43 Viral diseases

Hans W. Doerr, Gregor Caspari, Wolfram H. Gerlich

43.1 Viral infection and infectious disease

Infection is the adhesion, invasion and multiplication of a cellular or subcellular microbes in a cellular macro organism. Viruses are subcellular infectious agents. They consist of nucleic acid (DNA or RNA), which makes up the genome of the virus, and an envelope, which always contains proteins and often lipids and poly saccharides. Outside the cell, viruses are complexes of macromolecules that have no metabolism of their own. In their replication, they are obligate cell parasites. Some viruses contain enzymes. These are RNA and DNA polymerases as well as other enzymes of the nucleic acid metabolism, proteases and kinases, or neuraminidases found on the outer shell or envelope of viruses. If the infected host is vulnerable, the virus will develop an infectious disease.

Viruses must implant their genetic material into the host organism cells. The viral envelope is lost during or after entry of the virus into the cell. Viruses are ubiquitous throughout the cellular environment (prokaryotes, plants, animals). The individual types of viruses that infect animals and humans vary greatly in size, ranging from those which are just visible under a light microscope (Poxvirus) with a mean diameter of 300 nm) to very small particles measuring little more than 20 nm in diameter (Parvovirus). By way of comparison, spherical bacteria have a mean diameter of 1,000 nm and the elementary bodies of chlamydia (the smallest bacteria) a mean diameter of 250 nm. The smallest infectious agents are conformationally altered polypeptides called proteinaceous infectious organisms (prions). They do correspond to the term “virus” (Latin for poison) coined 100 years ago by Loeffler and Frosch, unlike non replication competent toxic (from Greek toxin = poison) molecules from bacteria or other living organisms.

Since prions, by definition, exist without a genome of their own, they are also called unconventional viruses /1/.

43.1.1 Virus classification

Viruses are classified according to the general biological classification scheme based on infection biology and structural criteria /2, 3/. This scheme groups viruses at different hierarchical levels of order (virales), family (Viridae), subfamily (virinae), genus, and species. Conventional viruses are divided into six groups based on their type of genome replication (Baltimore classification). In addition, viruses are categorized into types, subtypes, and variants. Serotyping (characterization of virus antigenicity) is increasingly being supplemented or replaced by genotyping (genome sequencing).

Besides the above taxonomic criteria, viruses are generally distinguished into DNA and RNA viruses based on the nature of their genome. The DNA of human pathogenic viruses is double-stranded (ds) (exceptions: Parvovirus and Annellovirus). DNA viruses replicate semi-conservatively in the nucleus (exception: Poxvirus in the cytoplasm). The genomes of RNA viruses are single-stranded (ss) (exception: Reovirus, rotavirus) and replicate in the cytoplasm of the infected cell (exception: Influenza virus and Bornavirus, which replicate in the nucleus).

The ssRNA genome is called:

• Plus stranded if it can function directly as an mRNA, as in Poliovirus, for example
• Minus stranded if the mRNA must first be transcribed by a viral RNA polymerase, as in Influenza virus, for example. Retrovirus with an RNA genome and the Hepatitis B virus with a DNA genome are unique in that their genome replication occurs by reverse transcription of a plus stranded RNA into DNA.

Animal viruses are more or less adapted to the specific host. Zooanthroponoses are infectious diseases that are transmitted from vertebrates to humans. New viral diseases emerge when a host specific virus enters a different type of host and adapts to it. This evolution is Darwinian in terms of the interplay between virulence and resistance factors (i.e., a natural phenomenon characterized by mutation and selection). It has been hypothesized that the large viruses may have evolved from degenerated bacteria and other prokaryote infectious agents. The small viruses may have originated from mobile genetic elements of the cells (e.g., bacterial plasmids or cell genome transposons). Tab. 43.1-1 – Structure-based classification of human viruses provides an overview of human viruses.

43.1.2 Structure of viruses

Besides some special types /1, 2, 3/, two structures have essentially established themselves in the evolution of animal viruses (Fig. 43.1-1 – Structures of viruses relevant to human medicine):

• Icosahedral viruses: the spherical icosahedron (polyhedron with 20 faces) is considered the most stable geometric structure relative to the structural material used. This icosahedral structure, where the genome associated with proteins and polyamines is fully enclosed inside the nucleocapsid, is common to a number of viruses. Only the smallest viruses are true icosahedrons. Larger viruses have a more complex structure, in which each (imaginary) triangular facet is enlarged by a triangular pyramid, which may itself also be enlarged in the same fashion. The number of new sub triangles per facet, as observed by electron microscopy, determines the characteristic triangulation number of the icosahedral virus.

Some viruses additionally have an envelope consisting of glycoproteins and lipids. The space between the envelope and the nucleocapsid is filled with protein (e.g., tegument phosphoprotein in the case of Herpesviridae). The structure of this envelope is comparatively unstable and, when studied under electron microscopy, often appears pleomorphic due to drying artefacts. Non enveloped (naked) nucleocapsid viruses tend to be considerably more resistant to the environment than enveloped viruses (exceptions: Rhinovirus, Poxvirus).

The envelope can easily be destroyed by lipophilic reagents. For laboratory diagnostic purposes, viruses are thus distinguished into ether resistant and ether sensitive viruses. When the viral envelope is destroyed, viruses lose their infectivity, which relies on the presence of an intact external structure.

• Viruses with a helical nucleoprotein: in contrast to icosahedral viruses, the nucleocapsid of helical viruses does not have an isometric structure, but encloses the genome (a single stranded ribonucleic acid) with its subunits (capsomeres), which are arranged helically around it. All these viruses are enveloped and thus ether sensitive.
• Special forms include the structures of rabies viruses, the poxvirus group and Filovirus (Marburg/Ebola), which are bullet, brick and filament shaped, respectively.

43.1.3 Viral replication

Unlike the replication of cells, viral replication does not occur by duplication, but according to a modular principle /4/. The viral genome introduced into the host cell induces the synthesis of its structural components, which are subsequently assembled. This assembly often leads to defects. Only some of the new viruses are infectious. These are called virions. When a cell is co infected with different virus variants with segmented genomes, the viral genes and components may reassort, as occurs in Influenza virus and Rotavirus, or recombine, as seen in retroviruses.

The replication of conventional viruses is traditionally divided into specific steps or stages, as shown in Fig. 43.1-2 – Important steps of virus replication in the cell. Research into viral replication has enabled the development and optimization of antiviral chemotherapeutics /1/. The new viruses are released either in a burst, killing the host cell, or gradually by budding from an internal or external cell membrane altered by the specific virus. In the latter case, the virus acquires an additional envelope, which is glycosylated or myristoylated in a specific way.

If the cell membrane is only slightly altered, the virus can “hide” from the immune system through budding (molecular mimicry).

At the same time, viruses can also potentially trigger autoimmune responses.

43.1.4 Viral pathogenesis: abortive infection

The abortive infection does not lead to efficient replication. Although the target cell of the infection has the right cell receptors for adsorption and penetration of the virus, it is not permissive for replication. The infecting viruses die.

43.1.5 Viral pathogenesis: productive infection

This type of infection produces new virus particles. If the release of progeny virus is accompanied by cell death, then the infection is lytic.

The new viruses may also exit the cell through budding. In subacute sclerosing pan encephalitis (SSPE), the release of measles virus from infected brain cells is blocked.

43.1.6 Viral pathogenesis: latent infection

As in abortive infection, initially no infectious complete viruses (virions) are produced. However, the viral nucleic acid is maintained and, in DNA viruses, persists in the nucleus in an episomal form. In retroviruses, the RNA genome is reverse transcribed into dsDNA by a special polymerase (retroviral reverse transcriptase) and even integrated into the cell’s genome. The integrated retroviral genome is known as provirus.

43.1.7 Viral pathogenesis: oncogenic infection

Some DNA and retrovirus infections stimulate replication of the cellular genome and are able to immortalize the infected cell (Tab. 43.1-2 – Human tumor viruses). Research has identified a number of viral genes whose expression regulates the synthesis of new viruses and interferes with the cellular replication cycle. DNA and weakly oncogenic retroviruses have genes that have a trans activating effect on cellular growth controlling oncogenes (proto-oncogenes) or interfere with cellular tumor suppressor gene products, in particular p53 and pRb. The genome of highly oncogenic retroviruses contains an additional oncogene that was acquired by the viruses from a cell in the course of their evolution and is integrated, during infection, into the genome of the current target cells of the viruses, where it becomes non physiologically active /1/.

43.1.8 Viral pathogenesis: slow virus infection

Some viral infections develop over years, causing gradual organ degeneration, preferably in the central nervous system. AIDS is a slow virus disease of the immune system and brain. Slow virus infections may not only be caused by conventional viruses but also by prions, which are the infectious agents of spongiform encephalopathy in humans and animals (Tab. 43.1-3 – Human slow virus diseases).

43.1.9 Clinical course of viral infection

Like other microbes, viruses usually enter the macro organism through body cavities or skin lesions. First, the cells of these portals of entry are infected, often with mild inflammation as a result of innate immune responses. Clinical signs include temporary sore throat, fever, and gastrointestinal symptoms. Within a characteristic incubation period, the viruses disseminate via the lymphatics and the bloodstream (viremia) and infect their respective target organs. Adaptive immune responses are triggered, which lead to the destruction of virus infected cells and elimination of free viruses. Thus, many viral diseases typically have two phases. However, depending on the individual extent of organ damage, only a variable proportion of infected individuals develop manifest symptoms of viral infection of the relevant organ. This manifestation index is a pathogen specific statistical mean. Many viral diseases show a lower manifestation index for infection during childhood than for infection in advanced age. This may be due to the following reasons:

• Young children have infections quite frequently and therefore have a higher level of resistance
• Cross and autoimmune responses become clinically manifest only after a certain age
• Infants are partially protected by transplacental maternal antibodies, which weaken the infection.

43.1.10 Time course of viral infection

In terms of time and clinical course, infections can be acute, subacute, or chronic. Acute infection usually has a productive lytic replication cycle. It causes a strong immune response, leading to good immunity (measles and other viral childhood diseases). Less lytic infections are more likely to persist (e.g., hepatitis B virus infections).

Some viral infections become latent (Herpes virus). By overcoming or evading immune defenses, a subclinical chronic productive viral infection can establish itself (cytomegaly). If there is little or no cellular immune response (innate or acquired immune defects), persistent viral infections are opportunistically reactivated, potentially leading to severe illnesses, such as are typical of AIDS. Persistent viral infections with slowly progressive organ damage have already been mentioned (slow virus disease).

Depending on the virus cell interactions, viral diseases may be caused by /1, 2, 3, 4, 5/:

• Direct cytopathogenicity of the virus. Examples: respiratory and gastrointestinal infections
• Direct, slowly progressive cytopathogenicity. Example: subacute sclerosing encephalitis
• Transformation of the infected cell into a benign or malignant tumor. Examples: papillomas, cervical carcinoma, liver carcinoma
• Specific (cytotoxic lymphocytes) or unspecific immune responses (cytokines). Examples: exanthems and hepatitis.

Some autoimmune diseases may be caused by yet unknown viral infections.

References

1. Doerr HW, Gerlich WH (eds). Medizinische Virologie: Grundlagen, Diagnostik, Prävention und Therapie virologischer Krankheitsbilder. Stuttgart; Thieme, 2.edition 2010.

2. Carter J, Saunders VA. Virology: Principles and applications. John Wiley & Sons, 2007; pp. 1–48.

3. Condic RC. Principles of virology. In: Knipe DM, Howle PM (eds). Fields Virology, volume 1. Lippincott-Williams & Wilkins, 2007; pp. 25–58.

4. Poirier EZ, Vignuzzi M. Virus population dynamics during infection. Curr Opin Virol 2017: 23: 82–7.

5. Lidsky PV. Andino R, Rouzine IM. Variability in viral pathogenesis: modeling the dynamic of acute and persistent infections. Curr Opin Virol 2017; 23: 120–4.

43.2 Diagnosis of viral diseases

Laboratory tests contribute to the diagnosis and monitoring of a transmissible disease by:

• Identifying or excluding an infection and assessing the immune status /1, 2, 3/.
• Diagnosing and predicting the disease as a direct or indirect (e.g. immunopathologic) consequence of the infection. This is the responsibility of the clinician, who generally relies on additional laboratory tests for examination of organ functions.

Before conducting a complex infection workup, the following general infection findings should always be tested:

• Viral diseases are typically associated with fever, elevated erythrocyte sedimentation rate (ESR), and relative lymphocytosis
• Bacterial superinfection is associated with the return of fever with leukocytosis
• The concentration of C-reactive protein (CRP) in the blood is only moderately elevated compared to bacterial infections.

A panel of laboratory tests for the diagnosis of viral diseases is available. In practice, testing is best performed in stages, beginning with basic and inexpensive routine methods, followed by more costly, special methods. For further information refer to:

• Tab. 43.2-1 – Methods of virologic laboratory diagnosis
• Tab. 43.2-2 – Methods approved for the diagnosis of viral diseases.

43.2.1 Tests in viral diagnosis

In order to prove an etiological relationship between an infectious microorganism and a transmissible disease, the following three diagnostic postulates formulated by Henle and Koch must be satisfied:

• The microorganism (inter alia) can be regularly isolated from every patient with the defined disease and
• grown in pure culture
• The cultured microorganism, when introduced into a human volunteer or experimental animal, should reproduce the defined disease.

In many infectious diseases, these postulates are not fulfilled in the strict sense, but can be demonstrated only indirectly (e.g., by observing infection chains).

Selection of test methods and specimens

An effective working relationship between clinicians and clinical pathologists is desirable in order to ensure that appropriate clinical specimens and test methods are selected. The clinician must define the objective of laboratory diagnostic testing by providing sufficiently detailed information in relation to the indication for testing. In general, a distinction must be made between the assessment of a patient’s immune or infective status and the workup of a disease process. The following information is always required: time of disease onset and sampling, as well as any therapeutic measures taken that may have an impact on the infection process or the immune status (antiviral therapy, blood products, immunoglobulins).

It is then up to the clinical pathologist, microbiologist and virologist to request, based on their professional knowledge and experience in the evaluation of the pathogenesis, suitable specimens in the correct form of transport (Tab. 43.2-3 – Specimen suitable for virus detection) and perform the relevant tests for the presence of the viral infections in question depending on the relevant epidemiological situation. Lists of tests to be performed for a suspected diagnosis based on epidemiological and statistical aspects, in particular during pregnancy are presented in:

• Tab. 43.2-4 – Information for test selection in the case of organ related clinical symptoms
• Tab. 43.2-5 – Virus infection and pregnancy.

43.2.2 Direct virus detection

Early diagnosis of an infection

This is best made by direct virus detection, especially in the case of diseases of the respiratory and gastrointestinal tract.

Specimen

Specimens used include sputum and nasopharyngeal swabs or stool samples. Special media are available for transporting respiratory material. In the simplest case, physiological saline solution is sufficient. If the material cannot be delivered within a few hours, it should be kept refrigerated during the entire transport process. For extended transport, refrigeration at below –20 °C is required. This also applies to all other types of specimen (cerebrospinal fluid, urine, stool).

For cerebrospinal fluid (CSF) diagnostic testing, the specimen should be refrigerated for transport to the virology laboratory, but not be refrigerated for transport to the cellular microbiology laboratory. If necessary, the specimen must be separated immediately after collection.

Besides nucleic acid testing, virus detection can also be carried out using an antigen test, electron microscopy, virus isolation, and virus culture.

43.2.2.1 Virus isolation

Viruses are usually isolated in cell cultures (Tab. 43.2-6 – Diagnostically important cell cultures). Only in exceptional cases are laboratory animals or incubated embryonated eggs used. The previously laborious method has been improved continuously, allowing the test to now be read within 24 hours: first, the specimen with the cell detritus and adhering viruses (skin or mucosal swab placed in solution, urine, stool fluid, aspirates, blood plasma with and without buffy coat, cerebrospinal fluid) is sedimented on a preformed cell layer by centrifugation (centrifugation culture; shell vial assay) in order to cause rapid adsorption of the viruses to be detected on the cultured cells and thus initiate the diagnostic infection process. After 24 hours, the immunohistologic test for the formation of early viral antigens in cells is performed. Correspondingly stained plaques are counted microscopically per visual field and provide information on the infectious dose of the inoculum (Fig. 43.2-1 – Immunoperoxidase staining of CMV infected fibroblasts). The advantage of this short term culture is that there is no interference by contaminating bacteria. Cultivating the virus until a typical cytopathogenic effect develops is generally only possible under antibiotic protection and usually takes between several days and 2 weeks.

