculosis
- Mycoplasma pneumoniae
- Neisseria gonorrhoeae
- Salmonella enterica
- Coxiella burnetii
- Francisella tularensis.
Infectious agents can be detected indirectly through determination of pathogen specific antibodies formed during the course of humoral immune response. Humoral immune response must be distinguished from passive immunity (i.e., passive transfer of antibodies):
- From mother to fetus via the placenta during pregnancy
- Following the administration of blood products
- After passive immunization.
The antibody titer is the reciprocal of the highest serum dilution in which an antigen antibody reaction is still detectable.
Units per milliliter (U/mL)
Most commercially available immunoassays use artificial manufacturer specific units for test quantification and, therefore, are not comparable regarding a given infection serology parameter . International standard preparations that generally allow the specification of results in international units (IU/mL) for correctly calibrated tests are only available for a small number of antigens (e.g., tetanus, pertussis, syphilis). In many cases, however, the quantitative results obtained with the corresponding, commercially available test systems are still hardly, or not, comparable, as demonstrated by the serological test results from inter laboratory proficiency testing .
The borderline titer/cutoff/threshold is the antibody titer/test value above which the result of a serological detection method such as IFT/ELISA is very likely to be specific. The borderline titer/cutoff is essential for the diagnostic sensitivity and specificity of a test and must usually be established for a given assay by comparative analyses of:
- Afflicted patients with unequivocal clinical symptoms
- A sufficiently high number (> 100) of healthy controls such as blood donors from the relevant region
- Samples of individuals with other infections from potentially cross reacting pathogens .
These cutoff values are not universally applicable for a variety of serological tests (e.g., Helicobacter sp., B. burgdorferi, Brucella sp.), but may have to be regionally evaluated in respect of their diagnostic sensitivity and specificity and adapted to local epidemiological conditions .
The gray area is a margin of uncertainty in result interpretation that is due to medical and methodological reasons and individually defined for each assay. The gray area implies the overlapping test results of negative and positive cohorts.
Diagnostic titer/test value
The antibody titer/test value is referred to as diagnostic if it is so high that, by itself, it very likely indicates the presence of an infection. Diagnostic titers of sufficient specificity relate to specific tests and have only been adequately established for a small number of microorganisms (e.g., VDRL > 16 in syphilis serology).
Significant change in serological test result/titer difference
Antibodies should be determined, if possible, on two samples collected at an interval of at least 10–14 days (or even 3–6 weeks for some pathogens such as Legionella sp. and Borrelia sp.).
The following constellations can be interpreted as being significant in parallel testing:
- The initial detection of specific antibodies in a previously negative sample (serological conversion)
- The analysis of both samples in the same assay procedure yields a difference in the test result by at least two geometric dilution levels i.e., 4-fold increase or decrease in titer or 2–3 fold increase or decrease in the quantitative ELISA value
- A decrease in the specific antibody titer near the detection limit in a previously positive sample (reconversion).
This only applies to parallel testing together with the previously analyzed sample in the same assay procedure.
Serological test methods are based on the antigen-antibody reaction, which provides evidence of humoral immune response to an infectious agent through the detection of pathogen specific antibodies by means of known antigens /, /.
Established serological test methods include:
- Direct agglutination (e.g., Widal)
- Indirect/passive agglutination such as indirect hemagglutination assay (IHA)
- Neutralization test (NT)
- Complement fixation (CF) test.
- Indirect immunofluorescence test (IIFT)
- Enzyme-linked immunosorbent assay (ELISA)
- Chemiluminescence immunoassay (CLIA)
- Multiplex fluorescence immunoassay
- Western blot (immunoblot)
- Line immunoblot.
A multiplex immunoassay is a type of assay that simultaneously measures multiple analytes in a single run/cycle of the assay. Multiplex bead immunoassays are based on tiny, antigen-coated polystyrene micro spheres that serve as the solid phase for a variety of detection reactions, similar to the Western blot and ELISA . The different sets of micro spheres are identified by a consistently defined fluorescent dye (blank dyeing) and coated, for example, with recombinant single antigens (e.g., Borrelia antigens). During the following incubation step, the individual micro sphere type binds to the corresponding antibodies that may be present in the patient samples to be analyzed. The next step is to label the antibodies attached to the micro sphere surface with a specific conjugate, thus visualizing them as an antigen-antibody complex. The amount of bound antibody to the micro sphere is directly correlated to the resulting fluorescence intensity and can thus be quantified. The analyzer individually detects, analyzes and quantifies the beads based on their binding specificity and blank fluorescence. The resulting multi antibody profiles combine the advantages of immunoblot analyses with the analysis principles of quantifiable immunoassays. Compared to conventional ELISAs, multiplex immunoassays using single or multiple antigens are diagnostically much more conclusive .
Interferon-γ release assay (IGRA)
The principle of these cell stimulation assays is based on the production of interferon-γ (INF-γ) by the patient’s immune cells (e.g., T cells) following exposure and specific stimulation with pathogen specific antigens. During incubation of the patient’s blood sample, IFN-γ is produced through antigen stimulation of specifically sensitized immune cells and, in a second test step, quantitatively determined by an ELISA. The test quality is essentially influenced by the choice of pathogen specific antigens and parallel testing of negative, positive and functional controls. IGRAs are primarily used in the diagnosis of tuberculosis .
Serological test results should be interpreted qualitatively and quantitatively. A qualitative result is interpreted as:
- Positive (reactive) if antibodies are detected
- Negative (non reactive) if no antibodies are detected.
Quantitative serological test results are specified, for example, as titers, in U/mL, IU/mL or as cutoff index or threshold. The results of immunoassays can also be specified semi quantitatively, or qualitatively with a relative quantitative statement, such as positive 42 U/mL, or positive approximately 320 U/mL.
In immunoblot interpretation, the class of detected antibodies, a description of the detected protein band pattern and an assessment of the test constellation must be communicated.
The diagnostic accuracy of test methods in infection serology and the interpretation of findings derived from the individual test results are decisively influenced by the technical options and the conception of a test. Only a small number of tests used in diagnostic infection serology have, by themselves, a sufficiently high diagnostic specificity and sensitivity. Accordingly, serological tests should best be combined in a tiered approach to achieve optimal sensitivity and specificity. The combination of a test with a high detection limit and a specific confirmatory test is recommended as the most reliable procedure.
Agglutination (Widal reaction) and complement fixation (CF) test
These tests detect antibodies of the IgG and IgM classes in the same test mixture (the Widal reaction detects mainly IgM antibodies); they do not differentiate between IgG and IgM antibodies in the immune response. Hence, the tests can primarily be used as screening tests and supplementary tests (CF) but are not suited as the sole diagnostic approach.
IIFT, ELISA, CLIA, LIA and immunoblot
Depending on the conjugate (polyvalent or monovalent secondary antibody), these tests allow the polyvalent detection of the pathogen specific immune response and detect pathogen specific IgG, IgM and IgA antibodies. Moreover, the immunoblot performs an antigen specific and antibody class specific analysis of the immune response. Hence, these tests are particularly suited as differentiation and confirmatory tests.
Qualitative interpretation of serological tests
A negative test result suggests the absence of specific antibodies. However, exposure to infection with the presumed pathogen can only be ruled out if the patient does not have an immunodeficiency and if there is a time interval of at least 7–14 days (even 3–6 weeks for certain pathogens, such as B. burgdorferi or Legionella) between the time of infection and blood collection.
A positive test result points to an acute infection or an infection in the recent or distant past. A prerequisite is that cross reactivity with antigens of other microorganisms can be ruled out.
Quantitative interpretation of serological tests
Quantitative results (titer, U/mL, IU/mL) obtained with serological tests depend on:
- The timing of the increase, peak level, persistence and decline of the titers or units
- The infectious pathogen
- The quality and timing of antibiotic treatment
- The immune status of the patient
- The antigen preparation
- The selected detection method.
Reliable quantitative result interpretation requires the monitoring of samples collected at an interval of at least 7–14 (–21) days and analyzed in the same assay. The following conclusions can be made:
- An increase by at least two geometric dilution levels (fourfold titer increase) in the conventional titer tests (IHA, IIFT, CF) and doubling to tripling of the quantitative ELISA results in consecutive, paired samples analyzed in the same assay suggest an acute infection
- Consistently elevated titers/values in consecutive samples point to an existing or recently resolved infection
- A 4 fold titer decrease in the conventional titer tests (IHA, IIFT, CF) or a 2–3 fold decrease of the ELISA result in two consecutive paired samples indicates an infection in the recent past.
However, a test result not exceeding the cutoff level does not rule out the presence of an acute infection. Such a situation may be caused by inadequate activation of the immune system due, for example, to:
- Recent exposure
- Early treatment
- Localized infection
- Impaired immune response.
Immunoglobulin class-specific analysis of antibody response
The detection of pathogen specific IgM antibodies (and in the case of Helicobacter and Chlamydia also of IgA antibodies) in a single sample usually points to an acute, existing or recently resolved infection. IgM antibodies are produced as the primary antibody immune response to an infection; they reach maximal levels after 2–3 weeks and, within a period of 2–3 months after the occurrence of the clinical symptoms, decline again to undetectable levels.
However, there are exceptions to this rule concerning some bacterial infection pathogens, such as Borrelia burgdorferi and Treponema pallidum. In such infections, specific IgM antibodies may persist for months or years after a resolved infection and also after treatment.
Specific IgG antibodies increase 2–3 weeks after an infection has occurred. Peak values are reached after several weeks. IgG antibodies persist for long periods of time, often for life.
The concentration of specific IgG antibodies can be a criterion of the immune status (persistence in serum, vaccination) and immunologic protection against reinfection with the same pathogen such as tetanus immunity. During follow up monitoring:
- Increasing IgG antibody titers point to an acute infection or an exogenous or endogenous reinfection
- Persistently high IgG antibody titers are indicative of a past but continuing infectious disease or a resolved infection in the past.
The following must be considered in serological test interpretation:
- Reinfections, for example in pertussis or syphilis, may cause an increase in titers/values despite the absence of detectable specific IgM in the CF, IHA, IIFT and ELISA
- In many cases, reinfections, for example in pertussis or syphilis, are associated with a delayed, or no increase in specific IgM antibody concentration
- Specific IgA antibodies are important in the diagnostic investigation of infections/reinfections by certain pathogens (e.g., Bordetella pertussis, Helicobacter pylori). However, these antigens may persist for long periods of time, show irregular patterns and be induced by vaccination. Hence, pathogen specific IgA determination is considered to be of limited value and no longer recommended in bacterial infection serology, especially since IgA assays turned out to be non reproducible in evaluation studies and during external quality assurance at standardized conditions.
- The detection of specific IgM antibodies in the fetus or newborn suggests a pre- or perinatal infection. It is not reliable to use umbilical cord blood in this context (beware of placental leak). A continuous decrease in pathogen specific IgG antibodies (half life: approximately 4 weeks) postpartum points to passive immunity.
- IgM and IgG antibody titers may rise after vaccination and make the assessment of a possible (re)infection more complicated.
Conclusions as to the effectiveness of pathogen specific antibiotic treatment based solely on qualitative or quantitative serological test results can only be made in exceptional cases, such as syphilis, and in consideration of additional established clinical information.
Depending on their type and quality, serological analysis techniques can be interfered by a number of factors. Such interference may be unspecific reaction failures or false positive results due to cross-reactions (shared epitopes) of the applied antigen with pathogens other than the one to be determined or with antigens of the afflicted host organism. Typical interference factors include:
- Rheumatoid factors
- Immune complexes
- Matrix effects.
Serological test results are most commonly caused by cross reactions (e.g., false positive Brucella serology in the presence of a Yersinia enterocolitica infection). The type and degree of the cross reactivity and susceptibility to interference are dependent on the test method and the quality of the antigen used.
Serological analyses are generally subject to the same pre analytic, analytic and post analytic requirements of good laboratory practice and quality assurance as in other fields of laboratory medicine. The serological laboratory must comply with all safety requirements mandatory for medical laboratories. It is imperative to establish a quality management system with internal and external quality assurance and documentation at regular intervals. For details, please refer to the relevant microbiological and infectiological quality standards. Infection serology utilizes biological test systems with a low degree of standardization and automation. At optimal conditions, however, it yields very good diagnostic results /, , /. Some aspects of infection serology deserving special attention are discussed in detail in the following.
Internal quality assurance in bacterial serology
The instructions for the applied test methods must always be kept up to date and should be compiled in a laboratory manual. As a rule, every diagnostic test should be subject to internal quality assurance. The kind and frequency of monitoring follows the manufacturer’s specifications and depends on the degree of susceptibility of the serological assay to interference.
As a rule, serum is used as specimen as long as sterile sample collection can be ensured. Hemolysis in the specimen should be avoided.
Serum samples can be stored in air-tight containers at 4 °C for approximately 3–4 days. For longer storage or for retained samples, the sera should be stored at –20 °C. Serum samples for use in IgM tests that cannot be performed within 3 days should optimally be deep-frozen at –60 °C or lower. If this is not possible, it must be kept in mind that the IgM fraction may no longer correspond to the original level .
High quality serological testing and the assessment of serum conversions and significant quantitative changes in results are decisively dependent on the availability of parallel testing with previously collected samples. Second sera should therefore be analyzed in parallel with the first serum. Accordingly, specialist laboratories are obliged to maintain a serum bank for the storage of reference samples to have negative and positive samples available for later parallel testing. Otherwise, valid serum diagnostics with clinically conclusive comments will not be possible.
Quality control sera
These sera must be portioned and adequately stored at –20 °C (IgM: –80 °C). As a rule, each test mixture should be accompanied by parallel testing of a positive (optimally quantifiable) control, a cutoff control and a negative control. In longer test runs, several controls must be tested in parallel in numbers corresponding to the test procedure.
Antigens, amboceptor, complement
The activity of new reagent lots for CF should be verified by defined controls in the corresponding previous tests . The verification can be general if the conformity and stability of the lot are guaranteed by the manufacturer.
Pipettes and dispensers, devices, microscopes
Calibration, maintenance and cleaning of such equipment must be performed and documented at regular intervals.
Whenever reasonable, tests should be performed in a qualitative and quantitative approach. In any case, defined positive, borderline and negative control sera must be run in parallel. In quantitative assays (IIFT, IHA, CF), the controls should be quantifiable and diluted, if required, to document inter assay variations and take these variations into account in diagnostic interpretation.
The ELISA result must be within the linear range of the calibration curve, especially if the data are used for further calculations (e.g., CSF/serum ratio).
In the case of lot changes, in-house conjugates intended for the ELISA, immunoblot or IIFT must be adjusted by checkerboard titration.
In immunoblot assays, the type and position of the immunodominant antigens in the blot must be verified and evaluation templates and well evaluated assessment keys must be available for the user. Each test mixture should be accompanied by parallel testing of defined positive and negative reaction controls and serum incubation controls. These tasks must be adequately represented by test-integrated functional controls such as incubation and/or conjugate controls. Cutoff controls (analytical or clinical cutoff) allowing the uniform assessment of the band color intensity for consideration in the test evaluation are of essential importance.
Reference sera and standard sera
Whenever possible, national and international reference standards should be tested in parallel and used for checking and improving uniform quantification. For an overview of the currently available national and international standard sera and how they can be ordered, please refer to: National Institute for Biological Standards and Control, PO Box 1193, Blanche Lande, South Mimms, Potters Bar, Herfordshire EN6 3QH, United Kingdom ().
External quality assurance
The objective of quality control in medicine is the review of medical procedure performance and, based thereupon, the improvement of treatment quality. Quality must be objectively verifiable and documented by certificates. A large number of serological parameters is subject to the provisions of national guidelines in regards to external quality assurance. In the poorly standardized fields of infection serology, inter laboratory proficiency testing is an indispensable important and independent tool for external quality control and, in particular, helps to detect systematic errors. Inter laboratory proficiency testing also allows a realistic evaluation of laboratory activities/services for a large number of laboratories.
Moreover, the entire spectrum of in-house tests and commercially available test methods can be checked against the well characterized sample panel. The correct interpretation and clinical significance of serological results as an important laboratory service can also be verified to a certain degree. Therefore, all quality-conscious laboratories consider the participation in inter laboratory proficiency testing a natural part of external quality control ().
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Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Bartonellosis afflicts humans and animals and manifests as subacute, acute or chronic persistent course. It comprises classical diseases such as five-day fever, Oroya fever and cat scratch disease. The course and manifestation of symptoms in humans are decisively influenced by the patient’s immune status. A host of new pathogens and disease varieties have first been described in HIV patients and other immunocomprimised patients. The infection is caused by facultative intracellular Rickettsia like bacteria. Some representatives of the genus Bartonella, for example Bartonella henselae, the causative agent of the catch scratch disease, were not definitely identified as being pathogenic to humans until the 1990s /, , /.
The genus Bartonella currently comprises more than 20 different species that differentiated at the molecular level, for example based on the 16S-23S (ITS) region or the ribC gene /, /. Phylogenetically, they are closely related to Rickettsia and Brucella /, /. Bartonella are fastidious, pleomorphic, Gram negative, rod-shaped bacteria that only grow on substrates containing boiled blood after a long incubation period (7–20 days) at microaerophilic conditions. B. quintana, B. bacilliformis and B. henselae are the main human pathogenic species (). Other zoonotic species, B. clarridgeiae, B. elizabethae, B. vinsonii, B. grahamii, B. koehlerae, B. washoensis, are presumably facultative pathogenic and have but recently come into the focus of medical interest. Man is the only known reservoir for B. quintana and B. bacilliformis. B. quintana is transmitted by the human body louse (P. humanus). Cats are the main reservoir host for B. henselae and B. clarridgeiae, the causative agents of the cat scratch disease. In many cases, the cats are afflicted by severe asymptomatic bacteremia. The vectors of B. henselae and B. clarridgeiae infections are not reliably identified and presumably include fleas and flies. B. bacilliformis is only endemic in certain regions of South America that correspond to the distribution area of its specific vector, the sand fly Lutzomyia. Transmission of various Bartonella species by ticks and other arthropods has also been discussed /, , , , , /.
Direct detection of Bartonella species by culturing is of no relevance in routine microbiological diagnostics.
Contrary to B. bacilliformis, which only occur in the Andean regions of Peru, Ecuador and Colombia, B. quintana and B. henselae can be found worldwide. Five-day fever is a disease very closely associated with poor hygienic conditions. For instance, Napoleon’s Grand Army suffered significant losses during the Russian campaign, and the Western Allies recorded more than one million afflicted soldiers during World War I alone (hence the name french fever) because of this infection . The pathogen has recently gained in significance as urban five-day fever and causative agent of endocarditis, especially in fringe groups. The prevalence of infection in certain risk groups (HIV, homeless and drug users) is 10–30% .
In the United States, the incidence of the cat-scratch disease is estimated to be 9/100,000 annually. The prevalence of antibodies in cats is 5–90% worldwide. In Germany, B. henselae causes up to 13% of the cases of lymph node hyperplasia . Potentially human pathogenic species have rarely been isolated to date. Hence, there are still many uncertainties in respect of their epidemiology, transmission and characterization of reservoir hosts and possible vectors /, , , /.
Approximately 7 days to several weeks.
Bartonella can cause a wide array of clinical syndromes. Besides purely local infections, almost all pathogens are also associated with systemic courses and septic manifestation /, /. The following typical disease patterns are distinguished depending on the underlying causative agent /, , , /:
- Cat scratch disease following infection with B. henselae or B. claridgeiae. Approximately 7 days after contact with cats, a papular or pustular efflorescence and subsequent regional lymphadenitis develop at the site of invasion. About 50% of afflicted individuals develop general symptoms associated with fever.
- Five-day fever caused by B. quintana infection: after several days to weeks, patients experience a sudden onset of fever and chills which, in the optimum case, last for 4–5 days. The periodic form of the disease is characterized by 5 to 8 fever attacks lasting for up to 5 days. Continuous fever for up to 6 weeks can occur in the chronic course of the disease. Oligosymptomatic afebrile forms of the disease are rare.
- Carrión’s disease caused by B. bacilliformis: this disease is endemic in South America and typically manifests at first by a high temperature infection (Oroya fever) associated with hemolysis (detection of intra erythrocytic bacteria in Giemsa stained blood smear) and considerable mortality in patients who are not treated with antibiotics. Following systemic feverish infection, immunocompetent patients may show persistent warty angiomatosis of the skin (Verruga peruana). Moreover, B. quintana and B. henselae , in particular, can be the cause of chronic infectious myocarditis and endocarditis associated with fever due to recurrent bacteremia. In addition, B. henselae is the causative agent for Parinaud’s syndrome (granulomatous conjunctivitis and ipsilateral lymph node hyperplasia) and is an important pathogen of uveitis, neuroretinitis and chorioretinitis. Neurological infections with a meningitic and encephalitic course of the disease have also been described /, , , , /.
Immunocomprimised individuals and HIV patients, in particular, may show a great variety of manifestations of the disease, including peliosis (angiomatous lesions in the liver and, more rarely, in the spleen) caused by B. quintana and also by B. henselae as well as bacillary angiomatosis. The vasoproliferative tumor lesions are triggered by Bartonella specific angiogenic factors of pathogenicity and are completely reversible after antibiotic treatment /, /.
According to the German Infection Protection Act (IfSG), these infections are not subject to mandatory reporting.
In clinically suspected infection, indirect pathogen detection should be done using serological tests after the first to second week of the disease.
Immunofluorescence test (IIFT)
For the analysis of the immune response for IgG, IgA and IgM to Bartonella, the conventional IIFT for the different species as well as the micro immunofluorescent test (MIFT) with several Bartonella spp. per application pad are available. The detection of specific IgG antibodies is the only established test /, , /. Bartonella specific antigen suspensions obtained from cell cultures (Vero cells) which adequately express important immunodominant antigens such as BadA have proved to be especially helpful. The detection limit can vary depending on the antigen used (Houston-/Marseilles strain) . In addition, the diagnostic sensitivity and specificity of the different assays depend on the relevant test and the manufacturer of the assay /, , /. Comprehensive evaluation studies with well characterized sera specify diagnostic sensitivities of 50–98% at specificities of 89–96% depending on the selected antigens and borderline titers . Solid culture antigens yield markedly lower titers compared to cell culture antigens.
ELISA and immunoblot
These tests use whole cell extract antigens or enriched outer membrane preparations of Bartonella. Antigens obtained from cell cultures have a higher sensitivity than preparations of Bartonella cultured on conventional solid culture media. ELISAs have generally been less sensitive for IgG and IgM detection than the IIFT, and very little objectionable performance data is available /, /. Commercially available immunoblots are very hard to find and have not been sufficiently evaluated in respect of clear, sensitive and specific criteria of interpretation and diagnostically relevant immunodominant proteins /, /.
Serum: 1–2 mL
Interpretation of serological test results
Most patients show seroconversion about 1–2 weeks after the onset of clinical symptoms. The specific IgM values reach their maximum after about 4 weeks and then decrease to below the detection limit within approximately 100 days. Specific IgG is detectable practically at the same time, reaches markedly higher values than IgM 7–8 weeks post infection, then decreases continuously and may persist at low titers for months (or years in some cases) post infection .
The formation of specific IgA antibodies is also an indication of a present or recently resolved infection. Depending on the duration of the infection and the course it takes (acute, subacute, chronic), positive findings of specific IgG and IgA in combination with IgM detection point to an infection. IgM may not be detectable in chronic courses of infection.
The IgM titers determined by IIFT are usually lower than the IgG and IgA titers. However, the interpretation of serological IgG detection (and not IgM and IgA detection) is currently the only serological interpretation established in routine diagnostics /, /.
- IgG titers above 64–128 in the IIFT are considered to be positive and indicate a present or resolved Bartonella infection
- IgG titers > 256 are diagnostically significant and usually indicate the presence of an infection at a positive predictive value of 90–100% /, /
- The titers of patients with clinical symptoms may be even higher (> 2048)
- A 4 fold increase in titer in parallel tested monitoring samples is considered to be diagnostic proof
- Seronegative findings in clinically and microbiologically confirmed bartonellosis have also been described.
Comments and problems regarding serology
Positive results in bartonellosis serology (including IgG detection) must generally be interpreted with care and always in the clinical context . The various Bartonella spp., especially B. henselae, B. quintana and B. bacilliformis, display marked mutual cross reactivity. Serological differentiation is only possible to some extent in the MIFT by parallel determined titers or by cross absorption .
False positive response due to cross reactivity with Treponema pallidum, Chlamydia, Mycoplasma, Coxiella, Ehrlichia, Anaplasma, Bordetella pertussis, Francisella tularensis and Toxoplasma gondii have been reported /, , , /.
Well evaluated commercial test kits are not available. Specialized laboratories have used molecular biological detection methods such as PCR and DNA hybridization for direct detection of Bartonella in clinical specimens (blood, CSF, biopsy material) under scientific research conditions.
For instance, species specific sequences of the 16S-rRNA gene, 16S–23S-inter genic transcribed spacer region (ITS), rpoB gene, htrA gene and citrate synthetase (gltA) gene have been described as diagnostic target sequences /, /. However, clinical evaluation studies performed by research laboratories have demonstrated significant variations in the detection limit of PCR depending on the patient cohort and method.
7. Eskow E, Rao RV, Mordechai E. Concurrent infection of the central nervous system by B. burgdorferi and Bartonella henselae: evidence for a novel tick-borne disease complex. Arch Neurol 2001; 58: 1357–63.
11. Slater LN, Welch DF. Bartonella species, including cat-scratch disease. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. Mandell GL, Bennett JE, Dolin R (eds). Philadelphia; Churchill Livingstone 2000: 244–56.
Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Borreliosis is a disease caused by spiral shaped mobile bacteria belonging to the family Spirochaetaceae. A relationship to treponemes and leptospiras therefore exists which is relevant for microbiological diagnosis. The genus Borrelia includes the so called relapsing fever, as well as genetically closely related members of the Borrelia burgdorferi complex, including the causative agents of Lyme borreliosis.
B. recurrentis plays a role worldwide as the causative agent of the epidemic louse borne relapsing fever. Moreover, various soft ticks transmit the endemic relapsing fever caused by B. caucasica, B. hispanica and B. hermsii, for example in Eastern Europe and the Mediterranean area /, /. The suspected clinical diagnosis of relapsing fever is usually confirmed by microscopic or molecular biological detection of the pathogen in the blood.
In the case of Lyme borreliosis as the most significant infectious disease transmitted by vectors, there are still major problems in both clinical and direct and indirect laboratory diagnosis in Central Europe. Hence, it is only the diagnostic possibilities in the case of suspected Lyme borreliosis that will be discussed in this chapter.
The causative agent of Lyme borreliosis was identified in the USA by W. Burgdorfer in 1982 and named B. burgdorferi according to its discoverer /, /. Sequence differences of the 5S–23S inter genic spacer region of the rRNA operon allow classification of the B. burgdorferi complex into different closely related genospecies () /, /. The B. burgdorferi complex currently comprises 18 species. Human pathogenicity has been discussed for B. valaisiana and B. bissetii. Human pathogenicity has only been confirmed to date for B. burgdorferi, B. afzelii, B. spielmanii, B. garinii and B. bavariensis .
The pathogens display some organotropism:
- B. burgdorferi for joint manifestation
- B. afzelii and B. spielmanii for skin manifestation
- B. garinii and B. bavariensis for the nervous system.
The spiral shaped bacteria are 4–30 μm long and 0.2–0.3 μm in diameter. The protoplasmic cylinder of the bacterial cell is surrounded by 7–11 flagellae . On the external surface, there is an outer membrane in which various immunodominant membraneous proteins (outer surface proteins: Osp A to Osp F) have been identified.
The human pathogenic species belonging to the B. burgdorferi complex show marked genetic and phenotypical heterogeneity, which may in some cases make microbiological diagnosis more difficult. Borrelia isolates can be typed by molecular biological methods (e.g., DNA-DNA hybridization, plasmid analysis, restriction fragment length polymorphism analysis) and by OspA specific monoclonal antibodies /, /. The currently most favored method to characterize new species is multi locus sequence typing (MLST) /, /.
Five human pathogenic genospecies of the B. burgdorferi complex occur in Europe (). In the USA, only B. burgdorferi sensu strictu is found. The natural Borrelia reservoir is predominantly in small rodents, but also wild and domestic animals. In Central and Eastern Europe, the transmission of Borrelia to humans occurs via ticks of the genus Ixodes (I. ricinus, I. persulcatus). In Germany, there is a risk of Borrelia infection in the case of a tick bite since approximately 20% of adult ticks, 10% of the nymphal stage ticks and 1% of the larvae are infected with Borrelia.
The high prevalence of Borrelia antibodies in the population (Europe: 1–36%; Germany: 3–17%) is evidence of a regionally varying risk of infection . However, only in about 1% of tick bites do symptoms occur consistent with the disease .
The incidence of infection is dependent on the biological activity of the ticks and reaches a maximum during the warmer seasons of the year (April to October). In Europe, the incidence of Lyme borreliosis varies between 155/100,000 in Slovenia and 0.6/100,000 in Ireland . In Germany, the estimation of 30–60,000 incident cases per year is based on older studies /, /. Mandatory reporting of new Lyme borreliosis cases has been established in the six new federal states of Germany. Up to 5,221 new cases have been reported for these states annually by the local public health authorities /, , /. However, newer studies have suggested up to 220,000 incident cases of Lyme borreliosis in Germany per year .About 60.000 cases are repoerted in2017 in the USA /.
Days to several months, in some cases even years.
Most infections (95%) with B. burgdorferi following a tick bite are clinically inapparent. Clinically manifest Lyme borreliosis, like syphilis, is a multiple system disease progressing in stages and primarily involving the skin, joints and nervous system ().
Early manifestation (localized: stage I)
Migrating erythema is the key symptom of this clinical stage. Following an incubation period ranging from days to weeks, stage I can manifest itself in the form of a localized, centrifugally spreading skin disease (erythema migrans).
Early manifestation (disseminated: stage II)
Within a period of days to months following primary infection, the pathogen can disseminate to various organs, especially the nervous system.
This is the most common type of disease manifestation, thus representing the key syndrome of clinical stage II. This meningoradiculoneuritis is characterized by burning, radicular pain (often occurring at night) without or with paralysis. Special types of stage II include facial palsy, multiple erythema, Lyme carditis and borrelial lymphocytoma. Facial palsy can present unilaterally or (more rarely) bilaterally and may manifest very early, especially in children, in some cases a few days following a tick bite without preceding erythema .
Late manifestation (disseminated, more than 6 months duration: stage III)
In the case of persistence of the pathogen, the disease may persist for months to years. Acrodermatitis chronica atrophicans (ACA) and Lyme arthritis are the most common disease manifestations during this clinical stage. ACA predominantly affects the lower extremities. After initially extensive inflammatory reactions, marked skin atrophy is observed during the final stage of ACA. Lyme arthritis predominantly affects the knee joint. Late neurological manifestation (encephalitis) is very rare /, /.
The natural course of Borrelia infection can vary significantly and is usually self limiting except for late manifestation (duration of more than 6 months). In most cases, a tick bite with subclinical infection will not lead to disease. Lyme borreliosis does not always progress from one clinical stage to the next. Even without antibiotic treatment, development of disseminated, early or late manifestations only occurs in about 20% of patients with erythema migrans. On the other hand, preceding erythema migrans is only observed in approximately 30% of patients with the Bannwarth syndrome /, /. Furthermore, the clinical diagnosis of Lyme borreliosis is complicated by the fact that symptoms of disease are frequently unspecific and a tick bite is remembered by only about 50% of patients /, , /. Because of these special difficulties surrounding the clinical diagnosis, the laboratory investigations in specific indications are of particular diagnostic importance () .
Nationwide mandatory reporting of Lyme borreliosis (B. burgdorferi) according to the German Infection Protection Act (IfSG) has not been established in Germany to date. However, according to Section 7 IfSG (laboratory), mandatory reporting applies to infection with B. recurrentis.
For the diagnosis of Lyme borreliosis, relevant clinical signs must be determined . Serology is only indicated in the presence of appropriate clinical symptoms. Confirmatory testing is only helpful after 2–3 weeks following possible infection or exposure. Substantiated indications for serological testing in suspected Lyme borreliosis are summarized in .
New immunoassays (ELISA and CLIA) using new antigens (VlsE, in particular) have improved diagnostic sensitivity and specificity. Recent serological research addresses whether one step tests are of sufficient sensitivity and specificity to replace the widely used two step approach (confirmation of positive screening results by subsequent immunoblots) . Relevant studies have demonstrated, however, that no single serological test used in Lyme borreliosis diagnosis has adequate diagnostic sensitivity and specificity /, , , , /. Therefore, most scientific associations recommend a screening test of high detection limit (ELISA) combined with a confirmatory test of high analytical specificity (immunoblot) /, , , /. Refer to:
ELISA and CLIA are both used as screening tests because they are suited for the polyvalent, selective and quantitative determination of IgG and IgM antibodies. Other serological tests are no longer appropriate /, /. In the assays, the patient’s serum is absorbed by T. phagedenis to increase the specificity of the screening tests. For selective detection of IgM antibodies, treatment of the serum with a rheumatoid factor (RF)-absorbent is necessary to avoid false positive results due to the RF. In many cases, recombinant test formats do not apply such pretreatment.
The antigen fractions for specific antigen detection in the ELISA and CLIA consist of ultrasonicate or borrelial cell extract (whole cell ELISA, extract antigen ELISA) or of recombinantly produced, purified proteins capable of stimulating a specific immune response in vivo, for example Osp17/p18 (DbpA), flagellin (p41), p39, p58, Osp C, VlsE (selective ELISA). Hybrid tests combining both types of antigen preparation are also available, for example enrichment of conventional antigen extracts with recombinant OspC or VlsE.
Studies on whether immunoassays, which use highly specific antigens (VlsE) or peptides (C6), have adequate diagnostic sensitivity and specificity to replace standard two-tiered serodiagnostic testing have left some questions unanswered do date. One reason is the presence of several human pathogenic Borrelia genospecies in Europe and the resulting heterogeneity of immunodominant antigens, making test approaches of this kind more complex /, , /.
CLIA and ELISA methods only differ in the type of detection they apply, but generally not in the antigen preparation they use. The diagnostic specificity of advanced immunoassays is 80–90%. Such assays are standard in routine serological laboratory testing for Borrelia because of automated reading, accuracy of measurement and ease of handling. The results of the available immunoassays can vary significantly depending on antigen composition and assay manufacturer. Hence, the results of different tests and/or different laboratories are only comparable to a limited extent /, , /, especially since almost no parallel testing with archived sera is performed.
Besides conventional whole cell lysate immunoblots (Western blots), immunoblots and line immunoblots using recombinantly produced antigens (recombinant blot) are employed as confirmatory tests. Hybrid tests combining both types of antigen preparation are also used to an increasing extent.
Whole cell lysate immunoblot
Whole cell lysate or whole cell antigen immunoblots use Borrelia ultrasonicate as antigen preparation (). Thus, all Borrelia antigens (i.e., specific immunodominant and unspecific proteins) are available for antibody detection. Optimal expression of the specific immunodominant antigens of the employed Borrelia strain is essential for the quality of such a blot.
Criteria for the interpretation of whole cell antigen immunoblots employing the strain PKo have been compiled under standardized conditions (). It is a disadvantage that certain important, new immunodominant antigens such as VlsE are only expressed in vivo and, therefore, not available under conventional conditions.
Hence, many manufacturers of conventional lysate blots selectively add certain recombinant proteins (VlsE, OspC) (hybrid tests) to close these diagnostic gaps. The use of lot specific evaluation templates and antigen localization verification with monoclonal antibodies in whole cell lysate immunoblots by the manufacturer are essential for diagnostic quality .
Highly specific immunoblots employing antigen preparations from recombinantly produced proteins, for example Osp17/p18 (DbpA), VlsE, p41 (flagellin, internal fragment), OspC, OspA, p39/BmpA, p41/i (flagellin), p83/100 for the detection of Borrelia antibodies are used to an increasing extent (selective blot) .
In addition, specific antigens of different genospecies can be used in the same test mixture to account for the immunological variability of the different borreliae. The diagnostic sensitivity of the recombinant test depends on the type and amount of antigens used.
The colored bands of the antigen pattern are more easily assigned to defined antigens in the recombinant immunoblot than in the whole cell lysate blot. The recombinant immunoblot is, therefore, recommended especially for laboratories with little experience in this serodiagnostic field.
According to studies on external quality control, inter laboratory inconsistencies became obvious from the variability of test results of the commercially available assays and the low recovery rate of specific immunoblot bands /, /. Therefore, the criteria presented in are only intended for orientation and cannot generally be applied to the evaluation of the currently available, differently designed commercial test kits with their array of different antigen preparations and evaluation criteria . There are, in particular, no recommendations for the standard handling of borderline findings.
In principle, line blots represent a modification of conventional recombinant immunoblots in respect of the production and processing of the antigens used. However, no electrophoretic separation step is necessary prior to the blotting procedure. For such tests, the individual antigens are gently and selectively sprayed directly onto the carrier membrane without previous denaturation (). Thus, several homologous and immunodominant antigens from different isolates of different genospecies with identical or similar molecular weight can be combined in diagnostic groups on a single strip. The line immunoblot achieves high levels of diagnostic sensitivity and specificity for the detection of Borrelia specific antibodies . The line immunoblot method has the advantage of revealing differences in the diagnostic reactivity of sera with highly specific immunodominant antigens (e.g., VlsE, OspC) from different strains /, , /.
Advantages of immunoblot and line immunoblot
Inter laboratory comparative studies point out the limitations of commercially manufactured conventional and recombinant immunoblots /, , /. Based on correct diagnostic indication, the immunoblot continues to be one of the most specific diagnostic tests for the detection of Lyme borreliosis antibodies. Immunoblots and/or line immunoblots using recombinant antigens, in particular, are especially suited and highly conclusive for most diagnostic concerns /, /. Taking into account the number and kind of Borrelia specific bands, these assays also provide information as to the quality and duration of the immune response, thus allowing the result to be classified more accurately in the clinical context /, , /.