Short and long term cultures allow the testing of antiviral drugs (virogram) and assessment of the phenotypic resistance to therapy /1, 2, 3/. The virus isolated in cell culture can be typed biologically, serologically or molecularly to identify the pathogen as well as infection chains. The biological methods are used for initial, rapid testing. For example, by adding suitable erythrocytes, it is possible to test whether viral antigens are incorporated in the membrane of the infected cell (hemadsorption test). Serotyping is performed with polyvalent or monovalent antisera, as previously described for short time culture. In scientific research, highly specific monoclonal antibodies are used for epitope analysis of the viral antigens.

43.2.2.2 Molecular biological methods

An overview of molecular techniques for diagnostic virology is shown in Tab. 43.2-7 – Molecular biological methods for the detection of viruses.

Electrophoretic visualization of viral genome segments

There are some RNA viruses whose genome does not form a continuous strand. For example, Rotavirus, which is the leading cause of severe gastroenteritis in children worldwide, has a genome consisting of 11 dsRNA segments. During an acute Rotavirus infection, large numbers of viruses are produced in the intestinal cells, so that a basic total nucleic acid extraction from stool fluid is sufficient to visualize the viral RNA segments with a basic agarose gel electrophoretic run. It was possible to demonstrate that the continuous mutation of these RNA viruses leads to a change in the size of the individual segments. The segmentation pattern is characteristic of a rotavirus strain and enables it to be identified, e.g. when investigating nosocomial infection chains.

Restriction fragment length polymorphisms

The genome of DNA viruses is not segmented. However, the isolated viral DNA can be segmented into defined fragments by suitable restriction enzymes, and then the RFLP of the individual viral strains and variants can be analyzed by electrophoresis. RFLP analysis is also used for viruses with a linear, non-segmented RNA genome. In such cases, the genome must first be transcribed in vitro into double-stranded complementary DNA (cDNA) by reverse transcriptase. Refer to Fig. 43.2-2 – Analysis of the segmentation/restriction fragment length polymorphism using the example of RNA segments of rotavirus and DNA PCR amplicons of HSV-1 from different patient isolates.

DNA or cDNA sequencing

This method, which is based on nucleic acid hybridization, is considerably more accurate, but also more laborious. At the same time, the use of automated platforms has standardized and considerably simplified the sequencing and reading process, resulting in the previously listed methods being increasingly replaced. DNA sequencing determines the order of nucleotides in a selected fragment of the viral genome or of a cDNA. Since the number of mutations increases over time, genetic trees can thus be constructed. Isolates with many differing mutations belong to branches that are far apart. While the electrophoretic visualization of segments and fragments of nucleic acids delivers robust results, the interpretation of DNA sequencing data requires a binding algorithm.

Sequencing is the method of choice for genotypic testing for resistant viral mutants that develop in the patient in the course of long term treatment of a viral infection (e.g., HIV, hepatitis B and C, and negatively impact the success of antiviral therapy. Sequencing is generally performed on genome fragments that were extracted in vitro directly from specimens, and amplified. This avoids the risk of an interfering in vitro mutation of the virus initially cultivated in cell culture.

Nucleic acid hybridization

All techniques for nucleic acid detection rely on the spontaneous hybridization of complementary deoxyribonucleic acid strands to double stranded nucleic acid, the hybridization occurs both with DNA and RNA /6/. With synthetic or cloned oligonuleotides used as nuclear probes, complementary sequences of the searched nucleic acid can be detected by hybridization. The hybridization is usually detected by radioactive or enzyme markers linked to the probe. However, for viral diagnosis, these hybridization technique lacks sensitivity. Therefore nucleic acids are multiplied in vitro by producing copies before the hybridization assay is used. Testing always involves the following steps in all methodological variants:

• Extraction of the total DNA from the specimen
• Raising the temperature in the reaction mixture to nearly 100 °C or to pH > 8 to cause separation (denaturation) of the dsDNA into two single strands
• Addition of the gene probe (DNA strand complementary to the viral gene, labeled with radioactive or enzyme marker)
• Lowering the temperature or the pH to the initial value to allow renaturation (hybridization) of the single strands to a double strand that incorporates the labeled gene probe
• Qualitative or quantitative measurement occurs using solid-phase based hybridization procedures, like blotting techniques and sandwich hybridization or the combination with solid phase immunoassays, where nucleic acids are bound by antibodies or by biotin-avidin interaction on solid phase.

The hybridization can also be performed in situ in tissue sections as part of a histologic evaluation, analogously to the immunohistologic demonstration of antigen. Ideally, specimens used for nucleic acid hybridization should be non fixed or gently fixed and possibly deparaffinized.

Polymerase chain reaction (PCR)

Viral genes of the nucleic acid extracted from a specimen are amplified through a rapid sequence of denaturation, hybridization and polymerase reactions with addition of nucleotides, thermostable polymerase and primer gene sequences. The PCR assay is performed qualitatively and quantitatively with both viral DNA and, after reverse transcription, viral RNA. The method greatly improves the sensitivity of detection of latent and productive infections. See also Section 52.3 – Amplification techniques.

As an alternative to amplification of the viral target nucleic acid sequence, some test methods make use of the possibility of amplifying the gene probe or hybridizing further labeled gene probes to it (signal amplification).

PCR is the standard method for determining the viral load in a specimen, especially in blood plasma. In all diseases in which the viral load correlates with disease progression, quantitative viral PCR or other nucleic acid amplification techniques represent an ideal marker for the indication and monitoring of antiviral therapy. In particular, this applies to HIV and HCV infection in the absence of other suitable laboratory tests for these infections. However, viral load determination is also routinely used in the treatment of hepatitis B and cytomegaly in immunocompromised patients.

Electron microscopy

Electron microscopy has unjustly taken a back seat to virus culture and genetic analysis. State-of-the art instruments allow rapid diagnosis and detection of multiple viruses at a time, if there is sufficient virus in the specimen (vesicle, stool diagnosis). Microscopic testing for the presence of viral inclusions is used in histopathology and allows accurate virus identification only in isolated cases (cytomegaly, Negri bodies characteristic of rabies virus). Viral antigen tests are considered part of viral serology testing /1/.

43.2.3 Viral serology

43.2.3.1 Development of antibodies

Antibodies developed in viral infections are polyclonal, of different immunoglobulin classes, and directed against many antigen determinants of the virus or the infected cell. Antibodies to specific viral infections usually only account for a very small proportion of total antibodies, except during the first 2 days of life of a newborn, when a serum IgM concentration above 200 mg/L is considered a marker of prenatal infection. IgM and IgA class antibodies are not transferred through the placenta. IgG antibodies, in contrast, are actively transferred from the maternal to the fetal circulation during the fetal period (i.e., from day 91 of pregnancy) and provide the newborn with maternal passive immunity for approximately one year. After birth, the newborn first produces IgM antibodies in response to an infection, followed immediately by IgG and then IgA antibodies.

• IgM antibodies disappear from the blood as soon as the pathogen has been eliminated or has become latent.
• IgG antibodies, however, may persist for a very long time, even for life, and are correlated with clinical immunity in many, although not all, viral infections
• IgA antibodies primarily protect the mucous membranes, because they are secreted with the mucus. In serum their kinetics is between that of IgM and IgG. In some mucous membrane infections, such as influenza, their diagnostic efficiency is higher than that of IgM. In other diseases, serum IgA can supplement IgM as a marker of recent or reactivated infection. Refer to Fig. 43.2-3 –Virus specific antibody kinetics.

During the course of an infection, antibody polyclonality declines slightly due to the B cells gaining a selective advantage (positive selection) with their membrane bound immunoglobulin receptors, which better match the relevant antigen (and which are then secreted as antibodies). Determining antibody avidity therefore also helps distinguish between acute and recurrent infection. Due to the heterogeneity of antibodies, clinical chemistry methods for determining the concentration (amount) of pathogen specific antibodies are of limited or no use. The serologist primarily measures antibody activity.

43.2.3.2 Qualitative antibody testing

Once disease has started, it often no longer makes sense to screen for the pathogen, since the disease only becomes active with the immune responses, and at this point the viremia and viral excretion stop. Here, antibody testing in serum/plasma and cerebrospinal fluid samples is important.

Sera and serum antibodies are stable for long periods, if bacterial contamination is prevented. Specimens can easily be shipped by mail, but must be stored at temperatures below –20 °C for long term storage. Cell mediated immune response tests (lymphocyte stimulation tests) are currently only used in science laboratories.

Infectious serology testing is carried out with traditional liquid phase tests (cell culture virus neutralization assay, hemagglutination inhibition) assay) or with state-of-the art (solid phase) immunoassays /1, 5, 6/.

43.2.3.3 Quantitative antibody testing

The traditional quantization method, which was introduced by Paul Ehrlich, is titration. The antibody titer is typically determined in a 2-fold serial dilution. Slightly deviating titers in repeat tests are accepted as variation. Greater deviations measured in successive serum samples of a patient are considered significant (indicative of infection).

Biological methods that are susceptible to interference, such as virus neutralization in cell culture, define the “significant rise in titer” according to system related biometric criteria in a slightly more complex manner and require several parallel tests. Since there is no (inter)national test standardization for many viral serology parameters, the significant rise in titer between two serum samples can only be determined in the same laboratory using the same assay system. The different serologic methods show different rates of detection of antibodies of the individual Ig classes and subclasses. Therefore, a serum antibody titer result is tied to the specific test method used. Tests must be interpreted by the clinical pathologist on an individual basis (i.e., specifically in relation to the method used). Because a patient’s immune responses can vary greatly, standard values can only be expressed as ranges (e.g., taking into account the patient’s epidemiology and age).

Note: the more detailed the indication for testing provided by the clinician, the easier it is for the clinical pathologist to select the appropriate test method for a specific situation and to interpret the laboratory result.

Titration by nature is material and labor intensive. Therefore, modern immunoassays are based on a one step principle, where only a single dilution step is required to analyze the serum sample /1/. This entails the risk of the prozone phenomenon occurring: if too many antibodies are present in an insufficiently diluted serum sample, they may block each other by competing for antigenic binding sites (false negative test result). A correct test result is obtained only when the serum is further diluted until the antibodies are also diluted.

Solid-phase immunoassay

There are many modifications of these assays. The most common one uses solid phase bound antigen, to which the serum antibodies and signal antibodies (tracers) directed against them are bound. Depending on the signal molecule (radioisotope, enzyme or fluorescent dye) linked to the tracer, the assay is called radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), or fluorescence immunoassay (FIA)/immunofluorescence test (IIFT). Sensitive detection systems are the amplified enzyme reactions using enzyme-substrate cascades and cycles, the time resolved fluorescence assays using europium chelates and the fluorescence and luminescence based reactions.

Cave: the expression of antibody activity in units/mL analogously to the measurement of enzyme activity is false precision. In the constant interplay of infection and immune activation due to a flood of related antigen determinants throughout the course of life, the immunobiological variation of the antibody titer can barely be reduced below a factor of two, since the B cells also proliferate through duplication.

There are a number of alternatives to the conventional immunoassay: for the detection of IgM antibodies, a reverse test procedure is used. First, (part of) the serum IgM is isolated from the sample with solid phase bound anti-IgM and then, in a second step, its antigen specificity is tested by adding an antigen which has a signal molecule attached to it (anti-μ capture assay). This method avoids the problem of competing IgG and IgA antibodies. It is important to reduce or eliminate IgG prior to assaying IgM antibodies, because often there are IgM autoantibodies which react with antigen bound IgG (even across animal species), resulting in a false positive result for pathogen specific IgM in sandwich assays. This commonly occurs in rheumatic diseases (rheumatoid factor).

43.2.3.4 Quantitative antigen testing

The antigen test is the easiest method for the direct detection of a virus and its structural elements. Outside immunohistology it is used in cases where particularly large amounts of viral antigen are present in the blood (hepatitis B surface antigen), stool (Rotavirus, Norovirus, Adenovirus) or respiratory tract (Influenza virus) during the course of infection.

Solid-phase immunoassay

This is the most commonly used assay for antigen detection. It is configured similarly to the reverse IgM assay. The antigen is isolated or immune adsorbed from the specimen by a specific, solid phase bound antibody that specifically targets the antigen. A signal antibody functions as a tracer. This sandwich assay could give a false positive result due to the rheumatoid factors, but this is rarely a problem today. Modern antigen assays, (e.g., for HCV core antigen) have sensitivities down to the pg/mL range and thus can sometimes replace PCR.

Antigen tests are easier to quantify than antibody measurements, since the immunological antigen affinity does not change in the course of infection. However, the variability of viral antigens and the plurality of viral genotypes must be taken into account.

43.2.3.5 Test interpretation

The results of diagnostic virology assays must be interpreted as follows /1/:

• Florid infection is demonstrated by determining viral nucleic acid sequences, by antigen testing, electron microscopy, or isolation of the virus directly in the patient specimen
• The quantitative results of these findings allow an evaluation of the course of infection and disease and of the treatment outcome
• Viral infection can be demonstrated indirectly by basic qualitative antibody testing in blood samples, provided there is no epidemic (at least not regionally) involving this infectious agent (tropical viral diseases, rabies). This also applies to the testing of special samples such as CSF or amniotic fluid, in which no antibodies are usually produced (cave: contamination with blood).
• If there is a high suspicion of infection, serology initially is only useful for exclusion diagnosis. A reliable diagnosis of acute or relatively recent infection can then be made by antibody quantization, if a significant rise in titer is detected in a second serum sample (excluding or taking into account active or passive immunization, blood transfusions, and maternal antibodies).
• In a single serum sample, elevation of virus specific IgM, or IgA, can be an indicator of recent, active or reactivated infection
• Compared to quantitative viral testing, serology is less suitable for monitoring the course of the infectious disease and the treatment outcome (slower clearance of IgM, decrease in IgA and IgG antibody levels).

Demonstration of protective immunity

In a number of viral diseases, protection from reinfection or at least from new infection is linked to the presence of infectivity neutralizing antibodies, (e.g., in poliomyelitis, hepatitis A, rubella, or measles). These antibodies prevent the virus from attaching to and entering the cell. In this case, immune protection can be conferred “passively” by administering (hyper-)immuno­globulin.

Sometimes a virus’ ability to adsorb to the target cells of the infection is closely correlated with its ability to bind to specific animal erythrocytes and to agglutinate them. In these cases the hemagglutination inhibition assay replaces the determination of neutralizing antibodies in the laboratory animal or cell culture (e.g., in rubella or influenza infection).

In some infections, the demonstration of antibodies, irrespective of their biological quality, is only a marker of past infection. If it is known that prior exposure to the infectious agent or inoculation for it leads to immunity, non biological antibody testing is sufficient for determining the patient’s immune status.

Diagnostic virology as part of epidemic control and for excluding the risk of infection

Many communicable diseases are subject to mandatory reporting to public health authorities. The German Protection against Infection Act distinguishes between mandatory reporting for general practitioners and clinicians (suspected diagnosis) and mandatory reporting for laboratories (detection of notifiable pathogen). A number of viruses (e.g., hepatitis viruses, enteroviruses, adenoviruses) play an important role in nosocomial infections /7/.

Many diagnostic viral tests are performed in transfusion medicine and to ensure the safety of biotechnological medicines. Although it is generally not possible to definitely exclude viral contamination, the risk can be drastically reduced.

References

1. Doerr HW, Gerlich WH (eds). Medizinische Virologie: Grundlagen, Diagnostik, Prävention und Therapie virologischer Krankheitsbilder. Stuttgart; Thieme, 2. Aufl. 2010.

2. Carter J, Saunders VA. Virology: Principles and applications. John Wiley & Sons, 2007; pp. 1–48.

3. Condic RC. Principles of virology. In: Knipe DM, Howle PM (eds). Fields Virology, volume 1. Lippincott-Williams & Wilkins, 2007; pp.25–58.

4. Dimitrov DS. Cell biology of virus entry. Cell 2000; 101: 697–702.

5. International Committee on Taxonomy of viruses. EC 46, Canada, July 2014.

6. Krech T. New techniques in rapid viral diagnosis. FEMS Microbiology Immunology 1992; 89: 299–304.

7. Rabenau HF, Kessler HH, Kortenbusch M, Steinhorst A, Raggam RB, Berger A. Verification and validation of diagnostic laboratory tests in clinical virology. J Clin Virol 2007; 40 (2): 93–8.

43.3 Adenovirus

Family: Adenoviridae. This family consists of several genera and subgenera in animals which are, however, generally not infectious across animal species /1/.

Genus: Mastadenovirus is differentiated in 7 species A–G

Viral structure: the Adenovirus consists of a regularly shaped icosahedral capsid containing a linear, double stranded DNA genome. The polypeptide molecules of the capsid are arranged in regular pentons and hexons. The pentons at the vertices of the viral capsid have projecting fibers, which are essential for adsorption of the virus as the first step of cellular infection. The virion has a diameter of 80–110 nm.

43.3.1 Epidemiology and clinical significance

Approximately 54 serologically distinguishable types of human Adenovirus have been described. Many infections run a subclinical course: Adenoviruses were originally isolated as an insignificant incidental finding from tonsillar tissue and nasal polyps (adenoids), where they can maintain persistent infections. Depending on the virulence of the viral strain and the body’s level of resistance (e.g. impaired resistance due to a cold), an infectious disease may develop, often of epidemic proportions /2, 3/.

Adenovirus associated diagnoses

In the respiratory tract (predominantly types 1–7, 14, 21):

• Acute febrile pharyngitis (all age groups)
• Pertussis like syndrome (infants)
• Bronchopneumonia (young children, military recruits).

In the ocular area (especially types 3, 4, 7, 8, 37, 53, 54):

• Adenovirus pharyngoconjunctival fever (young children)
• Follicular conjunctivitis (swimming pool conjunctivitis, differential diagnosis chlamydia)
• Epidemic (hemorrhagic) keratoconjunctivitis (often nosocomial).

In the gastrointestinal tract (types above no. 40, plus 1, 2, 5, 31):

• Acute gastroenteritis; main cause of diarrhea in children after Rotavirus.

In the urinary bladder:

• Acute hemorrhagic cystitis in babies and young children (types 11, 34, 35)
• Chronic in AIDS patients.

Genital ulcers: types 19, 37.

• Disseminated septic with strong immunosuppression Types 1, 2, 5, 31
• In rare cases, the brain (meningoencephalitis in children or immunocompromised individuals) and liver (hepatitis) are affected (types 1, 2, 5, 7, 11, 31, 34, 35).

Respiratory Adenovirus infections are highly prevalent in human populations. Reinfection with the same type is possible.

Adenovirus infections are increasingly recognized as important pathogens in immunocompromised hosts, especially in patients with severely suppressed T cell function. The recommended technique for monitoring of high risk patients is quantitative polymerase chain reaction (PCR) /4/.

Adenovirus is stable and a dreaded cause of nosocomial and smear infections. Adenovirus 8 is a common cause of epidemic keratoconjunctivitis, especially in eye hospitals.

43.3.2 Virus detection

To diagnose Adenovirus infection, direct virus detection is the method of choice. The following specimens may be used: throat swab or sputum (in pneumonia, sputum is most informative apart from broncho alveolar lavage), conjunctival swab, diarrheic stool sample, urine and cerebrospinal fluid. EDTA blood is used in suspicion of Adenovirus infection associated hepatitis. Due to the high stability of the virus, no preservation is required, except that the sample must be prevented from drying out. Tests for the antigen detection of Adenoviruses are:

• PCR has a high sensitivity for detecting all types of the virus and therefore must always be used for cerebrospinal fluid (CSF) testing in the workup of meningoencephalitis. Low viral loads (< 10³/mL) are occasionally also found in healthy individuals. Therefore a quantitative method should be used.
• The ELISA for the detection of all types of Adenovirus capsid hexon antigens is the most inexpensive and usually sufficient routine method of virus detection in stool samples
• Stool samples may also be examined under electron microscopy when screening for several viruses at a time
• Virus isolation in cell cultures takes more than 24 hours, usually several days, and works best for the A–E species. Once isolated, the pathogen can be typed serologically and with molecular techniques (DNA restriction analysis, DNA sequencing).
• DNA sequencing of PCR amplicons directly from the sample or cell culture allows fine typing for the evaluation of infection chains (e.g., in order to confirm or exclude a nosocomial infection).

43.3.3 Viral serology

The infectious etiology of Adenovirus may be confirmed serologically (i.e., via antibody determination) in blood samples two weeks after infection frequently in respiratory diseases but only sporadically in infections of other sites.

Type specific antibodies can only be detected with hemagglutination inhibition test and neutralization test, although often with inconsistent results.

Immunofluorescence test, enzyme immunoassay and immunoblot have not gained any importance for routine and Ig class differentiating antibody detection.

Overall, diagnostic serology is of little importance, but can be useful in severe respiratory infections without immune deficiency. The complement fixation test remains an inexpensive method for detecting all types of the virus. Titers of ≥ 1 : 80 are suggestive of recent infection.

References

1. Doerr HW, Gerlich WH (eds). Medizinische Virologie – Grundlagen, Diagnostik, Prävention und Therapie virologischer Krankheitsbilder. Stuttgart; Thieme, 2. Aufl. 2010.

2. Ginsberg HS, Price GA. The molecular basis of adenovirus patogenesis. Infectioud Agents and Disease 1994; 3: 1–8.

3. Hierholzer JC. Adenoviruses in the immunocompromized host. Clin Microbiol Rev 1992; 5: 262–74.

4. Matthes-Martin S, Feuchtinger T, Shaw PJ, Engelhard D, Hirsch HH, Cordonnier C, et al. European guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: summary of ECIL-4 (2011). Transpl Infect Dis 2012; 14: 555–63.

43.4 Alphavirus

Family: Togaviridae

Genus: Alphavirus genus comprises 29 different species (e.g., Chikungunya virus, O’nyong-nyong virus, Ross River virus, Sindbis virus, and Equine encephalitis virus. They are Arboviruses (arthropod borne, transmitted by arthropod vectors) /1/.

Viral structure: see Togavirus – Section 43.65.

43.4.1 Epidemiology and clinical significance

Alphavirus is distributed worldwide in different species specific, geographically delimited regions, but is not yet known to occur in Central Europe.

Pathogenic species of the virus are less common in the Old World; they occur more frequently in North and South America and in Australia. Alphavirus is occasionally transmitted to large domestic animals (horse) and humans through insect bites. After an incubation period of one week, it causes meningoencephalitis (equine encephalitis), usually with a good prognosis. A fatal outcome is rare (Tab. 43.4-1 – Viral infectious agents of Alphavirus caused encephalitides).

43.4.2 Laboratory diagnosis

The easiest method of detection is to test a serum sample for antibodies using a hemagglutination inhibition assay, whereby cross reactions between Adenoviruses must be considered (travel history). There are no cross reactions with flavi- (inoculation) viruses (e.g., yellow fever, dengue, West Nile, ESME) which are also transmitted by arthropods and cause similar disease /2/. During the viremic phase of acute illness, the pathogen can be detected by PCR in EDTA blood and cerebrospinal fluid, while isolation of the virus is successful only during the early stage of disease.

References

1. Doerr HW, Gerlich WH (eds). Medizinische Virologie: Grundlagen, Diagnostik, Prävention und Therapie virologischer Krankheitsbilder. Stuttgart; Thieme, 2. Aufl. 2010.

2. Schmaljohn AJ, McClain D. Alpha viruses (togaviridae) and flavoviruses (flavoviridae). In: Baron S (ed): Medical microbiology,.Galveston Texas.

43.5 Arbovirus

This term refers to a group of viruses that are transmitted by hematophagous arthropods (insects, ticks) (arbo, arthropod borne). They are classified into Flavivirus, Bunyavirus, Alphavirus and Reovirus, of which only few are human pathogenic. The Zika virus is predominantly a mild or asymptomatic dengue like disease that can cause Guillain-Barré syndrome and other neurologic conditions /1/. Apart from the Early summer meningoencephalitis virus, these viruses must be considered imported infectious pathogens (tropics, sub tropics, Mediterranean) (Tab. 43.4-1 – Viral infectious agents of Alphavirus caused encephalitides).

References

1. Fauci AS, Morens DM. Zika virus in the Americas – Yet another arbovirus threat. N Engl J Med 2016; 374: 601–4.

43.6 Arenavirus

Family: Arenaviridae

Genus: Arenavirus genus comprises more than 25 species, including Lassa virus and Lymphocytic choriomeningitis (LCM) virus in Africa and Europe. Further species are endemic to South America.

Viral structure: this family consists of pleomorphic viruses that have a genome consisting of two segments of single stranded negative sense RNA and contain ribosomes of a sandy appearance (Lat. “arena” = sand). The particles have a diameter of 110–130 nm.

43.6.1 Epidemiology and clinical significance

Arenaviruses are hosted by rodents and are zoonotic pathogens.

Lassa fever virus /1/

This virus is endemic in West Africa and causes infection (anthropozoonosis) which, in rare cases, may take the form of a hemorrhagic fever syndrome, often with a fatal outcome. The infection is transmitted directly from individual to individual by the bite of an infected rodent or through contamination with rodent urine, but also via the fecal-oral route.

LCM virus /2/

The virus causes lymphocytic choriomeningitis, a rodent borne zoonosis that also occurs in Central Europe. Humans may be infected via aerosol exposure. Since LCM infected hamsters are the primary source of the infection in humans, a history of exposure to hamsters should be sought.

The infection is often inapparent or may appear as a common cold. The CNS is rarely involved. The disease is triggered by an immunopathological mechanism.

43.6.2 Laboratory diagnosis

Laboratory diagnosis can be made by virus isolation from cerebrospinal fluid (LCM) or blood samples (lassa) in cell cultures (Lassa virus: level 4 safety laboratory is required), by PCR, or by serum antibody testing. Antibody detection (IgM, IgG) should be performed using indirect immunofluorescence. Any positive result is epidemiologically abnormal.

References

1. Yun NE, Walker DH. Pathogenesis of Lassa fever. Viruses 2012; 4 (10): 2031–48.

2. Lymphocytic choriomeningitis. Centers for disease control and prevention.

43.7 Astrovirus

Family: Astroviridae

Genera: Mamstrovirus (mammal), Avastrovirus (bird). In humans, there are 8 types (serologically and genetically distinguishable) /1/.

Viral structure: Astroviridae are small, icosahedral plus-stranded RNA viruses (28–34 nm diameter).

43.7.1 Epidemiology and clinical significance

Astrovirus infection is a differential diagnosis to be considered in patients with viral gastroenteritis. The most common mode of transmission is through contaminated food or via smear infection. Occasionally there are outbreaks in community facilities (nursing home) or nosocomial outbreaks in hospital units /2/. Astrovirus is of lesser clinical and epidemiological significance than Rotavirus, Adenovirus or Norovirus.

43.7.2 Laboratory diagnosis

The pathogen can be detected in diarrheic stool samples by PCR, antigen ELISA, or electron microscopy.

References

1. Schultz-Cherry (ed). Astrovirus research. Springer, New York 2013.

2. Jeong HS, Jeong A, Cheon DS. Epidemiology of astrovirus infection in children. Korean J Pediatr 2012; 55: 77–82.

43.8 Bocavirus

This virus is a member of the Parvoviridae family (Section 43.52 – Parvovirus). It has been detected in many children with bronchitis or pneumonia. It was also found in stool samples from patients with gastroenteritis. PCR testing is available for scientific purposes only in special laboratories.

References

1. Guido M, Tumolo MR, Verri T, Romano A, Serio F, De Giorgi M, et al. World J Gastroenterol 2016; 21; 22 (39): 8684–8697.

43.9 Bornaviruses

Family: Bornaviridae

Genus: Bornavirus

Viral structure: these are enveloped viruses, 70–130 nm in diameter, with a core containing an ssRNA genome.

43.9.1 Epidemiology and clinical significance

Bornavirus is widely distributed among vertebrates and cause chronic diseases of the central nervous system, which were first identified in horses in the area of Borna (Saxony) (1894/96) and later (1926) recognized as being caused by a virus.

A possible association of the virus with human disease such as depression, has been debated in recent years, but there is as yet no conclusive evidence of Bornavirus infection in humans /1/.

References

1. Carbone KM. Borna disease virus and human disease. Clin Microbiol Rev 2001; 14: 513–27.

43.10 Bunyaviruses

Family: Bunyaviridae

Genera: Bunyavirus, Hantavirus, Phlebovirus, Nairovirus.

Viral structure: Bunyavirus is an enveloped virus, spherical to ovoid in shape, with a helical nucleoprotein. The single stranded negative sense RNA consists of three segments. The virus has a diameter of 90–120 nm.

43.10.1 Epidemiology and clinical significance

The Bunyaviridae family comprises a multitude of (anthropo)zoonotic, arthropod or rodent borne viruses. In Central Europe, only Hantavirus is of relevance (Section 43.24 – Hantavirus). Occasionally, travellers from Southern Europe import pappataci fever, which is caused by Sandfly fever virus infection Section 43.49 – Pappataci (sandfly) fever virus). Exotic infectious diseases, which are usually transmitted by ticks or insects, include Rift Valley fever (Africa) and Crimean-Congo hemorrhagic fever /1/.

References

1. Alatoom A, Payne D. An overview of arboviruses and Bunyaviruses. Laboratory Medicine 2000; 40: 237–40.

43.11 Caliciviruses

Family: Caliciviridae

Genera: Norovirus and Sapovirus in humans with several gene groups or genome types. Two additional genera in the animal kingdom /1/.

Main genus and species: Norovirus with gene groups I and II.

Viral structure: Calicivirus contains a single stranded positive sense RNA genome. The icosahedral capsid shows characteristic cup shaped depressions (lat. “calix” = cup, chalice). The virus is 30 nm in size.

43.11.1 Epidemiology and clinical significance

Calicivirus has many variants and is highly prevalent in populations worldwide. Besides human Calicivirus there are those that cause disease in animals; all Caliciviruses are species specific.

In Central Europe, human Caliciviruses are associated with gastroenteritis: Norovirus (formerly norwalk virus) has become the leading cause of severe gastrointestinal diseases (gastroenteritis) in Central Europe, besides Rotavirus. While Rotavirus usually only infects young children, Norovirus infection can occur in all age groups /2/. Genotype 2 is considerably more prevalent than genotype 1. A less common cause of gastroenteritis is Sapporo-like virus (Sapovirus genus), which was first described in Japan.

43.11.2 Laboratory diagnosis

These gastroenteritis viruses are diagnosed by examining a diarrheic stool sample under electron microscopy, by the serotypic Norovirus antigen assay (ELISA), and especially by RT-PCR for genotypes 1 and 2 /3/.

References

1. Doerr HW, Gerlich WH (eds). Medizinische Virologie: Grundlagen, Diagnostik, Prävention und Therapie virologischer Krankheitsbilder. Stuttgart; Thieme, 2. Aufl. 2010.

2. Rockx B, de Witt M, Vennema H, Vinje J, de Bruin E, van Duynhoven Y, Koopmans M. Natural history of human calicivirus infection: a prospective cohort study. Clin Infect Dis 2002; 35: 246–53.

3. Chen H, Hu Y. Molecular diagnostic methods for detection and characterization of human noroviruses. Open Microbiol J 2016; 10: 78–89. doi: 10.2174/1874285801610010078.

43.12 Chikungunya virus

Family: Togaviridae

Genus: Alphavirus

Species: Chikungunya virus

Viral structure: see Togavirus – Section 43.65

43.12.1 Epidemiology and clinical significance

Chikungunya infection is a febrile viral infection transmitted by various mosquito species. Like dengue fever, it can lead to severe arthralgia. The prognosis is good. Chikungunya infection has historically been limited to Africa, the Indian subcontinent and East African islands. In 2006, several cases of Chikungunya infection caused by imported infected mosquitoes were diagnosed in Northern Italy. A year later, an outbreak was reported in Gabun (West Africa) /1, 2/.

43.12.2 Laboratory diagnosis

The diagnosis is accomplished by antibody detection (ELISA, immunoblot) and PCR. Due to the absence of widespread seropositivity in the European population, any positive antibody test is suspicious for infection.

References

1. Brighton SW, Prozesky OW, de la Harpe AL. Chikungunya virus infection. S Afr Med J 1983; 26: 313–5.

2. Morens DM, Fauci AS. Chikungunya at the door – deja vu all over the door again?. N Engl J Med 2014; 371: 885–7.

43.13 Coronavirus

Family: Coronaviridae

Genus and species: Coronavirus with three genetically defined groups and species specific strains. The human pathogenic Coronaviruses belong to groups 1 and 2. Chinese laboratories identified a new Coronavirus (SARS-COV-2) that causes the clinical disease COVID-19. Coronaviruses originate from animals. SARS-Cov-2 is transmitted person-to person primarily via respiratory droplets, but also indirect transmission through contaminated surface is possible /1, 2.