The identification of blot bands via lot specific evaluation templates and their adequate weighting as well as the parallel testing of positive controls and cutoff controls are essential to avoid false positive findings and confusion of antigens /, /.
The introduction of novel multiple parameter test systems based on single antigen Luminex technology allows to simultaneously perform analyses of a large number of analytes such as antigens in the same micro titer sample well in one and the same measuring process. This test method is based on tiny, antigen coated polystyrene beads that serve as the solid phase for a variety of detection reactions, similar to the Western blot and ELISA .The resulting multi analyte profiles combine the advantages of immunoblot analyses with the analysis principles of quantifiable immunoassays. Thus, MFIs using single or multiple antigens are diagnostically much more conclusive than conventional ELISAs.
- Serum: 1 mL
- Cerebrospinal fluid (in suspected Lyme neuroborreliosis, parallel to serum sample collection): 1 mL
The individual sensitivities and specificities of different test methods may diverge considerably depending on the test used and the assays of the manufacturers /, , /. Prior to the introduction of a new assay into a laboratory, internal proficiency control is necessary. For this purpose, serum samples from patients with clinically confirmed Lyme borreliosis and serum samples from healthy blood donors (control group) should be used.
In borreliosis serology, test results are reported as negative, positive or borderline (threshold) depending on the manufacturer’s specification. The definition of the borderline value for the ELISA and the assessment of specific bands by different manufacturers in the immunoblot do not yet follow general, standardized guidelines . Moreover, international or national reference preparations or standardized borderline values are not available. Hence, the qualitative and quantitative test results (e.g., U/mL in ELISA, band pattern in immunoblot) are not directly comparable, but depend on the manufacturer. This must be taken into account during monitoring and the interpretation of test results from different laboratories. The significance of changes in test results must always be verified by parallel testing with a previously collected serum sample /, /.
Stage dependent antibody kinetics in Lyme borreliosis
Antibodies to Borrelia antigens usually form 2–6 weeks after the onset of the Borrelia infection. In most cases IgM response precedes and IgG antibody production /, , /. Absence of an IgM response has been reported in some cases. An IgM response may also not occur in reinfection because reinfections are usually associated with significant IgG response without major IgM production. In the early infection stage, the immune response of both immunoglobulin classes is at first directed against a narrow range of Borrelia antigens, especially flagellin (p41), VlsE and OspC. Antibodies to VlsE and OspC are of special diagnostic significance because of their high specificity.
The introduction of the VlsE antigen has provided better diagnostic sensitivity in serodiagnostic testing for Borrelia . If used in early stages of disease manifestation (e.g., erythema migrans, Lyme neuroborreliosis), this antigen achieves a significant high detection rate regarding Borrelia specific IgG antibodies besides IgM antibodies. However, many patients still remain seronegative in the early stage of infection. The positivity rates investigated for various disease manifestations within the scope of studies on seroprevalence are summarized /, , /.
The number of seropositive patients increases as borrelial infection progresses and reaches almost 100% in late disseminated disease manifestations. In these cases, the immune response is directed against a wide range of Borrelia specific antigens.
Antibodies against antigens such as the p83/100 protein, p39 (BmpA) and Osp17/p18 (DbpA) are of particularly high diagnostic significance during the late stage. In contrast, antibodies against OspA, which is also specific, are rare and are observed, in particular, in patients with Lyme arthritis /, , /.
As with other infectious diseases, immunocomprimised patients may show delayed or no immune response. In all, however, seronegative Lyme borreliosis is extremely rare in immunocompetent patients, except in the very early course of the disease, but should be borne in mind in patients with a short duration of the disease. In such cases, direct detection of the pathogen should always be considered.
Interpretation in the case of a negative screening test
In the case of a negative screening test result, no further investigations are needed. However, a negative serological finding does not exclude Lyme borreliosis. Patients during clinical stages I and II often present negative test results. If infection continues to be suspected, the serological diagnosis can be repeated after 2–6 weeks. In individual cases, measurable immune response may have been suppressed by early initiation of antibiotic treatment causing seroconversion for IgG and IgM not to occur, with an abrogative effect on antibody response (absence of IgG seroconversion) /, , /.
Interpretation in the case of a positive screening test
A borderline or positive test result must be interpreted with care because false positive findings occur in cases of syphilis, other bacterial infections, EBV infection, despite absorption of cross reacting antibodies by T. phagedenis . In case of a borderline or positive result of the Lyme borreliosis screening test, a Treponema pallidum latex agglutination assay (TPPA) should be performed () to exclude present or resolved syphilis as a possible cause of false positive borreliosis serology /, , /. However, a confirmed, positive Borrelia test result gives strong evidence of Borrelia infection. If this serological finding unambiguously coincides with the suspected clinical diagnosis, further laboratory investigations are not required. In all other cases, a confirmatory test must be performed.
Interpretation in the case of a negative confirmatory test
A negative confirmatory test (immunoblot or line immunoblot) implies that the screening test provided a false positive result. Borrelia serology is to be reported as negative. Further investigations are usually not necessary. It must be kept in mind, however, that early infection stages (erythema migrans, Lyme neuroborreliosis) may produce false negative findings.
Interpretation in the case of a borderline confirmatory test
Borderline values in the immunoblot are particularly difficult to interpret because of the absence of the above mentioned criteria for the detection of Borrelia infection and the diagnosis of the disease. In these cases, performing a back-up test (e.g., verification of a whole cell antigen blot result by recombinant immunoblot) can be helpful ().
If screening and back-up tests provide divergent results in the presence of a borderline immunoblot result, this is strong evidence of false positive serodiagnosis.
In the case of a positive or borderline back-up test, the serological finding is consistent with Lyme borreliosis during its early stage. However, such results almost always speak against an existing, persistent infection or late manifestation of Lyme borreliosis. In suspected recent infection, it is recommended, depending on the clinical diagnosis, that the serological tests be repeated after 3–6 weeks. If the finding does not change, the presence of active Lyme borreliosis is unlikely /, , /.
Interpretation in the case of a positive confirmatory test
- Class of reactive antibodies (IgM and/or IgG)
- Intensity of the bands
- Number of bands (antigens) recognized and their molecular weight.
Suspected early or late stage of Lyme borreliosis: based on the following findings taking into account the clinical presentation:
- The isolated detection of IgM antibodies against p41 or OspC is an important marker of the early stage of Lyme borreliosis /, /. Although the isolated finding of the p41 band is no proof of Lyme borreliosis because the antigen is similar to the flagellin proteins of other bacteria, it is compatible with the clinical diagnosis of early Lyme borreliosis if corresponding clinical information is additionally present.
- By detecting antibodies against p41 or p 41/i, a Borrelia specific fragment of flagellin, and against VlsE or OspC, the diagnosis of early Lyme borreliosis is much stronger; at least one of the bands should be strongly present for confirmation of the diagnosis .
- The clinical diagnosis of late stage disease is only supported by laboratory testing if several bands of intensive staining are recognizable across a wide range of molecular weights. The p83/100 and Osp17/p18 bands are of particular diagnostic relevance because of their Borrelia specificity. In combination with a wide band pattern, they point to a late phase of immune response /, /.
For the diagnostic assessment of an immunoblot result as outlined above, detailed clinical information is essential:
- Conclusions as to the need for treatment cannot be made merely based on a positive immunoblot and ELISA because antibodies (including IgM class) may persist for a long time period (months to years) after resolved infection and even after treatment of the disease .
- The detection of specific antibodies by itself does not automatically confirm clinically manifest infection. Confirmation is provided by merging the result of the serodiagnostic test with the suspected clinical diagnosis, especially since an activity marker (similar to the VDRL reaction in syphilis serology) has to date not been available.
- Any reinfections can only be unambiguously diagnosed by verifying significant changes in the serological results by parallel testing with a previously collected serum sample
- In almost all cases, the isolated positive detection of IgM antibodies speaks against late manifestation of Lyme borreliosis /, , /.
Conventional antibody testing
Antibody production in the central nervous system is only detectable by parallel and quantitative testing of cerebrospinal fluid (CSF) and serum for Borrelia specific antibodies. For this purpose, the serum and CSF IgG concentration or albumin concentration is interrelated with the concentration of the pathogen-specific antibodies (IgG, IgM) determined in serum and CSF /, , /. This data is used to determine the CSF-serum index (CSI) according to the following formula:
Unless otherwise evaluated depending on the used assay, CSI values ≥ 2 in the ELISA corroborate intrathecal antibody production due to Lyme neuroborreliosis /, , /. CSI values of 1.5–1.9 are considered to be borderline results.
The diagnostic conclusiveness of positive findings must always be assessed in the context of other protein analysis and CSF serology data.
Lyme neuroborreliosis can be excluded by adequate tests if, in the absence of pleocytosis and the presence of normal CSF protein concentrations, neurological symptoms persist for more than 2 months /, , /.
CXCL13 as Lyme neuroborreliosis marker
The chemokine CXCL13 may be a useful parameter in early diagnosis of Lyme neuroborreliosis. Among other effects, this chemokine attracts B lymphocytes to the central nervous system /, /. The presence of B lymphocytes in CSF in the case of Lyme borreliosis (as well as in neurosyphilis) is an already established phenomenon. Recent studies have suggested that CXCL13 is reliably increased in the CSF of patients with well defined early Lyme neuroborreliosis and can precede specific antibody formation in the CSF /, /. Some studies have reported that CXCL13 shows high diagnostic sensitivity in early stages of Lyme neuroborreliosis when the Borrelia specific antibody index in CSF is still negative . The diagnostic sensitivity and specificity of CXCL13 are specified as 94–100% and 63–96%, respectively /, /. Moreover, the CSF CXCL13 concentration in treated patients decreases relatively fast and is, therefore, suggested as a potential biomarker for treatment response .
Information on the diagnostic specificity in respect of other infectious and inflammatory CNS disorders is still insufficient. It has been reported that elevated CXCL13 concentrations are also detectable in infections with related pathogens such as Treponema pallidum. However, the CXCL13 test is not yet available as a routine diagnostic tool and has not been sufficiently standardized to date .
The detection limit of PCR tests for Borrelia corresponds to that of detection by culturing /, , /. Patients with Lyme arthritis are an exception because, in their case, the PCR tests achieve higher detection limit than culturing and, using synovial fluid or joint biopsy material /, /.
The study results regarding external quality control for these diagnostic techniques are too heterogeneous to recommend a particular method . According to expert recommendation, the PCR should employ at least two different DNA target sequences to increase diagnostic reliability . Molecular detection in urine is not recommended because of inadequate analytical specificity /, /. Positive results should be confirmed by amplificate sequencing and identification of the detected genospecies .
According to the guidelines of the German Medical Association, the participation in inter laboratory proficiency testing in Borrelia serology twice a year is mandatory. The molecular biological detection of Borrelia burgdorferi is an option offered as part of the inter laboratory proficiency testing scheme on bacterial genome detection. The results of external quality control within the scope of these proficiency testing schemes demonstrate significant heterogeneity of the assays currently available on the market /, , /. provides a summary of the rates of passing for common test systems based on meta-analytical data from 2006–2008 as well as the clinical significance of proficiency test samples for serodiagnostic proficiency testing.
Non-recommended diagnostic tests
Besides the standard tests for Lyme borreliosis, other, poorly evaluated or unreliable investigation methods are also applied. They refer to:
- The detection of cystic forms of Borrelia
- CD57+/CD3– lymphocyte subpopulation tests
- The lymphocyte transformation test
- The detection of circulating immunocomplexes or the visual contrast sensitivity test (VCS).
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7. Wilske B, Preac-Mursic V, Göbel UB, Graf B, Jauris S, Soutchek E, et al. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J. Clin Microbiol 1993; 31: 340–50.
11. Poggensee G, Fingerle V, Hunfeld KP, Kraiczy P, Krause A, Matuschka FR, et al. Lyme borreliosis: research gaps and research approaches. Results from an interdisciplinary expert meeting at the Robert Koch Institute. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2008; 51:1329–39.
14. Ang CW, Notermans DW, Hommes M, Simoons-Smit AM, Herremanns T. Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots. Eur J Clin Microbiol Infect Dis 2011; 30: 1035–7.
15. Goettner G, Schulte-Spechtel U, Hillermann R, Liegl G, Wilske B, Fingerle V. Improvement of Lyme borreliosis serodiagnosis by a newly developed recombinant immunoglobulin G (IgG) and IgM line immunoblot assay and addition of VlsE and DbpA homologues. J Clin Microbiol 2005; 43: 3602–9.
17. Branda JA, Aguero-Rosenfeld ME, Ferraro MJ, Johnson BJ, Wormser GP, Steere AC. 2-tiered antibody testing for early and late Lyme disease using only an immunoglobulin G blot with the addition of a VlsE band as the second-tier test. Clin Infect Dis 2010; 50: 20–6.
18. Hunfeld KP, Stanek G, Straube E, Hagedorn HJ, Schörner C, Mühlschlegel F, Brade. In: Quality of Lyme disease serology. Lessons from the German Proficiency Testing Program 1999–2001. A preliminary report. Wien Klin Wochenschr 1997; 114: 591–600.
19. Hauser U, Lehnert G, Lobentanzer R, Wilske B. Interpretation criteria for standardized Western blots for three European species of Borrelia burgdorferi sensu lato. J Clin Microbiol 1997; 35: 1433–44.
20. Porwancher RB, Hagerty CG, Fan J, Landsberg L, Johnson BJ, Kopnitsky M, et al. Multiplex immunoassay for Lyme disease using VlsE1-IgG and pepC10-IgM antibodies: improving test performance through bioinformatics. Clin Vaccine Immunol 2011; 18: 851–9.
28. Reischl, U., W. Schneider, M. Maaß, E. Straube, V. Fingerle, and E. Jacobs. Bakteriengenom-Nachweis PCR/ NAT: Auswertung des Ringversuchs April 2010 von INSTAND e.V. zur externen Qualitätskontrolle molekularbiologischer Nachweisverfahren in der bakteriologischen Diagnostik. Der Mikrobiologe 2010; 20: 181–97.
Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Human brucellosis manifests as feverish acute, subacute or chronic, systemic bacterial infection. Brucellosis is primarily zoonotic, becoming zooanthroponotic by direct contact with infected wild or domestic animals or by the consumption of contaminated raw milk products .
Brucella species are small (0.6 × 0.7 μm), nonmotile, Gram negative, rod shaped bacteria. These pathogens are capnophilic, have lipopolysaccharides in their cell wall and are facultative intracellular bacteria capable of surviving within phagocytic cells .
Molecular genetic studies have suggested that the species described to date should be considered to be biotypes of a single genospecies, B. melitensis . Infected wild and domestic animals serve as pathogen reservoir. The pathogens can be cultured on special culture media; primary cultures must be incubated for up to 21 days. Because the bacteria are highly infectious, the handling of specimens and cultures often result in laboratory infections. Therefore, specific handling of Brucella is only permitted in laboratories under laboratory biosafety level L 3 .
Detection of the pathogen is always attempted in the acute stage of brucellosis. Primary cultures become positive after a week. Therefore, sufficiently long incubation of at least 3 weeks, especially of blood cultures, must be ensured.
Brucella are invasive and can infect humans by penetrating intact skin or mucosa and by entering the lungs following aerosol formation. Usually, humans become infected through contact with infected domestic animals or their excretions. Hence, individuals from certain occupational groups such as shepherds, farmers, animal caretakers, slaughterhouse workers, animal breeders, veterinarians, dairy farm workers, personnel in dairy companies and laboratories are especially at risk /, /. Infections among this category of persons are recognized as occupational disease. Inadequately heated milk or dairy products or foods derived from infected animals are another source of infection /, /. This route of infection is common in the Mediterranean area and Middle East and usually refers to patients with B. melitensis infections. The disease remains the world’s most common bacterial zoonosis, with over half a million new cases annually and prevalence rates in some countries exceeding 10 cases per 100,000 population /, , /. The animal stocks in Germany are considered to be free from Brucella. Therefore, human infections are in most cases acquired by travelers abroad or associated with food, such as imported unpasteurized cheese. In 2010, 22 cases of brucellosis were reported in Germany .
Usually 1–3 weeks; up to 3 months in the case of B. melitensis.
The disease starts with unspecific prodromic syndromes such as general malaise and melalgia and can then take an acute, subacute or chronic course. Patients may present with conjunctivitis, angina, bronchitis and skin lesions at the site of invasion (e.g., hands). The acute course of the disease is characterized by remittent fever with typical peaks > 39 °C reached in the evenings and paradoxically associated with relative bradycardia. Clinical manifestations are manifold /, , , , /. There are two forms:
- Bang’s disease caused by B. abortus which is a masked illness with intermittent fever attacks, splenomegaly and otherwise non characteristic clinical symptoms
- Malta fever caused by B. melitensis which is similar to typhus and takes an acute course with significant subjective symptoms, weight loss and typical undulant fever.
Acute brucellosis can be resolved spontaneously or develop towards chronicity and persistence capable of affecting any organ system. In this case, the facultative intracellular pathogens cause granulomatous inflammation. Organ manifestations typically include hepatosplenomegaly, lymph node hyperplasia, sacroiliitis, osteoarthritis, osteomyelitis and possible central nervous system involvement (neurasthenic syndrome, meningoencephalitis) /, , , , /. In children with non characteristic generalized symptoms and intermittent or undulant fever, brucellosis should be considered. Routine laboratory tests often detect mildly elevated LD and ALP and, less frequently, ALT and GGT serum levels. White blood cell counts are usually normal.
According to Article 7 of the German Infection Protection Act (IfSG), direct or indirect evidence of brucellosis is subject to mandatory reporting. Brucella infections among the above mentioned occupational groups must be reported to accident insurers for recognition as occupational disease /, /.
In the absence of a characteristic profile of the disease, diagnosis often relies on specific antibody detection. Agglutination tests are based on the detection of antibodies against lipopolysaccharide antigens. These antibodies may persist for a long time after resolved infection. Hence, the conclusiveness of serological tests in endemic regions is limited because of the high prevalence of infection. It is, therefore, important to evaluate serological test tools in the context of local epidemiological conditions /, , /. Approximately 85% of brucellosis cases are diagnosed by serological testing .
Clinically suspected acute or chronic brucellosis always warrants testing for specific antibodies 7–10 days after the onset of clinical symptoms.
Micro agglutination test (Card test)
The brucellosis card test uses the Brucella abortus strain 1119-3 (USDA) as antigen suspension (8%), stained with Rose Bengal dye (pH 3.65). Antigens to B. melitensis and B. abortus stained with Rose Bengal are commercially available for the sensitive card test. Positive results must be confirmed by the tube agglutination test (Widal) or other serological test methods /, /.
Widal type tube agglutination test
Inactivated Brucella suspensions are incubated with increasing dilutions of patient serum. The test can be performed as conventional Widal type tube agglutination of micro titer plate test with stained antigen and is considered to be the standard test for the indirect detection of Brucella. The diagnostic sensitivity is up to 95% in patients with clinically and culturally confirmed brucellosis ; the duration of the test is 2 days. The test detects IgG, IgA and IgM antibodies.
Complement fixation (CF) test
CF has a lower detection limit than the Widal reaction.
Fluorescence polarization immunoassay (FPA)
The FPA functions like a simplified ELISA, utilizing a Brucella polysaccharide antigen which is attached to a fluorescence labeled detector. Diagnostic sensitivity of the FPA is 96% compared to culture confirmed brucellosis. The diagnostic specificity is about 98%.
Brucella lateral flow assay (LFA)
The LFA is an immunochromatographic assay for specific IgM and IgG detection. The test can be performed as POCT without elaborate laboratory infrastructure. A drop of blood obtained by a finger prick is applied to the application pad. Any antibodies present are detected by immunochromatographic technique utilizing gold-labeled monoclonal antibodies. This assay achieves a diagnostic sensitivity and specificity above 95%. The LFA is suited for antibody detection at all stages of the disease.
Enzyme-linked immunosorbent assay (ELISA)
ELISA employing whole cell antigen is increasingly used as a sensitive and specific method to perform a class-specific analysis of the immune response.
The determination of IgG and IgM antibodies facilitates the differentiation between recent or past infections, also in low titer sera . Evaluation studies have specified diagnostic sensitivities of up to 97% and specificities of up to 97% for the combined detection of IgG and IgM /, , /. However, the quality of the commercially available test systems varies.
Serum: 2–4 mL
During the course of immune response, IgM antibodies are the first to become detectable (approximately 1 week after infection), followed by IgG antibodies in the second week of the disease. The maximum titer is reached approximately 4 weeks after the onset of the disease. Patients in the early stage of the disease can still be seronegative. Follow-up testing should be performed after about 2 weeks for verification. Successful therapy is usually indicated by a significant decrease in antibody titer within a few months /, /. Such investigations should use parallel testing with the same test system.
A rapid decrease in IgG antibodies is considered to be a good prognostic indicator of successful therapy. Persistently elevated IgG antibodies after treatment should, however, trigger close monitoring for recurrence /, /. The decline in specific antibody concentrations is slower in patients with local complications. In many cases, relapse is indicated by another increase in IgG specific antibodies. A number of efficiently treated patients still present high antibody titers for months or even years despite negative blood culture and the absence of clinical symptoms /, , , /.
Under study conditions, persistence of IgM antibodies was found in 25% and IgG specific antibodies were detected in almost 90% of the patients 12 months after efficient treatment. Hence, serology should be used with care for treatment monitoring. It is therefore not always possible in endemic regions to adequately distinguish between patients with an active infection and patients with relapse, pathogen persistence or resolved infection /, /.
Interpretation of special tests
The CF, dipstick test, FPA, LFA and Widal test are genus specific and do not allow any conclusion as to the etiologic pathogen.
Approximately 98% of patients have titers ≥ 160 at the time of clinical diagnosis /, , /. The titers reach a maximum in the first weeks of the disease and then decline slowly, sometimes over the course of years. A single titer result does not allow precise conclusions as to the time of infection.
Micro agglutination test
Brucellosis must be suspected in the case of titers ≥ 160 in a single sample. Seroconversion or a fourfold increase in titer after the onset of clinical symptoms, detected by parallel testing with a previously collected serum sample, are considered to be unequivocal evidence of infection. Co agglutination of other Brucella species in lower serum dilutions will always be detected and must not be taken into account /, /. A recent evaluation study states a diagnostic sensitivity of 84.6% for agglutination tests in patients with culturally confirmed brucellosis. At a threshold value of 1 : 160, the sensitivity was 64.7%, but at a threshold value of 1 : 320, it was only 47.1%.
Complement fixation test
The test usually becomes positive after week 4 of the disease (titers > 5). Low positive titers (20–80) persist in chronic infections or indicate the presence of cross reactions. However, in the case of justified clinically and anamnestically suspected infection in regions with a low incidence rate of brucellosis, titers ≥ 20 warrant further diagnostic testing and titer monitoring.
Diagnostic testing should always comprise IgG and IgM antibodies. In regions with a low incidence rate, each positive result should be interpreted as abnormal and investigated by further testing. Some authors suggest a lower specificity of ELISA compared to the agglutination test .
Comments and problems regarding serology
Card test and Widal test
False negative results are frequently encountered due to a prozone phenomenon induced by incomplete antibodies or very high antibody titers. Therefore, at least two serum dilutions should be examined. To detect a prozone phenomenon, the series dilution should be above 1 : 360 /, /.
In brucellosis, incomplete (blocking) antibodies are in many cases detected by using low to medium serum dilutions. These antibodies block Brucella antigens but do not cause visible agglutination. A brucellosis Coombs test is employed if brucellosis is suspected despite a negative Widal test /, /.
Brucellosis Coombs test
For the Coombs test, the Widal test reagent mixture with a serum dilution of 1 : 80 or 1 : 100 is centrifuged and the sediment composed of immune complexes, consisting of Brucella bound to incomplete antibodies, is washed by centrifugation. The washed sediment is resuspended and Coombs serum is added. In the presence of incomplete Brucella antibodies, agglutination occurs. Borderline titer ≥ 80.
False positive Widal titers are found in infections with Yersinia enterocolitica O9, in cases of tularemia, cholera and after cholera vaccination. Any positive response in serological tests for Brucella must therefore give rise to further serological tests for Yersinia and Francisella /, , , /.
Due to the heterogeneous and poorly specific clinical symptomatology and the risk of infection in laboratory workers associated with handling Brucella microorganisms, it has been attempted to establish molecular biological detection methods /, , , /. Scientific studies are under way to evaluate which specimen, serum or whole blood, is more suited for the molecular biological detection by Brucella specific PCR. For instance, some PCR test methods utilize the BCSP31 gene or conserved sequences of the 16S rRNA gene of Brucella /, /.
By employing conventional multiplex techniques, newer PCR methods are able to detect all Brucella species and the vaccine strain Brucella abortus RW 51, Brucella abortus S 19 and Brucella melitensis Rev1 /, , , /. In a worldwide multicenter study, the Bruce-ladder PCR assay proved to be suited for the rapid identification of Brucella isolates from clinical samples in the microbiological routine laboratory . The detection limit of some real time PCR assays is approximately 5 bacteria/test mixture. Under the specific conditions of research laboratories, these assays achieve high sensitivities and specificities (diagnostic sensitivity 94.9–100%; specificity 96.5–100%) /, , , /. The diagnostic sensitivity can be increased further by running several test mixtures per sample or by testing for multicopy genes (e.g., the insertion element IS711) of Brucella . However, such methods are not established and proven and should only be employed by special laboratories.
The PCR assay is not suited for monitoring treatment response because clinically efficiently treated patients may remain positive for Brucella DNA for months (or even years). The clinical significance of this phenomenon has not been elucidated to date. Hence, negative PCR results during follow-up monitoring suggest positive treatment response, while the persistence of positive results requires close clinical monitoring regarding possible recurrence or pathogen persistence .
1. Musallam II, Abo-Shehada MN, Hegazy YM, Holt HR, Guitan FJ. Systematic review of brucellosis in the Middle East: disease frequency in ruminants and humans and risk factors for human infection. Epidemiol Infect 2016; 2016; 144: 671–85.
Campylobacter are Gram negative curved or spiral, non spore forming, rod shaped bacteria with a polar flagella. The taxonomic family Campylobacteriaceae with the genera Campylobacter and Arcobacter includes 23 species of which only C. jejuni and C. coli play an essential, and C. upsaliensis and C. lari a minor role, as causative agents of human enteric infections. C. fetus subspecies fetus causes a number of extra intestinal infections mostly in patients with underlying chronic, immunocompromising diseases. A. butzleri and A. cryaerophilus are rare causative agents of enteric infections and both have also been found in conjunction with extra intestinal infections (bacteremia, endocarditis, gangrenous appendicitis) /, , /. The genus Sulfospirillum, which is also classified under the Campylobacteriaceae family, has no clinical significance. In practice, serological diagnostic testing for campylobacteriosis is only relevant to infections with C. jejuni and C. coli.
In 2010, the incidence of Campylobacter infections in Germany was 80.3 reported cases per 100,000 population . C. jejuni and C. coli are isolated, with regional differences, as the sole causative agent in 5–10% of microbiologically investigated diarrheal illnesses . Worldwide, thermophilic Campylobacter species are the most common causative agents of bacterial enteritis /, /. In Great Britain, the reported incidence in 2010 was 117/100,000 general population, thus taking a leading position in Europe , although epidemiological studies suggest an annual incidence of up to 1,100/100,000 . In Germany, the pathogen is more frequently encountered in rural regions.
Campylobacter infections are typical zoonoses. C. jejuni and C. lari often commensally colonize the intestinal tract of many birds and mammals; among farm animals, poultry are affected, in particular, and, to a lesser degree, cattle. Domestic animals, especially young cats and dogs, also are potential sources of infection for man /, /. The most common mode of infection in man by far is the consumption of contaminated food, primarily fresh poultry and raw milk /, , /. The routes of infection of C. fetus, C. upsaliensis, the Arcobacter species as well as the other rare Campylobacter species in humans still remain largely unknown.
Approximately 1–5 days for infections caused by C. jejuni and C. coli, otherwise unknown.
Intestinal Campylobacter infection
After a prodromal stage including malaise, chills, headache and melalgias, typically there is sudden onset of fever with abdominal cramps, nausea, dizziness and circulatory disturbances. Diarrhea usually sets in suddenly and increases to up to 20 bowel movements per day. Defervescence occurs after 1–3 days. At this stage, in about half of cases, the initially watery stools will reveal some mucus and blood mixed; sheets of leukocytes are usually detectable by microscopy. Diarrhea terminates for the most part spontaneously after 5–7 days /, , , /. After the acute stage subsides, pathogens are still excreted in the stool for 2–4 weeks /, /. Mild courses of the disease including asymptomatic excretion are observed as well.
After recovery from the disease, temporary immunity apparently develops against the causative strain leading to protection from disease but not from infection caused by the same pathogen . It is possibly the result of an IgA response to flagellin .
Extra intestinal Campylobacter manifestations
Relapsing bacteremia, thrombophlebitis, endocarditis, meningitis and extra intestinal abscesses are typical of infections with C. fetus, especially in patients with underlying chronic and immunocompromising diseases .
The most important late sequelae of Campylobacter enterocolitis are reactive arthritis, Reiter’s syndrome and Guillain-Barré syndrome (GBS). Reactive arthritis occurs in 1–2% of the infections. It starts 1–2 weeks after the acute dysenteric illness, more commonly in patients with the HLA antigen B27, and preferentially affects knee, hip and ankle joints as well as the small finger joints /, /. The arthritis is usually self limiting but may persist for up to one year. Up to 20% of patients with arthritis will develop Reiter’s syndrome . The GBS is a dangerous late sequela. It is presumed that up to one third of GBS cases result from preceding infection with Campylobacter /, /. For further information related to GBS refer to .
Campylobacter feature heat stable poly saccharides, oligosaccharides and heat labile protein antigens which are accessible on the surface and can be used for serotyping. Sequencing of the genome reveals pronounced phase variation, especially of the lipooligosaccharide (lipoglycan) antigens, which may pose a problem .
The species C. jejuni and C. coli are subdivided into at least 100 serotypes (protein antigens) according to Lior and approximately 90 serotypes (polysaccharide antigens) according to Penner. The former are determined by slide agglutination using absorbed antisera and the latter by passive hemagglutination. Overlaps exist between the two systems, thus accounting for the fact that the antigenic formula of a pathogen is always defined through the specification of both serotypes (LIO/PEN) /, /. Serotyping can be helpful in identifying chains of infections although it has not been accepted for routine diagnostic use and has meanwhile been replaced by molecular genetic typing methods .
For routine serodiagnostic investigation of late sequelae of Campylobacter infection, the ELISA and Western immunoblot have replaced the complement fixation (CF) test. Direct and indirect pathogen detection in stool samples and other specimens still have high priority in the diagnostic testing for Campylobacter infection.
Enzyme-linked immunosorbent assay (ELISA)
Originally, supernatants of sonicates /, / or of washed bacteria are employed, both after acid glycine buffer extraction from different C. jejuni strains. An ELISA using the recombinant proteins P18 and P39 has successfully been used achieving high diagnostic sensitivity and specificity . These proteins together with other recombinant surface proteins (PorA, Cbf1, Cbf2, PEB2) were used to develop an approved serological test kit (ELISA and immunoblot) which is approved and commercially available .
In an older study, however, significantly elevated IgG titers of up to 1 : 1280 were found in 89% of healthy controls , and according to another study, all consumers of raw milk showed significantly elevated IgG titers independently of acute Campylobacter infection . The diagnostic sensitivity of the test methods in both studies was 58.8–98% for IgG, 63–76.1% for IgA and 30–74% for IgM. The diagnostic specificity was 11–73.6% for IgG, 81.4–97% for IgA and 68.4–97% for IgM /, /.
IgA and IgM titers decline significantly 30–50 days after the onset of the disease, whereas IgG titers apparently persist for longer. In both studies, the greatest diagnostic significance was ascribed to the detection of significantly elevated IgA antibody levels /, /.
The test characteristics improve significantly by using recombinant protein antigens, achieving a diagnostic sensitivity of 92%, a diagnostic specificity of 99% and positive and negative predictive values of 97%, each . This ELISA is not available for routine diagnostic testing. Using the ELISA approved to date for commercial use in 310 patients with culturally confirmed Campylobacter infection, IgG and IgA antibodies were detected in 86% and 40% of the cases, respectively and in 16% and 3%, respectively, of healthy blood donors.
In a commercially available line immunoblot , in patients with confirmed Campylobacter infection, IgG and IgA antibodies were detected in 80% and 37% of the cases, respectively and in 14% and 2.5%, respectively, of healthy blood donors.
Possible interference with the serodiagnosis of legionellosis (IIFT): the detection of a positive Legionella IIFT has been repeatedly reported in patients with Campylobacter infection. For instance, 71% of patients showed a positive reaction to Legionella spp. .
The laboratory diagnosis of intestinal and extra intestinal Campylobacter/Arcobacter infections is primarily based on the cultural isolation of the pathogen.
The detection of serum antibodies to date is of no practical use for the diagnosis of acute Campylobacter infections. A reliable, sensitive and specific serological test for diagnosing recent campylobacteriosis would be desirable as part of the etiologic investigation of late sequelae; however, a negative test result to date does not exclude such etiology, in part due to the large antigenic diversity of the pathogens.
3. Vandamme P, Vancanneyt M, Pot B. Polyphasic taxonomic study of the emended genus arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., and aerotolerant bacterium isolated from veterinary specimens. Int J Syst Bacteriol 1992; 42: 344–56.
27. Foley SL, Lynne AM, Najak R. Molecular typing methodologies for microbial source tracking and epidemiological investigations of Gram-negative bacterial foodborne pathogens. Infect Genet Evol 2009; 9: 430–40.
28. Herbrink P, van den Munckhof HAM, Bunkens M, et al. Human serum antibody response in Campylobacter jejuni enteritis as measured by enzyme-linked immunosorbent assay. Eur J Clin Microbiol Infect Dis 1988; 7: 388–93.
29. Ang CW, Krogfelt K, Herbrink B, et al. Validation of an ELISA for the diagnosis of recent Campylobacter infections in Guillain-Barré and reactive arthritis patients. Clin Microbiol Infect 2007; 13: 915–22.
32. Nölting C, Reichhuber Ch, Wassenberg D, et al. First recombinant antigen-based ELISA and Line-Assay for serodiagnosis of Campylobacter jejuni/coli infections. 14. International Workshop on Campylobacter, Helicobacter and Related Organisms, Rotterdam 2007 (Poster).
Klaus-Peter Hunfeld, Volker Brade, Eberhard Straube
Chlamydiae are obligate intracellular bacteria which cause infections in humans and animals. The spectrum of Chlamydia induced clinical symptoms in humans is manifold and comprises acute and chronic diseases including venereal infection, ocular infection and respiratory diseases. Chlamydiae have all the structural elements typical of Gram negative bacteria and contain both DNA as well as RNA. Their cell wall contains a specific lipopolysaccharide which is responsible for immunological cross reactions between various species . As obligate intracellular parasites, the bacteria utilize various of the host cell’s synthesis capacities which they would actually be able to implement themselves based on their genetic material. This also refers to ATP. Thus, chlamydiae are in active exchange with their host cell via transmembrane secretion mechanisms and transport systems /, , , , /.
- The infectious form is the elementary body of approximately 0.3 μm. After entry into a susceptible host cell by endocytosis, these elementary bodies in a next step transform into 0.8–1 μm small, metabolically active reticulate (or initial) bodies capable of replicating.
- Within the endosome, the reticulate bodies replicate by binary fission and fill the cellular inclusion with up to several hundreds of bacterial cells. The duration and extent of replication depend on the cytokine reactions of the host cell and the availability of various metabolites. After 48–72 h, the reticulate bodies transform again into elementary bodies (condensation).
- These elementary bodies, which are now infectious, are released by rupture of the host cell or by controlled exocytosis and are, in turn, capable of infecting other cells or individuals.
In 1999, a controversial reclassification of the genus Chlamydia (C ) was proposed based on the genetic analysis of ribosomal genes, DNA-DNA hybridization and phenotypic and morphologic criteria /, /. The order of chlamydial bacteria with different hosts has grown rapidly since then. The family Chlamydiaceae includes species identified as pathogenic to humans such as C. trachomatis, C. pneumoniae and C. psittaci, as well as a multitude of species pathogenic to animals and environmental chlamydiae, including C. abortus, a pathogen of rare, severe systemic infections and Simkania negevensis and Waddlia chondrophila as pathogens of respiratory tract infections in humans /, , , , /.
Detection of the organism can be accomplished by cell culture or in chicken embryo culture, but is irrelevant for routine laboratory work. Because of the high risk of infection, culturing of avian isolates of C. psittaci may only be performed in special laboratories under laboratory biosafety level L 3 .
C. trachomatis is subdivided into serotypes A to L based on the major outer membrane protein (MOMP). The MOMP gene allows an almost identical classification. C. trachomatis causes sexually transmitted diseases, newborn infections and ocular infections /, , /. Chlamydiae are the most common bacterial cause of sexually transmitted infections worldwide, with an estimated incidence of approximately 100 million new cases annually /, /. In Germany, an estimated 300,000 to 500,000 cases of C. trachomatis infection occur annually . They primarily affect women in adolescence and young adulthood, risk groups with frequently changing sexual partners, and newborn children of infected mothers /, , , , , /.
The implications of the infection pose a special risk to girls and young women where clinically unapparent infection can, especially when becoming chronic, lead to inflammatory lesions of the ovary, ectopic pregnancy and infertility (up to 20%) /, , /. In Great Britain alone, the estimated costs for the diagnosis and treatment of such infections are up to 50 million pounds/year .
Epidemiological studies in Germany have provided evidence that infections occur in 0.9–10% of female adolescents between 15 and 17 years of age and only in 0.1–4% of male adolescents of the same age group /, , /. The prevalence is 4–6% in women between 20 and 24 years of age and only 1–2% in women older than 30 years . Transmission peripartum is to be expected in 36–60% of infected mothers. Infected children mainly develop ocular and respiratory tract infections /, , /.
Approximately 10 days to several weeks.