Viral structure: Coronavirus is 80–120 nm in size. The large single stranded positive sense RNA genome is enclosed in a helical nucleocapsid, which is surrounded by a lipid membrane that incorporates 16 nonstructural proteins and 4 structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). The capsid has a characteristic crown like fringe of bulbous surface projections (Lat. “corona” = crown, halo). Angiotensin converting enzyme 2 (ACE2) appears to be the host-cell receptor for SARS-CoV-2. Significant greater numbers of ACE2-positive cells are in the lungs from patients with COVID-19 than in those from uninfected controls /4, 5/.

43.13.1 Epidemiology of Coronavirus

Coronavirus is widely distributed and causes species specific infection in humans (human Coronavirus), pigs, cats, dogs, poultry and rodents and is a feared contaminant in biotechnology. Coronavirus, which is transmitted by droplets and aerosols, causes respiratory infections (frequently common colds). Occasionally it may cause gastroenteritis, especially in premature infants with an immature immune system or in immunocompromised patients of any age group. Transmission occurs via the fecal-oral route (smear infection). The diseases have a short incubation period of a few days. Many infections run a subclinical course.

Patients are infectious (via saliva droplets and stool) only after becoming symptomatic (fever above 38.5 °C). Therefore, the concerted action taken by the WHO and the consistent isolation of patients were able to initially contain the spread of SARS.

43.13.2 SARS-COV-2: Corona virus disease 2019 (COVID-19)

The coronavirus disease 2019 (Covid 19) pandemic caused by the infection of acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has dramatically changed the world. In 1 year more than 110 million cases have been confirmed, with more than 2.5 million deaths /6/.

The two key steps of SARS-Cov-2 are /7/:

• The infection is mediated by the corona­virus spike protein S. The S protein is found on the viral envelope membrane and that of its host receptor. The S protein is present in all coronaviruses that infect humans.
• The host receptor is one of the angiotensin-converting enzyme receptors (ACE 2). The receptor is present mainly but not exclusively in the lung alveolar epithelium and intestinal enterocytes.

Viral infectivity and transmission is mainly person-to-person primarily via respiratory droplets, but also indirect transmission through contaminated surface is possible. The incubation period ranges from 2–14 days with most patients showing symptoms 4–5 days after exposure. Those infected exhibit fever (> 38.5 °C) at the beginning of the illness.

SARS-COV-2 is associated with a broad spectrum of clinical respiratory syndromes, ranging from mild upper airway symptoms to progressive life-threatening viral pneumonia. The hallmark of the early phase of ARDS is diffuse aveolar damage with edema, hemorrhage, and intraalveolar fibrin deposition. Clinically, patients with severe COVID-19 disease have labored breathing and progressive hypoxemia. The presence of pulmonary intussusceptive angiogenesis and other vascular features in the lungs have been reported.The typical findings in the chest CT images are multifocal bilateral patchy ground-glass opacities or consolidation with interlobular septal and vascular thickening in the peripheral areas of the lungs /3, 4/.

43.13.2.1 Cognitive deficits in long Covid-19

Some patients who have recovered from an infection with SARS-COV-2, including those with mild disease report deficits in attention, execute functioning, language, processing speed and memory. The symptoms are collectively referred to as “brain fog”. Together with increased anxiety, depression, sleep disorder, and fatique, this syndrome of cognitive impairment contributes substantially to the morbidity of post-Covid-19 conditions /13/.

43.13.3 Laboratory testing

Besides RT-PCR rapid diagnostic antigen tests for the detection of acute SARS-COV-2 infection have been developed.

43.13.3.1 Reverse transcription polymerase chain reaction (RT-PCR)

The current diagnostic gold standard for the diagnosis of acute SARS-COV-2 infection is the RT-PCR with various methods demonstrating limit of detection from 10² to 10⁵ copies/mL /8/. According to a review /7/ amplification of Covid 19 viral RNA in a nasopharyngeal swab involves an initial conversion of RNA to complementary cDNA (i.e converse transcription) followed by the use of primers which anneal to the specific segments under consideration; this provides a DNA template for amplification by thermostable polymerase. RT-PCR is essentially then an RNA-directed DNA polymerase process of the primed viral RNA within a cDNA. RT-PCR reduces significantly the detection time of Covid-19 in samples. RT-PCR is highly sensitive because each sample undergoes 20–40 thermal cycles of exponential amplification. The false negative rate of RT-PCR in new hospital admissions has been reported at about 15% /9/. Although RT-PCR are highly sensitive and accurate, they can remain positive for weeks to months after infection /8/.

43.13.3.2 Antigen-based assays

Diagnostic testing for acute SARS-CoV-2 can be performed with either RT-PCR or antigen based assays. Antigen based assays (immunoassays) detect domains of the surface proteins, including nucleocapsid, spike, and receptor binding domains, that are specific to SARS-CoV-2 /8/. The antigen based assays recommended from the European Union fulfill the following criteria:

• Diagnostic sensitivity > 80% to detect SARS-COV-2
• Diagnostic specificity 98% (WHO 97%)
• Diagnostic sensitivity > 90% in infected patients with a Ct-value < 25 in RT-PCR
• Analytic sensitivity in the Ct-range 25–30.

SARS-COV-2 RT-PCR positive and symptomatic patients showed diagnostic sensitivity of antigen based assays was proportional to the SARS-COV-2 burden and referred to 10⁵–10⁶ viral copies/mL.

43.13.3.3 Antibody tests

Antibody tests to detect host IgG or IgM antibodies to SARS-CoV-2 should not be used to diagnose acute infection /8/. The determination of antibodies using serological tests (SARS-COV-2 antibody tests) use antibodies directed:

• against nucleocapsid antigen: indicates active infection or gone through infection
• against spike protein: binding antibodies units (BAU) are determined, BAU indicate vaccination
• against inhibition of spike protein binding to the ACE-2 receptor (% inhibition): indicates the proportion (%) of virus neutralized by the antibodies (marker of humeral immunity).

EDTA-blood and serum biomarkers

Biomarkers for prediction of mortality in admission of patients with SARS-CoV-2 may be significantly increased or decreased /10/.

Increased are: leukocyte count, neutrophile granulocyte count, C-reactive protein, procalcitonin, ferritin, D-dimer, IL-6, lactat dehydrogenase, aminotransferases, creatine kinase, bilirubin, and creatinine.

Decreased are: lymphocyte count, thrombocyte count, and serum albumin.

43.13.3.4 Antibody titer after Covid-19 mRNA vaccination

To specifically detect IgG antibodies against the SARS-CoV-2 spike protein receptor binding domain enzyme immunoassays are used.

Approximate serum values of IgG-antibodies are before and after BNT162b2 vaccination /11/:

• Baseline: Below 1 AU/ml
• 10 days after first vaccination about 10 AU/ml
• 35 days after first vaccination about 300 AU/ml
• 180 days after first vaccination about 10-100 AU/ml
• 265 days after first vaccination about 20 AU/ml
• 15 days after the second vaccination about 400 AU/ml
• 15 days after the third vaccination about 25,000 AU/ml.

Compared with two doses of the vaccine administered at least 5 months before, adding a third dose was estimated to be 93% effective in preventing Covid-19 related admission to hospital, 92% in preventing severe disease, and 81% in preventing Covid-19 related death, as of 7 or more days after the third dose /12/.

References

1. Arabi YM, Arifi AA, Blalkhy HH, Najm M, Aldawood AS, Ghabashi A, et al. Clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection. Ann intern Med 2014; 160: 389–97.

2. Berger A, Drosten C, Doerr HW, Stürmer M, Preiser W: Severe acute respiratory syndrome (SARS) – paradigm of an emerging viral infection. J Clin Virol 2004; 29: 13–29.

3. Ackermann M, Verleden SE, Kuehnel M, Haverich A, Welte T, Laenger F, et al. Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid 19. N Engl J Med 2020; 383 (2): 120–8.

4. Vabret N, Britton G, Gruber C, Hegde S, Kim J, Kuksin M, et al. Immunology of COVID-1: Current state of the science. Immunity 2020; 52 (6): 910–41.

5. Irsara C, Egger AE, Prokop W, Nairz M, Loacker L, Sahanic S, et al. Evaluation of four commercial, fully automated SARS-COV-2 antibody tests suggests a revision of the Siemens SARS-COV-2 IgG assay. Clin Chem Lab Med 2021; 59 (6): 1143–54.

6. Johns Hopkins University and Medicine. COVID 19 dashboard. https://coronavirus.jhu.edu/map.html

7. Ismail AA. SARS-CoV-2 (Covid 19): A short update on molecular biochemistry, pathology, diagnosis and therapeutic stategies. Ann Clin Biochem 2022; 59:(1): 59–64.

8. Drain PK. Rapid diagnostic testing for SARS-CoV-2. N Engl J Med 2022; 386: 264–72.

9. Kucirka LM, Lauer SA, Laeyendecker O, et al. Variation in false-negative reverse transcriptase polymerasechain reaction-based SARS-CoV2-tests by time since exposure. Ann Intern Med 2020; 173: 262–7.

10. Loomba R, Villarreal EG, Farias JS, Aggarwal G, Aggarwal S, Flores S. Serum biomarkers for prediction of mortality in patients with Covid-19. Ann Clin Biochem 2022; 59 (1): 15–22.

11. Kozakai R, Hoshi K, Izumi Y, Takahashi S. Assessment of antibody titer after third doses of Covid-19 mRNA vaccination in healthy volunteers. J Lab Med 2022. doi: 10.1515/labmed-2022-0008

12. Barda N, Dagan N, Cohen C, Hernan MA, Lipsitch M, Kohane IS, Reis BY, Balicer RD. Effectiveness of a third dose of the BNT162b2 mRNA Covid-19 vaccine for preventing severe outcomes in Israel: an observational study. Lancet 2021; 398: 2093–9.

13. Venkataramani V, Winkler F. Cognitive deficits in long Covid-19. N Engl J Med 2022; 387: 1813–5.

14. Schlenger RL. Antigentests auf SARS Cov-2. Was die Aussage betrifft. Dtsch Ärztebl 2022; 119 (51, 52): 2297–2301.

43.14 Coxsackievirus

Family: Picornaviridae

Genus: Enterovirus

Species: Human enterovirus A–C with members of the Coxsackievirus A and B subgroups

Serotypes: A1–24, B1–6

Viral structure: see Picornavirus – Section 43.54

43.14.1 Epidemiology and clinical significance

Named after the town where they were first isolated, Coxsackievirus is divided into group A and group B viruses based on early observations of the pathogenicity in newborn mice. Approximately 21 non cross-protective serotypes of group A and 6 serotypes of group B are recognized /1, 2/. Not all A types can be isolated in cell culture. Coxsackievirus infections are widely distributed in Central Europe, with a higher seasonal incidence in late summer. Although the manifestation index is low at < 5–10%, epidemic outbreaks are typical (summer cold, meningitis, myocarditis especially in young children). The possible association with juvenile diabetes mellitus remains conjectural. The pathogenicity spectrum of Coxsackievirus is shown in Tab. 43.14-1 – Clinical spectrum of enterovirus infections. The virus has an incubation period of only a few days. Picornavirus is stable and a feared cause of nosocomial infections, especially in maternity wards. Neonatal meningoencephalitis has a poor prognosis.

43.14.2 Laboratory diagnosis

The laboratory diagnosis is described in Section 43.56 – Poliovirus using the example of poliovirus. ELISA assays have so far failed to become established for serologic testing. Coxsackievirus infections are reportable if meningitis or encephalitis is present.

References

1. Buxbaum S, Berger A, Preiser W, Rabenau H, Doerr HW: Enterovirus infections in Germany: Comparative evaluation of different laboratory diagnostic methods. Infection 2001; 29: 138–42.

2. Rabenau H, Richter M, Doerr HW. Hand, foot, mouth disease: seroprevalence of Coxsackie A 16 and Enterovirus 71 in Germany. Med Microbiol Immunol 2010; 199: 45–51.

43.15 Cytomegalovirus

Family: Herpesviridae

Subfamily: Betaherpesvirinae

Genus: Cytomegalovirus

Species: Human cytomegalovirus with several genotypes (but only one serotype)

Viral structure: see Herpesvirus – Section 43.33

43.15.1 Epidemiology and clinical significance

Cytomegalovirus (CMV) infection can occur at any age, beginning from the embryonic period /1/. Due to special immune evasion mechanisms, the infection is poorly productive (i.e., subclinical) in many epitheloid tissues and endothelial cells, with occasional major reactivations and potential disease exacerbation.

Primary infections and major relapses are associated with viremia, desquamation of endothelial cells, and phagocytosis by granulocytes. The typical histology (cytomegalic cells with intranuclear inclusions; owl’s eyes) has been known to pathologists for over a century. It has been suggested that the virus persists by hiding within monocytes, thereby establishing proviral latency. From the monocytes/macrophages the virus is constantly disseminated to all organs.

The potential damage by a primary vertical infection during the first four weeks of pregnancy must be assessed and diagnosed in the same way as in the case of rubella, even if there is no accurate data on the risk of damage to the fetus /2/.

Secondary, reactivated cytomegaly can also be transmitted vertically. However, the clinical consequences are less serious and the infection has a good tendency to self resolve.

In Europe and North America, 0.1–0.5% of all newborns are thought to be infected with CMV. Only one tenth of them will suffer damage, mostly of a minor nature (Fig. 43.15-1 – Forms of progression of intrauterine CMV infections) /3/. Approximately 0.5–2.5% of infants are infected congenitally or peri natally in the birth canal or via breast milk. Immunologically immature premature infants in particular may develop interstitial pneumonia, often accompanied by Pneumocystis jirovecii infection.

CMV may also be transmitted postnatally, especially through close physical contact. After infancy, adolescence is another period of rapid acquisition of CMV. In a broader sense, cytomegaly is also a sexually transmitted infectious disease. In Central Europe, approximately 50% of young adults are infected with CMV. In the older population, about 70–80% are positive for anti-CMV antibodies. Postnatally, the manifestation index of CMV infection is below 1% in immunocompetent individuals /4/.

Occasionally, a CMV mononucleosis similar to glandular fever is observed in association with hepatitis, blood dyscrasia, splenomegaly and lymphadenopathy, but often without tonsillitis. It must be differentiated from toxoplasmosis or viral hepatitis /5/.

In the past, transfusion with leukocyte containing fresh blood occasionally led to a post transfusion syndrome. The incubation period is approximately 7 weeks. Guillain-Barré syndrome has been described as a late consequence occurring 3–4 months after primary infection with CMV.

Primary as well as reactivated cytomegaly is a feared opportunistic infectious disease (sepsis, pneumonia, retinitis, blood dyscrasia, transplant rejection) in immunocompromised individuals /6/. Following the emergence of AIDS, the entire spectrum of CMV pathology was recognized (Tab. 43.15-1 – Manifestations of CMV infection in AIDS and organ transplanted patients) /7/. These patients, less frequently those with a normal immune response, are often found to carry several genotypes of the virus. Exogenous reinfection is also possible. This finding poses a problem for the development of a vaccine, even if the genotypes have no corresponding immunologically different serotypes /8/.

43.15.2 Viral serology

The diagnosis of CMV infection is made or excluded by testing for serum antibodies, which are stimulated throughout life due to the persistence of the virus. ELISA and IIFT are inexpensive routine methods /9/. The quantization of neutralizing antibodies in the cell culture infection assay confirms only partial immunity. In addition, there are ELISA assays which use neutralization relevant envelope antigen. The production of these antibodies is less stimulated, so that their absence (e.g. in a positive conventional ELISA) is indicative of acute primary infection /9/. These tests use an antigen mixture from infected cell cultures or defined recombinant antigens from the tegument or capsid of the virus.

A positive ELISA or IFT for IgM antibodies is evidence of active primary or recurrent infection.

The IgA antibody test is often superior to the IgM antibody test for the detection of CMV recurrence in AIDS patients.

The disadvantage of serology is that antibody formation or boosting takes some time, resulting in a time lag of up to several days before laboratory diagnosis can be made. The delayed antibody kinetics also hinders rapid monitoring.

After an intrauterine infection, the IgM or IgA CMV antibodies in a blood sample collected from the newborn during its first days of life is positive only in 50% of cases.

43.15.3 Virus detection

Quantitative RT-PCR

Quantitative RT-PCR is best suited for early diagnosis, follow-up, and for determining the plasma (not heparinized blood) viral load. Viral load quantification is indispensable in the monitoring of bone marrow and organ transplanted patients who are at risk of CMV infection. It is the best marker of outcome during treatment with gancyclovir and foscarnet. In the case of resistance to therapy, DNA sequencing in the gene of the UL97 kinase which phosphorylates the gancyclovir and/or in the UL54 gene of the viral polymerase which is inhibited by gancyclovir triphosphate or foscarnet is indicated in order to detect relevant point mutations. PCR is also the method of choice for prenatal testing for CMV in the amniotic fluid and for cerebrospinal fluid analysis when the CNS is affected.

Detection of CMV antigen phosphoprotein (pp)65 in leukocytes

This method is less sensitive, but more closely correlated with the clinical manifestation, since this tegument protein of the virus is only produced during active infection.

Virus isolation in cell culture

Owing to the shell vial technique (rapid centrifugation culture), this method has significantly gained in importance as it is inexpensive and able to provide a result within 24 hours. It is still being used in diagnostic urine testing; pre- and peri natally infected newborns often eliminate high titers of the CMV (cave: nosocomial spread). The analysis of broncho alveolar lavage in CMV pneumonia is another area where rapid virus isolation is of value beyond the genome detection by PCR. The shell vial technique also allows the early determination of neutralizing antibodies.

References

1. Halwachs-Baumann G, Genser B, Danda M, Engele H, Rosegger H, Fölsch B, et al. Screening and diagnosis of congenital cytomegalovirus infection: a 5-y study. Scand J Infect Dis 2000; 32: 137–42.

2. Buxmann H, Hamprecht K, Meyer-Wittkopf M, Friese K. Primary human cytomegalovirus (HCMV) infection in pregnancy. Dtsch Arztebl Int 2017; 114: 45–52.

3. Bruggeman CA. Cytomegalovirus and latency: an overview. Virchows Archiv B Cell Pathol 1993; 64: 325–33.

4. Jahn G, Plachter B. Diagnostics of persistent viruses: human cytomegalovirus as an example. Intervirology 1993; 35: 60–72.

5. Varani S, Landinin MP. Cytomegalovirus as an hepatotropic virus. Clin Lab 2002; 48: 39–44.

6. Müller GA, Braun N, Einsele H, Müller CA. Human cytomegalovirus infection in transplantation. Nephron 1993; 64: 343–53.

7. Tendero DT. Laboratory diagnosis of cytomegalovirus (CMV) infection in immunodepressd patients, mainly in patients with AIDS. Clin Lab 2001; 47: 169–83.

8. Görzer I, Kerschner H, Redberger-Fritz M, Puchhammer-Stöckl E. Human cytomegalovirus (HCMV) genotype populations in immunocompetent individuals during primary HCMV infection. J Clin Virol 2010, 48: 100–103.

9. Weber B, Braun W, Cinatl Jr. J, Doerr HW. Humoral immune response to human cytomegalovirus infection: diagnostic potential of immunoglobulin class and IgG subclass antibody response to human cytomegalovirus early and late antigens. Clin Investig 1993; 71: 270–6.

43.16 Dengue virus

Family: Flaviviridae

Genus: Flavivirus

Species (group): Dengue with four serotypes

Viral structure: see Flavivirus – Section 43.21

43.16.1 Epidemiology and clinical significance

The Dengue virus is the most common virus of the Flavivirus genus in humans. The infection can be caused by four different serotypes of the virus and is found in tropical and subtropical areas in America, Africa and Southeast Asia. Dengue cases have also been reported in the Southern USA. Transmission occurs through the bite of various types of mosquitoes. Primary infection is generally harmless. Following an incubation period of 2–7 days, the virus spreads by viremia, predominantly targeting capillary endothelial cells. Clinical symptoms are sudden onset fever with two peaks, arthralgia, and rash /1/.

Secondary infection with a different serotype increases the risk for severe disease in the form of dengue hemorrhagic fever, which develops as a result of the virus forming immune complexes with non neutralizing antibodies, and complement activation. In this case, susceptibility to the virus is even increased (antibody dependent enhancement).

43.16.2 Viral serology

In addition to the hemagglutination inhibition assay, there are commercial immunoassays for IgG and IgM antibodies (immunofluorescence test, ELISA, immunochromatographic ELISA). The assay for IgG antibodies, which are an indicator of past infection, is non specific and shows broad cross reactivity with other flavi(arbo)virus infections or inoculations (see Yellow fever virus – Section 43.23, ESME virus – Section 43.22), which must be taken into account when evaluating the results /2/. The same applies to the detection of IgM antibodies as an indicator of relatively recent infection.

43.16.3 Antigen tests

Rapid immunochromatographic tests with good sensitivity are available for detecting the viral antigen in serum samples. Dengue antigen is detectable from the onset of illness, whereas antibodies do not appear until several days later.

PCR

This is the method of choice during the initial viremic phase of dengue fever. No commercial test is available as yet.

Electron microscopy

The viremia is often high enough to allow the diagnosis to be made by electron microscopy. Due to the risk of infection, the test sample should be inactivated with suitable disinfectants or fixatives immediately after being collected for virus detection.

Virus culture

Virus culture may only be performed in a high containment laboratory. The most sensitive method is cultivation of the virus in a laboratory animal (intracerebral inoculation of mice) using EDTA blood collected during the viremic phase. Cell cultures may also be used.

References

1. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, et al. Dengue: a continuing global threat. Nature Rev Microbiol 2010; december, S7–S16. doi: 10.1038/nrmicro2460.

2. Allwinn R, Doerr HW, Emmerich P, Schmitz H, Preisser W. Cross-reactivity in flavivirus serology: new implications of an old finding? Med Microbiol Immunol 2002; 190: 199–202.

43.17 Echovirus

Family: Picornaviridae

Genus: Enterovirus

Species: Human enterovirus B, echovirus subgroup

Serotypes: 1–7, 9, 11–21, 24–27, 29–33

Viral structure: see Picornavirus – Section 43.54

43.17.1 Epidemiology and clinical significance

The term echovirus is derived from the acronym ECHO, which stands for “enteric cytopathic human orphan”. The virus was originally an “orphan virus” as it could not be associated with any particular disease.

The clinical spectrum of Enterovirus infections is shown in Tab. 43.14-1 – Clinical spectrum of enterovirus infections. They have a similar epidemiology and pathogenicity to Coxsackievirus infections /1/. The manifestation index is low.

Gastrointestinal co morbidities are more frequent in Echovirus infections than in infections with other Enteroviruses.

43.17.2 Laboratory diagnosis

The diagnostic methods correspond to those described for suspected poliomyelitis (see Section 43.56 – Poliovirus). The ELISA has so far failed to become established in diagnostic serology.

References

1. Crennan JM, van Scoy RE. Echovirus polymyositis in patients with hypogammaglobulinemia: failure of high-dose intravenous gammaglobulin therapy and review of the literature. Am J Med 1986; 81: 35–42.

43.18 Enterovirus

Family: Picornaviridae

Genus: Enterovirus

Species: Enterovirus A–D, Poliovirus (molecular taxonomy). In practice better known as Poliovirus, Coxsackievirus, Echovirus and new enterovirus 68–71.

Viral structure: see Picornavirus – Section 43.54

43.18.1 Epidemiology and clinical significance

Enterovirus is a genus of the Picornaviridae family. Besides the three classic groups of polio, coxsackie and ECHO, further human specific Enteroviruses were identified, which are numbered in the order in which they were discovered /1/.

Enterovirus type 68 and type 69: so far they have not been associated with a specific disease.

Enterovirus type 70: causes acute hemorrhagic conjunctivitis (Apollo disease). The infection mainly occurs in tropical and subtropical highly populated urban areas.

Enterovirus type 71: causes hand-foot-and-mouth disease (like Coxsackievirus A16).

The new Enteroviruses have occasionally also been associated with the diseases caused by the classic Enterovirus infections such as meningitis.

43.18.2 Laboratory diagnosis

See Section 43.56 – Poliovirus. Swabs from the site of inflammation or vesicle puncture and virus isolation in cell cultures yield pathognomonic results /2/. Specimens must be shipped in moist medium. Since the Enterovirus is very stable, no special preservation is required.

References

1. Rotbart HA (ed). Human enterovirus infections. American Society for microbiology. Washington.

2. Dunn JJ. Enteroviruses and parechoviruses. Microbiol Spectr 2016. doi: 10.1128/microbiolspec.DMIH2-0006-2015.

43.19 Epstein-Barr virus (EBV)

Family: Herpesviridae

Subfamily: Gammaherpesvirinae

Genus: Lymphocryptovirus

Species: Epstein-Barr virus

Types: A and B

Viral structure: see Herpesvirus – Section 43.33

43.19.1 Epidemiology and clinical significance

The EBV is a B lymphotropic herpesvirus specific to humans /1/. It spreads easily through close contact and enters the host through the lymphatic epithelial cells of the nasopharynx, where it establishes persistent infection.

The infection is transmitted via throat secretions, which is reflected in a bimodal infection rate, with peaks in young children due to mother-to-child contact and again during post pubertal adolescence due to intimate relationships ("kissing disease"). Prenatal infections have not been described. EBV can infect both susceptible B lymphocytes and non susceptible epithelial cells. Viral tropism analyses have revealed two intriguing means of EBV infection, either by receptor mediated infection of B cells or by cell-to-cell contact mediated infection of non susceptible epithelial cells. A new mechanism for EBV infection of non susceptible endothelial cells is the formation of cell-in-cell structures /2/.

43.19.1.1 Acute infection

In Central Europe, 70–80% of children and 80–90% of adults are infected with the virus. Within 4–6 weeks after virus acquisition, a high level viremia occurs and the EBV infects the B cells, which are attacked and largely eliminated by cytotoxic T cells. A blood count shows the large number of activated T cells that are typical of infectious mononucleosis. In infants (with an immature T cell response), the virus host interaction has a subclinical course, especially since infants are still partially protected by transplacental maternal antibodies. In adolescents and adults, however, the infection leads to generalized lymphadenopathy, hepatomegaly and splenomegaly (EBV hepatitis) and tonsillitis in 30–50% of cases. Occasionally a rash may occur, which can be mistaken for rubella /3/. This syndrome was described as early as 100 years ago (glandular fever).

EBV infection has an autoimmune pathogenic component, as evidenced by the presence of pathognomonic (heterophile) antibodies that cross react with bovine erythrocytes. The antibodies are detected by hemagglutination. The disease can run a severe, recurrent course for weeks or even months. However, the long term prognosis is good and progression to chronicity is rare.

43.19.1.2 Latent infection

While most B cells undergo productive, lytic infection or are eliminated by cytotoxic T cells, some are latently infected and immortalized. In immunocompetent individuals these cells are under the control of the T cells throughout life. Reactivated infections usually remain subclinical. The disease can be fatal in primary infected individuals with an immune deficiency (e.g., X-linked proliferative syndrome in children). If immune deficiency occurs later, an immortalized B cell clone may grow into a lymphoma, as is often the case in AIDS patients. In 1962, Denis Burkitt, a British tropical doctor, described a special lymphoma, which Epstein and Barr associated with lymphotropic herpesvirus infection. EBV has also been implicated in other lymphomas and in Hodgkin’s lymphoma.

The association of EBV infection with lymphoepithelial nasopharyngeal carcinoma (Schmincke’s tumor) and with oral hairy leukoplakia in AIDS patients is well understood. While extremely rare in Europe, Schmincke’s tumor is very common in South China. The geographic prevalences of Burkitt’s lymphoma and Schmincke’s tumor suggest that there may be local co factors contributing to the oncogenesis (e.g. malaria).

In rare cases, EBV infection can manifest neurologically as meningitis, encephalitis or, several weeks later, as Guillain-Barré syndrome. An even rarer EBV associated disease is EBV myocarditis.

EBV may cause a post transplant lymphoproliferative disorder after bone marrow or allogeneic stem cell transplantation, which can prove fatal if left untreated.

Chronic active EBV infection in apparently immunocompetent hosts is characterized by chronic recurrent infectious mononucleosis like symptoms that persist for a long time and by an unusual pattern of anti-EBV antibodies. The probability of 5-year survival was 0.45 for older patients, 0.94 for younger patients and 0.38 for patients with thrombocytopenia (platelet count < 12 × 10¹⁰/L at diagnosis) /4/. Proposed guidelines for diagnosing chronic active Epstein-Barr virus infection are published /5/.

43.19.2 Virus detection

Virus detection can be performed by experimental cell culture infection/immortalization (using umbilical cord lymphocytes), by testing for Epstein-Barr nuclear antigen (EBNA), and with molecular methods (detection of EBV DNA by PCR) /6/.

Virus isolation

Isolation of the virus from throat swabs or blood lymphocytes is of little diagnostic value due to the persistence of the virus and the method being too time consuming.

Antigen test

This is the easiest and most inexpensive way of testing tumor material. Commercial monoclonal antibodies to EBNA 1–6 are available. Molecular analysis of the gene EBNA 2 allows a differentiation between the type A and type B strains of EBV. In Europe type A predominates.

PCR for DNA amplification

As long as no test kits are available, an in-house method must be established with commercial primers. PCR is the fastest and most sensitive method of EBV detection. It can easily be accomplished in throat swab specimens during acute infection, but also during subclinical reactivated infection, which limits its usefulness for diagnosis. This also applies to detection in broncho alveloar lavage samples in pneumonia. Consequently, only high viral titers are of relevance. PCR is the preferred laboratory method for testing cerebrospinal fluid samples. A positive result is pathognomonic. In immunosuppressed individuals, the viremia (viral load) determined by quantitative PCR has become a valuable indicator of reactivated infection. Since latent EBV infection occurs in B cells, plasma/serum samples should be preferred over whole blood samples.

43.19.3 Serologic tests

The easiest method is the detection of disease specific but not virus specific heterophile antibodies in the hemagglutination assay. This rapid test is available in a multitude of commercial variants (Hanganatziu-Deicher, Paul-Bunnell or Wöllner). These heterophile antibodies appear at the onset of illness and disappear after its resolution. Like the blood count of infectious mononucleosis, they are a marker of pathogenicity. This is consistent with the fact that young children, in whom the stimulation of autoantibodies is low and the infection usually remains subclinical, often test negative.

Virus specific antigens that are produced in the course of an infection can be visualized in experimentally infected cells fixated in different ways in order to demonstrate the relevant antibodies.

The following antigens are distinguished: early antigens (EA), viral capsid antigens (VCA), nuclear (tumor) antigens (EBNA), and membrane antigens. A wide range of commercial assays (EIA, IIFT) are available. Modern immunoassays use genetically engineered single antigens /7, 8/.

An overview of possible serologic approaches to EBV-associated diseases can be found in

• Fig. 43.19-1 – Course of antibody titers to the main EBV antigens
• Tab. 43.19-1 – Characteristic antibody patterns in patients with EBV associated diseases.

43.19.3.1 Steps involved in EBV antibody testing

Step 1: detection of antibodies to Epstein-Barr nuclear antigen 1 (EBNA 1). This viral tumor antigen is present in the nucleus of all persistently infected cells. It is expressed relatively late in an immunogenic form. Antibodies to EBNA-1 are not produced until several weeks or months following infection. An unequivocally positive test result is evidence of past infection. Antibodies to other Epstein-Barr nuclear antigens e.g. anti-EBNA-2 antibodies) appear earlier. However, this test is of no relevance to the current laboratory diagnosis.

Step 2: if the test for anti-EBNA-1 is negative or only weakly positive (first titer level of the test), screening for antibodies to the viral capsid antigen (VCA) is performed.

Step 3: if VCA is also negative, EBV infection can be excluded in the differential diagnosis of an EBV associated disease, since the VCA antigen is highly immunogenic. Most patients develop IgM and IgG antibodies at the onset of illness.

Step 4: elevated anti-VCA titers or measurements (equal to or greater than 1 : 160) with negative or borderline anti-EBNA-1 are associated with acute or relatively recent or reactivated infection. In most cases of primary infection this can be confirmed by a positive IgM anti-VCA test (consider cross reaction/stimulation with CMV). The IgM antibodies usually persist for no more than 10 weeks. The decrease in titer may show an undulating pattern. Active infection rarely has a protracted or chronic course. About 10–20% of young children test negative for IgM.

43.19.3.2 Differentiating primary infection from reactivation

Differentiating primary infection, which runs a more severe course in adults, especially in those who are immunosuppressed, from reactivated infection, which is usually clinically irrelevant, can be a challenge.