In affected women, the infection at first manifests as cervicitis, urethritis or proctitis and then, by further spreading, leads to adnexitis from which inflammatory scarring and tubal occlusion can develop. Pelveoperitonitis or perihepatitis and infertility are possible complications. In about 70% of cases, the course of infection is asymptomatic or only mildly symptomatic. Unspecific symptoms include vaginal discharge, dysuria, abdominal and back pain. Endometritis and pelvic inflammatory disease may also occur /, , , , , /.
Lymphogranuloma venereum (LGV)
Serotypes L1 to L3 of C. trachomatis are the causative agents of LGV. The disease is sexually transmitted and occurs mainly in the tropics and sub tropics. Increased incidence of autochthonous infections in homosexual, mostly HIV-positive men has been observed in Europe in recent years. Typical symptoms include urogenital mucosal ulceration associated with fever and pronounced swelling of the urogenital and/or perianal lymph nodes /, /.
- Serotypes D–K can cause swimming pool conjunctivitis by way of smear infection /, , /
- Serotypes A, B and C can cause trachoma and chronic follicular conjunctivitis in many countries of the tropics and sub tropics. Granulomatous conjunctivitis in the long term course of the disease and reinfection can cause pannus formation and scarring and, finally, loss of vision due to corneal opacity. The disease is transmitted by smear infection in endemic regions. It is estimated that 146 million individuals are infected with the disease worldwide and 6 million have lost their vision .
Infected pregnant women can transmit C. trachomatis to their newborn perinatally. Up to 40% of children born to infected mothers develop inclusion conjunctivitis (blennorrhea neonatorum), approximately 20% develop infant pneumonia. Chlamydia induced pharyngitis and otitis media have also been seen. Screening for Chlamydia has been a routine part of prenatal care in pregnant women since 1995 /, , , , , /.
Secondary (post and para infectious) symptoms can include reactive arthritis (in 1% of patients with C. trachomatis urethritis: 80% HLA-B27 positive) as well as Reiter’s syndrome (Reiter’s triad: urethritis, conjunctivitis, arthritis) /, , /.
Different regulations in different federal states of Germany apply.
Antigen detection employs direct immunofluorescence, enzyme immunoassay and rapid immunological tests. The direct immunofluorescence method uses monoclonal antibodies against antigens such as lipopolysaccharide (LPS) or Chlamydia pneumoniae outer membrane protein (MOMP), but is too expensive for handling large amounts of samples. Optimal collection of the smear sample is decisive for the quality of the specimen. Diagnostic sensitivity of direct immunofluorescence by comparison with culture is only 80–90% at changing specificity . Monoclonal antibodies to MOMP are preferred because of their higher specificity in regard to the potentially cross reacting anti-LPS antibody /, , /.
Antigen detection by ELISA employs monoclonal or polyclonal antibodies to LPS. Therefore, cross reactions with Gram negative bacteria can yield false positive results. In test positivity, higher specificity can be achieved by using blocking monoclonal antibodies. Urethral and endocervical smears are particularly suited as specimens, whereas urine is not. The diagnostic sensitivity of such tests is usually markedly lower than that of molecular detection methods. Diagnostic specificity is approximately 92–97% . Due to the resulting low positive predictive value at low prevalence, these methods are not suited for screening /, , /.
Immunochromatographic assays in the form of point of care test (POCT) are also available for antigen detection. Comparative studies have reported an inadequate diagnostic sensitivity and specificity for these rapid tests. The sensitivities and specificities specified for different commercially available tests vary between 12% to 83% and 69% to 100%, respectively, depending on the specimen used (swabs, urine). Therefore, these tests are not recommended by the relevant guidelines /, /.
Nucleic acid amplification tests are now considered the method of choice to detect Chlamydia. These assays achieve the highest detection limit and very high specificity in diagnostic testing. In many cases, the detection limit of molecular test methods is higher by one logarithmic level than that of ELISA . For instance, the genes pCCT1, Omp1, pCCT1 dnaB like region, 16S-rRNA gene and 23S-rRNA gene serve as specific targets for C. trachomatis. Other methods besides classical PCR include strand displacement amplification, transcription mediated amplification and ligase chain reaction /, , , , , , , /.
Clinical studies comparing these methods found good correlation between the different tests. Discrepancies are primarily found in samples with low pathogen concentrations. Methods employing multi copy targets such as 16S-rRNA or pCCT1 (cryptic plasmid) often achieve higher detection limits than those using single copy targets. A mutation in the PCR target region which amplifies a fragment of the cryptic plasmid led to the unnoticed spread of a C. trachomatis strain of serotype E (nvCT) in Scandinavia. Since such mutations may happen again, the applied PCR protocols should be regularly verified against a PCR using a target sequence contained in the relevant chromosome. The detection limit of commercially available assays ranges between 6 and 15 elementary bodies (EB/mL) or 0.025–2 inclusion forming units (IFU)/mL .
The detection limit and diagnostic sensitivity of different tests vary. Hybridization tests are reported to achieve diagnostic sensitivities of 65–96% and specificities of 96–100%. Diagnostic sensitivities of 90–100% and specificities of 99 –100% have been described for amplification methods . Thus, molecular methods have replaced culturing as the gold standard for C. trachomatis detection /, /.
Cervical or vaginal swabs in women and urethral swabs or first morning urine samples in men are suitable specimens for molecular detection methods. Urine of women is also mentioned as a cost saving specimen for screening. Ocular swabs as well as secretions of traces of secretions are also suitable test specimens. Screening for Chlamydia has been part of prenatal care in pregnant women in Germany since 1995. Since 2007, the annual examination for C. trachomatis by nucleic acid amplification has been a standard benefit of statutory health insurance companies for sexually active women up the 25 years of age .
Besides the detection of C. trachomatis, some commercially available assays also allow the concurrent detection of N. gonorrhoea /, /. provides an overview of the performance of the different diagnostic methods.
Comments and problems
Using PCR, false positive findings are possible due to contamination, whereas false negative findings sometimes occur in the presence of inhibitors. False negative findings are also seen in infections with specific C. trachomatis variants with deletion in the cryptic plasmid (pCCT1) . However, most commercially available assays have been revised as to also detect this so called Swedish C. trachomatis variant .
Molecular biological tests are not suited for therapy monitoring.
The specific detection of IgG, IgM and IgA antibodies to C. trachomatis primarily employs ELISAs as well as immunoblots and line assays besides the Micro Immuno Fluorescence Test (MIFT). The MIFT uses purified formalinized elementary bodies fixed onto glass slides as distinct dots of antigen. The test is labour-intensive and, because its interpretation is subjective, has not been standardized /, , /. The MIFT has been replaced by ELISA in routine diagnostic testing. The ELISA predominantly use LPS, MOMP as well as recombinant antigens. LPS based ELISA are thought to enable antibody detection early in the infection. However, such tests do not allow to differentiate between the various species of the ever growing family of chlamydiae /, , /. The genus specific tests should therefore no longer be used in routine diagnostics. Species specific ELISA should only be used based on synthetic peptide preparation. Immunoblots employing recombinant protein preparations are poorly evaluated, but presumably present the only solution in Chlamydia serology considering the difficulties in interpreting the test results due to unspecific cross reactions /, /.
Specific antibodies in urogenital infection are detectable after 6–8 weeks. Therefore, they are not suited to assist primary diagnosis. Antibody tests for C. trachomatis are also diagnostically insignificant in acute local or superficial infection of the lower genital tract because antibody response may be delayed, mild or does not occur at all . Molecular biological detection in infection of the lower genital tract should be performed according to the recommendations of the relevant guidelines /, , , , /.
Serology should only be done in persistent, ascending or invasive infection when C. trachomatis crosses the epithelium (pelvic inflammatory disease, lymphogranuloma venereum and reactive arthritis), usually causing pronounced antibody response. In these specific cases, a marked increase in IgG antibodies or individual, severely elevated IgG levels are of diagnostic significance because the pathogen is usually no longer detectable in the swab specimens /, , /. The diagnostic significance of IgA antibodies remains ambiguous because their role in the diagnosis of infections caused by C. trachomatis has to date not been unequivocally demonstrated in clinical studies.
The detection of specific IgM antibodies is useful in diagnosing neonatal pneumonia caused by C. trachomatis besides the molecular diagnostic analysis of respiratory tract specimens. IIFT titers ≥ 32 are considered to be diagnostically relevant in these cases .
Comments and problems
Species specific tests should be used to avoid cross reactions in high prevalences of C. pneumoniae infection (70–80% seroprevalence in adults). The standardization of the ELISA and MIFT still leaves much room for improvement /, , /. IgG antibodies to C. trachomatis can persist for months (or even years) after resolved infection. Moreover, reinfection or chronic infection may occur where no increase in IgG, IgM or IgA is detected and unequivocal serological differentiation is not possible. A resolved and an active infection can only be distinguished to a very limited extent or not at all. Hence, molecular detection is the method of choice /, , , /.
C. pneumoniae occurs worldwide and is a common cause of infection of the respiratory tract in man. The pathogen is considered the cause of up to 2% of all community acquired pneumonias and approximately 5% of all cases of bronchitis and sinusitis in adults /, , , , /. The epidemiology for C. pneumoniae infections in Germany is far from being comprehensive. The prevalence of antibodies is about 50% at 20 years of age and 70–80% at 60–70 years of age. Based on generally high infection prevalence, almost all humans can expect to be infected at least once during their lifetime. Reinfections are frequent, and infections may become chronic. The majority of infections take an asymptomatic or mild course /, , , , , , /.
Days to weeks.
Typical clinical findings are subacute to mild infections of the respiratory tract. Subacute onset is commonly encountered with symptoms ranging from sore throat, persistent cough and otitis media to rare manifestations such as endocarditis, myocarditis, erythema nodosum and reactive arthritis. Severe systemic infections and fever of unknown origin have occasionally been described /, , , /.
The pathogen C. pneumoniae is reported to account for 6–20% of Community Acquired Pneumonia (CAP), although data are, against the usual practice, based on a combined analysis of antibody prevalence and pathogen detection . Molecular epidemiological studies in Germany suggest that C. pneumoniae is the cause of community acquired pneumonia in less than 2% of cases . The clinical course may vary from mild, self limiting illnesses to severe forms of pneumonia, particularly in elderly patients and those with coexisting diseases. This agent participates in co infection involving other bacterial agents in approximately 30% of adult cases of CAP .
Other manifestations of respiratory tract infections include bronchitis, sinusitis and exacerbation in preexisting underlying disease (COPD, asthma). Involvement of C. pneumoniae in other diseases (atherosclerosis, multiple sclerosis, Alzheimer’s disease) has also been discussed. However, causal association between C. pneumoniae infections and these diseases has not been confirmed to date /, , , /.
Infections with C. pneumoniae are not subject to mandatory reporting.
The use of PCR, probe hybridization or real time PCR are especially attractive in the light of the above mentioned difficulties. Several studies have demonstrated that molecular biological methods achieve much higher diagnostic sensitivity in the diagnosis of C. pneumoniae infections than culture. In many cases, the results of molecular biology do not correspond with the serological test results . Target structures comprise the Pst-1 fragment (437-bp), specific fragments of the 16S-rRNA gene, the 53-kDa protein and the 60-kDa protein genes of C. pneumoniae for conventional or nested PCR protocols /, , , /. Real time PCR methods seem to be superior to conventional, non nested PCR protocols. A multiplex PCR test has been developed that allows simultaneous identification of L. pneumophila and M. pneumoniae in respiratory specimens as an atypical pneumonia panel. These assays are commercially available. The standardization and performance of methods employed outside special laboratories vary considerably /, /.
Serology testing for C. pneumoniae infection includes the MIFT and various ELISA. Serology is generally poorly standardized and does not yield reliable results. MIFT is considered by the CDC to be the gold standard for serodiagnosis despite significant limitations /, , , /.
Numerous ELISA are commercially available . It is essential to use species specific ELISA for antibody detection to avoid cross reactions with other species, especially C. psittaci. For instance, antigens depleted of cross reactive LPS are employed in C. pneumoniae ELISA. None of the methods have been standardized and use different quantification approaches. This has resulted in a wide variation of inter laboratory test performance and unreliable diagnostic interpretation /, , , /. Immunoblots and immunochromatographic tests are also available besides MIFT and ELISA. However, standardized interpretation criteria have to date not been available and the diagnostic quality of such tests has not been adequately evaluated . The usefulness of these assays for diagnosis, also in regards to generally higher cost, is still controversial.
Assessment of serologic results
In general, the currently available serodiagnostic methods provide little conclusive information. The detection of IgM antibodies, seroconversion or multi fold increase in IgG antibody titer are considered to be indicators in primary infection /, /. Paired serum analysis, though rarely performed in practice, is necessary in the course of the infection. IgM antibodies are detectable within 2–3 weeks and IgG antibodies within approximately 6–8 weeks after primary infection . In reinfection, IgM antibody formation may not occur at all or be present at low titers. An increase in IgG antibody titer is detectable earlier (approximately 1–2 weeks after reinfection). The status of infection is impossible to assess based on single serum samples . High IgG titers > 512 or above 640 in the MIF are considered to be epidemiologically abnormal /, , , /.
The detection of antibodies at an infection prevalence in the population of up to 80% is only useful to identify primary infection /, /. The role of serodiagnosis in the identification of frequently encountered reinfections is insignificant in most cases. The level of antibody titers does not correlate with the results of cultural and molecular biological direct pathogen detection methods . Moreover, high to medium IgG and IgA antibody titers can persist for months or even longer /, /.
According to the German S3 guideline on ambulant acquired pneumonia, Chlamydia serology plays no role in the primary diagnosis of pneumonia . Attempts have been made to calibrate species specific ELISA tests against MIFT to allow conclusions as to the status of infection based on antibody titers . The results of Chlamydia serology tests provide little conclusive information as long as they are evaluated against existing, poorly performing test systems. The diagnostic significance of Chlamydia serology will only increase if the tests are evaluated against clearly defined cases of infection. This is also confirmed by the results of external quality control .
Therefore, in suspected infection with C. pneumoniae, molecular biological testing is the method of choice for pathogen detection.
Comments and problems
- Difficulties in the acquisition and analysis of paired serum samples in the course of the disease
- High antibody prevalence in the adult population
- A significant rate of cross reactions
- Poor diagnostic sensitivity and specificity in the identification of acute infection and reinfection
- The MIFT, though considered to be the gold standard, generally does not achieve adequate diagnostic sensitivity and specificity and, except in primary infection, cannot reliably differentiate between persistent infection, reinfection or resolved infection .
A larger scale study analyzed 11 serodiagnostic test kits as to their retrieval rates in healthy blood donors. The study found a wide range of variation in diagnostic sensitivity (78–98%) compared to the MIFT. The diagnostic sensitivity of species specific tests varied between 58 and 100% . Therefore, species specific ELISA are only considered to achieve reliable sensitivity and specificity in childhood when seroprevalence is relatively low. Tests employing species specific antigens with high specificity, but low immunogenicity (e.g., MOMP) or high immunogenicity, but low specificity (Omp2, CrpA) yielded a great variety of results. It is possible that, in the future, the proteome analysis of C. pneumoniae will provide better means of identifying suitable antigens to promote serodiagnostics toward more conclusive results .
The pathogen was identified in Israel as the causative agent of respiratory diseases in inhabitants of the Negev desert. Infections, though sporadically, seem to occur worldwide. According to seroepidemiological studies, the infection prevalence is between 7 and 18%. Humans are thought to function as reservoirs. The pathogen is transmitted by droplet infection /, /.
Days to weeks.
The pathogen has primarily been detected in patients with bronchiolitis, asthma and community acquired pneumonia.
Infection with this pathogen is not subject to mandatory reporting.
Corresponding to the variability of the MOMP antigen and/or the ompA gene, the pathogen is subdivided into 8 serotypes or 9 genotypes /, /. Zoonotic infection with C. psittaci occurs worldwide in livestock and wildlife.
Avian strains are considered to be pathogenic to humans and, due to their high infectivity, may only be handled and reproduced in laboratories under laboratory biosafety level L3. Isolates of non-avian livestock or wildlife are not considered to be pathogenic to humans. Pandemic outbreaks of human psittacosis in the years 1929 and 1930 were linked to the import of infected tropical birds from South America to Europe and North America. During the 1980s and 1990s, psittacosis outbreaks were reported in the USA and European poultry industries /, /. C. psittaci has in the last decade been reported to be nearly endemic in the turkey industry in many European countries. Evidence of human infections associated with outbreaks in poultry as well as parrots and pigeons exists /, , , /. Typical risk groups include bird owners, animal attendants, veterinarians and individuals working in pet stores, on poultry farms or in slaughterhouses. Person-to-person transmission has not been described to date .
Transmission to humans predominantly occurs through inhalation of contaminated aerosols and dusts from urine, secretions or feces from diseased birds //. Even dried feces, secretions and plumage can remain infectious for about 4 weeks /, /. Between 12 and 155 human cases of psittacosis were reported in Germany from 1996 to 2007 /, /. A declining tendency has been observed in recent years. In 2010, 20 cases of psittacosis were reported in Germany . A significant number of undetected cases must, however, be assumed.
The incubation period of ornithosis is 5–14 (–30) days.
The infection can have a subclinical course, present as a flu like infection, mononucleosis or typhoid like disease as well as atypical pneumonia. Manifestation as atypical pneumonia is characteristic, with a sudden onset, shaking chills, high fever, dry cough and headache. Myalgias, arthralgias, hepatomegaly, gastrointestinal symptoms and fever of unknown origin have also been observed. Splenomegaly is seen in up to 70% of patients. Disease severity ranges from oligosymptomatic to systemic infection with a fatal outcome. If untreated, the mortality rate is up to 20% . The pathogen is also linked to MALT lymphoma in the lacrymal ducts /, , , , /.
Meningitis, encephalitis, endocarditis, myocarditis, conjunctivitis, reactive arthritis, skin rashes, hepatitis, renal involvement, venous thrombosis, pancreatitis or thyroiditis have been described in rare cases.
White blood cell count is usually normal to slightly lowered during the acute phase of the disease, occasionally developing into leukopenia (in approximately 25% of cases). Augmented C-reactive protein (CRP) and elevated aminotransferases seem to be related to the severity of the infection /, , /.
Molecular detection (e.g., using nested PCR based on ompA gene and incA gene detection) from BAL, blood and tissue samples is available . Moreover, besides the species, genotypes can be distinguished by sequencing genotype specific real time PCR or micro arrays /, , /. None of the described molecular detection methods are commercially available /, , /.
Commonly used serological tests include complement fixation (CF), ELISA and MIFT. CF and ELISA usually employ LPS based antigen preparations and, therefore, are susceptible to cross reactions /, , /. The CF test is considered to be the least sensitive method and is no longer recommended for lack of standardization. ELISA is deemed to be the most sensitive serological method . The MIFT is regarded as the reference standard for the detection of specific antibodies. However, antibody testing is hampered by poor standardization and diagnostic sensitivity. The tiered combination of the sensitive ELISA as a screening test and the MIFT as the confirmatory test would be a sensible approach /, /.
Comments and problems
False negative MIFT findings in patients with confirmed psittacosis primarily occur in the early phase of infection. Most serological tests use whole cell chlamydial organisms to capture antibodies. As some of the chlamydial LPS or HSP can cross react with antibodies to other bacteria, caution is needed when interpreting positive results /, /. Moreover, cross reactions between different species of the Chlamydiaceae family can also occur.
A new infection is considered to be reliably confirmed by a fourfold increase in antibody titer in the MIFT between paired serum samples collected at a 2-week interval /, /. A reciprocal titer of 1 : 16 for IgM in the MIFT is considered an indication and needs to be confirmed by IgG seroconversion during follow-up. Treatment of patients with antibiotics may, however, impair bacterial growth and lead to undetectable antibody response /, /.
When there is a low prevalence of C. trachomatis and C. psittaci antibodies in the patient group under investigation (less than 5%), consideration must be given to the fact that a high proportion of false positive results may occur, depending on the specificity of the antigen detection test utilized . Sera with high specific antibody titers to other Chlamydiae may be cross reactive in the different tests and can only be reliably interpreted when using the MIFT /, /. A positive ELISA or CF result should be confirmed by another method such as PCR or MIFT to avoid false positive results due to cross reactions /, /.
1. Jones RB, Schlossberg D, Graystone JT, Chlamydial diseases. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. Mandell GL, Bennett JE, Dolin R (eds). Philadelphia; Churchill Livingstone: 2000.
7. Everet KD, Bush RM, Andersen AA. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol 1999; 49: 415–40.
8. Price MJ, Ades AE, Soldan K, Welton NJ, Macleod J, Simms I, et al. The natural history of Chlamydia trachomatis infection in women: a multiparameter evidence synthesis. Health Technol Assess 2016; 20: 1–250.
11. Ahmadi MH, Mirsalehian A, Bahador A. Association of Chlamydia trachomatis with infertility and clinical manifestations: a systematic review and meta-analysis of case-control studies. Infect Dis (Lond) 2016; April 11: 1–7.
14. Weissenbacher TM, Kupka MS, Kainer F, Friese K, Mylonas I. Screening for Chlamydia trachomatis in pregnancy: a retrospective analysis in a German urban area. Arch Gynecol Obstet 2011; 283: 1343–47.
19. Kerndt PR, Ferrero DV, Aynalem G, et al. First Report of performance of the Versant CT/GC DNA 1.0 Assay (kPCR) for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. J Clin Microbiol 2011; 49: 1347–53.
21. O’Neil D, Doseeva V, Rothmann T, Wolff J, Nazarenko I. Evaluation of Chlamydia trachomatis and Neisseria gonorrhoeae detection in urine, endocervical, and vaginal specimens by a multiplexed isothermal thermophilic helicase-dependent amplification (tHDA) assay. J Clin Microbiol 2011; 49: 4121–5.
23. Reischl U, Straube E, Unema M. The Swedish new variant of Chlamydia trachomatis (nvCT) remains undetected by many European laboratories as revealed in the recent PCR/NAT ring trial organised by INSTAND e.V. Germany. Euro Surveill 2009; 14: pii 19302.23.
24. Dowell SF, Peeling RW, Boman J, Carlone GM, et al. Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada). Clin Infect Dis 2001; 33: 492–503.
26. Coste O, Müller I, Brade V, Hunfeld KP. Ergebnisse des bakteriologisch-infektionsserologischen INSTAND Ringversuchs 2007: Ein zusammenfassender Bericht – Beitrag der Qualitätssicherungskommission der Deutschen Gesellschaft für Hygiene und Mikrobiologie (DGHM). GMS Z Förder Qualitätssich Med Lab 2009; 2: 1–22.
35. Clad A, Prillwitz J, Hintz KC, Mendel R, et al. Discordant prevalence of Chlamydia trachomatis in asymptomatic couples screened using urine ligase chain reaction. Eur. J Clin Microbiol 2001; 20: 324–8.
Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
The genus Anaplasma is one of four distinct genera in the family Anaplasmataceae, which are obligate intracellular pathogens vectored by ticks and found exclusively within parasitophorous vacuoles in the host cell cytoplasm. The 2001 reclassification of the order Rickettsiales expanded the genus Anaplasma, which previously contained pathogens that were host specific for ruminants (A. marginale, A. centrale and A. bovis), by adding A. phagocytophila and the unnamed agent of human granulocytic ehrlichiosis .
The diseases subsumed as ehrlichiosis and anaplasmosis manifest as feverish systemic infections of various severity in humans and animals. During the last decades, numerous cases of human ehrlichiosis in the USA /, / and later also in Europe have document the medical relevance of such zooanthroponotic diseases to travelers and autochthonously tick exposed individuals in Europe.
Bacteria of the genus Anaplasma are 0.4–1.5 μm small, obligate intracellular Gram negative organisms which preferably reside in hematopoietic host cells. Immunodominant proteins are expressed on the outer membrane, for example P42–44 of A. phagocytophila and p120 as well as p22–29 of E. chaffeensis. Antigenic variants of these immunodominant proteins have been identified. The cell wall lacks important components such as lipopolysaccharide or peptidoglycan. During the pathogen replication cycle in the target cell, up to 40 daughter cells may accumulate forming micro colonies called morulae (from the Latin word morus for mulberry) within modified phagosomes /, /.
Molecular biological analyses based on the 16S-rRNA gene and the groESL operon documented close relationship, especially among the granulocytic Ehrlichia themselves and with the genus Anaplasma and gave rise to reclassification of the pathogens. Those members of the genus which develop in granulocytic target cells (e.g., E. phagocytophila, E. equi), were grouped and renamed Anaplasma phagocytophilum /, /. Moreover, E. sennetsu was assigned to the genus Neorickettsia and renamed Neorickettsia sennetsu .
Pathogen detection by culture in special cell cultures (HL-60) or animal models plays no role in microbiological routine diagnostics. As a rapid method for the detection of intracellular inclusions in the acute stage, diagnosis can be accomplished by Giemsa stained smears from peripheral blood or bone marrow /, /.
More than 5000 cases of human granulocytic anaplasmosis transmitted by Ixodes spp. as the primary tick vector and monocytic ehrlichiosis transmitted by Amblyomma americanum have been reported in the United States since 1986 (incidence rate of 16–58/100,000) /, /. In addition, human ewingii ehrlichiosis occurs in the south of the United States. Reported cases of human Sennetsu fever (West-Japanese infectious mononucleosis), which is caused by Neorickettsia sennetsu, appear to be limited to southeast Asia (western Japan and Malaysia) /, /.
Approximately 100 well documented cases of human granulocyte anaplasmosis have been reported in northern, central and southern Europe, with the majority coming from Scandinavia and Slovenia /, , /. The predominant causative agent of the infection in northern and central Europe is A. phagocytophilum. The regional occurrence of infection corresponds to the geographical distribution of the vector ticks of the genus Ixodes (I. ricinus, I. persulcatus). The risk of infection increases with the duration of the tick’s sucking action. Molecular epidemiological studies in Europe show a regionally different detection rate of 0.8–45% (Germany 3%) in the ticks Ixodes ricinus. Small mammals and red deer are thought to serve as animal reservoirs for most Anaplasma and Ehrlichia species /, , /. Causative agents of monocytic ehrlichiosis also occur in southern Europe (E. canis). Here, the primary vector is Rhipicephalus sanguineus . However, no cases of human monocytic ehrlichiosis have been reported from southern Europe.
The prevalence of specific antibodies is significantly higher in collectives at increased risk of tick exposure (Lyme borreliosis patients, forestry workers) than in the normal population (blood donors) /, , /. Seroprevalence in the relevant risk categories varies between 2–24% in Europe /, /. The clinical relevance of human ehrlichiosis and anaplasmosis is difficult the assess because the spectrum of clinical symptoms may range from influenza like courses to severe feverish infections. Moreover, specific microbiological tests are not generally available and there is a lack of comprehensive information as to the incidence and prevalence of the disease in Europe /, /.
Approximately 4 days to 4 weeks following tick bite (median of 7 days).
Human ehrlichiosis and anaplasmosis have similar clinical presentations /, /. Typical symptoms of acute human granulocytic anaplasmosis and human monocytic ehrlichiosis primarily include chills and fever, headache and myalgia within a few days to up to 4 weeks following tick bite.
The differential diagnostic between human granulocytic anaplasmosis, human monocytic ehrlichiosis and other tick borne diseases such as Lyme borreliosis and early summer meningoencephalitis can be difficult for the following reasons /, /:
- Most infections take a subclinical course
- Strain specific efflorescences of the skin occurs in 11–36% of cases /, /
- Gastrointestinal symptoms, arthralgia, meningitis and pneumonic infiltrates may be additionally present in severe courses (e.g., in immunocompromised patients)
- Laboratory findings are unspecific and include, for example, markedly elevated CRP (not in borreliosis and human monocytic ehrlichiosis), leukopenia, anemia, thrombocytopenia and variably elevated aminotransferases /, , /.
The duration of the disease is rarely longer than 14 days. Chronic courses have not been documented. The mortality rate is reported as about 3% in human monocytic ehrlichiosis patients and below 1% in human granulocytic anaplasmosis patients.
According to the German Infection Protection Act (IfSG), these infections are not subject to mandatory reporting.
In clinically suspected acute or recently resolved human anaplasmosis or ehrlichiosis, serological testing for specific antibodies, in particular, should be performed after the second week of infection.
The IIFT for IgG and IgM antibodies is the gold standard for indirect pathogen detection in suspected human granulocytic anaplasmosis or human monocytic ehrlichiosis. Anaplasma inoculated cell cultures serve as a source of antigens. Commercial tests are available, for example, for the detection of antibodies to E. chaffeensis (HME) and A. phagocytophilum (HGA).
ELISA using recombinantly produced immunodominant membrane proteins from Anaplasma (e.g., p44 from A. phagocytophilum) achieve good sensitivity and specificity, but have only been available to date in special laboratories /, /.
Serum: 1 mL
In the acute phase of the disease, only 18–45% of patients show positive antibody response. In such cases, blood smears or PCR methods should be employed for direct pathogen detection. Most patients (80%) seroconvert 7–30 days after the onset of clinical symptoms and can therefore be diagnosed through indirect detection based on pathogen specific antibodies /, , , /. Highest titers are found 4–6 weeks following infection and elevated titers are detectable in about 50% of patients for at least 18 months .
A resolved infection is suspected in IgG titers of 64 and/or 80 depending on the IIFT method. Titers from 160 to above 256 in combination with IgM positivity (titer > 20) suggest recent or recently resolved infection. Seroconversion and a fourfold increase in titers verified by parallel testing with a previously collected serum sample provide unequivocal evidence of infection /, , , /.
In the immunoblot, evidence of infection is provided by the detection of specific antibodies (IgG and IgM) to immunodominant outer membrane proteins p42–44 of A. phagocytophilum (human granulocytic anaplasmosis) and the proteins p120 and p28–29 of E. chaffeensis (human monocytic ehrlichiosis), respectively /, , /. The immunoblot is not as well evaluated, but considered to be highly specific and is used as confirmatory test by some laboratories. Results at variance with the IIFT are also obtained in patients with clinically confirmed ehrlichiosis and anaplasmosis and are due to the antigen variability of different isolates and/or varying quality and batch stability of commercially available immunoblots .
Tiered diagnostic testing as practiced in Lyme borreliosis has not been established as standard in human ehrlichiosis and anaplasmosis.
Comments and problems regarding serology
Molecular biological pathogen detection in EDTA blood, sodium citrate blood or cerebrospinal fluid plays an important role in the acute phase of infection. Relevant diagnostic tests are commercially available but are usually only employed by special laboratories and are poorly standardized /, /.
False negative and false positive results are possible. Diagnostic sensitivities of 85–90% and specificities of approximately 100% have been reported /, , , /. The target sequences employed for the diagnosis of ehrlichiosis infection by PCR consist of specific fragments of the 16S-rRNA gene and the epank1 gene for A. phagocytophilum (human granulocytic anaplasmosis) and the omp1, the gp120 the TRP32 or the 16S-rRNA gene of E. chaffeensis (human monocytic ehrlichiosis) /, , /. If the primer is adequately selected, specific target sequences of the 16S-rRNA gene can be used to theoretically detect all human granulocytic anaplasmosis and human monocytic ehrlichiosis pathogens in one mixture.
5. Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ”HGE agent” as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 2001; 51: 2145–65.
11. Dumler JS, Aguero-Rosenfeld M. Microbiology and laboratory diagnosis of tick-borne diseases. In: Cunha AB (ed). Tick-borne infectious diseases: diagnosis and management. New York; Marcel Dekker 1999: 39–42.
Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Gonorrhea (GO) is caused by Neisseria gonorrhoeae. The genus Neisseria is classified under the beta subgroup of Proteobacteria. GO belongs to the most common sexually transmitted bacterial infections globally besides infection caused by C. trachomatis .
The causative agents of gonorrhea, N. gonorrhoeae, are Gram negative, bean shaped, mostly intracellular diplococci. In diagnostic testing, they are primarily detected in purulent secretions by microscopy and culture. Pili on the gonococcal surface enable attachment to the mucosal cells in the urogenital tract, where the bacteria, absorbed by phagocytosis, replicate and destroy the host cells. These processes result in acute purulent infection /, /.
For direct microscopy, two slides with smear specimens (urethral smear, cervical secretion, anorectal smear) are prepared and, after drying, stained with methylene blue and according to Gram.
Direct microscopy on clinical specimens (primarily urethral smear from men) achieves a diagnostic sensitivity above 95% and a specificity above 99%. The detection limit of microscopy is insufficient for other clinical specimens or asymptomatic individuals .
Cultural gonococcal identification is the gold standard in microbiological diagnostic testing mainly because, based on detection limit, in addition the culture allows conclusions as to the most effective antibiotic therapy /, /. For pathogen culturing, mucosal specimens should be collected using Dacron or Rayon swabs and, unless immediately plated onto chocolate agar, stored in a transport medium /, /. A sentinel study performed by the Robert Koch Institute in Germany on the use of diagnostic tests in suspected N. gonorrhoeae infection showed microscopy (64%) and culture (60%) to be the methods most frequently used. PCR was used in 33%, antigen testing in 26% and serological techniques in 26% of cases .
The pathogen globally causes an estimated 60 million new cases of gonococcal disease annually . About 700,000 new cases of N. gonorrhoeae infection occur annually in the United States . Comprehensive, reliable information on the resistance and incidence regarding N. gonorrhoeae is not available in Germany since the infection is no longer subject to mandatory reporting . The incidence rate of gonorrhea in Germany was approximately 10/100,000 population in the year 2000, corresponding to only about 10% of reported cases.
Humans are the only reservoir for N. gonorrhoeae. Sites of entry are the mucous membranes of the urogenital tract, the pharynx, rectum and anal canal and, in newborns, the conjunctivae which are infected peripartum /, /.
An increase in penicillin resistant strains (penicillinase-producing N. gonorrhoeae) has been observed worldwide. Sentinel studies from the Rhine/Main region in Germany in 2008 show penicillin resistance in 25%, ciprofloxacin resistance in 64%, azithromycin resistance in 20% and tetracycline resistance in 16% of cases. However, third-generation cephalosporins continue to be effective . Approximately 25% of infected men and 50–80% of infected women are asymptomatic carriers of the pathogen, thus representing an important unrecognized source of infection.
Typical symptoms include painful purulent urethritis in men and often asymptomatic cervicitis in women.
Classical forms of manifestation:
- Women: cervicitis, urethritis, bartholinitis, pharyngitis, proctitis
- Men: urethritis, pharyngitis, proctitis.
- Women: endometritis, adnexitis, peritonitis
- Men: prostatitis, epididymitis
- Women and men: infertility, reactive arthritis (in many cases monarthritis of the knee joint), gonococcal sepsis, endocarditis
- Neonates: conjunctivitis.
According to the German Infection Protection Act (IfSG), these infections are not subject to mandatory reporting.
Various serological test methods such as complement fixation, latex agglutination, immunofluorescence tests and immunoblot as well as antigen detection have been developed and are partly available. These methods have become increasingly irrelevant since the introduction of molecular biological methods . Chronic or disseminated infection may indicate the need for serological pathogen detection /, , /.
Enzyme-linked immunosorbent assay (ELISA)
For direct pathogen detection monoclonal antibodies to gonococcal antigens are bound to a solid phase. Antigen eluted from the swab binds to the antibody of the solid phase. The immune complex thus formed is labeled using an enzyme bound, second antibody. The amount of bound enzyme labeled antibody is determined by enzymatic reaction.
Complement fixation (CF) test
For indirect pathogen detection in the CF test, a N. gonorrhoeae strain cultured in serum free culture medium is used as CF antigen.
- Serum (indirect pathogen detection): 1 mL
- Swab specimens from urethra, cervix (direct pathogen detection).
Interpretation of test results
Depending on the patient group and study, the antigen detection test in comparison to culture achieves a diagnostic sensitivity of 67–96% at a specificity of 94–98% . The positive predictive value of the test in female and male low risk patients is only about 50% . Such tests are currently not recommended because of their partly significant diagnostic shortcomings .
An at least 4-fold increase in titers between two sera from the acute and re convalescence phases suggests gonococcal infection. In the presence of corresponding clinical symptoms, CF titers ≥ 20 can indicate gonococcal associated sequela, such as arthritis.
Comments and problems regarding serology
In many cases, cross reactions with antibodies to antigens of different origin lead to false positive results in CF testing. Therefore, the results must be interpreted with care.
Direct antigen detection methods yield false positive test results in the presence of high counts of other bacteria (N. catarrhalis, Bacteroides spp., Enterobacter spp., Proteus spp.), especially in female patients .
Culture independent methods also detect vancomycin sensitive gonococci, which are inhibited on selective media, as well as dead or damaged microorganisms, for example, following antibiotic treatment. There is a whole series of commercially available molecular detection methods using different genetic target sequences for the identification of N. gonorrhoeae. Some of these methods also allow a combined approach including the detection of C. trachomatis to determine coinfection /, , /.
To reduce molecular biological testing costs, some international evaluation studies have investigated pooling specimens . It must be noted, however, that the pooling of 10 specimens has the potential to decrease the detection limit of the molecular biological detection test by a factor of 10. In general, the diagnostic sensitivity and specificity as well as the positive and negative predictive values are decisively influenced by the employed methodology and the prevalence of the pathogen (i.e., the local epidemiological situation). Moreover, such methods have to date not been capable to provide antibiotic resistance data /, , /.
Most commercially available tests are primarily approved for detection based on urethral and/or vaginal and cervical swabs and urine. It has been recommended that, in the absence of evidence based data, detection from extragenital specimens should primarily be performed by culture /, /.
Molecular biological detection initially does not provide any information as to the viability of the detected Neisseria and is not suited as a primary means of therapy monitoring. According to the United States Center of Disease Control, monitoring by molecular biological test methods is only prudent 3 weeks after termination of antibiotic treatment . When to perform a test of cure for Gonorrhea is under discussion .
Depending on the employed method, false positive results are obtained due to the close genetic relationship between N. gonorrhoeae and non-pathogenic Neisseria and due to the exchange of mobile genetic target sequences between pathogenic and non-pathogenic Neisseria. False positive results can also occur due to carryover contamination in the sample. False negative results due to the presence of inhibitors in the sample and also due to pronounced sequence variations between N. gonorrhoeae subtypes with subsequent failure of the primer binding to the species-specific target have been observed . Depending on the test and study protocols, molecular biological detection methods achieve diagnostic sensitivities of 65–100%, specificities of 94–100%, positive predictive values of 31–100% and negative predictive values of 95–100% /, , /.
8. Gilsdorf A, Hofmann A, Hamouda O, Bremer V. Highly variable use of diagnostic methods for sexually transmitted infections-results of a nationwide survey, Germany 2005. In: BMC Infectious Diseases 2010; 10: 98.
9. Dickson C, Arnason T, Friedman DS, Metz G, Grimshaw JM. A systematic review and appraisal of the quality of practice guidelines for the management of Neisseria gonorrhoeae infections. Sex Trans m Infect 2017; 93 (7): 487–92.
10. Zollinger WD, Boslego J. Immunologic methods for diagnosis of infections by gram-negative cocci. In: Rose NR, Hamilton RG, Detrick B (eds). Manual of clinical laboratory immunology. Washington; American Society for Microbiology 2002: 435–46.
11. Priest D, Ong JJ, Chow EFP, Tabrizi S, Phillips S, Bissessor M, et al. Neisseria gonorrhoeae DNA bacterial load in men with symptomatic and asymptomatic gonococcal urethritis. Sex Transm Infect 2017; 93 (7): 478–81.
12. Smith DW, Tapsall JW, Lum G. Guidelines for the use and interpretation of nucleic acid detection tests for Neisseria gonorrhoeae in Australia: a position paper on behalf of the Public Health Laboratory Network. Commun Dis Intell 2005; 29: 358–65.
13. Kerndt PR, Ferrero DV, Aynalem G, Monga D, et al. First Report of Performance of the Versant CT/GC DANN 1.0 Assay (kPCR) for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae. J Clin Microbiol 2011; 48: 1347–53.
Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Gram negative bacteria of the genus Helicobacter (H) are members of the class α-proteobacteria and the family Helicobacteriaceae. These pathogens have the structure of a curved or spiral, motile, flagellated rod. More than 30 different species have been described to date, including many occurring in animals and rarely transmitted to humans, such as H. heilmannii. The individual strains of varying human pathogenicity can cause very different diseases /, , /.
H. pylori, the most prominent species pathogenic to humans, is etiologically associated with type B gastritis, peptic ulcer disease, MALT lymphoma and gastric carcinoma. Humans are the major, if not exclusive, reservoir. The discovery of the pathogen in 1983 and the etiologic investigation of classic gastric ulcer by Warren and Marshall revolutionized the concepts of gastroenterology .
Infection with H. pylori is one of the most common chronic infections in humans. The gastric mucosa is the primary habitat of H. pylori. The bacteria are transmitted by the gastro-oral and fecal-oral routes, directly or indirectly via contaminated food /, , , /. Within familial transmission and outbreaks are well established . Infection with H. pylori occurs worldwide, with the infection prevalence increasing inversely proportionally to hygienic standards. Prevalence of infection with the pathogen varies between more than 90% in developing areas and approximately 30% in industrialized countries. Prevalences in Germany increase with age, are gender dependent and have been reported as 5–7% in children, 12–24% in adolescents and adults below 30 years of age and approximately 30% in adults above 35 years of age (increase by about 1% per year of life). Prevalence in the industrialized countries is currently gradually declining /, /.
Most H. pylori infections are clinically asymptomatic. Symptomatic courses manifest as:
- Chronic superficial gastritis
- Duodenal and, more rarely, gastric ulcers
- Chronic atrophic gastritis with potential development into gastric cancer.
Microbiological diagnostic testing for H. pylori comprises cultural, serological and molecular biological detection methods. Direct test methods, in particular, have been evaluated to such an extent that they can be used for infection diagnostics (histology, rapid urease test, urea breath test, stool antigen test). However, no test method by itself, with the exception of culture, is absolutely accurate /, /. If the probability of infection is low, a high rate of false positive test results must be anticipated. Two positive test results should exist for reliable diagnosis. In results at variance, additional diagnostic testing should be done . After the first therapy failure, it is recommended to employ endoscopy and a microbiological test for H. pylori with pathogen culture and susceptibility testing /, , , /.
Stool antigen ELISA
This assay employs monoclonal or polyclonal H. pylori capture antibodies bound to micro titer plate wells. Qualitative H. pylori antigen detection is performed by adding diluted stool samples and correspondingly labeled monoclonal or polyclonal antibody. A meta-analysis of studies on antigen detection in stool samples from children and adults showed significant superiority of ELISA based on monoclonal antibodies by comparison with test systems based on polyclonal antibodies /, /. In patients with no previous treatment, the tests achieve diagnostic sensitivities of 96% at specificities of 97% .
The stool antigen test is indicated as a screening test in patients < 45 years suffering from dyspepsia without alarm symptoms. The test should be considered as an alternative during therapy monitoring if no breath test is available (see also further diagnostic testing in ).
Serological tests for indirect pathogen detection
Individuals colonized by or infected with H. pylori usually show local antibody response to surface antigens of the pathogen. Immune response is also detectable in serum in approximately 95% of cases. Local antibody response at the gastric mucosa primarily involves IgA while circulating antibodies are more frequently of the IgG type. IgM antibodies are infrequently detectable with controversial specificity /, /. If the probability of a H. pylori infection is low, a high rate of false positive results must be anticipated. Serology is often not validated, does not allow a statement as to whether an infection or colonization is present or not and does not provide the basis for the diagnosis of active infection or therapy decision .
Tests and POCT assays for the detection of IgG and IgA antibodies in saliva, urine and whole blood achieve limited diagnostic sensitivity and specificity and are not recommended for the detection of H. pylori infection or colonization /, /.
ELISA for indirect pathogen detection
The ELISA method is employed for the quantitative determination of H. pylori antibodies. Detergent extracts of a H. pylori strain expressing cytotoxin associated protein (CagA) and vacuolating cytotoxin (VacA) are used as antigens. Recombinantly produced, specific antigen preparations are also used.
Immunoblot for indirect pathogen detection
Depending on the assay, the immunoblot uses purified whole cell lysate extracts and recombinant antigens of H. pylori. Immunoblots are available for specific diagnostic IgG and IgA testing, serve as confirmatory tests in ambiguous ELISA results and enable the detection of antibodies to pathogenicity associated proteins such as CagA and VacA. These proteins of H. pylori are suspected to increase the probability of developing ulcer or gastric cancer. However, the knowledge of the pathogen’s pathogenicity and virulence factors has no significant implications on patient management. Therefore, immunoblots and/or testing of pathogenicity associated proteins should not be performed on a regular basis .
- Serum (indirect pathogen detection): 1 mL
- Stool: a hazelnut sized amount of stool collected in a stool specimen tube for direct pathogen detection in the laboratory.
Antigen detection in the stool
The detection of H. pylori antigen in the stool confirms the diagnosis of H. pylori infection/colonization. If control endoscopy is not necessary after therapy, eradication control may be done using a breath test or a monoclonal antigen stool test. Therefore, the period between the end of therapy and control must be at least 4 weeks .
Serum antibody detection
Evidence of specific IgG indicates that the body was exposed to H. pylori where the IgG titer does not reflect the current status of infection. High titers detected for years can suggest the presence of chronic infection. According to some studies, the concurrent determination of IgG and IgA antibodies increases diagnostic sensitivity because IgA are the only antibodies detectable in 2–7% cases of H. pylori associated gastric ulcer and duodenal ulcer. However, IgA antibody tests are poorly standardized and therefore generally not recommended for diagnostic testing .
After successful H. pylori eradication, the IgA antibody titer may decrease more strongly than the IgG titer during the first six months. A decline in IgG titer is not detectable before 3–12 months after eradication /, , /. High positive titers of specific antibodies can persist for months (or even years) in successfully eradicated patients. Serological test results would only be useful if, compared to pre-therapeutic tests with an identical kit, a significant titer reduction by more than 50% were detected. Therefore, IgG antibody determination as a clinical follow-up control of H. pylori eradication is generally not recommended /, /.
Comments and problems regarding serology
Because of the inhibitory effect on H. pylori, the intake of antibiotics, bismuth preparations or proton pump inhibitors before stool antigen testing can cause a false negative result. Test kits for the detection of H. pylori antibodies should be evaluated against the given geographical environment to avoid false negative results based on the pathogen’s large antigen variation .
Nucleic acid amplification techniques allow the specific and, depending on the specimen used, more or less sensitive detection of H. pylori /, /. The detection of specific genomic DNA in feces using nucleic acid amplification methods implies the problem of inhibition and has not become established in practice due to being labor and cost intensive by comparison with antigen detection. Detection by PCR testing of gastric secretion and biopsies achieves high diagnostic specificity and sensitivity . PCR testing of H. pylori from gastric biopsy material is controversial regarding added value by comparison with other methods (culture, histology) and is considered a back-up test .
If culture is not successful in the case of positive urease reaction or positive histology, molecular biological methods (PCR or real time PCR with probe hybridization) are used as ”rescue” tests. This applies, in particular, to the culture independent detection of existing antibiotic resistances . These tests allow the reliable identification of the genetic determinants of resistance to clarithromycin, tetracycline, chinolone and rifamycin. However, resistance determination by culture is the preferred method because it enables exact phenotypic determination of the minimum inhibition concentration as well as an amoxicillin and metronidazole sensitivity testing .
5. Fischbach W, Malfertheiner P, Hoffmann JC, Bolten W, Bornschein J, et al. S3-guideline ”helicobacter pylori and gastroduodenal ulcer disease” of the German society for digestive and metabolic diseases (DGVS) in cooperation with the German society for hygiene and microbiology, society for pediatric gastroenterology and nutrition e.V., German society for rheumatology, AWMF-registration-no. 021/001. Z Gastroenterol. 2009; 47: 1230–63.
10. Gisbert JP, de la Morena F, Abraira V. Accuracy of monoclonal stool antigen test for the diagnosis of H. pylori infection: a systematic review and meta-analysis. Am J Gastroenterol 2006; 101:1921–30.
12. Dunn BE. Serodiagnosis of Helicobacter pylori infection and eradication. In: Rose NR, Hamilton RG, Detrick B (eds). Manual of clinical laboratory immunology. Washington; American Society for Microbiology 2000: 462–7.
15. Hoang TT, Rehnberg AS, Wheeldon TU, Bengtsson C, Phung DC, Befrits R, Sörberg M, Granström M. Comparison of the performance of serological kits for Helicobacter pylori infection with European and Asian study populations. Clin Microbiol Infect 2006; 12: 1112–7.
Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Legionellosis (Legionnaires’ disease) was first identified as a respiratory disease caused by Legionella when a pneumonia epidemic occurred during a convention for war veterans in Philadelphia in 1976 /, , /.
Legionella is the only genus of the bacterial family Legionellaceae and comprises more than 40 species with more than 60 serogroups. Legionellae are Gram negative, aquatic, obligate aerobic, rod shaped bacteria which are motile with a single mono polar flagellum. They are found in freshwater environments worldwide. Legionellae are capable of intracellular multiplication in amebae and other protozoa at temperature ranges of 25–55 °C. Stagnant water favors Legionella growth. They do not replicate at temperatures below 20 °C and die at temperatures above 60 °C. Less than half of the Legionella species are pathogenic to humans /, /. One species, L. pneumophila, causes approximately 90% of all reported cases of legionellosis in humans, with serogroups 1, 4 and 6 having the highest relevance /, , /. Other Legionella species such as L. longbeachae, L. bozemanae or L. micdadei have also been suggested as causative agents of infection, especially in immunocompromised risk groups /, /.
Legionella infection occurs through inhalation of contaminated aerosols produced by water systems such as hot water distribution systems in residential buildings, hotels and hospitals, from shower heads, air conditioning systems, whirlpools, inhalation systems, dental units or cooling towers. There is no risk of person-to-person transmission .
Diseases associated with Legionellae occur worldwide and particularly as sporadic, epidemic outbreaks. Infections are reported throughout the year, reaching a peak in the summer and fall months. An estimated 15,000–30,000 cases of sporadic Legionella pneumonia occur in Germany annually . Approximately 1–5% of the cases of pneumonia treated in hospitals are diagnosed as legionelloses /, /. The number of cases reported to the CDC in the United States increased by 70% from 2002 to 2003; more than 2,000 cases are reported to the CDC every year . A significant increase in the incidence of legionellosis in the United States and Europe has been documented over the last years. For the years 2003 to 2004, almost 4,000 community-acquired cases of Legionella pneumonia and 1,150 cases associated with travel abroad were reported in Europe .
In Germany, studies of the Community Acquired Pneumonia (CAP) Competence Network showed that Legionella pneumonia was diagnosed in 3.8% of all cases of CAP observed. Approximately 90% of cases were caused by L. pneumophila and 10% were caused by other Legionella spp. /, /. Legionella species have been detected in virtually all sources of fresh water. However, air conditioning systems and little used faucet water in buildings are considered to be the most frequently suspected sources of human infection . Regular checks of faucet water systems in public and apartment buildings are required by law in Germany. Definitive diagnosis of legionellosis is based on culture of the pathogen from respiratory secretions or pleural fluid on buffered charcoal yeast extract (BCYE) agar . Since Legionellae have never been isolated from healthy individuals, a positive culture result is always considered to be diagnostic proof of legionellosis (100% specificity at low sensitivity) /, /.
- Legionella pneumonia (Legionnaires’ disease): 2–10 days
- Pontiac fever: 1–2 days.
Classical Legionnaires’ disease starts with uncharacteristic, influenza like prodromal symptoms such as headache, myalgias and initially unproductive, later productive, cough. Features developing within a few hours include high fever, chills, myalgias (especially in the thorax region) and occasionally abdominal pain with diarrhea, vomiting as well as encephalopathy with drowsiness and delirium. The mortality rate is 15% and can increase to 80% in untreated, immunocompromised patients .
According to Article 7 of the German Infection Protection Act (IfSG), direct or indirect detection of Legionella spp. is subject to mandatory reporting.
Direct immunofluorescence test
The direct immunofluorescence test does not achieve satisfactory detection limit. To visualize a significant number of organisms, approximately 104 Legionellae per mL of specimen are necessary, a density of organisms not often reached. Direct immunofluorescence with polyclonal or monoclonal enzyme labeled antibodies for the detection of Legionellae from broncho alveolar lavage specimens has a diagnostic specificity of up to 95% and a sensitivity below 60%.
Bronchial secretion obtained via bronchoscopy is the preferred specimen.
ELISA employed for antigen detection in urine does not achieve the same detection limit for all Legionella species and their serogroups. The diagnostic sensitivity of the ELISA is estimated to be 50–70% in patients with defined legionellosis. The assay was most sensitive in cases in which L. pneumophila of serogroup 1 was isolated (94.6%) and less sensitive (86%) when including samples with serogroups 2, 3, 4, 6 and 10. No detailed information is available as to the detection limit for other Legionella species /, /.
Rapid immunochromatographic test
Nitrocellulose adsorbed antibodies to L. pneumophila serogroup 1 allow the rapid detection of Legionella antigen in urine. The assay is only designed for L. pneumophila serogroup 1 and does not detect other serogroups or other Legionella species .
These tests are commonly employed in diagnosing legionelloses, though they are more suited for epidemiological purposes than for the diagnosis of individual cases. The serological confirmation of a clinical diagnosis can only be made retrospectively. It is unreliable due to many possible cross reactions .
Indirect immunofluorescence test (IIFT)
Smear preparations of L. pneumophila mixtures of various serogroups are incubated with diluted patient serum. In positive samples, specific antibodies bind to the bacterial antigens. In a second incubation step, bound antibodies are stained with fluorescein labeled anti-human antibodies and rendered visible in the fluorescence microscope.
The detection limit achieved by the IIFT is significantly hampered by the variety of existing Legionella antigens. In many cases, this problem is tried to be avoided by using polyvalent antigen pools consisting of several serogroups of L. pneumophilia or different Legionella species. In doing so, however, it must be noted that the specificity of the immunological tests decreases with increasing number of different antigens. Therefore, positivity of a pool antigen must be verified by confirmatory testing with monovalent smear preparations to allow a valid diagnostic statement /, /.
- Serum (for indirect pathogen detection): 3 mL
- Urine (for direct pathogen detection): 5–10 mL
- Lower respiratory tract secretions (for PCR): 5–10 mL
Direct pathogen detection
Antigen detection in urine by immunochromatographic assay or ELISA offers simplicity and rapidity in diagnosing legionellosis. In a systematic review to asses the diagnostic reliability of antigen detection by commercially available and in-house test systems, it was found that such tests achieve a pooled diagnostic sensitivity of 74% (95% CI, 68–81%) at a pooled specificity of 99% (95% CI, 98–99%) /, /. A positive result of Legionella urinary antigen testing is proof of legionellosis. The test only detects antigens of serogroup 1. In some cases, cross reactions may also lead to the detection of other serogroups, albeit at low sensitivity /, /. Thus, most test systems are only adequately reliable for the detection of L. pneumophila serogroup 1 antigens in urine. Urinary antigen excretion is already detectable 24 h after infection and usually persists for several days or weeks, but rarely for months. However, a negative test result does not rule out the presence of Legionella infection.
Direct pathogen detection in urine is to be preferred over direct pathogen detection in bronchial secretion. The urinary antigen test achieves higher analytical sensitivity and is easier to handle by comparison with the immunofluorescence test employing bronchial secretion .
Serological tests for indirect pathogen detection
To identify a recent infection, indirect pathogen detection by serological testing requires a fourfold rise in antibody titers to at least ≥ 128 or seroconversion. For detection of L. pneumophila by IIFT, an at least fourfold rise in titers is considered to be diagnostically significant. The fact that in the non outbreak setting an acute-phase, single antibody titer ≥ 256 is in practice considered to indicate presumptive Legionella infection has been discussed controversially /, , /. In many cases, a single titer of 256 is not thought to achieve adequate specificity considering different prevalences of infection depending on the region and population group .
Since antibody formation does not start until the early stage of infection, equivocal clinical pictures can, as a rule, only be clarified in retrospective by serological detection with a rise in titers using paired sera. Clinical utility of serodiagnosis is limited, and the test is mainly useful as an epidemiological tool . In most legionellosis patients, seroconversion occurs up to 3 weeks after the onset of the disease. It should be monitored for a total of 9 weeks /, /. Some patients, including those with culture-confirmed legionellosis, never seroconvert during serological testing. Serodiagnosis achieves a diagnostic sensitivity of approximately 80% and a diagnostic specificity of approximately 95% /, , /.
Up to one third of all individuals tested in Germany have low Legionella antibody titers. This is not surprising considering the ubiquity of numerous Legionella species and serogroups at aquatic sites and the unclear prevalence of non-pneumonic Legionella infections such as the Pontiac fever. The prevalence of the Pontiac fever is estimated to be a hundred times higher than that of legionellosis /, , /.
Comments and problems regarding serology
The use of antigen pools in the IIFT for indirect pathogen detection will lead to a summation effect of low Legionella antibody titers, thus hampering clear threshold definition. In addition, a significant amount of immunological cross reactions between Legionella and other bacteria such as P. aeruginosa, P. fluorescens, P. alcaligenes, Xanthomonas spp., as well as Flavobacterium spp., Bacillus cereus, Bacteroides fragilis, B. pertussis, Campylobacter spp. and various enterobacteria will be detected. This can have a significant effect on the infection immunodiagnostic test outcome /, , /.
The difficulties in rapid Legionella detection testing by culture and the limitations of antigen detection for non L. pneumophila species have led to the development of various molecular biological methods for the rapid diagnosis of Legionella sp. from clinical specimens. Target regions include, for example, the 5S RNA and 16S RNA genes and the mip gene /, , /. Sputum and secretions of the lower respiratory tract are recommended as specimens. The usefulness of urine and other non respiratory tract samples in diagnostic testing for legionellosis has not been sufficiently evaluated. The great advantage of molecular detection methods is their high diagnostic sensitivity and specificity for L. pneumophila and other rare pathogenic Legionella sp., which are not sufficiently detected by the available antigen tests.
Studies on the accuracy of real time PCR-assisted detection methods with subsequent probe hybridization for the identification of Legionella sp. reported sensitivities of 97.4% at specificities of 98.6%. The studies involved patients with community acquired pneumonia and samples of patients with confirmed legionellosis . The objective is to achieve precise molecular identification of the species by probe hybridization, sequencing or melting point analysis of the relevant amplicon. This is important to avoid clinically irrelevant, false positive results due to environmental contamination by apathogenic non L. pneumophila sp., particularly since Legionella can also occur in solutions and laboratory equipment.
1. Edelstein PH. Detection of antibodies to Legionella spp. In: Rose NR, Hamilton RG, Detrick B (eds). Manual of clinical laboratory immunology. Washington; American Society for Microbiology 2002: 468–76.
3. Kümpers P, Tiede A, Kirschner P, Girke J et al., Legionnaires’ disease in immunocompromised patients: a case report of Legionella longbeachae pneumonia and review of the literature. J Med Microbiol 2008; 57: 384–7.
5. von Baum H, Ewig S, Marre R, Suttorp N, Gonschior S, Welte T, Luck C. Community-acquired Legionella pneumonia: new insights from the German competence network for community acquired pneumonia. Clin Infect Dis 2008; 46: 1356–64.
10. Plouffe JF, File TM, Breiman RF, Hackman BA, Salstrom SJ, Marston BJ, Fields BS. Reevaluation of the definition of legionnaires’ disease: use of the urinary antigen assay. Community based pneumoniae incidence study group. Clin Infect Dis 1995; 20: 1286–91.
13. Avni T, Bieber A, Green H, Steinmetz T, Leibovici L, Paul M. Diagnostic accuracy of PCR alone and compared to urinary antigen testing for detection of Legionella spp.: a systematic review. J Clin Microbiol 2016; 54: 401–11.
Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Leptospirosis is an acute to chronic, partly clinically inapparent zoonosis caused by pathogenic Leptospira /, , /. The genus Leptospira belongs to the family Spirochaeta and currently comprises approximately 20 species including 8 which are pathogenic to humans. Based on different antigenic characteristics, the species are classified serologically into at least 320 serotypes:
- The apathogenic species L. biflexa with about 60 serotypes
- The pathogenic species L. interrogans, which can be differentiated further into at least 260 serotypes, (e.g., L. interrogans serotype icterohemorrhagiae, L. interrogans serotype grippotyphosa).
Leptospiremia is detectable by blood cultures in the early stage of infection. For pathogen culture tests, please refer to the relevant literature .
Direct pathogen detection by microscopy and culture from clinical specimens is possible but not recommended as a routine laboratory procedure. Most infections are identified by serodiagnosis /, , , , , , , /.
Leptospira occur worldwide causing anthropozoonoses of various severities . The morbidity of leptospirosis has decreased because of improved hygiene levels in Western industrialized countries but has become an important public health problem in developing countries in the tropics and subtropics. It is estimated that 300,000 to 500,000 cases of new infections occur annually. Climatic and environmental changes promote spread of the infection. For instance, major outbreaks due to increased introduction of the pathogens to the environment, especially during flooding, have been observed. There is a significant risk of local outbreaks of infection associated with recreational activities in industrialized countries (swimming, canoeing, rafting) as well as after large international competitive events in addition to travel /, , /.
Infected animals, especially rats and mice, as well as domestic animals such as dogs and pigs are always the primary sources of infection. Humans become infected through contact with secretions of the animals (urine, snuffling), meat or surface water contaminated with Leptospira /, /. Bacteria enter the body through lesions in the skin and mucous membranes, conjunctiva and though inhalation of contaminated aerosols. Specific occupational groups at risk include hog farmers, butchers, workers in sewer and waste water facilities, field hands, veterinarians (to be reported as occupational disease) as well as anglers and water sportsmen. The former presumption that specific diseases are linked to specific serotypes, for example hemorrhagic jaundice to the serotype icterohemorrhagiae, Canicola fever to the serotype canicola, swineherd’s disease to the serotype tarassovi, slime and field fever to the serotype grippotyphosa, is no longer sustainable /, , , /.
3–30 days, usually 5–14 days.
Depending on the serotype and patient’s immune system, the clinical presentation of leptospirosis varies, ranging from inapparent to mild to severe fulminant infection. Approximately 90% of symptomatic patients present with mild, influenza like symptoms. Leptospirosis is under diagnosed in up to 70% of cases even in highly endemic regions /, /.
Clinical symptoms range from mild to severe courses of the disease with hemorrhage, renal insufficiency, jaundice and fatal complications /, /. The incubation period is 5–14 days. Acute renal failure occurs in 10% to more than 60% of cases. Thrombocytopenia has been observed in up to 50% of cases.
Leptospirosis associated severe pulmonary hemorrhagic syndrome with symptoms such as thoracic pain, cough, dyspnea and pulmonary hemorrhage is a just recently described complication. Respiratory symptoms show 4–6 days after the onset of the disease and may ultimately lead to death within 72 h . The disease, especially in severe infection, may take a dual-phase course with an early septicemic phase followed by a phase of organic disease (). The mortality rate in severe courses is more than 20% /, /.
In general laboratory tests, the erythrocyte sedimentation rate is elevated, white cell counts range from normal to moderately elevated and aminotransferases, bilirubin and alkaline phosphatase show mild elevation. Urinalysis shows abnormal findings. Serum bilirubin levels can reach up to 20 mg/dL (342 μmol/L). Associated neurological symptoms are generally reflected by a CSF cell count of less than 500 cells/μL while protein and glucose are normal or only slightly abnormal .
According to Article 7 of the German Infection Protection Act (IfSG), direct or indirect detection of Leptospira interrogans is subject to mandatory reporting.
The determination of specific antibodies 1–2 weeks after infection (during the organic disease phase) serves as guidance in suspected leptospirosis.
Antigen detection in urine is performed by dot blot ELISA, using monoclonal antibodies directed against a 35 kDa component of pathogenic leptospires. The assay detects the antigen in the urine of patients whose sera tested negative in IgM serological tests. Major evaluation studies are pending .
Direct immunofluorescence test
The detection of L. interrogans in body fluids (e.g., blood) by direct immunofluorescence using labeled antibodies allows pathogen identification in the early stage of infection but achieves controversial sensitivity /, /.
Microagglutination test (MAT)
The sensitive and specific, but time consuming and potentially infectious MAT is available as a serological reference method. The MAT employs live cultures of a wide spectrum of serotypes. The agglutination response is interpreted under the dark field microscope. The end point of the MAT is the final dilution at which more than 50% of the leptospires are agglutinated /, , , /.
Indirect hemagglutination (IHA) test
Ovine RBCs sensitized with Leptospira cell components of the serotypes icterohemorrhagiae and grippotyphosa are incubated in micro titer plates with diluted patient sera. The RBCs agglutinate in the presence of specific antibodies against leptospires. The RBCs form a cell layer at the bottom of the well in positive sera and a button shaped sediment in negative sera. The test is characterized by multiple cross reactions with other Leptospira serotypes (L. bataviae, L. australis, L. pomona, L. sejroe, L. hardjo) /, , , /.
ELISA, lateral flow test, Dri Dot test, immunoblot
These tests are used to detect genospecific IgG and/or IgM antibodies. Some are commercially available. Antigen preparations comprise lipopolysaccharide antigens as well as recombinant protein preparations (rLIP1 41, rOmpl 1, leptospiral immunoglobulin-like (LIG) protein) . The reliability of these test systems in routine laboratory settings is poorly evaluated. The ELISA is reported to achieve diagnostic sensitivities between 84% and above 90% at specificities of 88–99%. Lateral flow assays have a diagnostic sensitivity of 81% at a specificity of up to 96% . However, the results of evaluation studies vary. A WHO guideline provides an overview of commercially available test systems /, /.
Serum: 1 mL
Specific antibodies are detectable 5–7 days after the onset of clinical symptoms and reach a maximum in week 3–5 /, , /. The MAT is considered to be the gold standard . In the MAT, native patient sera are titrated with Leptospira cultures and incubated for 2–4 h at 30 °C or room temperature. The MAT detects serogroup-specific IgM and IgG antibodies /, /. The panels of live leptospires should include, as a minimum, representative serovars of important and locally occurring serogroups and serotypes /, /.
The WHO recommends 19 serovars from 16 serogroups . There is only limited correlation between serovar specific antibody detection and the serovar actually causing the infection. In many cases, the antibodies agglutinate several serovars /, /. The MAT can be negative at the onset of infection. Therefore, serological monitoring is required /, /. The MAT is reported to achieve a diagnostic sensitivity of 90% and a specificity above 90% . Serologically confirmed leptospirosis is defined by a fourfold rise in titer verified by parallel testing with serum samples collected at an interval of 8–14 days and/or serum from the acute and convalescence phase /, , /. Positive titers may persist for months (or years), especially in endemic regions /, /.
Interpretation of MAT results
Positive: titers ≥ 400; a titer ≥ 400 in the presence of the corresponding clinical symptoms is evidence of leptospirosis.
Suspected: titers of 100 and/or 200; these titers point to current infection.
Interpretation of indirect hemagglutination (IHA) test
A positive result (titer > 160) suggests the presence of Leptospira infection. A negative result cannot entirely exclude an infection. The test cannot differentiate between the individual serovars. The IHA by comparison with the MAT achieves a diagnostic sensitivity of 79% at a specificity of 96% /, /.
Interpretation of rapid slide agglutination test
A positive result of rapid testing must be verified by other serological test methods such as the MAT. A negative result does not exclude Leptospira infection.
Interpretation of ELISA
A 4-fold increase in the ELISA value in two paired serum samples is diagnostic evidence of infection . Comparative testing indicates a diagnostic sensitivity of 86.5% and a specificity of 97% for the IgM ELISA /, /.
Interference in serological tests
Cross reacting antibodies have been observed in syphilis, borreliosis, recurrent fever and legionellosis.
Up to 10% of patients do not seroconvert within 30 days after the onset of clinical symptoms.
In some cases, elevated antibody titers (≥ 100) can persist for years after a resolved infection.
The highly variable clinical symptoms and the problems in direct pathogen detection have led to the development of a number of molecular biological detection methods /, /. Some of these methods are described in the WHO guideline .
The nucleic acid amplification techniques allow the detection of pathogen specific DNA. They employ primer sets to ideally differentiate between pathogenic and apathogenic leptospires /, /. Genetic targets include 16S genes and 23S-RNA genes or insertion sequences (IS1533) secY, the flagellin gene, rrs, flaB, rrl genes as well as the genomic locus LA 3521 in Leptospira interrogans /, /. Another PCR protocol with molecular targets in the secJ and flagellin genes is described in the WHO manual.
The diagnostic sensitivities and specificities of these methods vary due to the lack of major evaluation studies . Diagnostic sensitivities of 100% at specificities of 93% have been reported for real time PCR /, , /. The detection of leptospires in blood, urine, CSF and tissue by nucleic amplification assay is possible. Little information is available on the duration of DNA shedding after infection and/or the asymptomatic shedding of leptospiral DNA in endemic regions .
4. Schütt-Gerowitt H. Die Familie der Spirochaetaceae-Spirochätosen. In: Köhler W, Eggers HJ, Fleischer B, Marre R, Pfister H, Pulverer G (eds). Medizinische Mikrobiologie. München; Urban & Fischer 2001; 452–64.
5. Tappero JW, Ashford DA, Perkins BA. Leptospira species. In: Mandell GL, Bennett JE, Dolin R (eds). Principles and Practice of Infectious Diseases. Philadelphia; Churchill Livingstone 2000; 2425–501.
9. Pope V, Bragg SL, Schriefer ME, Larsen SA. Immunologic methods for diagnosis of spirochetal diseases. In: Rose NR, Hamilton RG, Detrick B (eds). Manual of clinical laboratory immunology. Washington; American Society for Microbiology 2002; 477–93.
10. Lizer J, Velinesi S, Weber A, Krecic M, Meeus P. Evaluation of 3 serological tests for early detection of Leptospira-specific antibodies in experimentally infected dogs. J Vet Intern Med 2018; 32 (1): 201–7.
Udo Reischl, Ruxandra Enzensberger, Borris Boeddinghaus, Klaus-Peter Hunfeld, Jürgen J. Wenzel
The genus Mycobacterium (M) can be classified into three main groups for the purpose of diagnosis and treatment:
- M. tuberculosis complex with the obligate pathogenic species M. tuberculosis, M. bovis, M. microti and M. africanum, the causative agents of tuberculosis
- Non tuberculous Mycobacteria (NTM; also known as Mycobacteria other than tuberculosis, MOTT) as facultative pathogenic causative agents of atypical mycobacterial infections
- M. leprae, another obligate pathogenic species not discussed in this chapter.
Tuberculosis is one of the most dangerous infectious diseases worldwide, with the highest mortality rates after AIDS. Effective therapy of tuberculosis is nowadays available but implies long treatment times and frequent side effects. Antituberculosis drug resistance in strains of M. tuberculosis is a major problem. The epidemic occurrence of these strains has placed the focus on the significance of rapid diagnostics.
Non tuberculous Mycobacteria cause mycobacteriosis of the lung, skin, cervical lymph nodes and, in immunocompromised individuals, disseminated infection. Incidence of these diseases has increased considerably in the past two decades in connection with the AIDS epidemic.
Culture based testing and resistance testing take several weeks. The introduction of nucleic acid based methods in diagnostic testing has made numerous molecular biological techniques available for rapid direct detection in clinical samples and has enabled the differentiation of Mycobacteria species.
Mycobacteria are slowly growing, spore less, obligate aerobic, rod shaped bacilli. They are characterized by their specific cell wall structure which consists of many layers of peptidoglycan as well as numerous long chain, wax like mycolic acids. In special staining, the lipid-rich double layered cell wall makes the Mycobacteria resistant to aggressive destaining with a hydrochloric acid alcohol solution. Therefore, Mycobacteria are also referred to as acid-fast rods. Due to this unique structure, they can persist in the body within phagocytes in a dormant state for long periods of time, with the possibility of endogenous exacerbation. More than 140 species have been identified to date under the genus Mycobacterium. However, only some of these species are pathogenic to humans.
The species M. tuberculosis is the most important representative of the tuberculosis bacteria. It is primarily transmitted by droplet infection from individuals with active tuberculosis disease. By comparison, M. bovis has become rare since the elimination of bovine tuberculosis in Germany. However, the consumption of raw cow milk has remained a source of infection in Third World countries. In addition, M. africanum is a species of Mycobacterium most commonly found in Africa. Domestic cats, for example, are considered to be the reservoir of M. microti in Europe .
Nontuberculous Mycobacteria are ubiquitous as part of the environmental flora in soil, dust and water. Poultry and other animals also serve as reservoir of the pathogen. Direct person to person transmission only occurs in exceptional cases. The clinical significance of these opportunistic organisms in clinical samples can only be assessed in the context of individual clinical symptoms ().
It is estimated that approximately 1/3 of the world population are infected with tuberculosis. About 10 million new cases of active lung tuberculosis occur and approximately 2 million individuals die of the disease annually.
Incidence of tuberculosis in Germany was 5.3/100,000 population in 2010. It is twice as high in large cities like Frankfurt, Berlin or Hamburg because of the high fraction of migrants. Incidence is highest, however, in developing countries, primarily in Asia and Africa, with up to 200/100,000 population annually. A higher probability of infection and the possibility of antibiotics-resistant M. tuberculosis isolates must be anticipated in immigrants from the former Soviet Union, especially Kazakhstan, and from former Yugoslavia in Germany /, /. Morbidity of infection with multi-resistant pathogens was low (2.1%) in Germany in 2009, but associated with increased mortality (7.6% compared to 3.6%) and higher cost /, /.
Studies on the epidemiology of non tuberculous Mycobacteria (NTM) documented an incidence of 0.4 and 3.9/100,000 population in industrialized countries . NTM caused approximately 20% of cases of pulmonary mycobacteriosis in HIV negative patients. The fraction of NTM as the causative agents of mycobacterioses generally increased from 9% (1989) to 31% (1997). Certain patient groups are at higher risk of NTM infection: 25–50% of all AIDS patients and up to 10% of all patients who received bone marrow transplants or are severely immunocompromised suffer from NTM infection, most commonly in the form of disseminated disease. NTM infection involving the lung has frequently been reported in mucoviscidosis patients.
Incubation period and duration of infectiousness
The incubation period of tuberculosis can be a few weeks to many months. Clinical symptoms usually occur 6 months after infection. However, the disease may also manifest much earlier, even before a positive turberculin test result. The risk of active disease is highest in the first two years after infection. However, a latent nidus can still be reactivated after decades.
In the most infectious stage of active pulmonary tuberculosis, acid fast rods are detectable in sputum, bronchial secretion or gastric secretion by microscopy. Patients under effective, antituberculous combination therapy, who are infected with a sensitive strain, are usually no longer infectious after 2–3 weeks . Closed lesions such as, for example, spinal abscesses, are not considered to be infectious; hence, mandatory isolation does not apply to affected patients.
Predisposition to the occurrence of tuberculosis is promoted by social factors such as unbalanced diet and by various forms of immunodeficiency: alcoholism, HIV infection, hemato-oncological disease and treatment with cytostatic agents or cortisone. Only 10% of all infected patients develop manifest tuberculosis during their lifetime. Such cases usually are post-primary tuberculosis.
The clinical symptoms of active pulmonary tuberculosis (approximately 80% of manifestations) are ambiguous in most cases and indicate the presence of chronic, consumptive disease: chronic productive cough, hemoptysis in typical cases, thoracic pain in pleural involvement, sub febrile temperatures, night sweats, weight loss and sudden decline in performance.
Extra pulmonary forms of the disease (approximately 20%) most frequently include lymphadenitis (8%), followed by pleural involvement (4%), urogenital tuberculosis (3%), tuberculous arthritis (2%) and the life threatening tuberculous meningitis (0.5%) .
HIV patients take a special position by being exposed to a 5–10-fold higher risk of tuberculosis reactivation, in many cases in the form of extra pulmonary and disseminated clinical symptoms.