VCA IgM is often, but not regularly, negative in reactivated infection.

A more sensitive assay is the test for antibodies to early antigens (EA). These antigens, which are produced early in the infected cell, are less immunogenic than VCA, resulting in delayed detection of the primary infection by the antibodies induced against these antigens. By contrast, reactivated infection is generally detected early and usually with sufficient sensitivity to allow restriction to IgM class antibodies. EBV does not only persist as a latent infection, but also as a productive lytic infection in the course of, usually subclinical, recurrences. Here, the immunogenic expression of EBNA-1 is limited, which explains the decrease in the titers of these antibodies, while the titer of anti-VCA IgG, which persists for life, usually rises.

When testing a patient individually, only the result of an anti-VCA IgG test performed prior to the onset of disease can reliably differentiate between primary and reactivated infection.

In most cases, the differentiation can be made by testing for heterophile antibodies, which usually only appear during primary and clinically manifest infection. The hemagglutination assay is often a better marker for evaluating disease progression than anti-VCA IgM, which remains positive for longer and sometimes decreases only in an undulating fashion.

43.19.3.3 Antibody avidity

It has frequently been proposed to use antibody avidity testing to distinguish between the early, polyclonal low avidity antibodies, which are indicative of a primary infection, and the less polyclonal high avidity antibodies, which are indicative of reinfection. This can be done using an urea elution of the antibodies bound, with variable strength, to the carrier bound antigen in the immunoassay. In the individual case, this method can be used to mostly, but not definitely, exclude a primary infection, which is more severe than recurrent infection /9/.

43.19.3.4 Supplemental tests for EBV-associated tumors

Schmincke’s tumor stimulates the synthesis of IgA-class EBV antibodies, especially to VCA. Apart from VCA, special non structural antigens from the early EBV replication cycle are also recommended for serodiagnostic tests (DNA polymerase, DNA nuclease, main DNA binding protein). However, since these antibody levels also increase in common EBV infections and recurrences, only elevated titers are of diagnostic value (anti-VCA IgA 1 : > 80). Anti-VCA IgM is usually negative, and IgG titers are high.

Burkitt’s lymphoma greatly stimulates the production of antibodies to a component of the many EBV specific EA, which is not denatured by methanol fixation. Therefore, elevated titers of antibodies to these restricted antigens (restricted early antigen, EA-R) can be more useful for diagnostic purposes than the normal tests. The other serologic tests are not pathognomonic, even if they usually give high anti-VCA IgG titers.

PCR in plasma is indicated for the detection of post transplant lymphoproliferative syndrome after bone marrow and stem cell transplantations.

43.19.3.5 Cerebrospinal fluid serology

The diagnosis of EBV infection of the CNS, which is rare, can be accomplished by demonstrating the presence of intrathecally produced antibodies to the VCA, provided that blood contamination has been excluded. Oligoclonal antibodies are produced by lymphocytes that have migrated into the CSF via the choroid plexus. Only a delayed diagnosis can be made here. For early diagnosis, viral DNA detection by PCR is performed.

References

1. Cohen JI. Epstein-Barr virus infection. N Engl J Med 2000; 343: 481–92.

2. Ni C, Chen Y, Zeng M, Pei R, Du Y, Tang L, et al. In-cell infection: a novel pathway for Epstein-Barr virus infection mediated by cell-in-cell structures. Cell Res 2015; 25: 785–800.

3. Schuster V, Kreth HW. Epstein-Barr virus infection and associated diseases. Eur J Pediatr 1992; 151: 718–25.

4. Kimura H, Morishima T, Kanegane H, Ohga S, Hoshino Y, Maeda A, et al. Prognostic factors for chronic active Epstein-Barr virus infection. J Infect Dis 2003; 187: 527–33.

5. Okano M, Kawa K, Kimura H, Yachie A, Wakiguchi H, Maeda A, et al. Proposed guidelines for diagnosing chronic active Epstein-Barr virus infection. Am J Hematol 2005; 80: 64–9.

6. Hess RD. Routine Epstein-Barr virus diagnostics from the laboratory perspective: still challenging after 35 years. J Clin Microbiol 2004; 42: 3381–7.

7. Wiedbrauck DL, Bassin S. Evaluation of five enzyme immunoassays for detection of immunoglobulin M antibodies to Epstein. Barr Virus viral capsid antigens. J Clin Microbiol 1993; 31: 1339–41.

8. Hofmann H, Popow-Kraupp Th. Diagnosis of EBV infection by means of ELISA. Serodiagn Immunother Infect Dis 1994; 6: 135–9.

9. Vetter V, Kreutzer L, Bauer G. Differentiation of primary from secondary anti-EBNA-1-negative cases by determination of avidity of VCA-IgG. Clin Diagn Virol 1994; 2: 29–40.

43.20 Filovirus / Ebolavirus / Marburg virus

Family: Filoviridae

Genus: Ebolavirus, Marburgvirus

Species: Ebolavirus 5 species, Marburgvirus

Viral structure: Marburg and Ebola viruses have a filamentous structure (Lat. “filum” = filament) when viewed under an electron microscope. The filaments are very variable in length (600–800 nm) and only 80 nm in diameter. The envelope proteins of Marburg and Ebola viruses do not have any cross reacting antigens. The single stranded negative sense RNA genome is contained in a helical capsid measuring 50 nm in diameter.

43.20.1 Epidemiology and clinical significance

Fruit bats are considered the natural host of the virus. The infection is transmitted from individual to individual through direct contact with blood and through respiratory droplets and can cause severe hemorrhagic fever after 5–9 days /1, 2/. If high level viremia is present, infection occurs, resulting in the destruction of the capillary endothelial cells of all organs, with the pathogenesis initially being exacerbated by the production of antibodies through the deposition of immune complexes and complement consumption. Death then results from hemorrhagic shock.

Ebola virus

Three major Ebola virus outbreaks (1978 and 1995, 2015) have been described in Central Africa, with a high fatality rate (60–80%). The disease is characterized by fever, severe diarrhea, vomiting, and a high fatality rate /3/. The most important measures of infection control are hand and respiratory hygiene in healthcare workers, especially when dealing with patients with hemorrhagic pneumonia /4/.

Marburg virus

The infection was first seen in 1967 in the German towns of Frankfurt am Main and Marburg and was traced to infected imported monkeys, which themselves were only primary hosts of the infection /5/. Several fatal cases were reported in researchers and laboratory staff who had become infected through contact with contaminated monkey kidney cell cultures.

43.20.2 Laboratory diagnosis

Laboratory diagnosis can be made by electron microscopic analysis of a serum sample inactivated with glutaraldehyde to screen for various tropical viruses. PCR on an EDTA blood sample is considerably more sensitive. In addition, antibody testing in special laboratories and virus isolation in cell cultures in high containment laboratories (WHO biosafety level 4) have become established methods. However, they do not allow an early diagnosis. Survivors of disease outbreaks caused by Ebola or Marburg viruses exhibit cross reactive and long lived antibody responses /6/.

All investigations are performed in special laboratories (Bernhardt Nocht Institute, Hamburg; Virological Institute, University of Marburg).

References

1. Moghadam SRJ, Omidi N, Bayrami S, Moghadam SJ. Alinaghi S. Ebola virus disease: a review of literature. Asian Pacific J Trop Biomed 2015; 5: 260–7.

2. Baize S, Leroy EM, Georges-Courbot MC, Capron M, Lansoud-Soukate J, Debre P, et al. Defective humoral immune responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola infected patients. Nature Medicine 1999; 5: 423–6.

3. Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet 2011; 377: 849–62

4. Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba NF, et al. Emergence of Zaire Ebola virus disease in Guinea. N Engl J Med 2014, 371 (5): 1418–25.

5. Slenczka W, Klenk D. Forty years of Marburg virus. J Infect Dis 2007; 196: 131–5.

6. Natesan M, Jensen SM, Keasay SL, Kamata T, Kuehne Al, Stonier SW, et al. Human survivors of disease outbreaks caused by Ebola or Marburg viruses exhibit cross-reactive and long -lived antibody responses. Clin Vaccine Immunol 2016; pii: CVI.00107-16.

43.21 Flavivirus / Zika virus

Family: Flaviviridae

Genus: Flavivirus with over 50 species

Viral structure: Flavivirus has a positive-sense RNA genome contained within an icosahedral capsid, which is surrounded by a glycoprotein containing lipid envelope. The virus measures 40–60 nm in diameter. The Flavivirus genus includes yellow fever virus, Dengue virus, ESME virus, West Nile virus, looping III virus, Japanese encephalitis virus, Zika virus, and many other viruses that are transmitted by arthropods /1/.

The Zika virus is transmitted by aedes mosquitoes and potentially by transfusion, perinatal and sexual transmission /2/. Neonates with microcephalus attributed to Zika virus maternal infection associated with an epidemic of Zika virus in South America has been reported /3/.

References

1. Solomon T. Flavivirus encephalitis. N Engl J Med 2004; 351: 370–8.

2. Musso D, Ko AI, Baud D. Zika virus infection – after the pandemic. N Engl J Med 2019; 381: 1444–57.

3. Schuler -Faccini L, Ribeiro EM, Feitosa IM, Horovitz DD, Calvalcanti DP, Possa A, et al. Possible association between Zika virus infection and microcephaly. MMWR Morb Mortal Wkly rep 2016; 65: 59–62.

43.22 Early summer meningoencephalitis virus

Family: Flaviviridae

Genus: Flavivirus

Species (group): Tick-borne Encephalitis virus (TBE)

Viral structure: see Flavivirus – Section 43.21

43.22.1 Epidemiology and clinical significance

The natural reservoir of the Early summer meningoencephalitis (ESME) virus are rodents. The virus is taken up by ticks (Ixodes ricinus; wood tick) and spread transovarially. Ticks mainly reproduce during early summer but also, to a lesser extent, during late summer. The virus can spread to humans through a tick bite. Virus carrying ticks are widely distributed in Southern and Central Germany. Outside Central Europe, many variants of the virus can be found, predominantly in Eastern Europe (Far Eastern tick-borne encephalitis virus, TBE-FE, Russian ESME virus) /1/. After a tick bite, the virus can spread to the nasopharynx through an initial viremia during an incubation period of 7–14 days and manifest as a prodromal common cold. A secondary viremia disseminates the virus to the central nervous system, where it leads to meningitis with or without encephalitis /2/. This occurs rarely in children, but relatively often in adults (20–30%). The prognosis is good, if strict bed rest is adhered to. Residual damage is rare. Adults and adolescents should be inoculated with inactivated virus (immune protection period following triple immunization according to the tetanus schedule: at least 5 years). Passive immunization, which should only be used immediately after a tick bite in adults in high risk areas, has been abandoned.

Besides the ESME virus, wood ticks can also transmit Borrelia burgdorferi. Borrelia carrying ticks are endemic, (e.g., throughout Germany). There is also a sheep tick (Ixodes marginatus), which transmits Coxiella burnetii, the pathogen that causes Q fever. This tick does not inoculate the pathogen with saliva during the bite, but excretes it in feces (infection by inhalation of dust).

43.22.2 Virus detection

Although not commonly performed, virus isolation is possible in cell cultures from a throat swab, from blood or cerebrospinal fluid 1–3 weeks after the tick bite. The test takes several days, whereas pathogen detection by PCR can be accomplished within a few hours.

43.22.3 Viral serology

The least expensive method of choice is the antibody test. Any positive result is considered abnormal because of the low prevalence even in endemic areas or in forestry workers. After an incubation period of approximately 3 weeks, antibodies to the virus can be detected in blood and subsequently also in cerebrospinal fluid in the hemagglutination inhibition assay or IgM ELISA and, a little later, in the IgG ELISA. Cave: crossreactions with other species of the Flavivirus genus, especially when assessing the immune status /3/.

References

1. Mickiene A, Laiskonis A, Günther G, Vene S, Lundkvist A, Lindquist L. Tickborne encephalitis in an area of high endemicity in Lithunia: disease severity and long-term prognosis. Clin Infect Dis 2002; 35: 650–8.

2. Schmolck H. Neurologic, neurophysiologic, and electroencephalographic findings after European tick-borne encephalitis. J Child Neurol 2005; 20: 500–8.

3. Allwinn R, Doerr HW, Emmerich P, Schmitz H, Preisser W. Cross-reactivity in flavivirus serology: new implications of an old finding? Med Microbiol Immunol 2002; 190: 199–202.

43.23 Yellow fever virus

Family: Flaviviridae

Genus: Flavivirus

Species: Yellow fever virus with nine genotypes

Viral structure: see Flavivirus – Section 43.21

43.23.1 Epidemiology and clinical significance

Yellow fever virus is the prototype of the Flavivirus genus, whose name is derived from it (Lat. “flavus” = yellow). The infection is found in tropical and subtropical areas of Africa and Central and South America, but not in Asia.

There are two types of transmission cycles /1/: the sylvatic and the urban cycle. In the sylvatic cycle, the virus circulates in the jungle between primates and mosquito species of the hemagogus and aedes genera. If humans enter this habitat, they can participate in this transmission cycle as primates and pass the virus to other humans via the vectors. In the urban cycle, the virus circulates between domestic mosquitoes and viremic humans. The urban cycle is responsible for the major outbreaks of yellow fever.

After replicating in the regional lymph nodes, the virus subsequently spreads through viremia to the liver, the spleen, heart and skeletal muscle, bone marrow, and the brain. Severe cases of infection are associated with extensive abnormal clotting and coagulation. The clinical spectrum is variable, from inapparent (fever and headache ≤ 48 h) to severe disease.

Yellow fever has an incubation period of 3–6 days and begins with an abrupt onset of symptoms consisting in fever (up to 40 °C), chills, severe headache, nausea, vomiting, and injection of the sclera. The initially rapid pulse slows to a rate that is too low in relation to the fever (Faget’s sign). In mild cases, the disease ends at this stage after 1–3 days. In more severe cases, the fever falls 2–5 days after onset in a period of remission lasting from several hours to several days, before returning again. These severe cases are characterized by jaundice, intense albuminuria and hematemesis. Other symptoms occurring in this phase include renal failure and, frequently, petechiae and mucosal hemorrhages.

43.23.2 Virus detection

During the first phase (acute stage) of the disease, the diagnosis can be confirmed or excluded only by nucleic acid amplification (PCR in special laboratories) or direct hybridization /2/. Virus isolation in cell culture may only be performed in a high containment laboratory. Electron microscopy, which is rarely employed today, can be used with some success during the acute, high viremia phase, but does not distinguish between different members of the Flavivirus group.

43.23.3 Viral serology

Antibodies are only produced during the second phase of the disease and are best detected with EIA or IFT. For special investigations, a neutralization test can be used. Cave: cross-reactions with other Flaviviruses, especially when assessing the immune status /3/.

References

1. Barnett ED. Yellow fever: epidemiology and prevention. Clin Infect Dis 2007; 44: 850–6.

2. Bae HG, Nitsche A, Teichmann A, Biel SS, Niedrig M. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay. J Virol Methods 2003; 110: 185–91.

3. Vasquez S, Valdes O, Pupo M, et al. MAC-ELISA and ELISA-inhibition methods for detection of antibodies after yellow fever vaccination. J Virol Methods 2003; 110: 179–84.

43.24 Hantavirus

Family: Bunyaviridae

Genus: Hantavirus

Species and types: the following types are associated with hemorrhagic nephritis: Hantaan, Seoul, Dobrava (Belgrade, Crimea) and Puumala. The following types are associated with acute, non cardiogenic pulmonary edema (Hantavirus pulmonary syndrome, HPS): Sin Nombre, Black Creek Canal, Bayou, New York-1, Prospect Hill.

Viral structure: see Bunyaviridae – Section 43.10

43.24.1 Epidemiology and clinical significance

Hantavirus is distributed worldwide, and there are many types. The virus is named after the Hantaan River in Korea, where thousands of US soldiers fell ill with an infectious hemorrhagic nephritis during the Korean War. Similar diseases are caused by other types of virus in other parts of the world. In 1993, in the Southwestern USA, a different disease manifestation, a severe pneumonia, was linked to infection by the Sin Nombre Hantavirus (Hantavirus pulmonary syndrome).

Hantavirus causes persistent infection in different rodents /1/. The virus is excreted in rodent urine, feces or saliva. Humans are infected by inhaling contaminated aerosols. The reported incubation period is 2–3 weeks. The most common disease in the Far East are Korean hemorrhagic fever and epidemic hemorrhagic fever, which are characterized by fever, muscular pains, hemorrhagic manifestations, and proteinuria; the complication of shock and renal failure may lead to death /2/. The European disease is much less severe, as hemorrhagic manifestations do not occur.