In immunocompetent individuals, non tuberculous Mycobacteria (NTM) cause tuberculosis like pulmonary disease, for example M. kansasii. Moreover, they can cause soft tissue infections (M. marinum, M. chelonae) or lymphadenitis in children (M. avium subsp. hominissuis, M. intracellulare, M. malmoense) /, /. In some cases, they are inoculated by trauma (e.g., M. marinum). The increased importance of NTM due to the rising number of severely immunocompromised patients in recent years necessitates a stronger focus on these opportunistic organisms in microbiological diagnostic testing. Disseminated infections are the most frequently encountered manifestations in immunocompromised patients, especially including M. avium intracellulare and M. kansasii infections as the most common bacterial complications . Depending on the pathogenicity, however, only approximately 10% of patients with detectable NTM also show clinical symptoms of infection. In many cases, they merely function as carriers.
According to Article 6 of the German Infection Protection Act (IfSG), all cases of disease and death from tuberculosis requiring treatment, even in the absence of bacteriological evidence, are subject to mandatory reporting by the treating physician. The laboratory first reports the detection of acid-fast rods in sputum, in particular, according to Article 7 IfSG, the cultural and nucleic acid-based detection of Mycobacteria from the M. tuberculosis complex and subsequently the result of resistance determination. Atypical Mycobacteria are not subject to mandatory reporting.
Despite much progress in the field of diagnostics, microscopy continues to be the essential method in tuberculosis control because it allows the rapid and cheap identification of all kinds of infectious cases based on the Ziehl-Neelsen or auramine-rhodamine staining methods. However, microscopic detection methods do not allow to differentiate between tuberculosis bacteria and NTM . Moreover, they achieve insufficient detection limits to permit exclusion of mycobacterial infection ().
The definitive diagnosis of tuberculosis and other mycobacterioses requires bacteriological pathogen detection which, however, is time intensive due to the long generation time of some (slowly growing) Mycobacteria species. The combined use of solid and liquid culture media has proven to be the gold standard for detection . Special liquid culture media reduce the mean detection time to 14 days. However, the cultures must be incubated for 8 weeks to allow definitive exclusion of mycobacteriosis /, /. Any new isolate from the M. tuberculosis complex must be tested against first choice tuberculostatic agents.
Species within the M. tuberculosis complex are considered to be risk group 3 pathogens. Thus, all work steps involving cultured pathogens must be performed in strict compliance with adequate hygienic precautions (L3 laboratory).
A broad spectrum of molecular biological methods is available for direct detection of Mycobacteria in clinical samples, species identification and resistance testing. While some nucleic acid based methods achieve poorer detection limits than culture, they can reduce the time to diagnosis from weeks to hours or days. Although more expensive, they are much more sensitive and specific than microscopy and allow the specific detection of pathogens of the M. tuberculosis complex at nucleic acid level. This feature is especially important as the positive predictive value of microscopy decreases in high incidence of non tuberculous mycobacteria (NTM) in a hospital population. The use of nucleic acid amplification techniques in diagnostic mycobacterial testing comprises /, /:
- Direct detection of M. tuberculosis and/or NTM in clinical specimens
- Species identification of Mycobacteria multiplied by culture
- Molecular resistance testing
- Molecular genotyping for epidemiological concerns.
Nucleic acid amplification (NAA) is a method yielding the greatest benefit with medium probability of tuberculosis. Earlier laboratory confirmation can lead to earlier treatment initiation and increased opportunity for the early introduction of hygienic measures to interrupt transmission. NAA is indicated in /, /:
- Reasonably suspected tuberculosis in the presence of microscopically negative sputum
- Microscopically positive sputum if rapid differentiation between tuberculosis pathogens and NTM is necessary
- Severe clinical symptoms
- Patients at especially high risk (AIDS, immunocompromised individuals, infants).
Method of determination
All NAA methods are based on nucleic acid isolation and subsequent exponential multiplication of specific DNA fragments within the mycobacterial genome or RNA. The most common in vitro amplification methods comprise the polymerase chain reaction (PCR), transcription mediated amplification (TMA) and strand displacement amplification (SDA). Many commercially available NAA tests for the rapid and sensitive detection of nucleic acids of M. tuberculosis from clinical specimens have been approved by the United States Food and Drug Administration. The validity and reliability of commercially available test kits is documented in relevant studies and in the recent evaluations of the regularly performed inter laboratory proficiency tests on the NAA based detection of M. tuberculosis. As a rule, such tests achieve values of 92–97% . The variations in mean performance of the commercially available NAA test systems are found to be but marginal.
The further development of two test methods should be noted:
- The reverse dot blot (strip blot) method: special strip blot test formats are commercially available for the rapid differentiation between representatives of the M. tuberculosis complex and NTM. These tests are performed subsequently to a classical PCR to detect the relevant amplification products via specific hybridization reactions and color detection (comparable to Western blotting). Depending on the design of the PCR and strip blot, the resulting band pattern can by comparison with the corresponding reference pattern be assigned to a specific Mycobacteria species, a specific genotype or a mutant expressing antibiotics resistance. NALC-NaOH decontaminated pulmonary, microscopically positive, direct samples or culture isolates are recommended as specimens for these test systems.
- PCR test cartridges: some new test concepts use a sealed ”all-in-one” cartridge where the individual steps of DNA isolation, amplification and real-time detection of specific PCR products are processed. As the test is largely automated, there is no need for occasionally error-prone manual work steps, test processing is no longer restricted to specially trained personnel and result interpretation is performed by inherent software with a laboratory embedded display port (EDP) interface. Proper processing based on this closed-circuit concept can rule out contamination by PCR products. Decontaminated, direct respiratory tract specimens and culture isolates are recommended as specimens. Following chemical inactivation, specimens from the respiratory tract can also be directly used in the test system. However, this will have a detrimental effect on the limit of detection.
Samples from the respiratory tract, cerebrospinal fluid (CSF) and lymph nodes, biopsies, aspirated fluid, gastric secretion, urine and, in disseminated infection, whole blood.
The selective use of Nucleic acid amplification (NAA) methods for rapid diagnostic testing and resistance determination helps to improve the survival rate of patients and reduce costs . NAA techniques detect tuberculosis in almost all microscopically positive samples as well as in about half of microscopically negative, culture positive cases . They also yield similar results in various specimens as long as the nucleic acid preparations stem from non respiratory specimens without the presence of inhibitory substances.
In all, the diagnostic accuracy of NAA test results is superior to microscopy and only slightly inferior to culture . If NAA techniques are used to ask the question whether an untreated patient has active pulmonary tuberculosis, the answer will be correct in 92–95% of cases as compared with 80% of cases if smears are used. Thus, NAA assays could and should replace sputum smears for diagnostic purposes. Given the much higher cost, however, this may not be economically feasible and clinically useful in all cases .
Testing by culture should always be attempted because the rapid detection of antibiotic susceptibility provides the basis for efficient and selective treatment.
NAA tests achieve a diagnostic sensitivity of 80–90% at a specificity of approximately 99% (). The resulting positive predictive value is 94–98%. Smear negative samples, however, only achieve a diagnostic sensitivity of 60–80%. Considering the significance and consequences, a positive result should be confirmed by second analysis of a different sample . All detection methods may yield false positive results due to laboratory contamination, the detection of non infectious or non replicable pathogens or cross reactions.
Treatment should be initiated if tuberculosis is clinically suspected. Another sample should be analyzed for confirmation or the culture result should be awaited.
Comments and problems
Specimens from the respiratory tract are primarily recommended for diagnostic testing. The approval of numerous commercially available test systems is restricted to the use of such specimens.
Diagnostic testing for tuberculosis should use the first of three sputum samples collected on different days or the culture positive sample for Nucleic acid amplification (NAA) . Numerous studies have documented that the tests also yield conclusive results when using other specimens. Refer to .
It should be noted that the clinical diagnosis of extra pulmonary tuberculosis is ambiguous in many cases and can be guided by the microbiological test result. CSF and lymph nodes as well as biopsies, aspirated fluid, gastric secretion, urine and, in disseminated infection, whole blood are specimens suited for diagnostic testing.
Biopsy and abscess specimens include a high amount of inhibitors which may have an interfering effect on the test.
Fixed specimens can also be used, but imply much reduced detection limit.
All samples should be stored in a cool place and protected from drying.
Microscopy is usually employed for analyzing the infectiousness and monitoring patient therapy.
Direct molecular biological detection is especially well suited for initial diagnosis of active, untreated tuberculosis. It does not detect the intact, replication competent pathogens, but only their nucleic acids. These methods have not been sufficiently evaluated for assessment of infectiousness of the detected pathogens.
Test results must be evaluated in their entirety. Sole molecular biological testing of a patient sample is insufficient because culture always achieves higher detection limit and is indispensable for making an antibiogram.
As a rule, commercially available Nucleic acid amplification (NAA) tests are provided with a control system to indicate the presence of inhibiting substances in the analyzed nucleic acid preparations. Thus, a negative NAA result is only valid if inhibition control is positive. The findings of a test where inhibition events are observed are not suited for interpretation.
The fraction of inhibited samples is 1–5% depending on the type of specimen and the NAA test used. It is higher in known problematic specimens such as stool, formalin-fixed tissue and purulent samples. In such cases, the entire NAA test should be repeated or new specimen requested .
Inhibition control can also be negative in NAA test systems employing inter species primer sequences and M. tuberculosis specific probe sequences in the presence of great amounts of non tuberculous Mycobacteria in the specimen. In these rare cases, it is recommended to use an alternative test system or wait for the culture test result.
The widespread tuberculin skin test was historically the first immunological diagnostic tuberculosis test. The test principle was developed more than 100 years ago. It is based on the provocation of local skin reaction to tuberculoprotein as a classical sign of delayed type IV immune response according to Coombs and Gell.
Common tests included the tine test, in which antigen is introduced into the skin by an intracutaneous multiple-puncture device, and the Mendel-Mantoux test, in which tuberculin solution is injected into the skin. Histologically, positive reaction manifests as palpable, flat, irregular cutaneous induration with surrounding red halo caused by perivascular mononuclear cell infiltration. The test is read after three days. If the measured diameter of the induration exceeds 6 mm, the result is classified as positive.
By comparison with modern immunological methods, the relatively economic tuberculin skin test includes the following disadvantages:
- The test does not distinguish between response due to infection with M. tuberculosis and response due to previous BCG vaccination
- Test performance and interpretation are to a great extent dependent on the examiner; the patient must present again to read the test result
- Cross reactions with NTM are common, but insufficiently evaluated to date.
The IGRA has been commercially available for several years and partly compensates the limitations of conclusive information provided by the tuberculin skin test. Currently available test systems include the T-SPOT.TB and the QuantiFeron Gold In-Tube. The results of these assays are not influenced by previous BCG vaccination. Cross reactions with most NTM do not occur (except in infections with M. szulgai, M. marinum and M. kansasii).
For the T-Spot test, one lithium heparin 6–8 mL tube of whole blood is collected from adults to prepare the peripheral blood mononuclear cells (PBMC). For the QuantiFeron Gold In-Tube, 1 mL of blood is collected in three specially coated collection tubes each. All other test steps take place in the laboratory. The patient needs not present again in the hospital or doctor’s office for test interpretation. However, these advantages are opposed by higher cost by comparison with the simple tuberculin skin test. Moreover, it should be noted that differentiation between active and latent infection with M. tuberculosis is not possible with these assays.
The principle of both assays is based on the measurement of interferon gamma (IFN-γ) production from T cells in response to specific stimulation with two or three M. tuberculosis specific antigens (ESAT-6, CFP-10, TB 7.7; the latter only in QFT) after 16–24 hours of incubation. Although the tests share a common principle, they use different methodology for IFN-γ detection.
This assay is based on the enzyme linked immunospot technique. In a first step, mononuclear cells are isolated from peripheral blood and then washed and counted. For each sample, four reactions with approximately 250,000 cells each are prepared in pre-coated micro titer plate wells: negative control, stimulation with ESAT-6, stimulation with CFP10 and positive control (phytohemagglutinin as mitogen). During the incubation period, the specifically stimulated or mitogen stimulated cells release IFN-γ which binds to the membrane at the bottom of the well pre-coated with IFN-γ antibody and, on the next day, is rendered visible by using secondary antibody conjugates in a color reaction. Each color dot on the membrane represents an individual, cytokine producing T cell. The amount of dots correlates with the amount of M. tuberculosis specific T effector cells in the patient’s peripheral blood. The T-Spot assay is relatively complex. Under certain clinical conditions (e.g., strong immunosuppression, HIV infection), however, the use of a standardized amount of washed mononuclear cells is associated with an advantage over other methods.
QuantiFeron Gold In-Tube assays (QFT)
For this test, 1 mL of peripheral blood is drawn in three coated tubes each (negative control; stimulation with ESAT-6, CFP10 and TB 7.7; positive control). Incubation usually takes place in the laboratory. During this period, the cells in the whole blood are stimulated by the antigens in the tube wall coating. After incubation and centrifugation, the IFN-γ produced by the T cells is measured in the supernatant by ELISA. Both assay formats employ negative control, where no stimulation by antigens takes place, to exclude unspecific IFN-γ production. The general capability of the cells for IFN-γ production is verified by mitogen positive control.
The assessment of IGRA assays as to their diagnostic sensitivity and specificity for the detection of latent or culture negative active tuberculosis is hampered by the fact that there is no reference method for this clinical concern. Therefore, studies were performed to assess, as an alternative, the detection accuracy of the assays against populations with culture confirmed active tuberculosis. Specificity of the assays was accordingly assessed against populations unlikely to be infected with M. tuberculosis. Thus, the data related to diagnostic sensitivity and specificity, which are examined in studies, can only to a limited extent be applied to the diagnosis of latent tuberculosis. In a multitude of studies, the diagnostic sensitivity of the IGRA was compared with that of the tuberculin skin test and found to be equivalent or slightly superior (80–90%). In most cases, the diagnostic specificity of the IGRA (80–90%) was found to be superior to the tuberculin skin test (approximately 85%) .
In a systematic review directly comparing QFT and T-Spot, QFT was reported to achieve slightly higher specificity in non BCG vaccinated populations (99% vs. 96%) and BCG vaccinated populations (96% vs. 93%), whereas T-Spot appears to be more sensitive .
Comments and problems
Both IGRA formats may yield non interpretable results. Pre analytical and technical interferences may include, for example, longer transport times than permissible before the incubation or isolation of mononuclear cells, improper sample storage, insufficient mixing or overloading of the QFT sample tubes. Moreover, non interpretable results may also be encountered in immunocompromised or HIV infected patients with a CD4 T cell count below 200 cells/μL.
Rapid detection and differentiation of NTM is desirable in certain cases when therapy is guided by the detected species. Indication includes, for example:
- The detection of M. avium, M. intracellulare, M. kansasii and M. malmoense in the blood of HIV patients or immunocompromised patients
- Rapid determination of a mycobacterial species in specimens with sufficiently high bacterial counts (e.g., biopsies with acid-fast rods detected by microscopy).
Reverse dot blot (strip blot) method
Special strip blot test formats are commercially available for the differentiated NAA based detection of a number of clinically relevant NTM species. These blots have superseded the formerly common gene probes due to better practicability, differentiation and superior analytical specificity. They allow, in combination with a previous NAA based amplification step, the direct detection of M. avium, M. intracellulare, M. kansasii, M. malmoense and the M. tuberculosis complex in decontaminated, pulmonary and non pulmonary clinical samples.
Some specialized laboratories use the amplification of marker genes, subsequent gene sequencing and comparison with analyzed sequences in databases for the molecular genetic identification of known, as yet non described mycobacterial species. In contrast to ready-made strip blots with a defined range of species, the advantage of this approach is to cover and identify all mycobacterial species.
A disadvantage of these inter-species detection and differentiation techniques is their method related low limit of detection. However, they are excellently suited for the rapid identification of pathogens in primarily sterile, microscopically positive specimens such as biopsies.
The choice of specimen is species dependent:
- Lymph nodes in children: M. avium complex, M. malmoense
- Blood: M. avium complex
- Cutaneous abscess: M. marinum.
Direct NTM detection allows early adaptation of therapy and/or termination of unnecessary isolation. Sufficient data is only available for some clinical concerns. The tests generally achieve high specificity. Their detection limit depends on several factors (e.g., the method employed, specimen used and species to be detected). Considering the fact that the bacteria are facultative pathogenic, positive findings should be confirmed by quantitative detection .
PCR based detection and differentiation methods
A broad spectrum of test systems is available for the molecular biological differentiation of cultured mycobacterial species. The majority of infectiologically relevant non tuberculous Mycobacteria can be rapidly identified at high sensitivity and specificity within 2 h using pure cultures or single colonies after amplification of the corresponding sequences of the bacterial genome (so-called species markers) . However, the commercially available test systems do not identify all pathogenic species.
Large scale experience has only been gained to date with some of the test system manufacturers. The relevant test systems are able to detect and differentiate the M. tuberculosis complex as well as several other clinically relevant mycobacterial species .
Pure cultures or single colonies of Mycobacteria can also be used for the molecular identification of all mycobacterial species based on specific PCR protocols and subsequent sequencing of the amplified fragments within established marker genes (such as 16S rDNA, rpoB, gyrA, ITS). As already described in , this procedure is also suited to characterize as yet non described species.
A reliable comparison of the obtained sequence data requires the availability of comprehensive, possibly fee-based databases that have been validated specifically for Mycobacteria (e.g., RIDOM, MicroSeq 500 or SmartGene ). Therefore, the qualified interpretation of sequence data is in most cases left to laboratories specialized on molecular species identification.
A decisive, method related disadvantage of DNA sequencing is the partial overlapping of sequencing signals in mixed pathogen samples. Therefore, these techniques are strictly limited to species differentiation in pure pathogen cultures or the detection of bacterial or fungal mono-infection in usually sterile specimens. These molecular biological tests are rapid, specific and relatively economic by comparison with the classical biochemical methods.
MALDI-TOF for species identification
Some laboratories routinely use the biophysical MALDI-TOF technique and correspondingly comprehensive databases for the rapid differentiation of cultured bacteria and fungi. These databases include comprehensive collections of specific fragment patterns of all clinically relevant mycobacterial species. The availability of high capacity equipment provided, these fragment patterns can be used for reliable species differentiation of all pure culture based mycobacteria.
Comments and problems
Final identification of the mycobacterial isolates, as in all other bacterial species, can only be achieved by the critical synoptic interpretation of all cultural, biochemical, biophysical and/or molecular genetic results.
The efficient control of diseases caused by Mycobacteria requires rapid and reliable species differentiation and sensitivity testing. The increasing spread of resistant M. tuberculosis isolates is a serious threat to patients and their environment. Conventional culture methods for mycobacterial diagnostics and resistance testing are very time consuming. Unless the presence of resistant M. tuberculosis isolates in a patient is confirmed, the administration of ineffective antibiotics may lead to the further spread of resistant bacteria and promote the acquisition of additional resistance. Moreover, nonadherence to the therapy plan or premature termination of therapy can lead to the development of multi-drug resistant tuberculosis .
Multi-drug resistant tuberculosis is present if the M. tuberculosis isolate is resistant to the two most important, first line antituberculosis drugs rifampicin and isoniazid. Resistance related information should be obtained early considering the rapid increase in infections with multi-drug resistant isolates worldwide. Delayed diagnostic testing and the resulting inadequate therapy in multi-drug resistant tuberculosis patients can also promote the development of extensively drug-resistant (XDR) M. tuberculosis isolates. Infections with XDR tuberculosis pathogens have been observed around the globe. In some countries, 20% of multi-drug resistant tuberculosis cases are extensively drug resistant.
A number of NAA based test systems are commercially available for molecular resistance testing. The Xpert MTB/RIF real time PCR test system, for example, allows the detection of M. tuberculosis DNA and also determines the presence of mutations encoding rifampicin (RMP) resistance . The practical significance of RMP resistance detection is its useful role as an indicator of the presence of multi-drug resistance and resulting adaptation of therapy.
If DNA preparations of smear positive samples or pure cultures are available, certain GenoType strip blots can be used for further molecular testing for resistance to first and second line antituberculosis drugs.
For instance, potential resistance to the following drugs can be determined:
- Rifampicin by detection of the most frequent mutations in the rpoB gene
- Isoniazid by detection of the most frequent mutations in the genes katG and inhA
- Fluorochinolones by analysis of the gyrA gene
- Antibiotics such as viomycin, kanamycin, amikacin or capreomycin by analysis of the rrs gene
- The first line antituberculosis drug ethambutol by analysis of the most frequent mutations in the embB gene.
As a rule, concordance between PCR results and the results of conventional phenotype sensitivity testing is above 90%. By using the molecular biological assay, however, 20 to 30 days can be saved in the time to diagnosis .
The use of molecular resistance testing methods is recommended if a positive M. tuberculosis result was obtained during primary diagnostic testing in regions with a high resistance rate (some mega cities in industrialized countries and/or emerging countries). This allows the rapid detection of potential resistances and early interruption of transmission of multi-drug resistant M. tuberculosis isolates .
However, the molecular resistance test methods can only reflect the recent level of knowledge due to:
- The multitude of possible biochemical resistance mechanisms
- The partly or entirely identified complexity of resistance mechanisms
- The dynamic change in resistance encoding mutations in the bacterial genome.
Molecular resistance test methods provide useful indication of the probable presence of resistance to specific antibiotic drugs. Final assessment must, however, await the result of conventional antibiotic sensitivity testing.
Restriction fragment length polymorphism (RFLP) and Spacer oligonucleotide typing (spoligotyping) are molecular biological techniques for genotyping individual M. tuberculosis complex strains. These methodologically complex typing methods are useful to detect epidemic transmission or false positive laboratory findings. However, they are mostly only practiced by reference centers since they require relatively complex equipment as well as expertise in evaluation and interpretation .
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Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Pertussis (whooping cough) is a bacterial infection of the respiratory system characterized by severe, paroxysmal cough of several weeks. The infection is caused by Bordetella pertussis, a Gram negative rod shaped bacterium . Transmission occurs in close contact by the inhalation of airborne droplets. The probability of infection in non immune individuals is approximately 90% . Mediated by various adhesins such as filamentous hemagglutinin, the Bordetella bacteria localize and multiply among the cilia of respiratory epithelial cells. B. pertussis, in particular, produces a variety of toxins and virulence factors, which include the pertussis toxin filamentous hemagglutinin, the tracheal cytotoxin, pertactin, heat labile toxin and adenylate cyclase toxin. These toxins help to bypass the host defense and cause local tissue damage, for example tracheal cytotoxin /, , , /.
A zoonotic reservoir for B. pertussis does not appear to exist; humans are its only known host. B. parapertussis and B. bronchiseptica are found in humans and animals . Infection with B. parapertussis can also cause pertussis like disease, which frequently take a milder and shorter course. B. bronchiseptica causes respiratory disease in animals and is only in rare cases found to cause respiratory disease in immunocompromised patients. Other Bordetella such as B. avium, B. hinzii or B. holmsii are also detected in rare cases in respiratory, local and systemic infections in immunocompromised patients /, , /.
Culture detection using special culture media only provides sufficient evidence at the onset of the disease (incubation period, catarrhal stage, beginning of paroxysmal stage). Detection rates by culture vary between 5–70% at 100% specificity. Thus, the rates of direct culture detection are significantly limited in daily practice due to the sensitivity of the pathogens /, , , , /.
Pertussis is encountered throughout the year, and especially frequently in the fall and winter. Every 3–4 years, a wave of pertussis infections occurs in Germany /, , /. Incidence in Europe ranges from < 1 (Portugal) to 200 (Switzerland) registered cases per 100,000 population annually.
In Germany, the incidence of registered pertussis cases in 2006 ranged from 12 in Saxony to 68 in Mecklenburg-West Pomerania per 100,000 population . A study conducted in the cities Rostock and Krefeld pointed to a frequency of 165 cases per 100,000 population annually . The general recommendation to vaccinate newborns and infants in Germany and the use of combination vaccines including the acellular pertussis components has led to a continuous increase in vaccine coverage and achieved approximately 93% of school-aged children in 2007 /, /. However, reinfection in adolescents and adults is possible following natural disease (15–20 years) and/or complete vaccination (about 10 years) because of the limited duration of immunity /, , /. The number of pertussis notifications has increased continuously especially among adolescents and adults in Germany, and the mean age of patients with pertussis infections has risen from 15 years in 1995 to 41.7 years in 2008. The cause of this trend is a continuous decrease in protection by vaccination in the general population above 30 years of age /, /. It has therefore been recommended since 2009 to include pertussis in due boosters against tetanus and diphtheria in adults .
7–14 (–20) days.
Pertussis usually lasts for several weeks to months. The infection is transmitted from the end of the incubation period until up to 3 weeks after the onset of the paroxysmal stage. Patients are contagious up to five days after the start of antibiotic treatment.
- Catarrhal stage (duration 1–2 weeks): influenza like symptoms with cold, mild cough, qualm and moderate fever
- Paroxysmal stage (duration 4–6 weeks): paroxysmal cough (staccato cough) followed by inspiratory whoop
- Convalescent stage (duration 6–10 weeks): gradual decrease of respiratory symptoms.
Atypical course (especially in adults):
- Persistent cough without typical fits of coughing.
Clinical symptoms of pertussis infections can show age-dependent variations:
- Newborns and infants present with atypical paroxysmal cough as well as pneumonia and apnea (75% pneumonia, 25% apnea, 14% spasms, 5% encephalopathy) in 90% of cases
- School aged children present with typical symptoms
- Pertussis infection is the underlying cause of chronic cough persisting for more than 7 days in 10–20% of all adolescents and adults .
Cough persisting for more than 14 days and one of the following criteria: paroxysmal coughing, inspiratory stridor, post-tussive vomiting (also in infants).
Positive result for at least one of the four following methods: isolation of pathogen, detection of bacterial nucleic acid (e.g., PCR), detection of markedly elevated IgG (ELISA), detection of IgG/IgA antibodies by marked change in two samples (e.g., ELISA).
According to the German Infection Protection Act (IfSG), these infections are not subject to general mandatory reporting. Mandatory reporting to state authorities exists in the federal German states Brandenburg, Mecklenburg-West Pomerania, Saxony, Saxony-Anhalt and Thuringia. According to Article 1 of the IfSG, personnel in public facilities such as nurseries and daycare centers who are diagnosed with pertussis must be suspended from duty and may return to work no earlier than five days after antibiotic treatment has been initiated. Without antibiotic treatment, affected personnel must wait at least three weeks after symptom onset before resuming work. Healthcare facilities should follow the same measures /, /.
The diagnosis of pertussis in the presence of classical symptoms is usually based on clinical manifestations. Serological testing should be performed in children, vaccinated individuals, adolescents and adults suffering from persistent coughing without typical coughing fits. Serological diagnostic testing for B. pertussis antibodies is primarily done by ELISA using filamentous hemagglutinin (FHA) and pertussis toxin (PT) as antigens. Pertussis toxin (PT)-based ELISAs are to be preferred /, , , /.
Enzyme-linked immunosorbent assay (ELISA)
ELISA is used for the semi-quantitative and qualitative detection of IgG, IgM and IgA antibodies. Modern ELISA use purified or recombinant antigens, primarily pertussis toxin and filamentous hemagglutinin.
Only pertussis toxin is specific for B. pertussis. ELISA using other antigen preparations do not have a broad linear range by comparison with the WHO standard. However,pertussis toxin based ELISA do not detect infections with other Bordetella.
Therefore, for the detection of B. pertussis antibodies by ELISA should use purified PT as antigen and be standardized to the 1st WHO International Reference Preparation for B. pertussis antibodies /, , , , /.
The immunoblot can be used for two tiered diagnostic testing in the case of equivocal ELISA results. Electrophoretically fractionated proteins of B. pertussis, including PT and FHA, are used to detect specific immune response. Testing for adenylate cyclase toxin antibodies, which is additionally offered by some commercial test kits, does not reliably differentiate between infection and vaccination . Diagnostic sensitivity and specificity vary considerably among commercially available test systems. The immunoblot is not superior to ELISA. Comparative studies showed that semi-quantitative blot results do not correlate with quantitative ELISA results . Thus, blot tests for IgA and IgG antibodies only play a limited role in the serological diagnosis of pertussis /, , /.
Pertussis toxin neutralizing antibodies can be detected by a complex method involving a neutralization test with Chinese hamster ovary cell culture. However, this test is less sensitive than ELISA . It plays no role in routine diagnostic testing.
Complement fixation (CF)
Complement fixing antibodies are, even in the advanced stage of the disease, only detected in 30–50% of patients, whereas specific antibodies are almost always detectable by ELISA /, /. CF is no longer recommended for routine diagnostic testing because of the lower diagnostic sensitivity compared to ELISA /, /.
Serum (indirect pathogen detection): 1 mL
In primary infection of under-vaccinated individuals with B. pertussis, antibodies are not detected until relatively late (i.e., 1–2 weeks after the onset of symptoms). The sole detection of specific IgG points to a status following pertussis vaccination, while the concurrent detection of IgA and IgM in the presence of corresponding symptoms in an under-vaccinated patient points to primary infection /, /.
IgG antibodies: the antibodies are detectable in serum 2–3 weeks after the onset of the disease. Maximum antibody production is reached 6–8 weeks after the onset of the paroxysmal stage . Testing is standardized to a WHO reference preparation /, /.
IgA antibodies: in natural infection, these antibodies are measurable 7–14 days after the onset of the disease and persist for 6–24 months . IgA antibodies are also produced following vaccination or in the course of natural, symptomatic or asymptomatic reinfection and may therefore even be detected in healthy adults /, /.
IgM antibodies: the antibodies are in the short term detectable in some vaccinated individuals and mainly in primary infection 5–10 days after the onset of the paroxysmal stage and persist for 6–12 weeks. IgM antibodies indicate acute disease. However, testing is not well standardized /, , /.
Comparative studies involving commercially available pertussis toxin (PT)-based ELISA document:
- Diagnostic sensitivities of 84–100% at specificities of 81–93% for IgG determination
- Diagnostic specificities of 51–59% for ELISA using mixed antigen
- Diagnostic sensitivities of 53–73% at specificities of 67–94% for IgA determination .
- Indication of recent pathogen contact: IgG above 100 IU/mL
- No indication of recent pathogen contact: IgG below 40 IU/mL
- Confirmed specificity: IgG above 40 IU/mL and below 100 IU/mL (analysis of 2nd sample or determination of antibodies to other antigens).
Note: serological evidence of an abnormal single value is not usable up to 36 months following vaccination with acellular pertussis vaccine.
- The first sample was collected within the first 2 weeks after the onset of paroxysmal cough and
- the second sample was collected 3–5 weeks later.
Comments and problems in serodiagnostics
Hyperlipemic, hemolytic or microbially contaminated samples can lead to false positive or false negative results. Tests employing filamentous hemagglutinin or whole cell lysates are not specific to B. pertussis. Positive results may also be obtained in infections with other Bordetella or Mycoplasma /, /.
The nucleic acid amplification (NAA) techniques allow the detection of pathogen specific DNA. Established target regions include the Adenylate cyclase gene, the promoter region of the pertussis toxin gene (ptxA-Pr), the Porin gene and the presumably highly sensitive insertion sequences (e.g., IS481). Ideally, the employed primer systems should detect B. pertussis, B. para pertussis and B. bronchiseptica and differentiate between the different species. IS481 based detection is not strictly B. pertussis specific. In a major study involving children under 5 years of age, PCR positive results were typically found within 21 days from the onset of initial symptoms and 14 days from the onset of paroxysmal cough/. In young, under-vaccinated children, culture and PCR achieve a diagnostic sensitivity of approximately 70%. Diagnostic sensitivity of PCR in school-aged children, adolescents and adults is 10–30%. PCR detection rates decrease as a function of increasing duration of the disease. Thus, PCR is no longer useful 4 weeks after the onset of the disease /, , , /.
Nasopharyngeal aspiration or throat swabs, ideally with dacron swabs, without transport medium.
5. Podbielski A, Berger A, et al., In: Podbielski A, Herrmann M, Kniehl E, Mauch H, Rüssmann H (eds.). MIQ Qualitätsstandards in der mikrobiologischen Diagnostik – Infektionen des Mundes und der oberen Atemwege (Teil II). München; Urban & Fischer 2010; 98–106.
9. Guiso G, Berbers G, Fry, n K, He Q, et al. EU Pertstrain group. What to do and what not to do in serological diagnosis of pertussis: recommendations from EU reference laboratories. Eur J Clin Microbiol Infect 2011; 30: 307–12.
10. Pawloski LC, Plikaytis BD, Martin MD, Martin SW, Prince HE, Lape-Nixon M, et al. Evaluation of commercial assays for single-point diagnosis of pertussis in the US. J Pediatric Infect Dis Soc 2017; 6 (3): e15-e21.
11. Riffelmann M, Thiel K, Schmetz J, Wirsing von König CH. Performance of commercial Enzyme-Linked Immunosorbent Assays for detection of antibodies to Bordetella pertussis: J Clin Microbiol 2010; 48: 4459–63.
12. Kennerknecht N, Riffelmann M, Schmetz J, Wirsing von König CH. Comparison of commercially available immunoblot assays measuring IgG and IgA antibodies to B. pertussis antigens. Eur J Clin Microbiol Infect Dis 2011; 30: 1531–5.
13. Trollfors B, Krantz I, Singurs N, Taranger J, Zackrisson G, Roberson R. Toxin-neutralizing antibodies in patients with pertussis, as determined by an assay using chinese hamster ovary cells. J Infect Dis 1988; 158: 991–5.
16. Xing D, Wirsing von König CH, Newland P, et al., Characterization of reference materials for human antiserum to pertussis antigens by an international collaborative study. Clin Vaccine Immunol 2009; 16: 303–11.
18. Lee AD, Cassiday PK, Pawloski LC, Tatti KM, Martin MD, Briere EC, et al. Clinical evaluation and validation of laboratory methods for the diagnosis of Bordetella pertussis infection: culture, polymerase chain reaction (PCR) and anti-pertussis toxin IgG serology (IgG-PT). PloS One 2018; 13: e0195979.
Treponema pallidum infections cause different types of disease in humans (). In endemic treponematoses (bejel, yaws, pinta), late manifestations of disease are rarely seen and the rate of spontaneous full recovery is reported to be almost 100%.
T. pallidum is the causative agent of syphilis. Only 60% of cases affected by this disease recover spontaneously. In a large proportion of cases, if the infection is not treated in time or is treated inadequately, it will progress to a secondary stage after a long latency period and will finally reach a clinically recognizable tertiary stage (or neurosyphilis).
Syphilis is an important disease worldwide. According to WHO estimates, approximately 12 million cases of new infections occur annually, primarily in Southeast Asia, Africa and South America. In Eastern Europe, a strong increase to up to 263 per 100,000 population of new syphilis cases was observed in the successor states of the Soviet Union in the late 1990s. In 2008, 59 new cases were recorded in the Russian Federation and between 4.7 and 10.3 in Central Europe per 100,000 population. The incidence of new syphilis infections has increased again in Western Europe and the United States since the year 2000. The current peak incidence of syphilis infections in the Western industrialized countries is approximately 2–4 per 100,000 population annually. In Germany, 3.7 cases per 100,000 population were recorded in 2010. The disease primarily affects men in sexual contact with men. For instance, the incidence of syphilis was 14 times higher in men (7 cases per 100,000 population) than in women (0.5 cases per 100,000 population) in 2010. The problem of syphilis and HIV co infection is of special clinical relevance. Congenital syphilis is currently reported as 1 case per 100,000 annually and, thus, plays almost no role in Germany anymore due to the generally low rate of infection in women and the consistently implemented prenatal care /, , , , , /.
At the site where the pathogen enters the body, a papule initially occurs which develops into a vesicle and finally into a usually painless, firm and round chancre, referred to as the primary complex. With dissemination of the infection, the regional lymph nodes become involved. The location of the primary complex is not limited to the genital area. It may also be situated in the oral or anal region, depending on sexual practices. Clinical diagnostic is impeded by possible atypical forms of manifestation. Non indurated lesions with indistinct margins, multiple and/or painful ulcers, especially in the anal region and in HIV infected patients, are seen. The primary complex may not occur, may not be diagnosed if occurring in locations where the lesions are difficult to find or may be mistaken for lesions of other origin (venereal lymphogranuloma, chancroid, genital herpes). The diagnostic method of choice at this early, primary stage of syphilis is direct pathogen detection in lesion samples. In most cases, however, pathogen specific antibodies of the IgM and IgG types are already detectable when the patient sees the doctor.
Approximately 9–10 weeks after the onset of untreated infection, a maculopapular rash commonly erupts as the result of hematogenous spread of the pathogen. The secondary stage lasts for weeks to months. Recurrence may occur after symptom free intervals. At this stage, detection of the pathogen by conventional methods is no longer possible. Instead, serological tests usually reveal characteristic antibody patterns which also provide reliable information as to the need for treatment.
Depending on the definition /, / symptom free intervals of infection are referred to as early latency period during the first and/or second year and as late latency period later on. In the absence of anamnestic information, latent syphilis can only be diagnosed based on Treponema antibody screening tests.
The third stage of syphilis occurs after a symptom free latency period of 1–20 years. It usually presents as cardiovascular syphilis, gummatous lesions being rare nowadays. A multitude of complexes of neurological symptoms are possible in the central nervous system. Meningo vascular syphilis is the most common form. Its clinical manifestation is uncharacteristic. The classical form of neurosyphilis, progressive paralysis and tabes dorsalis, also referred to as quaternary syphilis or lues, are now only rarely seen. A wide range of neurological diseases can give the same clinical presentation as neurosyphilis. It is frequently necessary to perform a T. pallidum screening test to detect late stages of the disease.
Reinfection may occur following curative treatment of the infection despite persistence of IgG antibodies. It starts with the clinical manifestations of primary syphilis and the tests appropriate for this stage should be carried out.
Syphilis neonatorum or congenital syphilis
A T. pallidum screening test should be performed as part of the routine prenatal care program in order to exclude this infection and thus avoid congenital syphilis. In the case of a positive test, further evidence for the presence of active infection and resulting appropriate treatment must be sought by obtaining a history of previous infections and treatment and/or by serological tests. Infants born to women who had syphilis during pregnancy should generally be tested for acquired T. pallidum infection.
Dark field microscopy
Principle: direct detection of T. pallidum. Using a sterile loop, material sampled from a suspicious primary lesion of the early stage is applied to a slide, covered by a cover glass and examined with the dark field microscope at a magnification of about 400 × for the presence of motile spirochetes of characteristic morphology. The detection limit is approximately 1 × 105 treponemes/mL. The diagnostic sensitivity of dark field microscopy is 80–95% at a specificity of 77–100% .
Nucleic acid amplification (NAA) technique
Principle: detection of T. pallidum by use of PCR targeting the 47 kDa protein membrane gene of T. pallidum (tpp47-Tp-PCR) and targeting the DNA polymerase I gene (polA-Tp-PCR). The detection limit is reported as 1–65 treponemes. PCR is more sensitive than microscopic methods. It achieves a diagnostic sensitivity of up to 95% in the primary stage of syphilis and up to 80% in the secondary stage . Molecular strain typing is based on characterization of 3 variable treponemal genes arp, tpr and tp0548, respectively.
In smears obtained from suspicious looking skin and mucous membrane lesions:
- Fresh sample for dark field microscopy
- Dried sample on a slide for the detection of T. pallidum by immunofluorescent technique.
NAA techniques are possible from a wide range of specimens (e.g., smears, blood, cerebrospinal fluid), aspirated fluid, tissue samples, amniotic fluid or eye chamber water. A volume of 2–5 mL should be collected from liquid specimens because the detection limit depends on the amount of sample from which the pathogen specific nucleic acid can be extracted.
All liquid samples should be sent to the laboratory in separate, sterile tubes without preservatives. Smears should be sent on swabs in 0.9% NaCl solution. Tissue samples can be formalin fixed or paraffin embedded, although tissue samples in 0.9% NaCl solution without preservatives are preferred. The laboratory should receive the samples as soon as possible. Interim storage at 4–8 °C for up to 24 hours is possible.
Interpretation of test results
Shortly after infection T. pallidum is detectable in a syphilitic lesion by direct diagnostic testing while serological tests are still negative. The diagnostic sensitivity of dark field microscopy is 80–95% at a specificity of 77–100% . In a systematic review and meta-analysis the diagnostic accuracy of PCR was investigated using dark field microscopy as reference test :
- Diagnostic sensitivities were 79.8% (95% CI 72.7 to 85.4%) and 71.4% (46.0 to 88.0%) for tpp47-Tp-PCR and polA-Tp-PCR, respectively
- Diagnostic specificities were 95.3% (93.5 to 96.6%) and 93.7% (91.8 to 95.2%), respectively.
Laboratory diagnosis of neurosyphilis is complicated especially when it is asymptomatic and no single laboratory test result is appropriate to diagnose central nervous system infectivity caused by T. pallidum. In patients with neurosyphilis T. pallidum DNA was detected amplified in 29.8% and 24.2% of CSF samples with the 47-PCR and polA-PCR, respectively. Diagnostic sensitivities were 75.8% and 69.7% and specificities 86.8% and 92.3% for 47-PCR and polA-PCR techniques, respectively .
In a Danish study molecular strain typing based on characterization of the treponemal genes arp, tpr and tp0548 in 197 fully typeable specimens 22 strain types were identified, with one type, 14d/g, accounting for 54% .
It is evident from the published literature that PCR is useful as an adjunct test to direct fluorescence assay and dark field microscopy, and is useful in confirming syphilis in genital ulcer, tissue and other body fluid samples, providing more sensitive detection. Serological tests remain the mainstay tests since T. pallidum is non culturable and also because blood collection is easy .
Serological screening tests specific to T. pallidum antibodies are used to identify and/or exclude syphilis. For economic reasons, tests are recommended which accomplish the concurrent detection of T. pallidum-specific antibodies of types IgG and IgM. Syphilis antibody tests are based on various antigen concepts, ranging from ultrasound homogenates or detergent extracts from T. pallidum, subsp. pallidum (Nichols strain) to the application of singular recombinant antigens. Most new ELISA and chemiluminescence assays employ a combination of the antigens Tp15, Tp17 and Tp47 which are accepted as highly specific .
Treponema pallidum hemagglutination assay (TPHA)
Principle: indirect hemagglutination. Fragments of T. pallidum (Nichols strain) subjected to ultrasound or sodium dodecyl sulfate (SDS) are fixed onto the surface of sheep or other animal erythrocytes. Macroscopically recognizable agglutination occurs in the presence of homologous antibodies. In negative serum, the red blood cells settle by sedimentation in the wells of the micro titer plates. Non sensitized red blood cells are used as control antigens. Titer quantification of the antibody findings is possible and recommended for reactive samples. Diagnostic sensitivity is 76 (69–90)% in the primary stage of syphilis and 100% in the subsequent infection stages. Specificity is reported as 98–100%. The test detects IgM and IgG antibodies /, , /.
Treponema pallidum particle agglutination assay (TPPA)
Principle: as in TPHA. Instead of red blood cells, gelatin or bentonite pellets are used as antigen carrier. The advantage of this test system is that heteroagglutinating antibodies do not interfere with the reaction. Sensitivity and specificity correspond to those of TPHA.
Treponema pallidum latex agglutination assay (TPLA)
Principle: as in TPHA. Latex particles are used as antigen carrier. Diagnostic sensitivity and specificity correspond to those of TPHA. Heteroagglutinating antibodies do not interfere.
Treponema pallidum enzyme immunoassay (Tp-ELISA)
Principle: competitive or indirect antibody ELISA. Fragments of T. pallidum or relevant antigens are attached to a solid phase and coated with patient serum. After washing, the IgG and/or IgM antibodies attached to the solid phase are determined in the patient serum.
In competitive tests, the antigen-antibody complexes are quantitatively determined using an enzyme labeled antiserum treated with Reiter treponemes (T. phagedenis) for pre adsorption of T. pallidum by measuring the enzymatic activity.
In indirect ELISA, the antibodies attached to the solid phase are determined in the sample by using enzyme labeled antihuman immunoglobulins. Semi-quantitative assessment of the antibody content in the sample is done by calculating the index value (absorption value of the sample divided by the absorption value of the cutoff control value).
Further ELISA modifications for the detection of specific IgG and IgM antibodies are commercially available (e.g., μ-capture-ELISA) but usually used for confirmation and not as screening tests.
Treponema pallidum chemiluminescence assay
Principle: as a rule, these syphilis screening tests are used as one-tier or two-tier immunoassays in large equipment. Single, or a combination of, recombinant antigens attached to a solid phase of micro particles (Tp15, Tp17, Tp47) are used as test antigens. After washing, antibodies attached to the solid phase are determined in the patient sample by induced chemiluminescence reaction.
These tests also allow semi-quantitative determination of the antibody concentration in the sample. Little information has been available to date as to their diagnostic sensitivity and specificity. Experience so far gathered with these tests demonstrates a diagnostic sensitivity and specificity comparable to those of TPHA/TPPA and ELISA,
Rapid diagnostic tests for syphilis
Principle: these tests use recombinant antigens for the immunochromatographic detection of T. pallidum antibodies. The test can be performed with whole blood, heparinized capillary blood, plasma or serum as specimen. The test quality and nature of the sample have a decisive influence on diagnostic sensitivity. The relative sensitivity of serum based rapid tests by comparison with TPHA/TPPA is 67–100% and, thus, adequate, but declines to 64–82% when whole blood samples are used .
Compared to conventional detection methods, rapid tests do not enable earlier diagnosis. They do not differentiate between active syphilis infection in need of treatment and past, resolved infection. The test finding alone is insufficient to initiate treatment.
The specificity of positive or ambiguous screening test results must be verified by confirmatory tests. Special tests can be used for this purpose which are not employed for syphilis screening. However, the serological screening tests described in , except for the rapid tests for syphilis, are also suited as confirmatory tests if their methodology differs from the screening test performed.
Fluorescent treponemal antibody absorption test (FTA-ABS)
Principle: indirect immunofluorescence after absorption of the patient’s serum using cross reacting antigens of non pathogenic treponemes.
Non viable treponemes of the pathogenic Nichols strain are used as antigens for the FTA-ABS test either fixed on slides or, in the case of Treponema suspensions, air dried on slides. Prior to the application of patient serum to the coated slide, it is treated with an extract of T. phagedenis (Reiter) in order to remove cross reacting antibodies. The pre-adsorbed samples are then incubated on the slide wells. Following washing, coating is performed using Fluorescein isothiocyanate (FITC)-conjugated, polyvalent or IgG specific anti-human globulin with subsequent incubation. After thorough rinsing, the preparation is dried and examined under the fluorescence microscope at a wavelength of 480 nm and approximately 400-fold magnification.
Diagnostic sensitivity of the FTA-ABS test with the standard serum dilution of 1 : 5 (one part serum to four parts absorption medium) is 84% (70–100%) in patients with early primary stage syphilis, whereas in later stages of the infection it is 100%; diagnostic specificity is 94–100% /, , , , /.
Treponema pallidum immunoblot
Principle: the T. pallidum polypeptides separated in the electric field by sodium disulfate (SDS) polyacrylamide gel electrophoresis are bound to nitrocellulose strips. In addition, test kits alternatively using recombinant antigens directly sprayed onto nitrocellulose strips and referred to as immunoblot or line assay are commercially available for the detection of Treponema specific IgG and IgM antibodies. IgM and/or IgG specificity is ensured by appropriate horseradish peroxidase labeled antisera. Diagnostic sensitivity of the IgG immunoblot is approximately 99%; the diagnostic specificity is 98–99% /, /, but various diagnostic sensitivities have been reported for the IgM immunoblot.
In seropositive primary stage syphilis and secondary stage syphilis, the immunoblot achieves a diagnostic sensitivity of almost 100%, whereas in latent infection, tertiary stage syphilis and reinfection, variances in the results of various IgM antibody assays as well as false negative results are possible .
The nomenclature of the polypeptides of the Nichols T. pallidum, subsp. pallidum, reference strain separated by SDS-PAGE is well defined . It includes 16 proteins or polypeptides with a molecular weight of 190–15.5 kDa. For the immunoblot, only those with a molecular weight of 47, 17 and 15.5 kDa are of diagnostic significance due to their T. pallidum specificity. In many cases, the 44.5 kDa antigen (TmpA) is also considered in the test assessment.
Fractionated IgM antibody assay (19 S IgM antibody test)
Principle: separation of the IgG from the IgM antibodies in patient serum by column chromatography or ultracentrifugation. Alternatively, the IgG fraction can be precipitated by anti-human IgG serum (RF absorbent). Antibodies can be detected using FITC conjugated anti-human IgM serum with μ-chain specificity /, /.
T. pallidum IgM antibody ELISA
Principle: there are several ways of performing the T. pallidum IgM antibody ELISA. The most common method is the sandwich ELISA where the solid phase is coated with T. pallidum fragments or recombinant antigens. The patient serum is precipitated with anti-human IgG serum prior to testing and the test is developed using anti-human IgM antibody conjugated with horseradish peroxidase /, /.
Alternatively, a μ-capture enzyme immunoassay with anti-human IgM antibody attached to the solid phase is used. Detection of the treponemal specific IgM antibodies is achieved by adding treponemal antigen and a monoclonal antibody to an antigen of the axial filament of T. pallidum /, /.
T. pallidum IgM immunoblot
See description above.
Venereal Disease Research Laboratory test (VDRL), card microflocation test (CMT)
Principle: quantitative detection of anti-lipid antibodies by means of precipitation. Such antibodies, of the IgM and IgG type, occur in patients with Treponema infections but are not specific for them. The international designation of the test is VDRL, also referred to as CMT in Germany. For the VDRL (CMT) test as well as the cardiolipin CF test, a mixture consisting of cardiolipin crystals, lecithin and cholesterol as the antigen is added to serial dilutions of patient serum. The test is performed on special glass slides with cut wells and, after incubation on a rotary shaker, read under the microscope at approximately 100-fold magnification. In the presence of anti-lipid antibodies, flocculation of the crystalline antigen ensues. The test can also be performed using micro titer plates. The addition of a small amount of dye to the antigens and the use of a mirror to enlarge the view of the precipitate allow good recognition of the dilution giving a 2+ reaction.
The origin of the anti-lipid antibodies found in infections has not been elucidated to date. It is assumed that they represent immune reaction products of inflammation induced cellular destruction. Lipid containing mitochondria are released from affected cells, which may not be recognized as self antigens, and antibodies are formed against them.
Rapid plasma reagin (RPR) test
Principle: solutions from patient serum and cardiolipin/lecithin/cholesterol/charcoal particle suspension are mixed on plastic coated or cardboard cards. The test antigen can also be dried onto the cards. In this case, the patient serum solutions are dropped onto the card. After incubation on a rotary shaker, a positive test result is indicated by macroscopically detectable large black stains from agglutination. This test is easier to handle than the VDRL test. Both tests yield largely comparable qualitative and quantitative results . The RPR test is widespread throughout Anglo-American countries where it is also used as a screening test for syphilis.
Cardiolipin complement fixation (Card-CF)
Principle: the binding of antigen to lipid antibodies in patient serum results in the consumption of guinea pig complement which is no longer available in the test mixture to react with the hemolytic indicator system (sheep erythrocytes + amboceptor). If lysis does not occur, complement was consumed (i.e., the Card-CF test result is positive). If lysis does occur in the indicator system, no lipid antibodies are present (negative Card-CF test result). This method is rarely used because of its complexity.
The following tests are used:
- 19S IgM antibody test: see description above.
- Quantitative VDRL, CMT, RPR, Card-CF: see description above.
Suspected primary or secondary syphilis
Shortly after infection, T. pallidum is detectable in a syphilitic lesion by direct diagnostic testing (dark field microscopy, immunofluorescence assay, nucleic acid amplification) while serological tests are still negative. Patients usually seroconvert within a few days after showing initial clinical symptoms. Therefore, if the T. pallidum screening test is negative in cases with clinical symptoms suggestive of primary or secondary syphilis, further tests need to be done at weekly intervals. Treponema specific antibodies are detectable in all subsequent infection stages of syphilis. The screening test should be repeated at intervals of several weeks for up to 90 days after the time of potential infection if the patient’s history points to early infection in the absence of clinical symptoms or presence of atypical symptoms.
A positive T. pallidum screening test must be verified by one or several diagnostic tests to confirm the result specificity. Once infection is confirmed by screening and confirmatory tests, its potential activity is assessed by further diagnostic testing (e.g., a quantitative cardiolipin test: VDRL, RPR or Card-CF). In initial diagnosis and if no history of infection or treatment is available, the positive serum should be tested for Treponema specific IgM antibodies. Lipid and Treponema specific IgM antibody tests complement one another and should not be considered to be alternative methods. During the early stage of initial syphilis infection, IgM anti-treponemal antibodies are detectable earlier than anti-lipid antibodies. In late latent infection, in the tertiary stage and especially in reinfection, lipid antibodies are found at high titers, while the test results for T. pallidum-specific IgM antibodies are negative or only mildly positive.
A positive result in the Treponema specific IgM antibody assay in untreated patients indicates the need for treatment. During the early stages of untreated, initial T. pallidum infections, as well as in some patients after inadequate therapy, IgM anti-treponemal antibodies are found at a high titer (> 1 : 320). The lipid antibody titer can be negative or positive in these cases. Lipid antibody titers > 1 : 8 in untreated patients also point to active infection. In the case of positive screening and confirmatory tests, a negative IgM and lipid antibody assay usually suggests that the patient had treponematosis in the past and was adequately treated or spontaneous recovery occurred (immunological IgG scar).
When interpreting the results, it is important to keep in mind that if the titer of IgG anti-treponemal antibodies is very high, in vivo inhibition or even complete suppression of IgM antibody synthesis may ensue. Thus, a negative IgM antibody test result does not always exclude active treponemal infection.
Suspected latent treponemal infection
Highly positive screening test results (e.g., in TPHA/TPPA) at titers > 1 : 5,120 and positive confirmatory test suggests pathogen persistence in asymptomatic patients (latent infection). In these cases, specific IgM antibody titers and lipid antibody titers can vary from highly positive to negative. Lipid antibodies are usually detectable at titers > 1 : 8. Such cases are often characterized by a negative IgM antibody assay result and a markedly positive lipid antibody test result. It is important to note in this context that very high titers of Treponema specific antibodies can persist for long periods of time in reinfection even after resolving antibiotic treatment.
Approximately 3.5% of patients with clinical or ophthalmologic features of syphilis have neurosyphilis on the basis of serum and cerebrospinal fluid findings. Neurosyphilis can be categorized as asymptomatic or symptomatic and as early (1 to 2 years after primary infection or late).In patients with HIV co infection may have earlier development of neurologic features than people without HIV infection. Three types of neurosyphilis are differentiated :
- Meningo vascular syphilis causes strokes and many types of myelopathy and is usually interposed temporally between early and later forms of primary infection. Typically occurring 1–10 years after the primary infection.
- General paresis a structural brain disorder that simulates many forms of mental disease. The time since primary infection is between 5 and 10 years.
- Tabes dorsalis is characterized by gait ataxia with Romberg’s sign (falling or stepping to one side when standing with feed together and eyes closed).The time between primary ’infection is about 10 years.
Neurosyphilis is not an isolated infection of the central nervous system (CNS). If the condition is clinically suspected, examination of the serum is sufficient for excluding the disease. To confirm CNS involvement, however, concurrent examination of serum and cerebrospinal fluid (CSF) samples obtained on the same day is mandator /, , /.
Isolated examination of the CSF does not provide any diagnostic information because the immunoglobulin content and thus the level of T. pallidum specific antibodies within the CSF may be influenced by intrathecal factors and functional conditions
Functional condition of the blood-CSF barrier. In the case of increased permeability, more serum proteins will spill over into the CSF resulting in their relative increase within the CSF
- Local immunoglobulin synthesis within the CNS. Immunoglobulin synthesis may result in a relative increase in antibody concentration within the CSF independent of the blood-CSF barrier function
- Immunoglobulin concentration within the serum. An increase in the serum level of immunoglobulins or a rise in the titer of specific antibodies leads to an increase in the CSF.
Functional disorders of the blood-CSF barrier
Disorders in the function of the blood-CSF barrier can be recognized by calculating the albumin CSF/serum ratio using the following formula:
The upper reference interval values of the ratio (quotient Q) beyond the sixth month of life are (5–8) × 10–3.
Intrathecal immunoglobulin synthesis
A more precise assessment of whether T. pallidum specific antibodies in the CSF originated in the serum or the CNS can be made by concurrent determination of the total IgG concentration (mg/L) and the pathogen specific IgG antibodies (titer or ELISA value) in serum and CSF based on the calculation of the ITpA index (index of T. pallidum-specific antibodies). The assumption is that the fraction of Treponema specific IgG relative to total IgG is identical for serum and CSF if the antibodies exclusively originate in serum. Calculation based on the formulas
reveals a ratio (quotient Q) of 1.0 if the antibodies are from the serum with a range of 0.5–2.0. If pathogen-specific IgG is synthesized locally within the CNS, this value increases to ≥ 3.0.
Concurrent titration of serum and CSF is also possible after adjustment of the serum IgG concentration by dilution against the CSF IgG concentration. To calculate the ratio, the CSF TPHA/TPPA titer is divided by the serum TPHA/TPPA titer. Interpretation of the resulting CSF/serum ratio corresponds to that of the ItpA index.
These formulas may only be applied if no intrathecal IgG synthesis is detectable in the CSF (QlimIgG > QIgGtotal). For details refer to . Otherwise, the calculated antibody index values are too low. If intrathecal IgG synthesis is present (QlimIgG < QIgGtotal), the maximal calculated CSF IgG proportion originating from the serum is to be inserted into the formula instead of the measured CSF IgG proportion. This is calculated using the formula:
The determination of intrathecal IgM antibody synthesis is also possible using the formulas if adequately sensitive tests for the determination of total IgM and pathogen specific IgM in the CSF are chosen.
The detection of specific intrathecal IgG antibody synthesis does not imply the diagnosis of active neurosyphilis because it remains detectable for years, and in many patients for life, even after adequate therapy. For assessment of disease activity, non specific parameters such as pleocytosis, elevated total CSF protein and function of the blood-CSF barrier must be considered.
Lipid and/or Treponema-specific IgM antibody positivity in CSF also suggests active neurosyphilis /, , /. Lipid antibodies detectable in CSF usually do not originate from serum. This can be explained by the fact that, in most cases, the proportion of lipid antibodies from serum is below the detection limit of lipid antibody tests.
Assessment of the success of treatment
To assess the success of treatment, it is recommended to perform a follow-up test 2–4 weeks after completion of therapy as a baseline for further follow-up tests. The antibody titers should then be determined at intervals of three months for a year or, if required, for a longer period of time depending on the post therapeutic antibody kinetics .
The baseline result has an essential influence on the choice of method for the antibody kinetics monitoring in each individual case. If concurrent tests initially reveal high titers of pathogen specific IgM and lipid antibodies or only lipid antibodies, it is generally sufficient to verify the success of treatment by quantitative cardiolipin measurement. If the baseline result shows a high T. pallidum specific IgM antibody titer but a negative or low lipid antibody titer, treatment success should be verified by determining the IgM antibody kinetics.
After successful therapy of initial, primary stage or secondary stage syphilis infection, a significant decline in titers by three or more dilution levels is observed within a few months to one year . In persistent syphilis and reinfection, the decline in lipid specific and Treponema specific IgM antibody titers can be significantly delayed or the titers can persist at a certain level for a long period of time.
Post therapeutic kinetics of the lipid antibodies (as well as the pathogen specific IgM antibodies) is influenced by the time interval between infection and start of treatment /, , /. If the anti-lipid antibody titers fail to decrease or rise again, treatment was either not successful or the patient was re-infected in the interim. Final success of treatment is assessed by performing a T. pallidum specific IgM antibody assay and a lipid antibody test approximately 12–24 months after completion of therapy, depending on the status of infection before the start of therapy and the course of antibody kinetics.
The documentation of the total Treponema antibody titer (e.g., in the TPHA/TPPA) has proven useful for monitoring. This also applies to the index value in other polyvalent syphilis screening tests such as ELISA and chemiluminescence assay. A continuous decline in total Treponema antibodies usually ensues after initial infection. A significant increase in titers during follow-up suggests reinfection.
Syphilis and HIV infection
Numerous interactions exist between syphilis and HIV infection. On the one hand, genital ulcerations facilitate the entry of the HIV while, on the other hand, the normal course of syphilis is influenced by the HIV infection. This does not affect the advancement of stages, but rather causes a more aggressive clinical course in the early stages, such as ulcerating syphilis (malignant syphilis) in the secondary stage and more rapid progression to the late stages, especially neurosyphilis /, , , /.
The serological pattern in patients with co infection is variable. In many patients with HIV infection and secondary syphilis, high titers of lipid specific and IgG specific antibodies are found. These results are most likely due to the common occurrence of reinfection in this risk group. At the same time, there are also patients in the identical stage of syphilis in whom immune response is delayed or absent . These different patterns probably result from variable damage to the immune system.
Following treatment of syphilis in patients with co infection, TPHA and FTA-ABS tests may be negative depending on the reduction in CD4+ T cell count. In most cases, syphilis serology provides reliable diagnostic information in patients with concurrent HIV infection.
The majority of laboratories still perform the traditional algorithm (syphilis screening algorithm begins with a non treponemal immunoassay). However, a minority starts with the reverse algorithm (begins with a treponemal immunoassay). Although the non treponemal immunologic response typically wanes after cure and becomes undetectable, treponemal immunoassays typically remain positive for life .
Titer specification: when reporting quantitative TPHA/TPPA results, some laboratories indicate the final titer (dilution) after the addition of reagents (initial dilution 1 : 80), while others state the individual serum dilution (initial dilution 1 : 20). This should be standardized. It is advisable, as for other serological tests with the exception of bacterial agglutination assays, to specify the titer relative to the individual serum dilution.
Results from inter laboratory surveys have raised the question as to which extent antigen preparation has an influence on specificity and antibody titers in the quantitative assay. In a comparative study, it has been shown that test systems using ultra sonicated antigens tend to be more non specific than those using SDS pretreatment. Direct titer comparisons between antibody levels determined using different TPHA kits are not possible.
If equivocal (±) results are observed at the first level of serum dilution (1 : 20), such as small ring formation of the sensitized red blood cells associated with button like sedimentation of the control cells, underlying reasons may include:
- The batch dependent tendency of the sensitized red blood cells to non specific sedimentation
- The fact that sensitized red blood cells are subject to more marked agglutination by heterophilic antibodies than non sensitized ones
- The characteristics of the micro titer plates in use (e.g., chemical composition and electrostatic charge, may influence the process of sedimentation).
Evaluation of results which are not clearly negative by a confirmatory test is mandatory
A disadvantage of TPHA is the fact that heterophilic antibodies to red blood cell membranes, which are commonly found in human serum, result in false positive reactions which, however, may be detected in the control test. Differentiation between heteroagglutinating antibodies and T. pallidum specific antibodies can be accomplished by comparative titration using sensitized and non sensitized red blood cells. A specific reaction is probable if the sensitized red blood cell titer is at least two titer levels higher than that with non sensitized cells. Verification of the reaction by means of a confirmatory test is mandatory /, /.
T. pallidum enzyme immunoassay
False negative results in polyvalent screening tests must be anticipated in patients having borderline, residual TPHA/TPPA antibody results (immunological scars). A variety of different results may also occur if various tests are applied during the early seroconversion phase in acute, initial syphilis infection /,, /.
IgM ELISA, IgM immunoblot
Various methods for the detection of T. pallidum specific IgM antibodies yield discrepant results especially in patients with late latent syphilis, neurosyphilis and reinfection. These groups of cases have more positive results in the 19S-IgM-FTA-ABS test than in IgM-EIA or IgM immunoblot /, , /.
The diagnostic sensitivity of cardiolipin tests is low during the early stage of syphilis. During the secondary stage, the tests are positive in all patients, with a diagnostic sensitivity > 99% . The sensitivity starts to decrease to about 30% with increasing interval between infection and testing. The high rate of T. pallidum nonspecific results is a disadvantage of these tests /, , , / and explains their limited value as part of the diagnostic evaluation. In the case of syphilis diagnosed clinically and/or by T. pallidum specific testing, the detection of cardiolipin antibodies and the subsequent course of the antibody kinetics are important parameters for monitoring therapy of T. pallidum infection due to their low cost.
Fluorescent treponemal antibody absorption test (FTA-ABS, IgG FTA-ABS)
Statements on false positive results of Treponema specific antibody tests in the literature usually refer to the FTA-ABS test and its modifications. Atypical fluorescent images are primarily encountered in systemic, discoid, drug induced lupus erythematosus. False positive reactions have been described in Hansen’s disease, feverish conditions and advanced age. Technical errors in the laboratory also play a role.
The cause of the results is often not elucidated. Some of the findings may be based on cross reactions in the presence of Borrelia infection. A wide range of borrelial and treponemal antigens are closely related. The specificity of the FTA-ABS test is decisively dependent on the sorbent used in the test to remove all cross reacting antibodies. Experience has shown that false positive results due to borrelial antibodies are more frequently encountered in the FTA-ABS test than in TPHA/TPPA. These cross reactions play no role in modern immunoassays based on recombinant antigens. Equivocal results can be verified by immunoblot /, , /.
19S-IgM fluorescent treponemal antibody absorption test (19S-IgM FTA-ABS)
In the original test, T. pallidum specific IgM antibodies were separated from the IgG antibodies by means of labor and time intensive procedures (ultracentrifugation, gel chromatography, HPLC). Another procedure for fractionation is the use of mini columns in which protein G is usually used as an adsorbent which binds the four IgG subclasses. Experience suggests that up to 50% of IgM antibodies are lost during this procedure. Low IgM antibody titers may have an adverse effect on the final test result. A control must be used in parallel to every patient sample for detecting T. pallidum specific residual IgG in order to exclude false results.
In many laboratories, the IgG antibodies are precipitated using goat anti human IgG, and the supernatant after centrifugation is examined using the FTA-ABS test with a μ-chain specific, FITC conjugated antiserum in order to detect IgM antibodies. However, even using this technique, a control should be run with every sample to exclude contamination by pathogen specific IgG antibodies .
Treponema pallidum PCR
The causative agent of syphilis is almost exclusively transmitted during sexual intercourse by direct contact with infectious efflorescences of the primary and secondary stages. The infection occurs following the penetration of T. pallidum through micro-injuries of the skin or through intact mucous membranes. At the infection site, a vesicle forms within a mean incubation period of 21 (10–90) days, and then a papule and finally an ulcer with a firm edge (chancre or hard ulcer) develop. At the same time, the pathogens migrate into the regional lymph nodes (local lymphadenitis). Healing of the primary complex (chancre and local lymphadenitis) is the result of a local cellular immune reaction.
Immune response in initial infection
The activation of the cellular and humoral immune system takes place during the incubation period. Approximately 4 days after invasion by the pathogen, T. pallidum specific IgM antibodies are synthesized. At 10–21 days post infection, these antibodies reach titers which can be detected in the patient serum by various IgM antibody assays. IgG antibodies are detectable a few days later at the same pathogen specificity.
The antibodies detected by different serological tests are probably not identical but are targeted against different T. pallidum polypeptides. Thus in some patients, the screening tests such as TPHA, will be negative at a time when the FTA-ABS is already positive. Our observations in patients with primary syphilis indicate that anti-lipid antibodies, initially of the IgM type and later of the IgG type, are not detectable in patient serum until later in the course of the infection than Anglo-American publications would suggest. Cardiolipin tests are not very useful for early diagnosis (). Their main role, besides the IgM antibody assay, is for quantitative follow-up after completion of therapy.
It has not been entirely elucidated why symptoms of secondary syphilis occur about 3–6 weeks after the primary complex, at a time when the patient appears to have immunological control over the local infection. During this stage of infection, hematogenous spread of the treponemas throughout the body, eruption of skin rashes and the development of systemic immunity occur. The immune response triggers premunition, a phenomenon frequently seen in parasitoses. Although the body is protected against reinfection during this stage, it is unable to eliminate the pathogen. The manifestations of the secondary stage, such as recurrent skin eruptions lasting from weeks to months, seem to be directly related to the development of systemic immunity.
Since direct person-to-person transmission of the pathogen is only possible from superficial skin lesions, a patient is considered to be infectious for the first two years after the infection. Following this early phase, the patient is considered non infectious.
The phase, in which the host has immunological control over the T. pallidum infection to the point where no further clinically apparent eruptions occur, is referred to as late latency. During this phase, a delayed type hypersensitivity reaction against the pathogen is detectable. This reaction, in combination with the humoral immune response, is considered to be crucial for the suppression of infection. The elimination of the pathogen from the host is not linked to this.
The mechanism of T. pallidum persistence in the infected patient has not been elucidated. It is possible that:
- The pathogen survives within so-called immunological niches such as in CNS tissue
- The low metabolic activity of persistent pathogens plays a role
- The amount of persistent pathogens is too low (critical antigenic mass) to activate the immune system /, /.
After another latency period, which may last more than 20 years, clinical manifestations of tertiary syphilis may occur. The immunological mechanisms leading to this are unknown.
Immune response after specific therapy of initial syphilis infection
Curative treatment of initial, primary or secondary stage syphilis infection results in a significant decline in titers or even complete disappearance of T. pallidum specific IgM antibodies and non specific lipid antibodies from the patient serum within 3–12 months. The longer the time intervals between infection and start of therapy, the slower the decline in lipid and pathogen specific IgM antibody titers /, , /. The reduction in IgG antibodies (decline in titer) depends on the time interval between infection and first antibiotic treatment. If this interval is short, the infection may resolve without immunological scars (i.e., all serological tests may again have negative results). If the time interval amounts to several months or even years, many clones of memory cells will have developed, containing the information for the production of T. pallidum specific IgG antibodies.
Under these circumstances, it is possible that the antibody concentration will not decrease below the lower limit of detection in some assays. In such cases, there is a residual, so-called, immunological IgG scar (i.e., persistence of IgG) which may remain detectable for life.
Immune response after the second or multiple reinfections
In the case of second or multiple reinfections, different pathophysiological mechanisms control the synthesis of antibodies. Second or multiple contacts with the antigen complex boost production of preformed IgG antibodies. Immediately after infection, these antibodies show a steep rise in titer. IgG synthesis simultaneously leads to in vivo inhibition or suppression of the de novo synthesis of specific IgM antibodies. These IgM antibodies are thus either not detectable in the serum of patients with second or multiple reinfections or only after a time delay of 2–4 weeks.
6. Schöfer H, Brockmeyer NH, Bräuninger W, Gross G, Hagedorn HJ, Handrick W, et al. Diagnostik und Therapie der Syphilis. Leitlinie der Deutschen STD-Gesellschaft. AWMF online-Leitlinien-Register Nr. 059/002, 2008. .
8. Hagedorn HJ, Brockmeyer NH, Hunfeld KP, Münstermann D, Potthoff A, Schöfer H. Syphilis. In: Podbielski A, et al (eds). MIQ 16 – Qualitätsstandards in der mikrobiologisch-infektiologischen Diagnostik. München; Elsevier Urban & Fischer, 2012.
11. Gayet-Ageron A, Combescure C, Lautenschläger S, Ninet B, Perneger TV. Comparison of diagnostic accuracy of PCR targeting the 47-kilodalton protein membrane gene of Treponema pallidum and PCR targeting the DNA polymerase I gene: systematic review and metaanalysis. J Clin Microbiol 2015; 53: 3522–9.
13. Salado Rasmussen K, Cowan s, Gerstoft J, Laesen HK, Hoffmann S, Knudsen TB, Katzenstein TL, Jensen JS. Moleculat typing of Treponema pallidum in Denmark: a nationwide study of syphilis. Acta Derm Venereol 2016; 96: 202–6.
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18. Sambri V, Maragoni A, Simone MA, Antuono AD, Negosanti M, Cevenini R. Evaluation of recomWell Treponema, a novel recombinant antigen-based enzyme-linked immunosorbent assay for the diagnosis of syphilis. Clin Microbiol Infect 2001; 7: 200–5.
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Bacteria of the genus Salmonella (S) are classified as members of the taxonomic family Enterobacteriaceae. They are facultative anerobic, non spore forming, Gram negative, rod shaped organisms which are all motile except for S. gallinarum-pullorum. The genus consists of the species S. enterica, S. bongori and S. subterranea; the latter is of no significance to humans . Biochemically, 6 subspecies of S. enterica can be differentiated. Among these, the subspecies enterica is the most important pathogen in humans. Due to various combinations of their heat stable O (somatic) antigens (polysaccharides of the outer membrane) and heat labile H (flagellar) antigens (flagellar polypeptides), Salmonellae can be classified into approximately 2500 serotypes according to the Kauffmann-White scheme . As part of this classification, 46 O serogroups altogether encompass groups of Salmonellae each sharing the same O group antigen.
Among the large number of serotypes, the following types play, as pathogens in humans, a role in the diagnosis of enteric infections:
- Salmonella enteritidis. Although worldwide, and especially in tropical countries, a large range of serotypes is found, in Central Europe S. enteritidis is the most frequently isolated serotype followed by S. typhimurium. S. enteritidis causes inflammation of the intestinal mucosa but does not usually give rise to systemic, periodic infection. However, septicemia with a typhoid like clinical picture may occur in 1–2% of cases
- The pathogens of systemic infection. They will cause general infections in certain species to which they are adapted: S. typhi and S. paratyphi A, B, C, for instance, are adapted to humans, while S. cholerae-suis and S. dublin are adapted to pigs and cattle, respectively; S. gallinarum-pullorum causes septic infections in chickens.
Although only a relatively small number of serotypes have been regularly identified as pathogens in humans, all Salmonellae are to be considered as potentially pathogenic to humans and various animal species.
In 2010, a total of 25,307 cases of salmonellosis, but only 57 cases of paratyphoid fever and 71 cases of typhoid fever were reported in Germany. Thus, the incidence per 100,000 population was 30.9 for Salmonella gastroenteritis and less than 0.1 for typhoid and paratyphoid fever . The number of unreported cases of salmonellosis is estimated to be 10–25-fold higher. According to Article 7 of the German Infection Protection Act (IfSG), Salmonella isolates are subject to mandatory laboratory reporting of transmissible pathogens.
Salmonella enteric infections are anthropozoonoses, (i.e., farm animals), especially poultry, are the most important source of infection while food of animal origin is the most important infection vehicle for S. enteritidis /, /. Many farm animals including those to be slaughtered are asymptomatic Salmonella excreters. For instance, the prevalence of infection with S. enteritidis has clearly increased in poultry within recent years.
In Europe, shell eggs have thus become the number one vehicle of infection for human salmonellosis . Further sources of infection include poultry meat, salads prepared with mayonnaise, ground meat as well as baked goods containing inadequately heated eggs.
S. typhi and S. paratyphi A, B and C are pathogenic exclusively to humans and therefore are transmitted from person to person via the following routes of infection:
- By direct fecal-oral contact with affected patients or chronic carriers
- Much more frequently by indirect transmission involving fecally contaminated drinking water or food not reheated prior to consumption.
The seasonal distribution pattern of Salmonella infections shows a peak incidence during late summer.
Salmonella infections occur in all age groups. Small children aged 1–4 years are especially affected; Salmonella infections in this age group are detected several times more frequently than in the population at large .
Typhoid fever and paratyphoid fever, 1–4 weeks; Salmonella gastroenteritis, a few hours up to 3 days.
S. typhi and S. paratyphi A, B and C typically give rise to systemic infection characterized by the clinical picture of typhoid or paratyphoid fever. Bacteremia, bacteriuria and septic dissemination occur. During the first week, pathogens are detectable in blood cultures while pathogens can be found in the feces from the second week, and specific serum antibodies finally occur from the third week of illness .
S. enteritidis usually causes febrile gastroenteritis with the inflammatory reaction limited to the intestinal mucosa. Diarrhea is the predominant symptom. During the course of this illness, bacteremia does not usually occur and serum antibody formation is not usually detectable. Salmonella gastroenteritis therefore can only rarely be diagnosed exclusively by serological testing.