43.24.2 Virus detection

Virus isolation or PCR from blood or urine can be successful only during the early, acute phase.

43.24.3 Viral serology

The laboratory diagnosis is made serologically by antibody screening by IFT or ELISA. In Central Europe, the Seoul and Puumala types should be used as antigens. Dobrava was also recently detected in Germany. The antigens only partly detect the antibodies through cross reactivity. Due to the low rate of infection in the population, a positive single test must be regarded with suspicion. A positive result should be confirmed by seeking a history of exposure and by performing a confirmatory test with a second test system. If acute infection is suspected, the specimen should be tested for IgM antibodies /3/.

References

1. Bi Z, Formenty PBH, Roth CE. Hantavirus infection: a new global update. J Infect Developing countries 2008; 2: 3–23.

2. Public Health Laboratory Service Communicable Disease Surveillance Center. Haemorrhagic fever with renal syndrome: Hantaan virus infection. BMJ 1985; 290: 1410–11.

3. Machado AM, de Figureido GG, dos Santos Jr. GS, Figueiredo LTM. Laboratory diagnosis of human hantavirus infection: novel insights and future potential. Future Virology 2009; 4: 383–9.

43.25 Hepatitis A virus (HAV)

Family: Picornaviridae

Genus: Hepatovirus

Species: Hepatitis A virus with 6 genotypes (but only 1 serotype)

Viral structure: naked, icosahedral, approximately 27 nm in diameter, positive stranded RNA

43.25.1 Epidemiology and clinical significance

HAV causes classic, infectious jaundice.

The virus is distributed worldwide. It is endemic in regions with poor hygiene standards. In Western industrialized countries the infection and naturally acquired immunity have become rare; the infection is usually acquired during overseas travel. In most cases transmission occurs through the fecal-oral route /1/.

The virus is excreted in the stool of infected persons. It is spread via the fecal-oral route as a result of poor hygiene practices of the carrier, or through contaminated drinking water and contaminated, insufficiently heated food. Shellfish can concentrate HAV from seawater. Nosocomial infections are possible. Parenteral infections through blood transfusion are very rare due to the short viremia compared to HBV and HCV. However, naked viruses are considerable more difficult to inactivate in plasma products than enveloped viruses. Parenteral transmissions are also observed in individuals addicted to intravenous drugs. Elevated seroprevalences are seen in individuals who have oro-anal sexual contact. The incubation period ranges from 15 to 50 days and averages 30 days.

In developing countries, the infection is usually asymptomatic in young children and in 25% of adults /2/. Prodromal symptoms include fever, vomiting, malaise with frequently severe lassitude, myalgias, and diarrhea. A few days after the onset of these symptoms, signs of hepatitis appear, including elevated aminotransferase levels, dark urine and light colored (discolored) stool as well as jaundice, which is best observed in the sclera (cave: tanning lotion).

The severity of the clinical course generally increases with the patient’s age, especially after maturation of the immune system in childhood, since unspecific and specific immune responses damage the hepatic cells (immunopathogenesis). It is unclear whether the aging process, liver damage, or comorbidities acquired in the course of life (e.g., chronic HCV or HBV infection) are responsible for the rising manifestation index.

Apart from rare cases of an acute fulminant, fatal course of infection in patients with preexisting chronic hepatitis B or C, the infection tends to resolve within 2 to 4 weeks. Protracted courses lasting several months have been described, but chronic infection is not known to occur. HAV infection provides lifelong immunity against hepatitis A.

43.25.2 Virus detection

HAV antigen

HAV antigen is excreted in stool in relatively large amounts prior to the onset of clinical symptoms. The test rapidly becomes negative at the onset of clinical or subclinical illness. It has been mostly replaced by the more sensitive RNA test.

Genome detection /3/

HAV RNA can be detected in stool or serum during acute infection by nucleic acid amplification testing, preferably by reverse transcription polymerase chain reaction (RT-PCR). However, a negative result does not reliably exclude acute infection. Due to the high detection limit of commercial test systems, the HAV RNA test in stool can be positive for weeks, although infectivity in normal everyday life is doubtful. Therefore the antigen test has been employed again recently.

43.25.3 Viral serology

Anti-HAV (total)

Immunoassays for anti-HAV are used to detect both IgM and IgG antibodies (Fig. 43.25-1 – Course of acute hepatitis A infection). If the infected individual is able to develop antibodies, the test will be positive immediately before or at the onset of clinical or subclinical illness and usually remain positive throughout life. Seroconversion can also be expected after hepatitis A immunization. Anti-HAV can be acquired passively through placental transfer or later from IgG-containing blood products, in particular immunoglobulin preparations. The presence of anti-HAV IgG indicates immunity.

Anti-HAV IgM

These antibodies are detectable for several weeks to several months after acute infection. Infections can have a protracted course, especially in adults, and then remain anti-HAV IgM positive for an extended period of time.

43.25.4 Diagnostic approach

Anti-HAV positivity in the absence of clinical or subclinical symptoms of hepatitis is evidence of HAV immunity acquired through previous exposure or immunization and thus protection from reinfection.

If hepatitis symptoms are present, a positive anti-HAV test must be followed up with an anti-HAV IgM test which, if positive, is indicative of acute or relatively recent HAV infection. It should be noted that such a result does not necessarily explain an existing liver pathology, since a mild HAV infection can be combined with liver damage of a different etiology. This investigation requires additional laboratory tests and possibly a tissue biopsy.

A single positive anti-HAV IgM result along with negative anti-HAV antibodies should be verified by follow-up testing.

To detect potential infection of contacts of an HAV infected individual at an early stage, RNA detection in stool or serum or antigen detection in stool can be used.

The safety of plasma to be used for manufacturing plasma derivatives is ensured with a sensitive test for detecting HAV RNA in pools of donor plasma.

References

1. Cuthbert JA. Hepatitis A: old and new. Clin Microbiol Rev 2001; 14: 38–58.

2. Jacobsen KH. The global prevalence of Hepatitis A virus infection and susceptibility. World Health Organization 2009.

3. Birkenmeyer LG, Mushahwar IK. Detection of hepatitis A, B and D virus by polymerase chain rection. J Virol Methods 1994; 49: 101–12.

43.26 Hepatitis B virus (HBV)

Family: Hepadnaviridae

Genus: Orthohepadnavirus

Species: (Human) Hepatitis B virus with nine genotypes (A–I)

Viral structure

The spherical 42 nm virion comprises an envelope that is outside a 30 nm nucleocapsid that encloses a circular 3.2 kb DNA genome. The minus-strand HBV DNA genome is synthesized by reverse transcription of the 3.5-kb pregenomic RNA (pg RNA.) after copacking of the pg RNA and HBV polymerase inside the icosahedral nucleocapsid /12/.

43.26.1 Epidemiology and clinical significance

HBV is a blood borne virus that causes classic hepatitis (serum hepatitis). Only later was it recognized that the virus is mainly transmitted sexually and perinatally.The minus-strand HBV DNA genome synthesized by reverse transcription of the 3.5-kb pregenomic RNA (pg RNA) after copacking of the pg RNA and HBV polymerase inside the icohedral nucleocapsid /12/. The hepatotropic DNA virus can replicate at high levels and can cause minimal disease or severe liver injury. HBV generally causes a self limiting disease in adults, with viral control mediated by the adaptive immune response.

Prevalence

HBV infection has a low prevalence and incidence in Central and Northern Europe and North America. In Germany, 5–8% of the population show signs of a past HBV infection (children less frequently, elderly individuals more frequently), 0.4–0.7% are chronic carriers of HBsAg. A higher prevalence is seen in Southern and Eastern Europe, a lower prevalence in Great Britain and Scandinavia /1/.

Different ethnic groups in the same region show different prevalences. The parents’ ethnic background is critical. Areas of high endemicity of HBV infection include sub-Saharan Africa, South East and East Asia and Oceania, where the majority of adults have been exposed to the virus and up to 10% of the population have chronic HBV infection or are infectious carriers of the virus.

Geno(sub)types

There are 9 genotypes (A–I) and numerous genosubtypes of HBV with a typical geographic distribution. Genosubtype A2 is prevalent in Central and Northern Europe and in the USA. Genosubtype D2 is also common in Germany as well as in the Middle East and North Africa /2/.

Transmission

In high prevalence countries, the virus is usually transmitted perinatally or during childhood; this route of transmission is becoming less common due to perinatal simultaneous immunization in many countries.

Depending on the background prevalence and hygiene practices, virus transmission in healthcare settings is frequent to rare and still occurs in Germany, too. Transmission by HBV tested transfusion blood has become very rare. Despite the testing of blood donors for HBsAg, anti-HBc and usually HBV DNA, the residual risk of HBV transmission through blood transfusion is 1 in 277,000, which is still 15 to 35 times higher than for HCV or HIV. If infection is suspected and no HBV infected donor can be identified, other sources of infection (e.g., in healthcare settings) must be considered. Often, sexual contact is likely the most common route of HBV transmission. The incubation period ranges from 40 to 200 days (on average 120 days).

Pathogenicity

HBV itself is not cytopathic. The hepatitis is caused by the cytotoxic immune response (immunopathogenesis). In contrast to HAV, HBV infection can be persistent. As the immune system matures, the manifestation index rises after birth, while the rate of persistence of the infection decreases /3/. Individuals who are immunotolerant to HBV or highly immunosuppressed show no clinical symptoms of infection, whereas the rate of persistence increases in these individuals.

Immunosuppression

In the setting of severe immunosuppression (e.g., due to neoplasia or transplantation) the virus may replicate again. Termination of immunosuppressive or cytostatic therapy can lead to fulminant hepatitis in patients with reactivated HBV infection.

HBV immunization

Immunization is performed with genetically engineered HBsAg. The vaccine can produce transiently detectable levels of HBsAg and thus lead to a false positive result for HBV infection. Therefore, individuals who have received HBV vaccination are currently required to wait one week before donating blood /7/.

43.26.2 Acute hepatitis B infection

Perinatal infection

Infants born to a HBV positive mother almost always become chronic carriers of the disease. The same applies to immunodeficient individuals. However, in infants (with an immature cellular immune system) the infection is generally subclinical.

Infection in older children and adults

With increasing age, the infection tends to be acute and transient, with variable degrees of hepatic cell damage, which (in mild cases) often remains undetected. During the prodromal phase, fever is less common than in HAV infection; typical manifestations include arthralgia, flu-like symptoms, and gastrointestinal problems. Hepatitis B symptoms cannot clinically be distinguished from those associated with HAV infection. The virus usually persists in the liver, even if serologic results indicate that the infection has resolved. Chronic HBV infection often (in approximately 20% of cases) leads to liver cirrhosis and liver cancer, although in most cases it initially presents with few symptoms.

Hepatitis B can be diagnosed and evaluated unequivocally only based on clinical symptoms, ultrasound, histopathology, laboratory markers of hepatic cell damage and serologic markers /4/.

43.26.2.1 Standardized interpretations of acute HBV infection /4/

• A hepatic pathology in the absence of anti-HBc (provided the patient is generally able to develop antibodies) indicates that the hepatitis is not caused by infection with HBV, because the disease essentially has an immunopathogenic etiology.
• If there is a hepatic pathology and anti-HBc is positive, then detection of HBsAg confirms active infection. A strongly positive result for anti-HBc IgM is suggestive of acute (recent) infection.
• Acute symptoms in the presence of HBsAg and anti-HBc are insufficient to support a diagnosis of acute hepatitis B, since a chronic infection may become acute or a hepatitis may be caused by other, additional hepatic noxae.
• Every patient with newly detected acute or chronic HBV infection should be tested for HDV RNA at least once and then every time the patient undergoes an exacerbation in chronic infection.
• A hepatic pathology in the presence of anti-HBs and anti-HBc is rarely due to HBV infection (investigation by nucleic acid detection after other causes have been ruled out).

43.26.2.2 Diagnostic procedure

Hepatitis B Antigen (HBsAg), anti-HBs and anti-HBe and aminotransferases are the established markers of acute hepatitis B. Refer to:

• Fig. 43.26-1 – Progression of various forms of HBV infection
• Tab. 43.26-1 – Serologic markers in suspected cases of acute HBV infection.

43.26.3 Chronic Hepatitis B virus (HBV) infection

Persistence of HBsAg for longer than 6 months indicates chronic HBV infection /4/. The clinical spectrum of chronic hepatitis B can ranges from no symptoms to progressive hepatic fibrosis, advanced cirrhosis, and hepatocellular carcinoma. About 206 million people worldwide have chronic hepatitis B. Several disease phases are diagnosed on the basis of aminotransferase concentration, antigen status of HBe (HBeAg), HBV-DNA concentration, and histologic characterization.

HbsAg, HBeAg, anti-HBc, and hepatitis B DNA are established markers of chronic hepatitis B (Tab. 43.26-2 – Markers of chronic HBV infection).

43.26.4 Laboratory diagnosis

43.26.4.1 HBsAg

HBsAg is a component of the viral envelope. In addition, in large excess the 20 nm particles and filaments of the virus are produced /5/. This is the reason of HBsAg tests high detection limit. The presence of HBsAg always indicates current acute or chronic HBV infection and potential infectivity. HBsAg appear 1 to 6 months following infection and several weeks prior to clinical or subclinical disease. Even earlier detection is possible by HBV DNA testing. HBsAg disappears as the infection resolves. By definition, the presence of HBsAg for longer than 6 months indicates chronic HBV infection. However, due to the high detection limit of HBsAg assays, HBsAg may remain detectable for 7 or 8 months even if the infection is resolving.

Commercial HBsAg tests show good detection limit (below 0.1 ng/mL; 1 ng corresponds to approximately 1.3 IU/mL). However, since HBsAg levels can be as high as 1000 μg/mL, a strongly HBsAg positive serum can contaminate numerous other sera, if handled improperly /8/. Isolated HBsAg, if specific, is the first serologic marker of HBV infection. Weakly positive (less than maximally positive) tests in particular are often false positive. Test manufacturers usually require a positive HBsAg test to be confirmed by an HBsAg neutralization assay. Since the latter is generally not required for tests that are strongly positive for HBsAg and is inconclusive for tests that are weakly positive for HBsAg, other serologic markers such as anti-HBc and HBeAg should be assayed first. If doubts remain, a sensitive qualitative HBV DNA test should be ordered. Isolated HBsAg positive results must be followed up after 4 and, if necessary, 8 weeks. HBsAg results sometimes diverge among different test kits due to the presence of escape mutants, which can be detected reliably only by HBV DNA testing.

Quantitative HBsAg testing is becoming increasingly popular, since in chronic infection serum HBsAg levels are an indirect marker of intrahepatic HBV cDNA levels, even if HBV replication is suppressed by therapy. Loss of HBsAg is associated with permanent resolution of the illness, especially in patients receiving interferon therapy. Because the HBsAg level is usually significantly higher than the measuring range of immunoassays, a suitable dilution (e.g. 1 : 400) has to be assayed in most cases.

43.26.4.2 Anti-HBs

The lipid bilayer of the virion envelope incorporates the 42 kD hepatitis B virus surface protein (HBs).The appearance of anti-HBs is usually preceded by an interval of several days to weeks, during which many tests are unable to detect either of the markers /6/. Because these are present in the form of immune complexes, very low titers of both markers are detected with other tests during this period. The appearance of anti-HBs at levels above approximately 100 IU/L is indicative of successful immune control of the virus. Provided the patient does not become immunosuppressed again later, he/she has overcome the infection and is very unlikely to become re-infected. However, unlikely previously, it is now thought that the virus is not completely eliminated even in the presence of anti-HBs. Long-term coexistence of HBsAg and anti-HBs can lead to immune complexes depositing in organs rich in capillaries, resulting in inflammatory responses through complement activation (e.g., nephritis or erythema nodosum).

The quantitative determination of anti-HBs in international units per liter (IU/L) is used to monitor the success of hepatitis B immunization. It is indicated in individuals who are at increased risk of infection. Titers > 100 IU/L are considered protective against hepatitis B infection; individuals with lower titers should have a booster dose /6/.

Past HBV infection

“Classic” past HBV infection with permanent immunity and protection from reinfection is characterized by the concurrent presence of anti-HBc and anti-HBs /4/. Administration of immunoglobulin can lead to false positive results. After immunization, only anti-HBs, but not anti-HBc, becomes positive.