In 1–3% of salmonellosis, bacteremia does occur ; under such circumstances, extra intestinal dissemination with involvement of organs such as the kidney, lung, meninges or bone is possible. Especially patients with an immature or impaired immune system are affected (i.e., infants and the elderly but also patients with AIDS or sickle cell anemia) .
Salmonella are defined by their antigenic structure, and their antigenic repertoire is expressed as an antigenic formula. All Salmonella serotypes are classified according to a serological table of determination, the so-called Kauffmann-White scheme which is annually amended by supplements to the Kauffmann-White scheme. More than 60 different O antigens and more than 90 different H antigens are known. The O antigens are labeled with Arabic numerals, while the H antigens are labeled partly with small Latin letters and partly with Arabic numerals. Therefore, the antigenic formula of a Salmonella (e.g., of S. typhi) is:
O 9,12: H d:–; the capitalized letters O and H in the formulas are mostly dispensable.
H antigens can only occur in one form of expression (monophasic Salmonellae) or reveal a regular pattern of phase changes between the 1st and 2nd expression (phase) (diphasic Salmonellae). H antigens of the 1st phase are labeled with small Latin letters, whereas H antigens of the 2nd phase are labeled with either Arabic numerals or with small Latin letters. Thus, the antigenic formula for a monophasic Salmonella (e.g., S. agona) is 1,4,12: f,g,s:– and for a diphasic Salmonella (e.g., S. typhimurium) is 1,4,5,12: i: 1,2.
Some Salmonellae such as S. typhi (antigenic formula: 1,9,12: Vi: d:-) feature an additional capsular polysaccharide antigen which is referred to as Vi antigen due to its presumed relationship to virulence and, anti genetically, is largely identical to the Vi antigen of C. freundii /, /. Furthermore, most Salmonellae have surface structures or so-called type 1 fimbriae and, in addition, string-like type 3 fimbriae may occur which cross react with type 3 fimbriae of other enterobacteria such as Yersinia . If their expression is strong, both Vi antigens and type 3 fimbriae may inhibit or interfere with the agglutination of Salmonellae by specific anti-O sera (Gruber reaction). They may be removed by boiling the bacterial suspension .
The Kauffmann-White scheme groups together Salmonellae with the same representative O antigen into serogroups labeled with Arabic numerals according to each of the O group antigens. The terminology currently in use still partly includes terms employing capitalized Latin letters.
For serotyping Salmonella isolates according to the Kauffmann-White scheme, the O (somatic), H (flagellar) and, if present, Vi (capsular) antigens are usually determined by slide agglutination . For the slide agglutination test, commercially available antisera containing specific polyclonal or monoclonal antibodies are dropped onto a glass slide (with ground edges, if possible). A small part of suspect Salmonella colony is taken from no-selectivity or low-selectivity culture medium with a loop and placed immediately next to the drop, homogenized (using the loop) with a small amount of antiserum from the drop and then mixed with the rest of the antiserum to obtain a smooth suspension. Then the slide with the milky suspension is gently tilted back and forth for further mixing and, within about 30 seconds, the solution is observed for macroscopically visible agglutination (fine granular clumping) under adequate lighting conditions. Continued smooth, milky turbidity of the suspension is regarded as a negative result.
Identification of O antigens
To identify the O antigens, agglutination is performed using an omnivalent antiserum or polyvalent antisera which react with one or several O antigens from a number of serogroups. Depending on the reaction, the agglutination procedure is continued with monovalent O-reactive antisera until a given serogroup can be unequivocally identified according to the formulas of the Kauffmann-White scheme.
Identification of Vi antigen
The Vi antigen is a heat labile capsular polysaccharide typical of S. typhi, but can also occur in S. dublin, S. paratyphi C and in (beware!) Citrobacter freundii strains. In suspected typhoid fever, Salmonella suspect colonies are agglutinated with both anti-O9 antiserum and Vi-specific antiserum. It must be considered that the Vi antigen may sterically interfere with the agglutinability of the O antigens. In this case, a dense suspension of suspected colonies are boiled in a pressure cooker for 15 min. to eliminate the Vi antigen. The O agglutination test is then repeated with the cooled suspension.
Vi agglutination is best achieved using fresh isolates and can be lost after repeated subculture.
Identification of H antigens
Flagellar H antigens are best identified in motile Salmonella isolates, especially in swarming colonies, by slide agglutination. Motility is promoted by growing the colonies on a semi-solid, low agar culture medium, for example swarming agar according to Gard . It is expedient to start the detection of H antigens with antisera which agglutinate with phase 2 H antigens yielding fewer variants than phase 1 H antigens. Based on the identified O group, the most frequent phase 2 H antigens and then the corresponding phase 1 H antigens according to the Kauffmann-White scheme are determined. Liquefied swarming agar cooled down to 50 °C is mixed with antisera to achieve suppression of a phase and, thus, detection of the complementary phase.
During immune response to Salmonella infection, usually two different antibody specificities are formed: O agglutinins directed against the heat stable somatic antigens of the outer membrane (O antigens) and H agglutinins against heat labile flagellar antigens (H antigens).
The detection of specific antibodies to O, H, and Vi antigens in suspected cases of disease is accomplished by means of the Widal reaction. Healthy chronic carriers of Salmonellae with Vi antigens such as S. typhi or S. paratyphi C, can be identified by detecting Vi antibodies in the hemagglutination assay .
Invasive salmonellosis caused by S. enterica serotype typhi or paratyphi A, B, C or non-typhoidal serotypes, are an immensely important disease cluster for which reliable, rapid diagnostic tests are not available . Commercially available serologic tests for typhoidal salmonella have a limited sensitivity and specificity. Blood culture remains the gold standard but is insensitive, slow, and resource-intensive.
Tube agglutination test (Widal test)
O, H and Vi agglutinins are examined in separate test mixtures. To determine H and O agglutinins, for each of the agglutinins, constant quantities of a formalized (H antigen) or boiled (O antigen) Salmonella suspension are added to each geometric serial dilution of the patient serum starting at 1 : 25. For the detection of H agglutinins, incubation follows for 2 h at 50 °V and for an additional 3 h at room temperature. For the detection of O and Vi agglutinins, the incubation period is 2 h at 37 °V followed by an overnight period at room temperature . Positive and negative serum controls are treated correspondingly. The resultant granular or floccular agglutination from O and H agglutinins, respectively, is read by use of an agglutinoscope.
Widal test on micro titer plates
This test is the preferred method in comparison to the tube agglutination test on account of lower serum and antigen requirements as well as the possibility for partial automation. Serum dilutions and antigen preparations correspond to those in the tube agglutination test; however, only 50 μL of either serum or antigen suspension per well are needed. Both test mixtures are incubated at 36 °C for 18 h. The result is read using a micro titer plate reader .
The composition of the antigen suspensions used in both test methods should reflect the prevalence of the typhoid Salmonella serotypes encountered in each particular geographical region. In Europe, typhoid fever is mainly caused by S. typhi and S. paratyphi B whereas in Africa and Asia, S. paratyphi A is important, in addition, and in the Middle East and Asia, S. paratyphi C is another possible serotype . The following antigen suspensions, for example, should be used for serodiagnostic tests in European typhoid Salmonella infections: S. typhi O, S. typhi H, S. paratyphi B–O and S. paratyphi B–H (1st phase).
Furthermore, a suspension with the unspecific 2nd phase (1, 2) of S. paratyphi B can be run simultaneously in order to detect antibodies to the 2nd phase of S. paratyphi B and possibly to other Salmonellae (e.g., S. typhimurium). In other regions of the world, the repertoire of antigens should be adapted to the particular Salmonella serotype spectrum occurring in each region .
- Borderline titers: O agglutination 1 : 100,
- H agglutination 1 : 100, Vi agglutination 1 : 10. Positive: O agglutination ≥ 1 : 200,
- H agglutination ≥ 1 : 200, Vi agglutination > 1 : 10. Negative: O agglutination ≤ 1 : 50,
- H agglutination ≤ 1 : 50, Vi agglutination < 1 : 10.
This test is used for the detection of healthy chronic Salmonella carriers with antibodies to Vi antigen (e.g., S. typhi). It employs sheep erythrocytes coated with highly purified Vi antigen of a C. freundii strain . Test kits are not commercially available.
- Borderline titers: ≥ 1 : 120.
Rapid tests suited for serodiagnostic testing for enteric (typhoid) fever and diagnosing S. enteritidis infection are based on the following principle:
- Inhibition of binding between an anti-O9 IgM monoclonal antibody conjugated to colored latex particles and S. typhi lipopolysaccharide conjugated to magnetic latex particles by corresponding antibodies in the patient serum /, , /
- S. typhi IgM dipstick test with binding of S. typhi specific serum antibodies to color labeled anti-IgM antibodies and binding of colored conjugate to dipsticks coated with S. typhi lipopolysaccharide .
Other Enterobacteriaceae also have O antigens similar to those encountered in Salmonella, therefore accounting for the fact that O agglutinin titers of up to 1 : 50 are found in 2–3% of the population . Cross-reacting antigens similar to the Salmonella H antigens do not exist in other bacteria. H agglutinin titers thus reflect a recent or past contact with Salmonella assuming that no vaccination took place. The prevalence of H agglutinins within the population of different geographical regions depends on the corresponding prevalence rate of Salmonella infections. For instance, rates of 1–2% have been reported for England , while in Central America corresponding antibodies were found so frequently that the Widal reaction is diagnostically useful only in children .
Salmonella infections may, on the one hand, induce broad polyclonal stimulation of the immune response; on the other hand, there are many antigenic cross reactivities between Salmonella serotypes with partly corresponding O and H antigens. This results in the production not only of specific antibodies to the causative agent but also of antibodies to cross reacting groups or other groups of O and H antigens. This explains the multifaceted picture of serological results which may often be encountered during serodiagnostic evaluations of Salmonella infections.
Suspicion of Salmonella infection is raised if the agglutinin titer of the H and/or O agglutination in the serum sample of the first examination is ≥ 1 : 100 or if there is an increase or decrease by at least two titer steps in the follow-up samples. The O agglutinin titer is more valuable than the H agglutinin titer in helping to establish the diagnosis of acute Salmonella infection since increases in the H agglutinin titer may also represent anamnestic responses.
Laboratories performing serodiagnostic evaluations of Salmonella infections usually employ S. typhi and S. paratyphi antigen suspensions. Thus, two problems arise in conjunction with the serological detection of febrile Salmonella gastroenteritis:
- The antibody response in Salmonella gastroenteritis is irregular
- The diagnostic spectrum is limited to non-typhoidal Salmonellae which share common antigens with S. typhi or S. paratyphi B or the unspecific 2nd H phase 1, 2. These prerequisites would, for example, apply to S. enteritidis (9, 12: gm:–) and S. typhimurium (1, 4, 5, 12: i: 1, 2).
O agglutinins are suited for the diagnosis of acute Salmonella infections. They rise from the 2nd week of disease but they may also not be elevated until significantly later in the course of the illness . They peak at a titer of approximately 1 : 400 and decline again within a few weeks after clinical recovery. Not all infections with S. typhi are associated with significantly elevated O agglutinin titers; in S. paratyphi infections, the increase is less reliable than in infections with S. typhi .
H agglutinins can be indicators of acute infection or infection which occurred in the recent or distant past. Following vaccination, H agglutinins are usually still detectable for years. H agglutination is more sensitive and more specific than O agglutination since other bacteria do not have any identical H antigens in common with Salmonellae. In acute infections, H agglutinins rise from the 10th day of illness and reach a peak after 3 weeks at a titer of up to 1 : 1600 and higher. After clinical recovery, the titers slowly decline but elevated titers may also persist for up to several years. Failure of H agglutinin titers to increase has been described in conjunction with typhoid or paratyphoid fever . Isolated, elevated H agglutinin titers, especially in the test mixture with H phase 2 antigen suspensions, may be detected up to a titer of 1 : 100 even in individuals not acutely infected. In such cases, they are often due to preceding Salmonella gastroenteritis (e.g., caused by S. paratyphi B or non-typhoidal Salmonellae such as S. typhimurium. If antigen suspensions of both S. typhi and S. enteritidis are used in the Widal reaction, agglutination of both antigen suspensions will ensue (e.g., in the presence of S. typhi infection) because they have O antigens in common. In such a setting, a higher H agglutinin titer against S. typhi antigen suggests the presence of S. typhi infection.
Given antigenic stimulation in diarrheal diseases not associated with Salmonellae, non specific increases in the titers of Salmonella antibodies may occur if the patient has previously had a Salmonella infection.
Vaccination against typhoid and paratyphoid fever results in a significant rise in H agglutinins to titers above 1 : 100 which persist for years. If O agglutinins are produced as well, they will decline to normal levels within weeks to a few months. Thus, in vaccinated individuals, only a significant elevation in O and H agglutinins but not in H agglutinins can be considered proof of an acute infection.
On the one hand, many Salmonella serotypes have O and H antigens in common (e.g., a common O9 shared by S. typhi and S. enteritidis or O12 shared by S. typhi and S. paratyphi B). On the other hand, many individuals have previously undergone clinically inapparent or overt Salmonella infections or they were previously vaccinated. Not infrequently therefore, by using serodiagnosis alone, is it difficult or impossible to identify the causative agent among typhoidal Salmonellae or to distinguish between typhoidal and non-typhoidal Salmonellae. The Widal reaction can, thus, help as an additional diagnostic criterion in order to verify the presence of Salmonella infection. However, under no circumstances can it replace the isolation of the causative agent during the acute stage of the disease.
Vi antibodies and Salmonella carriers
In various studies, excreters of S. typhi and S. paratyphi C could be identified by the detection of Vi antibodies (e.g., as revealed by titers of 1 : 160 or higher in the hemagglutination test) with high diagnostic sensitivity and specificity /, /.
Rapid tests seem to be especially suited to identify acute S. typhi infections in endemic regions /, /. The low incidence of typhoid fever in Central Europe does not necessitate the routine application of these methods for the time being.
The traditional methods currently used for detection of salmonella environmental samples require 2 days to produce results and have limited sensitivity. A real-time PCR salmonella screening method detects 55% more positives for Salmonella in half the time required for the traditional method .
The xTAG assay for determination of the serotypes of salmonella consists of two steps:
- Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of salmonella
- Magplex-TAG micro sphere hybridization to identify the specific PCR products of different antigens
17. Rohde R, Aleksic S, et al. Profuse fimbriae conferring O-inagglutinability to several strains of S. typhi-murium and S. enteritidis isolated from pasta products. Cultural, morphological, and serological experiments. Zbl Bakt Parasitenk Infektionskr Hyg I Abt Orig 1975; A230: 38–50.
18. Bopp CA, Brenner FW, Fields PI, Wells JG, Stockbine NA. Escherichia, Shigella, and Salmonella. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH (eds). Manual of Clinical Microbiology. Oxford; Blackwell 2003: 654–71.
22. Parker MT. Enteric infections: typhoid and paratyphoid fever. In: Parker MT, Collier LH. Topley and Wilson’s principles of bacteriology, virology and immunity, 8th ed, Vol 3. London: Edward Arnold, 1990: 423–45.
Shigellae belong to the family Enterobacteriaceae. They are Gram negative, non motile, non encapsulated, rod-shaped bacteria featuring neither flagellar nor capsular antigens. The genus Shigella (S) consists of the 4 subgroups A–D which can be distinguished by group specific poly saccharides and biochemical characteristics /, /:
- Subgroup A is composed of 13 serotypes of the species S. dysenteriae
- Subgroup B is composed of 6 serotypes and 14 subtypes of S. flexneri
- Subgroup C consists of 18 serotypes of S. boydii
- Subgroup D is composed of the species S. sonnei of which only one serovariety exists.
Shigellae cause intestinal infections mainly involving the colon. Extra intestinal manifestations are rare.
In 2010, 731 cases of shigellosis were reported in Germany; the incidence was 0.9 per 100.000 population . According to Article 7 of the German Infection Protection Act (IfSG), Shigella isolates are subject to mandatory laboratory reporting of transmissible pathogens.
Bacterial dysentery is rare in industrialized countries. Most cases are imported by travelers returning home or by immigrants. However, the illness does occur endemically and epidemically in developing countries with a warm climate and presents a significant health risk especially to infants and small children. For instance, in rural areas of Bangladesh, Shigellae were confirmed as the cause of half of all cases of bloody diarrhea which in that region is responsible for 60% of diarrhea related deaths . In Thai children, the proportion of shigelloses in cases of dysentery amounted to 46% . The mortality rate in hospitalized children in Dhaka reached 10% in cases of infection with S. dysenteriae 1 .
The worldwide distribution of the Shigella subgroups has shown pronounced fluctuations over longer periods of time: whereas before World War I S. dysenteriae type 1 prevailed, a clear increase in S. flexneri was observed between the two world wars. Since World War II, S. sonnei has been increasingly detected worldwide . Regarding geographical distribution, S. sonnei is now the leading causative agent of bacillary dysentery in Europe and North America. S. flexneri frequently occurs in the Southwest of the United States, but it is also detected at above average rates for example in Bangladesh and Africa , while S. dysenteriae type 1 presently plays an important role in Central Africa, Central America, Burma, Vietnam, Thailand, Bangladesh, Pakistan and Sri Lanka /, /.
Sources of Shigella infection are humans, especially those ill with the infection who excrete Shigellae in large amounts in the stool, as well as higher primates. Shigelloses are often transmitted directly person-to-person, although transmission by contaminated food and drinking water also occurs . The classical vehicles of infection in shigelloses include the fingers, feces, flies and contaminated fomites /, /. In the United States, direct person-to-person spread between children of preschool age in day care centers takes on special significance in regard to the epidemiology of shigelloses .
The morbidity peaks worldwide differ regionally (e.g., in Bangladesh it is during the dry season whereas in Guatemala it is during the rainy season) . In Central Europe, shigelloses are observed more frequently during the summer, in some regions also during the winter and during the tourist season when travelers visit endemic areas.
In endemic areas, infants and children less than 15 years of age are especially affected /, /; in industrialized countries with low shigellosis prevalence, travelers returning home from endemic areas are most at risk.
1–5 days, mean 48 h.
After initial, general symptoms such as fever, headache and arthralgias, lower abdominal cramps set in with intermittent bouts of tenesmus and diarrhea. A classical course of the illness initially features diarrhea with thin to watery stools which later change to teaspoon sized fecal portions made up of mucus with blood and pus mixed in. The inflammation is usually limited to the large intestine. In adults in endemic areas, the disease not infrequently takes on a clinically inapparent course or one with mild symptoms . Systemic complications are especially common in small children and in infections caused by S. dysenteriae 1. These complications include toxic megacolon, leukemoid reactions and, in particular, the hemolytic uremic syndrome which is associated with high mortality and often with permanent renal damage /, /. Reactive arthritis or Reiter’s syndrome apparently often occurs especially after infections with S. flexneri and in patients with the HLA-B27 antigen /, /.
Shigella infection is characterized by bacterial invasion of intestinal cells, dissemination within the colic epithelium through direct spread from cell to cell, and massive inflammation of the intestinal mucosa. The dissemination process primarily relies on actin assembly at the bacterial pole, which propels the pathogen throughout the cytosol of primary infected cells .
Shigellae only feature O (somatic) antigens (poly saccharides of the outer membrane) and, in contrast to other enteric bacteria, no H (flagellar) or C (capsular) antigens. However, they do share numerous cross reacting O antigens with E. coli, especially with entero invasive E. coli serotypes . S. sonnei is the exception, although it has total cross reactivity with the O antigen of Plesiomonas shigelloides . S. flexneri often carry fimbriae which, although they do not cross react with the fimbriae of other enteric bacteria, may interfere with serotyping if antibodies to fimbriae are contained in the patient sample. The diagnosis of acute shigellosis is accomplished by direct isolation of the pathogen from stool cultures.
Serodiagnostic evaluations for all practical purposes are irrelevant in acute shigellosis; serodiagnosis may, on the other hand, be helpful as part of retrospective investigation of outbreaks or in epidemiological prevalence studies . Indirect hemagglutination tests and ELISA are used for this purpose.
Widal tube agglutination test
The test is characterized by inadequate diagnostic sensitivity and specificity if inactivated suspensions of Shigellae are used. For the detection of S. flexneri agglutinins, better test performance can be achieved by using live strains as antigens .
Different antigen preparations containing Shigella lipopolysaccharides /, , , / and more recently also virulence associated, plasmid coded proteins /, / are used for the detection of IgG, IgA and IgM antibodies. For the determination of serum antibodies to S. sonnei, a lipopolysaccharide from Plesiomonas shigelloides is also used .
The Widal reaction is not suited for diagnosing acute shigellosis. Serum antibodies usually do not occur before the 2nd week after the onset of the disease, when the clinical symptoms have usually already subsided . Diagnostic sensitivity and specificity are low because not only are elevated agglutinin titers found in individuals without the disease, for example, in endemic areas, but also because agglutinins may often not be produced in acute shigellosis /, , /. In addition, cross reactions with O antigens, especially of E. coli strains, may occur .
The use of the Widal reaction is, if at all, only suited for retrospective investigations of outbreaks of bacillary dysentery, and is valuable only if live strains are employed as antigens (beware of laboratory infections) . In an investigation of an outbreak with S. flexneri 6, 4–12 weeks after the onset of the disease 100% of the patients with the infection had agglutinin titers > 1 : 4, whereas among 60 controls not a single case was detectable with an agglutinin titer > 1 : 2. The increase in agglutinins occurred between the 7th and 10th day of the disease .
Titers ≥ 1 : 40 are considered to be an indicator of active Shigella infection or infections in the recent or distant past , while titers ≥ 1 : 160 are diagnostic proof . In a study , a reliable conclusion could be drawn from the investigation of individual sera only in infections with S. dysenteriae 1, whereas infections with S. sonnei or S. flexneri 6 only resulted in significantly elevated mean titers in patient samples as compared to healthy controls. Titers usually increase after the 2nd week of the disease and reach their peak after 4–6 weeks. Elevated titers may still be detectable after one year.
In a study involving Swedish patients, it was shown that 80% of cases with S. flexneri infections and 79% with S. sonnei infections had significant antibody titers to Shigella LPS which declined to normal levels after 4–6 months. Parallel measurements of anti-Ipa-IgG titer were positive in only 60% or 43%, respectively, but they remained elevated for 4–6 months. Diagnostic specificity of the anti-Ipa ELISA was 90% while that of the LPS-ELISA varied from 84–90%. The determination of LPS antibodies was useful for detecting acute infection, whereas the determination of Ipa antibodies was suited for diagnosing past infections . The detection of Ipa antibodies, however, appears to be useful for establishing the diagnosis only in countries with low shigellosis prevalence . This also seems to apply to the detection of LPS antibodies which, in endemic areas, are also found quite frequently in individuals not actually ill with the disease .
2. Muthuirulandi Sethuvel DP, Deveanga Ragupathi NK, Anan dan S, Veeraraghavan B. Update on shigella new serogroups/serotypes and their antimicrobial resistance. Lett Appl Microbiol 2017; 64 (1): 8–18.
24. Hyams KC, Malone JD, Bourgeois AL, et al. Serum antibody to lipopolysaccharide antigens of shigella species among U.S. military personnel deployed to Saudi Arabia and Kuwait during Operations Desert Shield and Desert Storm. Clin Diagn Lab Immunol 1995; 2: 700–3.
25. Li A, Rong ZC, Ekwall E, et al. Serum antibody response against shigella lipopolysaccharides and invasion plasmid-coded antigens in shigella infected Swedish patients. Scand J Infect Dis 1993; 25: 569–677.
28. Munoz C, Baquar S, van de Verg L, et al. Characteristics of Shigella sonnei infection of volunteers: signs, symptoms, immune response, changes in selected cytokines and acute phase substances. Am J Trop Med Hyg 1995; 53: 47–54.
31. Ahmed A, Aziz KMS, Rahaman MM. An antibody assay in shiga dysentery by microtiter passive haemagglutination using human erythrocytes and chromium chloride as a coupling agent. Indian J Med Res 1980; 71: 12–21.
Thomas A. Wichelhaus, Klaus-Peter Hunfeld, Volker Brade
Staphylococcus aureus is the most important representative pathogenic to humans of the genus Staphylococcus (S) and is a common causative agent of both community acquired and nosocomial bacterial infections.
Diseases caused by S. aureus are classified in pyogenic, invasive processes and toxin mediated diseases. The course of disease in invasive processes is influenced by the interaction of a wide range of S. aureus virulence factors including α-hemolysin (staphylolysin), while there is only one specific toxin of pathogenic relevance in toxin mediated diseases. The site of toxin production can be clinically inapparent (menstrual toxic shock syndrome) or, in the case of enterotoxic gastroenteritis, be outside the patient in the food . Infective endocarditis has an estimated annual incidence of 3–9 cases per 100,000 individuals. Streptococci and staphylococci account for 80% of cases .
Serological detection of pathogen specific antibodies should only be performed in cases of suspected S. aureus infection in which direct pathogen detection appears to be difficult such as osteomyelitis, previous antibiotic treatment.
Hemolysis inhibition reaction (anti staphylolysin test)
Erythrocyte hemolysis by the pore forming α-hemolysin (staphylolysin) of S. aureus is inhibited by staphylolysin antibodies. The higher the serum anti-staphylolysin level, the greater the extent to which inhibition of hemolysis is shifted toward higher dilution levels. The test is usually performed in the micro titer format and allows quantitative interpretation in IU/mL.
Latex agglutination test
Latex particles are coated with staphylolysin. The addition of diluted patient serum leads to visible agglutination if the serum contains antibodies to staphylolysin (anti-staphylolysin). The detection limit is adjusted in such a way that agglutination only occurs in staphylolysin antibody concentrations above 2 IU/mL.
Serum: 1 mL
Anti staphylolysin > 2 IU/mL
Following infection with S. aureus, the antibody concentration usually increases to > 2 IU/mL after 2–3 weeks; staphylolysin antibody concentration reaches its maximum after 2–3 months and 5–6 months after the recovery from an infection declines to levels below the threshold value. In general, surface (skin and mucous membrane) infections result in low to moderately elevated concentrations whereas deeper processes and sepsis produce higher levels.
Anti-staphylolysin levels below the threshold value do not rule out the presence of S. aureus infection. Elevated concentrations (≥ 8 IU/mL) are considered to be of diagnostic relevance and point to staphylococcal infection.
A seroprevalence study analyzed the anti-staphylolysin level in culture confirmed S. aureus infections . 79.9% of the patients had anti-staphylolysin concentrations ≥ 8 IU/mL. Related to specific diseases, the findings were as follows:
- Sepsis: 93.7% of the cases
- Deep purulent infections: 70% of the cases
- Pyoderma 11% of the cases (79.8% > 2 IU/mL)
- Bone and joint diseases: 79% of the cases.
Comments and problems
The hemolysis result can be ambiguous in hemolysis inhibition tests. In such cases, the relevant test tubes or micro titer plates should be centrifuged at 150 × g for 2–3 min.
Up to the 5th week of life, staphylolysin antibody titers in the umbilical cord blood and in serum of neonates are higher than in adults.
1. Kloos WE, Bannerman TL. Staphylococcus and Micrococcus. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (eds). Manual of clinical microbiology. Washington;American Society for Microbiology 1999: 264–82.
6. Gaydos CA, Manhart LE, Taylor SN, Lillis EA, Hook EW, Klausner JD, et al. Molecular testing for Mycoplasma genitalium in the United States: results from the AMES prospective Multicenter Clinical Study. J Clin Microbiol 2019; 57 (11) e01125-e19
Thomas A. Wichelhaus, Klaus-Peter Hunfeld, Volker Brade
Streptococcus pyogenes (group A Streptococcus, GAS) is a bacterium exclusively pathogenic to humans and a common cause of purulent infections of the mucous membranes, skin and soft tissue. It is also of special relevance to rheumatic fever and acute glomerulonephritis as Streptococcus associated sequelae /, , /.
Pathogen detection by culture is the gold standard in microbiological diagnostic testing in purulent/invasive GAS infections . GAS antigen detection from pharyngeal samples is another direct detection method and achieves a diagnostic sensitivity of 85% and a specificity of 96% . A negative rapid antigen test result should be verified by culture confirmation .
St. pyogenes colonizes the oropharyngeal area and is detectable in 2–10% of the population /, /; thus, the detection of GAS in the oral cavity must be associated with the presence of symptoms of infection in order to suggest abnormal conditions. Diseases caused by St. pyogenes is classified into purulent invasive infections and non purulent sequelae () .
The peak incidence of ARF occurs in young individuals aged 5–15 years. Due to penicillin treatment, oropharyngeal streptococcal infection has become rare in industrialized countries (< 1–5 cases/100,000 children) /, /. By comparison, an unchanged high incidence is seen in developing countries (2–10 cases/1,000 children) /, /. ARF is exclusively caused by oropharyngeal Streptococci. Peak occurrence is found in the fall and winter months of the year /, , /.
ARF is a GAS allergic, inflammatory, systemic disease which may involve the heart (endocarditis, myocarditis, pericarditis) in 50–60%, joints (migratory polyarthritis) in 75%, CNS (chorea minor) in 10–15% and the skin and subcutaneous tissue (erythema annulare rheumaticum, erythema nodosum) in less than 2% of cases /, /.
The diagnosis of ARF is likely if:
- Previous GAS infection is confirmed by positive pharyngeal culture and/or antigen detection and/or by the detection of GAS antibodies
- Two major manifestations or one major and two minor manifestations according to the Jones criteria are fulfilled ().
Etiologically, AGN is associated with Streptococcus infections of group A (as well as C and G) of the oropharyngeal area and especially of the skin. In southern, warm climates, AGN primarily occurs following GAS pyoderma (peak occurrence in the summer months), whereas in the moderate climate zones, it is just as frequently associated with oropharyngeal infection. The incidence of AGN by comparison with ARF is low in industrialized countries .
The latency period between Streptococcus infection and AGN varies between 1–4 weeks, with a mean of 10 days.
Acute glomerulonephritis manifests as immune complex nephritis with hematuria, hypertension and edema (Volhard’s triad) as the main symptoms.
The determination of specific antibodies to the streptococcal metabolic products streptolysin, DNAse B, hyaluronidase, NADase and streptokinase is an essential diagnostic element in the identification of GAS sequelae because direct detection by culture is not successful in many cases. Serological testing plays no role in the pathogen detection of acute purulent and invasive infection.
Important clinical laboratory tests for the diagnosis and monitoring of streptococcal infections and sequelae are:
- Anti-streptolysin O (ASO, ASL or AST) test
- Anti-deoxyribonuclease B (ADB, anti-DNAse B, anti-streptodornase B) test.
Immunonephelometry and immunoturbidimetry for the quantitative determination
Agglutination occurs when streptolysin O coated polystyrene particles are added to samples containing antistreptolysin O. The intensity of light scatter measured by nephelometry and/or the change in turbidity measured spectrophotometrically depend on the ASO concentration. Thus, the ASO concentration in the sample can be determined by comparison with dilutions of a standard of known concentration. The measurement result is specified in international units (IU/mL) referred to a WHO standard. ASO concentrations can also be detectable in streptococcal group C and G infections because Streptococci of these groups also produce streptolysin O.
Hemolysis inhibition reaction for the quantitative determination of ASO
This test is based on the fact that streptolysin O hemolyzes erythrocytes and that hemolysis can be prevented by the neutralizing effect of ASO if these antibodies are in the patient serum. A serial dilution of patient serum is incubated with a constant quantity of streptolysin O and ovine or rabbit erythrocytes. The reciprocal value of the last dilution level, at which no hemolysis occurs, is considered to be the ASO titer. For the removal of unspecific streptolysin inhibitors, absorption of the patient serum with dextran is advisable. The results of the ASO test are related to a WHO reference preparation and specified in IU/mL.
Rapid latex test
On a black-bottom test plate, 1 drop of streptolysin O-coated latex particles is mixed with 1 drop of patient serum. The test system is adjusted to show visible white agglutinates at an anti-ASO antibody concentration > 200 IU/mL.
Immunonephelometry and immunoturbidimetry
A defined quantity of DNAse B is added to the test mixture. DNAse B binds in the presence of anti-DNAse B antibodies in the patient serum. Subsequent adding of anti-DNAse B coated latex particles does not lead to agglutination because all DNAse B is already bound.
Principle: if the patient serum does not contain any antibodies to DNAse B, agglutination of the latex particles by unbound DNAse B occurs.
Toluidine blue O method
Principle: diluted patient serum is incubated with a defined activity of DNase B and its substrate, DNA conjugated to toluidine blue O, is added. During the enzymatic degradation of the toluidine blue O-DNA substrate, the dye precipitates in a floccular manner and the supernatant decolorizes. If the patient serum contains antibodies to DNase B, degradation of the substrate is prevented and the solution remains blue.
DNAse is also produced by group B, C and G streptococci. The isoenzyme DNAse B, however, is specific of Streptococci group A. Care must be taken to ensure that pure enzyme is used.
Streptococci of serogroup A, as well as those of groups B, C, G, H and L, produce an enzyme which breaks down hyaluronic acids. Infection with hyaluronidase-producing Streptococci leads to the synthesis of antibodies which are capable of inhibiting the enzyme mediated degradation of hyaluronic acid.
Principle: detection of anti-hyaluronidase antibodies is achieved by a mucin clot prevention test, a turbidity reduction test or the viscosity reduction test as the indicator reaction.
Serum: 1 mL
In many cases, the determination of GAS antibody specificity will not reflect the complete immune response to GAS. Accordingly, at least two antibody specificities (usually ASO and ADB) are determined in serodiagnostic testing /, /. A mixed antibody reaction test offers no or only minor advantages because of the different titer patterns of the different antibodies and the resulting difficult interpretation . There are no universally usable threshold values for the assessment of normal GAS antibody titers /, , /. Hence, a comparison of two patient sera at an interval of 2–3 weeks is useful for a significant diagnosis based on a 4-fold increase in titers.
Anti-streptolysin O concentration
A positive ASO titer can be expected after an interval of 1–3 weeks following a GAS infection. The highest ASO titers are measurable 3–6 weeks after infection. They decline to normal levels after 6–12 months.
A positive ASO titer is relevant because it confirms a present or preceding GAS infection. A concentration above the threshold and/or a significant increase between a first and subsequent serum (analyzed in parallel testing) are considered to be a positive result.
A one time mild to moderate increase in antibody concentration (200–400 IU/mL) does not necessarily indicate a present or recent GAS infection.
An additional antibody test of different specificity (e.g., ADB) is of crucial importance because the ASO titer may already have declined below the threshold value at clinical manifestation of sequelae. The diagnostic sensitivity of the ASO test is reported to be 80–85%. It should be noted that elevated ASO titers are primarily found in oropharyngeal GAS infection, while no ASO reaction may occur in GAS associated pyoderma.
This circumstance is important to consider in the diagnostic testing for glomerulonephritis which is frequently encountered following GAS associated skin infections. The documentation of a decline in ASO titers is relevant for prognosis in patients with rheumatoid fever and positive ASO test result /, , /.
Anti-DNAse B concentration
During the course of the disease, the elevation of the streptococcal anti-DNAse B often occurs later than that of the ASO titer. Peak anti-DNAse B concentrations are measured 6–8 weeks following infection. Whereas elevated antistreptolysin titers are rare in GAS associated skin infection, elevated anti-DNAse concentrations are usually common.
Diagnostic sensitivity of the anti-DNAse B test is reported as 75–85%. However, combined determination of at least two antibody specificities (usually ASO and anti-DNAse B) achieves an increase in sensitivity to > 90%. The fact that anti-DNAse B concentrations increase later and persist longer by comparison with streptolysin O antibody titers makes anti-DNAse B especially useful for the identification of patients with Sydenham’s chorea, a sequela with a relatively long latency period /, , /.
A negative serological test result does not rule out an existing or resolved GAS infection, especially if only the antibody concentration to a single streptococcal product has been determined. On the other hand, a onetime mild to moderate increase in antibody titers does not necessarily point to present or recent GAS infection. ASO and/or anti-DNAse B concentrations should be checked again after 2–3 weeks, even if they were not elevated before.
Once a GAS infection has been recognized based on a high or increasing antibody titer, monitoring of the titers must be conducted in order to assess whether an antigenic stimulus persists, even if the signs of a florid illness have clinically subsided.
Comments and problems
The presence of non specific lipoprotein inhibitors in the sera of patients with chronic liver disease may result in false positive or elevated ASO titers. Such non specific activity can be prevented by precipitation of lipoproteins with dextran sulfate .
External quality control
External quality control is available as part of the bacterial-serological inter laboratory proficiency test. The results in Germany document partly poor standardization of commercially available, automated and non automated test systems in streptococcal serology .
1. Bisno AL, Stevens DL. Streptococcus pyogenes (including streptococcal toxic shock syndrome and necrotizing fasciitis). In: Mandell GL, Bennett JE, Dolin R (eds). Principles and Practice of Infectious Diseases. Philadelphia: Churchill Livingstone 2000; 2101–17.
2. Bisno AL. Nonsuppurative sequelae: rheumatic fever and glomerulonephritis. In: Mandell GL, Bennett JE, Dolin R (eds). Principles and Practice of Infectious Diseases. Philadelphia: Churchill Livingstone 2000; 2117–28.
3. Podbielski A, Lütticken R. Die Familie der Streptococcaceae. In: Köhler W, Eggers HJ, Fleischer B, Marre R, Pfister H, Pulverer G (eds). Medizinische Mikrobiologie. München; Urban & Fischer 2001; 260–76.
6. Ferrieri P. Immune responses to streptococcal infections. In: Rose NR, Friedmann H, Fahey JL (eds). Manual of clinical laboratory immunology. Washington; American Society for Microbiology 1986: 336–41.
7. Ayoub EM, Harden E. Immune response to streptococcal antigens: diagnostic methods. In: Rose NR, Hamilton RG, Detrick B (eds). Manual of clinical laboratory immunology. Washington; American Society for Microbiology 2002; 409–17.
14. Putnam SD, Gray GC, Biedenbach DJ, Jones N. Pharyngeal colonization prevalence rates for Streptococcus pyogenes and Streptococcus pneumoniae in a respiratory chemoprophylaxis intervention study using azithromycin. Clin Microbiol Infect 2000; 6: 2–8.
16. Steer AC, Vidmar S, Ritika R, Kado J, Batzloff M, Jenney AWJ, Carlin JB, Carapetis JR. Normal ranges of streptococcal antibody titers are similar whether streptococci are endemic to the setting or not. Clin Vaccine Immunol 2009; 16:172–5.
17. Danchin MH, Carlin JB, Devenish W, Nolan TM, Carapetis JR. New normal ranges of antistreptolysin O and antideoxyribonuclease B titres for Australian children. J Paediatr Child Health 2005; 41: 583–6.
18. Shet A, Kaplan EL. Clinical use and interpretation of group A streptococcal antibody tests: a practical approach for the pediatrician or primary care physician. Peadiatr Infect Dis J 2002; 21: 420–30.
Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Tularemia, also known as rabbit fever, lemming fever or deerfly fever, is a bacterial zoonosis causing regional endemic infections in numerous wild and domestic animals in large parts of the northern hemisphere. Various ectoparasites such as ticks and horseflies act as vectors of the infection. The routes of transmission to humans are direct contact with affected animals or inhalation of aerosols /, /. The disease is named after Tulare County, California, where the pathogen was first isolated in affected animals in 1912.
Francisella tularensis is an aerobic, poorly staining, Gram negative, rod shaped coccobacillus which requires host cysteine for culture growth. The pleomorphic, 0.3–0.7 μm small, non motile, facultative intracellular pathogens are able to survive in macrophages by escape from the phagolysosome . Francisella are members of the family Pasteurellaceae and have attracted special attention as organisms suited for use as a biological weapon because of their airborne transmission and high infectiousness /, /. The genus was named in honor of E. Francis, who, from 1919, made essential contributions to establishing microbiological diagnostic testing and elucidating the epidemiology of the pathogen .
The species F. tularensis comprises four subspecies: ssp. tularensis (biotype A), holarctica (biotype B), mediasiatica and novicida.
- The highly infectious F. tularensis which is predominantly found in North America and also in Northern Europe
- The less virulent F. holarctica and F. mediasiatica, which are predominantly found in Europe, the states of the former Soviet Union, in Japan and, rarely, in North America.
Differentiation and species assignment of Francisella are based on molecular genetic information /, /. The cell wall includes lipopolysaccharide and possesses a thin anti-phagocytic capsule. Because of its high pathogenicity and virulence, the pathogen may only be handled in safety laboratories (L3 laboratories) /, , /.
Although it is possible to culture the pathogen, the culture method only plays a minor role in routine diagnostic testing.
Tularemia occurs most frequently in the United States, the states of the former Soviet Union and in Japan (incidence USA: 5–36/1 million population). In Europe, cases of the disease are primarily reported from the Northern European states, the Czech Republic, Slovakia, Turkey, Austria and Switzerland /, /. Serological field investigations in Norway document a subclinical, low grade course for a number of infections. For instance, Widal tests performed on school aged children showed titers above 20 in 4.7%, above 40 in 4.2% and above 160 in 2.6% of cases in certain regions . In Germany, major outbreaks have been reported . The annual number of infected individuals varies significantly and has slightly increased recently . In 2010, 31 cases of tularemia were reported in Germany in compliance with the German Infection Protection Act (IfSG) .
1–10 days (mean 3–5 days).
Clinically manifest tularemia starts with sudden onset of fever, headache, nausea and vomiting. Tularemia spreads to humans primarily by direct exposure to infected animals (hares, rabbits), by inhalation of aerosols (in meat processing) and in individual cases also by insect bites (ticks or horseflies) (infective dose: 10–508 colony forming units) /, /. Francisella are highly invasive and capable of penetrating the smallest skin lesions and even intact skin. As a facultative intracellular pathogen, F. tularensis causes granulomatous inflammatory reactions. Groups especially at risk include hunters, farmers, butchers and veterinarians. Various forms of clinical manifestation develop depending on the route of entry of the bacterium into the body (skin, conjunctiva, oral, pulmonary) /, , , , /.
Extrinsic tularemia (local lesion with or without regional lymphadenopathy):
- Ulcerated skin lesion with swelling of the lymph nodes (ulceroglandular form)
- Glandular form (lymphadenopathy)
- Conjunctivitis (oculoglandular form)
- Pharyngotonsillitis (oroglandular form).
- Abdominal pain (typhoidal form, often in conjunction with pleuropneumonia)
- Atypical pneumonia (thoracic form).
If untreated, intermittent fever attacks and tachycardia can persist for months. The symptoms are caused by endotoxins of the pathogen. Person-to-person transmission is very rare.
According to Article 7 of the German Infection Protection Act (IfSG), direct or indirect detection of F. tularensis is subject to mandatory reporting.
The majority of infections are diagnosed by serological testing, and the WHO recommends serological test methods for indirect pathogen detection . In clinically suspected tularemia, indirect pathogen detection by serodiagnostic testing should be performed starting from the 2nd week of infection. Serodiagnostic tests use whole cell lysates, purified LPS, outer membrane proteins and Francisella membrane extracts as a source of antigens.
Constant volumes of F. tularensis suspensions are added to increasing dilutions of patient serum. The test is a common diagnostic method and performed as tube agglutination or, better, as micro agglutination test /, /. The first reading is performed after a 3 hours of incubation at 37 °C, the second one the following day after the sample has been allowed to incubate at room temperature .
Indirect hemagglutination (IHA) test
Human erythrocytes are coated with soluble bacterial extracts from F. tularensis which are produced by phenol-water extraction. A geometric serial dilution is prepared from each patient serum and a constant volume of coated erythrocytes is added. After incubation at 37 °C for 2 h and at room temperature for 18 h, the reagent mixtures are checked for the presence of agglutination .
Latex agglutination test
The agglutination technique uses sonicated antigens adsorbed to latex particles for titration.
Immunochromatographic tests (ICT)
ELISA and immunoblot
Sensitive sandwich ELISA and LPS-specific immunoblot are available for the detection of specific IgG, IgM and IgA antibodies /, /. Diagnostic sensitivities documented in the literature vary depending on the duration of the disease.
In a large evaluation study on sera from all over Europe, an LPS based ELISA and an immunoblot were documented to show diagnostic sensitivities of 99% and 100% and specificities of 97% and 99%, respectively /, , /.
Lymphocyte stimulation test (LCT)
The test principle is based on the measurement of γ-interferon from patient lymphocytes after 24 h of stimulation with F. tularensis lysate. The test is only available in research laboratories, but already shows a high proportion of positive results during the first 7 days after the onset of the disease. It has not been evaluated to date under routine conditions .
Serum: 1 mL
Widal type tube agglutination test
The Widal test is thought to be relatively insensitive in new infection. Agglutinins are usually detectable during the 2nd week of illness /, , /. In the first sample, titers above 80 are considered to be abnormal and requiring monitoring. Titers above 160 suggest new or recent infection. Detection of seroconversion or a fourfold titer increase or a titer decrease found in parallel titration with previously collected sera are also considered proof of infection. During the 3rd to 4th week of the disease, mean peak titers of 640 are generally reached. The agglutinins decline only slowly, thus accounting for the fact that even years later titers ranging from 20 to ≥ 160 are still measurable.
The presence of prozone phenomena due to low avidity, incomplete antibodies may cause false negative results in the Widal reaction . For this reason, negative test results should always be verified by a Coomb’s test of the first patient serum dilution and the controls. In the Coomb’s test, the bacterial suspension of both mixtures is centrifuged and the precipitate is washed repeatedly with normal saline. A drop of anti-human globulin serum is then added to the resuspended precipitate. Presence of agglutination means that the bacteria bind incomplete antibodies. During a period from the 3rd week of illness up to 3 months after the disease, marked pro zones (i.e., the absence of agglutination at low levels of dilution) are observed in the Widal type tube test even in the presence of high antibody titers. Pro zones may occur up to serum dilutions of 1 : 160 and are not recognized if the geometric serial dilution of the patient serum employs too few dilutions and/or the Coomb’s test is omitted.
A significant proportion of sera that agglutinate Brucella and Y. enterocolitica (serotype O9) also agglutinate F. tularensis at the same time and vice versa. In such cases, further tests have to be done such as an absorption test /, , , , /.
In 18 cases out of 20 tularemia infections, titers above 40 were detected during the first two weeks of the disease . From the 5th day after the onset of the disease, significant titers can be reached in the hemagglutination test. During the course of the disease, the hemagglutination titers are 3–7 steps higher than the agglutination titers in the Widal type tube test. Pro zones similar in extent to the ones seen in the Widal type tube test or a complete absence of the reaction are not found in the hemagglutination test . Cross-reactions, however, do occur as well because of the simultaneous reaction with Brucella and Yersinia. In this case, further evaluation by means of an absorption test is required /, , /.
Latex agglutination test
ELISA and immunoblot
Antibodies against F. tularensis in patients appear 6–10 days after the onset of symptoms. In most cases, IgG, IgM and IgA antibodies arise simultaneously . A measurable immune response may not be present after early treatment or early after infection. Antibody positivity can persist for years (up to 25 years) following vaccination or resolved infection /, /. In such cases, immunoglobulin class specific analysis or quantification tests can be useful to narrow down the time of infection .
Comments and problems
It must be kept in mind when interpreting positive titers, that a live attenuated vaccine not approved in Germany is available. The Widal type tube test and hemagglutination test show persistently elevated titers for years after vaccination /, , /. Cross reactions with Brucella have been described in agglutination tests. LPS-based ELISA and immunoblot are highly specific .
PCR methods are poorly standardized, not comprehensively evaluated under routine conditions and only available in special laboratories. Various specific gene sequences have been identified as target sequences (fragments of the fopA, tul4, 23 kDa protein and isfu2 genes) /, /. Direct detection by PCR from lymph node and biopsy specimens has proven useful in ulceroglandular and oculoglandular infections in humans. Some studies report positivity in whole blood samples /, /. Molecular detection is also part of multiplex assays for the detection of biological warfare agents in environmental samples. Molecular assays achieve high limit of detection (1–104 CFU/mL) in different specimens . False positive and negative results occur.
4. Schmitt P, Splettstoesser W, Porsch-Özcürümez M, Finke EJ, Grunow R. A novel screening ELISA and a confirmatory western blot useful for diagnosis and epidemiological studies of tularemia. Epidemiol Infect 2005; 133: 759–66.
13. Splettstoesser WD, Guglielmo-Viret V, Seibold E, Thullier P. Evaluation of an immunochromatographic test for rapid and reliable serodiagnosis of human tularemia and detection of F. tularensis-specific antibodies in sera from different mammalian species. J Clin Microbiol 2010; 48: 1629–34.
15. Cubero A, Durantez C, Almaraz A, Fernandez-Lago L, Gutierrez MP, Castro MJ, et al. Usefulness of a single-assay chemiluminescence test (tularemia VIRCLIA IgG+IgM monotest) for the diagnosis of human tularemia. Comparison of five serological tests. Eur J Clin Microbiol Infect Dis 2018; 37 (4): 38:643–9.
The genus Yersinia (Y), named after Alexandre Yersin, the discoverer of the causative agent of plague, belongs to the family Enterobacteriaceae. It currently includes 10 species, of which Y. pestis, Y. pseudotuberculosis and Y. enterocolitica are pathogens in humans. Yersiniae are short, facultative anerobic, Gram negative, rod shaped bacteria, which grow best at a temperature around 28 °C. Y. pestis has no flagellae while Y. pseudotuberculosis and Y. enterocolitica at temperatures below 30 °C form flagellae thus rendering them motile.
Due to the presence of O (somatic) and H (flagellar) antigens, Y. pseudotuberculosis and Y. enterocolitica can be serotyped. Yersiniae in part share antigens and show serological cross reactions with a number of other Enterobacteriaceae .
The causative agents of Yersinia infections, excluding plague, are Y. enterocolitica and Y. pseudotuberculosis, which occur worldwide in moderate and subtropical climate zones. Small mammals, especially rodents, and birds are the natural pathogen reservoir and source of infection .
Yersiniae cause intestinal as well as extra intestinal infections. The diagnosis of acute illness is primarily based on culture of the causative agent. Serodiagnostic evaluations supplement the diagnostic evaluation of acute infections and are essential for evaluating sequelae of the disease.
According to biochemical markers, 6 biotypes can be distinguished: 1A, 1B, 2, 3, 4 and 5 . Among the 60 or more O serogroups, primarily O:3, O:9, O:8 and, less frequently, O:5,27 and O:6,30 are isolated in Yersinia infections in humans .
The somatic antigen O:9 has characteristic cross-reactivity with the O antigen of the following bacteria: Brucella spp., E. coli O157, Morganella morganii O:43, Salmonella serogroup O:30, Stenotrophomonas maltophilia and V. cholerae serotypes Inaba and Ogawa .
More than 20 different H (flagellar) antigens have to date been detected, which are distributed among the entire spectrum of O serogroups . Cross reactivity is found between H:g (e.g., of Y. enterocolitica O:5,27 and H:a of S. paratyphi A) .
The pathogenicity of Y. enterocolitica in humans is closely correlated with the presence of the 70-kb pYV virulence plasmid which, under the regulation of calcium and temperature, encodes for 11 outer membrane proteins, the so-called Yops /, , /. During the course of infections with virulent Yersiniae, immune responses to plasmid encoded proteins are induced and can be used for diagnosis /, /.
Six serogroups of Y. pseudotuberculosis have been described. They are designated by the Roman numerals I–VI. Groups I, II, IV and V can be subdivided into 2 subgroups (A and B).
- Y. pseudotuberculosis serogroup II with the Salmonella O antigen 4,27
- Serogroup IV with the Salmonella O antigen 9,46, the E. coli O antigens O17 and O77 and also Enterobacter cloacae
- Serogroup V with the Salmonella O antigen 14
- Serogroup VI with E. coli O55.
Y. enterocolitica is detected as the causative agent in 1–4% of enteric infections in Germany. A total of 3368 enteral Yersinia enterocolitica infections were reported in 2010, corresponding to an incidence of 4.1 per 100,000 population . In comparison to this, Y. pseudotuberculosis is very rarely detected in Germany.
According to Section 7 of the German Infection Act (IfSG), both Y. pseudotuberculosis and the serotypes of Y. enterocolitica which are pathogenic to humans, are subject to mandatory laboratory reporting of transmissible pathogens.
The pathogenic serogroups of Y. enterocolitica show regionally different distribution worldwide . In Europe and Japan, O:3 and O:9 are primarily isolated in conjunction with human disease, whereas O:8 and a few other serogroups are almost exclusively detected in American patients. More recently, an increasing prevalence has been noted for O:3 in the USA .
Y. enterocolitica: this pathogen has been isolated in rodents, other mammals, birds, snakes, animal products, milk and surface water . Pigs appear to be a significant source of serogroups O:3 and O:9.
Inadequately cooked pork or pork related products, unpasteurized milk, contaminated uncooked food and contaminated drinking water play a role in the transmission to humans. Direct transmission to humans by infected dogs as well as human-to-human spread have also been observed /, /. The peak incidence falls in the cooler seasons of the year.
Y. pseudotuberculosis: a characteristic geographical distribution can be seen for this pathogen as well: serogroups I to V occur mainly in Europe and Asia including Japan, whereas in North America and New Zealand, serotypes I and II are frequently encountered and serogroup VI is almost exclusively found in Japan . Y. pseudotuberculosis is isolated in numerous animal species, especially rodents, cattle, sheep, goats, hares/rabbits, dogs and cats . The routes of transmission to humans are largely unknown.
Y. enterocolitica clinical symptoms
The infection is associated with a diverse spectrum of intestinal and extra intestinal manifestations which, in part, depend on age and immune status of the infected individual.
Patients affected by the disease include:
- Infants who usually present with enterocolitis featuring watery and, less commonly, bloody diarrhea
- School aged children and young adults who, in particular, present with pseudo appendicular signs and symptoms
- Adults who especially present with episodes of relapsing abdominal discomfort of undetermined origin accompanied by arthralgias, headaches and melalgias. Leukocytosis and a significantly elevated erythrocyte sedimentation rate are typical. The duration of the disease is 1–3 weeks . Intestinal complications include appendicitis, ulcerative enterocolitis, bowel perforation, peritonitis, toxic megacolon, cholangitis and mesenteric venous thrombosis . A host of extra intestinal complications have been observed, especially septicemia, endocarditis, meningitis, pharyngitis, pneumonia, osteomyelitis as well as pulmonary and renal abscesses .
In yersiniosis, post infectious sequelae take on special importance; they include reactive arthritis, erythema nodosum and Reiter’s syndrome, more rarely also glomerulonephritis, myocarditis, thyroiditis as well as sporadic clinical presentations with hemolytic anemia and sarcoidosis like pulmonary lesions .
In Scandinavia, reactive arthritis is observed in 10–30% of cases in adults . It is associated, in particular, with serotypes O:3 and O:9, starts 1–3 weeks after the intestinal infection with Yersinia and typically affects the ankle, knee and sacroiliac joints . Individuals with HLA-B27 are affected especially commonly . In the course of a year, yersiniosis patients develop arthritis 47 times more often than healthy controls. Thus, yersiniosis clearly takes a leading position compared to other enteric infections . Patients developing arthritis show indications of persistent Yersinia LPS and heat shock protein, but not of live bacteria and DNA in affected joints . This might maintain the persistence of IgA antibodies in these patients .
Y. pseudotubeculosis clinical symptoms
As with Y. enterocolitica infections, Y. pseudotuberculosis causes febrile diarrheal diseases. Mesenteric lymphadenopathy is encountered at a higher rate. Typical complications include sepsis, liver abscess, erythema nodosum and reactive arthritis. A few cases of hemolytic uremic syndrome and nephritis have been described .
In Japan, the occurrence of a special form of clinical presentation is seen which resembles so-called Izumi fever. It is characterized by high fever, scaly skin rash, raspberry tongue, conjunctivitis and lymphadenopathy /, /.
Agglutination test (Widal reaction)
The Widal reaction is performed as a macro agglutination test in tubes or as a micro agglutination test on micro titer plates. Boiled, washed bacterial suspensions (O antigen) as well as live or formalin inactivated bacteria (OH antigen) are used as antigens. Patient serum is serially diluted starting at 1 : 10 and, following the addition of the same volume of antigen suspension to each dilution, is incubated at 37 °C for 18 h. In Europe, depending on prevalence rates, strains of serogroups O:3 and O:9 are used /, , /.
- Borderline titers: O agglutination 1 : 40; OH agglutination 1 : 80.
- Positive: O agglutination ≥ 1 : 80; OH agglutination ≥ 1 : 160.
Preparations of Yersinia outer membrane proteins (Yops) stemming from pYV plasmid positive strains as well as recombinantly produced ones are used as antigens . The assessment is made based on classification into IgG, IgA and IgM antibodies.
ELISAs are based on Yersinia LPS, formalized pYV plasmid positive cells or a combination of LPS and expressed plasmid encoded proteins /, /. Using an LPS-bsed ELISA and a definition of positive as a positive result for either IgA, IgG or IgM antibodies a diagnostic sensitivity of 81% was achieved for samples received within 60 days after onset of symptoms . Standard tube-agglutination assay achieved a sensitivity of 60% on the same samples.
Complement fixation (CF) test
O antigen preparations are employed.
Borderline titers: ≥ 1 : 10.
Borderline titers: ≥ 1 : 160.
Agglutination test (Widal reaction)
The Widal reaction is performed as a macro agglutination test in tubes or as a micro agglutination test on microtiter plates. Boiled, washed bacterial suspensions (O antigen) are used as antigens. Patient serum is serially diluted starting at 1 : 10 and, following the addition of the same volume of antigen suspension to each dilution, is incubated at 37 °C for 18 h. Either strains of serogroup I alone or all 6 serogroups are used.
- Borderline titers: 1 : 40.
- Positive: ≥ 1 : 80.
The Widal reaction is the most commonly used method for serodiagnosing acute Yersinia infections. Significant OH titers are detectable from the second week after the onset of the disease.
It does not suffice to make the diagnosis of acute Y. enterocolitica infection based on the OH agglutinin titer alone since OH titers may remain elevated anamnestically for prolonged periods of time . Diagnosis therefore necessitates the additional determination of the O agglutinins.
O agglutinins appear with a delay of a few days, their titer remains below that of the OH agglutinins, they decline from the second month and are usually no longer detectable by 6 months after the onset of the disease. They are considered to be highly specific, and significant cross reactions between O : 3 and O : 9 occur only if antigen suspensions are contaminated by LPS rough strains (e.g., as seen after incubating the reference strains at 37 °C).
Based on the pattern of O and OH antigens, deductions can be made as to the certainty of the underlying yersiniosis; a relatively high O titer by comparison with the OH titer suggests that an acute infection is still present .
- OH titer of ≥ 1 : 160
- Elevated O and OH antigens together with titers of 1 : 40 (O antigen) and 1 : 80 (OH antigen).
In the case of repeat agglutinin titer determinations, a fourfold rise or decline in titers is considered to be significant.
The highest OH agglutinin titers are measured within the first 4 weeks after the onset of acute intestinal symptoms; a rapid decline suggests a favorable course of recovery. Given uncomplicated recovery from intestinal yersiniosis, OH titers will fall significantly within 3 months and by 6 months are usually no longer detectable. In the case of extra intestinal sequelae such as reactive arthritis or erythema nodosum, residual OH titers ranging from 1 : 40 to 1 : 80 may in a few cases apparently persist for years.
Because of common antigens between Y. enterocolitica O9 and Brucella species , serological cross reactivity in the Widal agglutination is observed. For instance, experience shows that the reaction of Brucella antigen is characterized by the same high titers in the presence of O:9 yersiniosis.
The following criteria may be helpful in identifying true brucellosis:
- The medical history and the typical clinical presentation of brucellosis
- The isolation of the causative agent from blood cultures and/or bone marrow specimens
- The comparison of both titers; in true brucellosis, the Brucella agglutination titer is markedly higher than the Yersinia OH titer since Brucella does not have any H antigens
- The brucellosis complement fixation test; in true brucellosis, it is usually elevated whereas in yersiniosis, it tends to be negative or borderline. If in doubt, cross-over serum absorption must be attempted .
Reactive bands at Yop M, Yop H, Yop D and Yop E are considered to be pathognomonic . The determination of IgA antibodies is of special significance because, in comparison to uncomplicated courses of the disease, they are more likely to persist if reactive arthritis occurs /, /. Therefore, immunoblotting is especially suited, as is the ELISA, to diagnose immunopathological complications of yersiniosis.
The ELISA is evaluated according to specific Ig classes, as is the immunoblot. It has not been clarified to date whether elevated IgM titers /, / or elevated IgA titers /32/ are useful indicators of acute infection. The detection of a persistent IgA response in the immunoblot or the ELISA is characteristic for underlying immunopathological complications /, , , , /.
Complement fixation test and indirect hemagglutination test
ELISA and immunoblot based on plasmid encoded virulence antigens (YOPS) are generally suited for detecting an immune reaction to Yersinia pseudotuberculosis . To date, adequate experience in regard to serodiagnosing Y. pseudotuberculosis infections is limited worldwide to the Widal type agglutination. Agglutinins are usually already detectable at the onset of clinical symptoms.
- Y. pseudotuberculosis II with Salmonellae of serogroup O4
- Y. pseudotuberculosis IV with Salmonellae of serogroup O9 and Y. pseudotuberculosis VI with E. coli O55.
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5. Heesemann J, Gross U, Schmidt N, et al. Immunochemical analysis of plasmid-encoded proteins released by enteropathogenic Yersinia sp. grown in calcium-deficient media. Infect Immun 1986; 54: 561–7.
8. Gaede K, Mack D, Heesemann J. Experimental Yersinia enterocolitica infection in rats: analysis of the immune response to plasmid-encoded antigens of arthritis susceptible Lewis rats and arthritis-resistant Fischer rats. Med Microbiol Immunol 1992; 181: 165–72.
20. Granfors K, Merilathi-Palo R, Luukkainen R, et al. Persistence of Yersinia antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3 infection with and without reactive athritis. Arthritis Rheum 1998; 41: 855–62.
27. Heesemann J, Eggers C, Schroeder J. Serological diagnosis of yersiniosis by immunoblot technique using virulence-associated antigen of enteropathogenic yersiniae. Contrib Microbiol Immunol 1987; 9: 285–9.
28. Paerregaard A, Shand GH, Gaarslev K, et al. Comparison of crossed immunoelectrophoresis, enzyme-linked immunosorbent assay, and tube agglutination for serodiagnosis of yersinia enterocolitica serotype O:3 infection. J Clin Microbiol 1989; 29: 302–9.
29. Maki-Ikola O, Heesemann J, Lahesmaa R, et al. Combined use of released proteins and lipopolysaccharide in enzyme-linked immunosorbent assay for serologic screening of Yersinia enterocolitica infections. J Infect Dis 1991; 163: 409–12.
30. Dalby T, Rasmussen E, Schiellerup P, Krogfelt KA. Development of an LPS-based ELISA for diagnosis of Yersinia enterocolitica O:3 infections in Danish patients: a follow up study. BMC microbiol 2017; 17 (1). .
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37. Granfors K. Measurement of immunoglobulin M (IgM), IgG, and IgA-antibodies against Yersinia enterocolitica by enzyme-linked immunosorbent assay: persistence of serum antibodies during disease: J Clin Microbiol 1979; 9: 336–41.
38. Larsen JH, Hartvig-Hartzen S, Parm M. The determination of specific IgA-antibodies to Yersinia enterocolitica and their role in enteric infections and their complications. Acta Path Microbiol Immunol Scand 1985; B 93: 331–9.
Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Volker Brade
Human babesiosis is a rare, zoonotic, high fever infection. The pathogens, tick transmitted hemoparasites (genus Babesia) similar to Plasmodia, are members of the Apicomplexa. Babesia play an important role in veterinary medicine (bovine babesiosis, Texas cattle fever). The first case of human babesiosis caused by B. divergens in a splenectomized cattleman was described in the former Yugoslavia in 1957. Cases of disease in humans have since then repeatedly been reported primarily from North America and sporadically also from Europe and Asia /, , /.
Babesia were first identified as causative agents of hemolytic fever in cattle by V. Babes in 1888. More than 100 different species have to date been identified. Based on their morphological and phenotypic characteristics, they are traditionally divided into small babesias (e.g., B. microti, B. divergens, B. gibsoni) and large babesias (e.g., B. bovis) . Recent classification is based on phylogenetic analyses of the 18S-rDNA and the β-tubulin gene .
Various Ixodes ticks act as vectors, while small mammals and wild and domestic animals are the reservoir hosts. The hemoparasites exclusively infect red blood cells and are occasionally confused with Plasmodia in blood smears. Inter erythrocytic tetrad forms found in high parasitemia and in immunocompromised patients are important for differential diagnosis. However, these cross shaped structures looking like a Maltese cross can also be absent . Whereas in malaria, parasitic pigment is usually detectable, Babesia lack pigment.
The most important Babesia species pathogenic to humans are B. divergens (mainly in Europe) and B. microti (mainly in North America). However, individual cases of human infections caused by Babesia spp. (B. venatorum, B. duncani) have been reported worldwide /, , / ().
The pathogens can be cultured in special laboratories; however, the culture method plays no role in diagnostic practice. The diagnosis of acute human babesiosis is based on the detection of the parasites in Giemsa stained thin blood smears or on PCR /, /.
In North America, more than 200 new cases of human babesiosis caused by B. microti are annually reported from the southern New England states . In Europe, approximately 40 cases of infection caused, in particular, by B. divergens in splenectomized patients and, recently, by other species (B. venatorum, B. microti) in immunocompromised individuals have been reported /, /. B. divergens as well as B. microti and other closely related Babesias are detectable in I. ricinus with conventional and molecular biological methods /, /. Only one well documented case of human autochthonous B. microti infection has to date been reported in Europe . Such infections must especially be considered in travelers returning from endemic regions (United States and Canadian east coast) /, /. Seroepidemiological studies show a significantly higher prevalence of antibodies against Babesia in patients exposed to ticks than in controls (blood donors) . Asymptomatic or influenza like infections also develop in immunocompetent individuals after tick bite. However, further investigation is necessary to finally assess the epidemiological distribution and medical significance of these tick transmitted pathogens in Europe.
5 days to 9 weeks.
The spectrum of clinical manifestations of human babesiosis ranges from asymptomatic or influenza like infection to life threatening, partly lethal disease depending on the individual predisposition and level of parasitemia /, , , /. Most infections occur in the period of main activity of the vectors between May and September. After a tick bite, patients present with faintness, headache, arthralgias, hemolytic anemia, hemoglobinuria and fever up to 40 °C. Abdominal pain, hepatosplenomegaly, renal insufficiency and unproductive cough are also seen.
Elevated bilirubin and LD (hemolysis) and procalcitonin and, typically, a positive direct Coomb’s test should prompt further diagnostic tests . B. microti infections usually resolve without complications, despite a prolonged course of the disease in some cases. Clinically manifest B. divergens infections primarily affect splenectomized patients (established by medical history) and almost always are life threatening medical emergencies /, , /. B. venatorum infections mainly affect immunocompromised patients with underlying hematological disease, but usually take a milder course than infections with B. divergens /, /. Co infections with other tick transmitted pathogens (Borrelia, Ehrlichia) are rarely reported and some of these patients have severe and persistent courses of infection /, ,/.
Typical risk factors for an aggravated course of the disease include underlying hemato-oncological disease, suppressed cellular immunity (e.g., application of rituximab, HIV, organ transplants and advanced age) . Post-babesiosis warm autoimmune hemolytic anemia can occur within 3 months after the diagnosis and treatment of babesiosis .
Babesia may persist in the blood of patients for weeks or months even after resolved infection. Blood donations from asymptomatic carriers can be the cause of repeatedly reported transfusion associated cases of babesiosis that may become manifest after 2 months .
Screening for Babesia microti is performed in the U.S. blood supply. In a study using an arrayed fluorescence immunoassay 35 of 89,153 (0.38%) donors were confirmed to be positive, of which 20% were PCR positive, 9 samples were antibody negative (arrayed fluorescence immunoassay) representing 13% of all PCR-positive samples. In conclusion screening for antibodies to and DNA from B. microti was associated with a decrease in the risk of transfusion-transmitted babesiosis.
Serodiagnostic testing should be performed in the 2nd to 3rd week of illness to confirm the diagnosis in ambiguous cases of suspected babesiosis. It is also useful in patients with low or chronic parasitemia and a masked course of the disease /, /.
Immunofluorescence test (IIFT)
The serological method of choice is IIFT for IgG and possibly also IgM antibody determination. Preparations of red blood cells of hamsters infected with human or animal Babesia isolates, for example, are used as a source of antigens. Test systems for the detection of antibodies against B. divergens and B. microti are commercially available. The test results may vary significantly depending on the test system and antigen used /, /. In US American studies on patients with confirmed babesiosis, the diagnostic specificity and sensitivity of the IIFT are specified as 88–92% and 90–100%, respectively . Such performance is to a limited extent available for Europe /, /.
ELISA and immunoblot
Serum: 1 mL
Most patients show seroconversion 7–10 weeks after the onset of clinical symptoms. Antibody titers reach a peak within 3 months after infection and then decline gradually to below the detection limit. High positive titers are usually observed in patients with persistently low parasitemia /, , /.
In the IIFT, IgG titers of 64 and/or 128, depending on the test, are suspicious and IgG titers ≥ 256 or results in combination with a positive IgM result (titer ≥ 20) are highly suspicious of a new or recent infection. Patients with clinically acute disease show partly extremely high titers (above 10,000). The detection of seroconversion and the fourfold increase in titers in parallel testing with previously collected serum is considered proof of babesiosis /, , /. The antibody response in immunocompromised patients may be delayed for weeks and the outcome of serodiagnostic testing in acute infection is not reliable /, /.
Comments and problems regarding serology
False positive results, especially for IgM, occur in Babesia serology in the case of autoimmune disease, CMV and EBV infections. Therefore, serodiagnostic testing should focus on IgG determination. IgM should only also be determined in positive test results /, , /.
Seroreactivity against B microti has been reported to be highly specific not only to B. microti lineages but also to sub lineages. A study confirmed that there is ow cross-reactivity between B. microti lineages and sub lineages.
Direct detection of the parasites in the blood by PCR is of high significance in the acute phase of infection and also in the diagnosis of chronic infection /, , , , , /. Relevant diagnostic PCR methods are considered to be highly sensitive and specific, but are only available in special laboratories and are poorly standardized.
The target sequences employed for the diagnosis of Babesia infection by PCR based on EDTA or sodium citrate blood specimens consist of genus specific fragments of the 18S-rRNA gene.
PCR may remain positive for months in persistent infection and also after therapy /, /. No specific information on the clinical and analytical performance of the test methods is available because of the small number of cases.
4. Dumler JS, Aguero-Rosenfeld M. Microbiology and laboratory diagnosis of tick-borne diseases. In: Cunha BA (ed). Tick-borne infectious diseases: diagnosis and management. New York; Marcel Dekker 2000; 39–42.
8. Hildebrandt A, Hunfeld KP, Baier M, Krumbholz A, Sachse S., Lorenzen, T, et al. First confirmed autochthonous case of human Babesia microti infection in Europe. Eur J Clin Microbiol Infect Dis 2007; 26: 595–601.
12. Krause PJ, Telford SR, Ryan R, Conrad PA, Wilson M, Thomford JM, Spielman A. Diagnosis of babesiosis: evaluation of serologic test for the detection of B. microti antibody. J Infect Dis 1994; 196: 923–6.
13. Häselbarth K, Tenter AM, Brade V, Krieger G, Hunfeld, KP. First case of human babesiosis in Germany – Clinical presentation and molecular characterisation of the pathogen. Int J Med Microbiol 2007; 297: 197–204.
14. Sayama Y, Zamoto-Niikura A, Matsumoto C, Saijo M, Ishiara C, Matsubayashi K, et al. Analysis of antigen-antibody cross-reactivity among lineages and sublineages of Babesia microti parasites using human babesiosis specimens. Transfusion 2018; 58 (5): 1234–44.
- Enteric pathogens e.g., Shigella sp. and Hepatitis A virus
- Pathogens spread by close contact e.g., Neisseria meningitidis
- Pathogens that can spread through sexual contact e.g., Zika virus
In addition, increase in antimicrobial resistance (e.g., Neisseria gonorrhea and Mycoplasma genitalium) have increased the concern about limited treatment options for sexually transmitted diseases. Refer to .
For identification of infections qualitative real-time multiplex PCR tests for simultaneous measurement of sexually transmitted infections (STI) have been developed. The overall agreement rate is about 99% .
- The pathogen e.g., antimicrobial resistance, infectiousness, virulence
- The environment e.g., access to biomedical interventions (chemsex, condomless sex), international travel, access to testing and treatment, social media and use on online dating apps
- The host e.g., previous immunity (to Hepatitis A, N. meningitidis), coinfection with other sexually transmissible pathogens, coexisting conditions (HIV infection).
In patients with sexual transmission of enteric pathogens by direct contact (oral-anal contact) or indirect contact (through contact with fecally contaminated fingers or objects) pathogens can be well recognized.
- Stool culture
- Antimicrobial susceptibility testing for relevant pathogen
- Stool PCR test for enteric pathogens (with reflex culture if bacterial pathogen is identified)
- Testing for HIV and other sexually transmitted pathogens, including rectal pathogens if proctitis is clinically suspected.
6. Gaydos CA, Manhart LE, Taylor SN, Lillis RA, Hook EW, Klausner JD, et al. Molecular testing for Mycoplasma genitalium in the United States: Results from the AMES Prospective Multicenter Clinical Study. J Clin Microbiol 2019; 57 (11) e01125-19.
9. Goldstein E, Martinez-Garcia L, Obermeier M, Glass A, Krügel M, Maree L, et al. Simultaneous identification of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis – multicenter evaluation of the Alinity m STI assay. J Lab Med 2021; 45 (4–5): 213–23.
NAA, nucleic acid amplification
*IgG, IgM and, if required, IgA; N, nephelometry; T, turbidimetry; I, immunoblot; W, Widal
Clinical case definition
(a) If < 5 cm in diameter, a history of tick bite, a delay in appearance (after the tick bite) of at least 2 days and an expanding rash at the site of the tick bite is required.
(b) As a rule, initial and follow-up samples have to be tested in parallel in order to avoid changes by inter assay variation.
(c ) In early cases, intrathecally produced specific antibodies may still be absent.
* The PCR is not yet accepted as a routine diagnostic method. EM, erythema migrans; ACA, acrodermatitis chronica atrophicans.
IgG positive: ≥ 2 bands
IgM positive: ≥ 1 band
IgG positive: ≥ 5 bands
IgM positive: ≥ 2 bands
IgG positive: ≥ 2 bands
IgM positive: ≥ 2 bands
* 13C is not radioactive; 14C is radioactive (cave children, pregnant women).
* Alarm symptoms: bleeding, weight loss etc.
1st septicemic phase
2nd organic disease phase
* % referred to clinically confirmed cases. CFU, colony-forming units.
NAA, Nucleic acid amplification.
Overall n = 7352
Mean overall results of studies involving respiratory and non respiratory specimens, referred to cultural an d clinical data. Date expressed in %. Sens, sensitivity; Spec, specificity; PV, positive predictive value; NV, negative predictive value.
Sexually transmitted syphilis
Bejel endemic syphilis
* Predelection time of infection in years
Name of test
* FITC-labeled anti-Tp antibody
** Venereal Disease Research Laboratory test
Antibody test result
* Weakly positive or equivocal test results always require follow-up retesting
Interpretation of test result
* The listed titers refer to the fractionated IgM-FTA-ABS test and the IgM FTA-ABS test after IgG precipitation. In the case of separation of the samples by gel or ion exchange chromatography, dilution factors are in the range of 1 : 5 to 1 : 30 and may have to be determined separately for the individual samples. When combining the IgM-FTA-ABS test with IgG precipitation, the output titer is 1 : 10.
* or comparable screening test; ** fractionated IgM-FTA-ABS test or other IgM antibody assays with comparable diagnostic sensitivity and specificity
Antibody test result
N, number of cases