43.26.4.3 HBcAg and anti-HBc tests

HBcAg cannot be detected during any stage of infection unless the blood or serum sample has been pretreated. Anti-HBc first appears when aminotransferase levels begin to rise (usually several weeks after HBsAg is detectable) and then remains positive for a very long time, often for life. In chronic infections, anti-HBc is detectable together with HBsAg, in serologically resolved infections it is present together with anti-HBs. During the window period between the clearance of HBsAg and the appearance of anti-HBs it is the only marker of HBV infection, apart from anti-HBe, which is not always assayed. Such a result can be confirmed by concurrent anti-HBe positivity. An isolated weakly positive anti-HBc result can also be indicative of remote HBV infection, because either anti-HBs or anti-HBc may disappear first long after infection /9/.

However, a single strongly positive anti-HBc test can also be a sign of chronic HBV infection in cases where HBsAg is expressed at levels too low to be detected even by very sensitive HBsAg tests (low level carrier) or where it is structurally altered such that it does not bind to the monoclonal antibodies used in the relevant test (escape mutant). It is also possible that the synthesis of HBsAg is temporarily suppressed by HDV interference /4/.

Findings of isolated weakly positive anti-HBc are often unspecifically reactive in that they only occur in one or few of the many approved tests, but not in those from other manufacturers. Their significance remains poorly understood. Prior to immunosuppression, a weakly positive, unconfirmed anti-HBc result should also be interpreted as a sign of possible occult HBV infection. Such a result should be an indication for HBV monitoring, since an undetected reactivation may result in the necessary antiviral therapy not being initiated.

43.26.4.4 Anti-HBc IgM assay

These antibodies can be detected in active HBV infections with marked HBcAg synthesis and viral replication. Titers are significantly higher in acute compared to chronic infections. Most commercial anti-HBc IgM assays are designed such that they become positive in acute infections but remain negative in chronic or recent, inapparent infections.

43.26.4.5 HBeAg and anti-HBe assays

HBeAg is not essential for HBV replication but acts as a modulator of the cytotoxic immune response to HBcAg. Anti-HBe indicates the breakdown of the HBeAg-induced immune tolerance to HBcAg.

The two markers were used in the past to distinguish highly infectious HBeAg positive HBV infection from anti-HBe positive HBV infection, which is usually less infectious. However, since some HBeAg positive HBV infections are not associated with elevated viral concentrations, while anti-HBe positive HBV infection can be associated with high viral concentrations in the presence of specific mutants /10/, a more reliable distinction can be made by HBV DNA testing. High levels of HBeAg negative mutants are associated with increased pathogenicity.

HBeAg and anti-HBe reflect different immune control of the infection. One of the goals of antiviral therapy for HBV infection therefore is seroconversion from HBeAg to anti-HBe.

HBeAg is an indicator of the specificity of an isolated HBsAg test, anti-HBe an indicator of the specificity of a positive anti-HBc test /4/.

43.26.4.6 HBV-DNA detection

The presence of HBV DNA in serum is indicative of active viral replication in the liver. An overview of assays for serum HBV DNA is presented in Ref. /11/.

The detection of HBV DNA by amplification of DNA target sequences, usually by PCR, is a sensitive test for detecting HBV infection. It is the earliest indicator of infection for individuals at risk of being infected, e.g. through needlestick injuries or by being in the same vicinity of known infected individuals. The sensitivity of detection depends on various test parameters, in particular the sample volume from which the DNA is extracted, and should always be reported together with the test result. It means that in 95% of cases a sample with the corresponding DNA concentration becomes positive in this test. While a positive test result is evidence of the presence of DNA, a negative result can never completely rule out the presence of DNA, but can only establish that DNA levels are below the level of detection with a likelihood of 95%. Levels of detection below 50 viral genomes/mL should be aimed for.

Quantitative DNA test

While having the same structure as the qualitative DNA test, the quantitative test is slightly less sensitive. It is important, because HBV infections can be associated with very low as well as with extremely high viral concentrations of up to 10¹¹/mL. High viral concentrations > 10⁸/mL are associated with increased infectivity of those infected, while low viral concentrations < 10⁵/mL are rarely associated with infectivity (including mother-to-newborn transmission) except where large amounts of blood are transferred, e.g. during transfusions.

Successful antiviral therapy is associated with a decrease in the HBV concentration by several powers of ten, while the development of resistance is associated with a renewed increase. Antiviral therapy is generally not required for viral concentrations < 10⁵/mL. Permanent recovery under antiviral therapy can be assumed only if HBV DNA has been cleared completely (viral concentration < 10/mL). There is a WHO standard that measures the HBV concentration in abstract IU/L, where 1 IU/L usually corresponds to between 3 and 7, most often 5, genomes/mL, depending on the test. Although there now is a WHO standard for the quantification of HBV DNA, the different methods of quantification still produce markedly different results in some cases.

DNA sequencing

Sequencing of the HBV DNA can be performed directly on the product of the PCR run or after cloning of the relevant DNA molecules. The first method detects only the main variants and does not work in strongly heterogeneous mixtures. Escape mutants are detected by sequencing the gene S and appear after unsuccessful immunization or after virus reactivation. Precore and core promoter mutants are found in HBeAg negative patients. The M204V mutation of the reverse transcriptase domain, in addition to several other mutations, confers resistance to lamivudine. Mutations in rt236 are associated with resistance to adefovir. When analyzing the chain of infection, any long sections of the genome are suitable for comparisons. In the case of HBeAg positive samples the variability within a chain of infection is minimal due to the absence of immune selection (except resistance to antivirals), with anti-HBe positive samples the variability can be as high as with HCV.

References

1. Margolis HS, Alter MJ, Hadler SC. Hepatitis B: evolving epidemiolpgy and implications for control. Sem Liver Dis 1991; 11: 84–92.

2. Schaefer S. Hepatitis B virus: significance of genotypes. J Viral Hepatitis 2005; 12: 111–24.

3. Immunopathogenesis of viral hepatitis. Clin Rev Allergy Immunology 2000; 18: 141–66.

4. Caspari G, Gerlich WH. The serologic markers of hepatitis B virus infection – proper selection and standardized interpretation. Clin Lab 2007; 53: 335–43.

5. Liang TJ. Hepatitis B: the virus and disease. Hepatology 2009: 49 (5 Suppl): S13–S21.

6. Lada O, Benhamou I, Poynard T, Thibault V. Coexistance of hepatitis B surface antigen (HBsAg) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of “a” determinant variants. J Virol 2006; 80: 2968–75.

7. Poland GA. Hepatitis B immunization in health care workers. Dealing with vaccine nonresponse. Am J Prev Med 1998; 15: 73–7.

8. Dufour DR. Hepatitis B surface antigen (HBsAg) assays– are they good enough for their current uses. Clin Chem 2006; 52: 1457–9.

9. Lau GKK. How do we handle the anti-HBc positive patient. Clinical Liver Disease 2015; 5, issue 2. doi: 10.1002/cld.399.

10. Chen WN, Oon CJ. Human hepatitis B virus mutants: significance of molecular changes. FEBS Letters 1999; 453: 237–42.

11. Ho SKN, Chan TM. An overview of assays for serum HBV DNA. Clin Lab 2000; 46: 609–14.

12. Dusheiko G, Agarwal K, Maini MK. New approaches to chronic hepatitis B. N Engl J Med 2023; 388: 55–69.

43.27 Hepatitis C virus (HCV)

Family: Flaviviridae

Genus: Hepacivirus

Species: Hepatitis C virus

Viral structure: enveloped virus with single-stranded positive-sense RNA

43.27.1 Epidemiology and clinical significance

Before HCV was identified, illnesses caused by this virus were called non-A-, non-B hepatitis. This term has become obsolete.

According to the WHO, an estimated 170 million individuals worldwide are infected with HCV. In Germany, approximately 0.5% of the population are affected. The very high prevalence in some countries, (e.g., Egypt estimated at up to 30%), is attributed to parenteral therapy and immunization programs with unclean injections /1/.

There are 6 genotypes and over 50 subtypes (a–n), which can be of importance for diagnosis and successful therapy. The distribution of the genotypes varies among regions. Genotypes 1–3 are distributed globally. In Germany, genotype 1b is prevalent. Here, the infection likely spreads in three waves:

• Through poor hygiene during parenteral injections and medical interventions, beginning as early as before World War II
• Through transfusion of contaminated blood products and plasma derivatives from World War II to the introduction of specific testing
• Through intravenous drug use and sharing of drug injecting paraphernalia among infected individuals.

Nosocomial infections still occur as a result of a lack of hygiene awareness, lack of implementation of existing regulations, and illegal practices. Since infection through blood products has become extremely rare in Germany, at least since the introduction of PCR testing of transfusion blood, nosocomial transmission must be considered as a potential source of infection, and infected carriers must be identified in the relevant healthcare setting.

HCV infection is highly prevalent among groups at risk for parenteral infection. Sexual transmission is rare compared to that seen with HBV. It has been suggested that other, parenteral mechanisms of transmission may also play a major causative role in a proportion of supposedly sexually transmitted infections.

Another risk factor, which is less common but nevertheless important due to the possibility of minor outbreaks, are the occult parenteral routes of transmission: tattooing, body/ear piercing, acupuncture and other forms of invasive alternative medicine treatment.

HCV infections are much less commonly clinically apparent than HBV infections /2, 3/. It is therefore difficult to estimate how long the incubation period will be; the formation of antibodies which, depending on the assay used, are detectable 6–8 weeks after infection, serves as a surrogate marker. As with HAV and HBV, an immunopathology develops, although this is usually comparatively mild, because the HCV infection can escape the immune system through various mechanisms (immune escape). Conversely, this often leads to persistent infection (> 80%).

Course of disease /4/:

• Symptoms are unspecific such as chronic fatigue or rapid fatigability
• The type 2 essential cryoglobulinemia, one of the potential immune complex related extrahepatic comorbidities of HCV infection, manifests as skin symptoms, purpura, vasculitis, and glomerulonephritis (more commonly than in HBV)
• The liver fibrosis, which can also occur in the absence of hepatitis, leads to liver cirrhosis and possibly liver cancer years to decades later.

43.27.2 Laboratory diagnosis

The serologic (ELISA) or genomic (PCR) markers only indicate whether an individual is infected with the hepatitis C virus; the actual hepatitis C disease may not necessarily result from the infection. It is absent especially in immunosuppressed individuals. The disease can only be diagnosed based on clinical symptoms, histopathology or laboratory markers of liver cell damage.

43.27.2.1 Anti-HCV

Screening tests for anti-HCV antibodies contain recombinant antigen mixtures of HCV core protein and various non structural proteins, which are important for replication of the virus (anti-NS3, anti-NS4, anti-NS5) /5/.

Even in immunocompetent patients a negative anti-HCV result shortly after the onset of hepatitis symptoms does not exclude HCV as the cause of such symptoms, since in some cases the test does not become positive until days or, less frequently, even weeks after the onset of clinical symptoms or elevated aminotransferase levels. In contrast to HBV infection, there is no risk of false positive results due to administration of immunoglobulin, since plasma derivatives for use in humans are not allowed to contain any HCV antibodies.

43.27.2.2 Anti-HCV confirmatory tests

Due to the very low prevalence of HCV infection in the normal population, the positive predictive value of a positive screening result is relatively low (between 1% and 30%), depending on the selection. Only a small proportion of individuals with a positive screening result are actually infected with HCV. As a precaution, such results should therefore be termed “reactive” rather than positive.

The confirmatory tests consist of membrane strips onto which the different HCV antigens are immobilized in different positions (strip immunoassays). This distinguishes them from real Western Blot assays, in which the antigen mixtures are first separated in a gel and then blotted onto the membrane /6/. Assay manufacturers usually require two or more positive antigen antibody reactions of a defined minimum strength for a positive test. Strip immunoassays, too, can sometimes give false positives or even false negatives as well as equivocal results.

43.27.3 HCV antigen tests

These tests have a high sensitivity. By performing these tests in serum panels from seroconverted patients, infections can be detected much earlier than by antibody screening alone. This makes the tests suitable for monitoring patients on dialysis, for example. The currently most sensitive test variants do not offer a price advantage over the even more sensitive nucleic acid test.

43.27.3.1 RNA detection

In HCV RNA detection, preferably the 5’-non-coding region is amplified, either by PCR after reverse transcription or as RNA by transcription mediated amplification /7/.

Genotyping can be performed by hybridization of the reaction product. Genotype 1 requires more intensive antiviral therapy.

Sequencing: by sequencing the HCV genome or suitable sections, the genosubtype and chains of infection can be identified.

43.27.4 Diagnostic approach

The best laboratory evidence to support a diagnosis of acute HCV infection is a positive HCV antibody test after prior anti-HCV negativity or a positive HCV RNA test (Fig. 43.27-1 – Course of chronic hepatitis C).

Workup:

• Diagnostic testing for acute or chronic HCV infection (Fig. 43.27-2 – Course of acute hepatitis C with resolution) begins by screening for the presence HCV antibodies
• A negative anti-HCV antibody screening test excludes HCV infection > 3 months in the past, provided the patient is generally able to develop antibodies. If necessary (suspicion of infection), this period can be reduced to about 2–3 weeks by a sensitive nucleic acid test or the HCV core antigen test. In some HCV infected patients the screening test may not be reactive until days or weeks after the onset of hepatitis symptoms.

If the suspicion remains, individuals who tested negative for anti-HCV antibody at the onset of hepatitis symptoms should be retested.

• If the anti-HCV antibody screening result is strongly positive and/or infection is strongly suspected, a RNA test can be performed immediately. Detection of HCV RNA confirms the presence of HCV infection; a result below the limit of detection largely, but not completely, excludes HCV infection.
• A positive HCV antibody confirmatory test along with a negative HCV RNA result indicates resolved infection (provided there was no antiviral therapy), in which the antibodies persists for several years after the clearance of HCV RNA
• An indeterminate result in the anti-HCV confirmatory test must be followed up with further tests
• If both the RNA test and the anti-HCV confirmatory test are negative, then the screening test was usually false positive. Nevertheless, donor units with such a result are not used because, depending on the method, the confirmatory test is slightly less sensitive, and it is feared that in very rare cases the screening test may indicate true infection.
• If the anti-HCV antibody screening test is only weakly reactive and/or suspicion of infection is low, the screening test should be followed up with the anti-HCV confirmatory test instead of the RNA test, since a negative HCV antibody test result would make further tests unnecessary.

43.27.5 Determination of the genotype

Due the potentially unfavorable long term prognosis of hepatitis C, it should be checked for as many patients as possible if therapy is indicated. This requires determination of the genotype and the initial viral concentration (quantification of RNA, e.g., by PCR).

43.27.6 Monitoring

To monitor antiviral therapy, an initial quantitative HCV RNA test and a repeat investigation after 2 months are recommended. If the initial concentration is high (> 10⁶ IU/mL), one year of therapy with interferon and ribavirin would appear advisable, especially for genotype 1. If the HCV RNA concentration does not decrease by a factor of at least 100 within 2 months, the therapy can be stopped.

References

1. Heitges T, Wands JR. Hepatitis C virus: epidemiology and transmission. Hepatology 1997; 26: 521–6.

2. Sharara AI, Hunt CM, Hamilton JD. Hepatitis C. Ann Intern Med 1996; 125: 658–68.

3. Dhillon AP, Dusheiko GM. Pathology of hepatitis C virus infection. Histopathology 1995; 26: 297–309.

4. Iwarson S, Norkrans G, Wejstal R. Hepatitis C: natural history of a unique infection. Clin Infect Dis 1995; 20: 1361–70.

5. Aoyagi K, Iida K, Ohue C, Matsunaga Y, Tanaka E, Kiyosawa K, et al. Performance of conventional enzyme immunoassay for hepatitis C virus core antigen in the early phase of hepatitis C infection. Clin Lab 2001; 47: 119–27.

6. Schröter M, Feucht HH, Schäfer P, Zöllner B, Laufs R. Serological determination of hepatitis C subtypes 1a, 2b, 3a, and 4a by a recombinant immunoblot assay. J Clin Microbiol 1999; 37: 2576–80.

7. Saldanha J, Lelie N, Heath A. Establishment of the first international standard for Nucleic acid Amplification Technology (NAT) assays for HCV RNA. Vox Sang 1999; 76: 149–58.

43.28 Hepatitis D virus (HDV)

Family: currently unassigned

Genus: Deltavirus

Species: Hepatitis D virus with 8 genotypes

Viral structure: same envelope as HBV. The core contains δ antigen and a viroid-like negative-sense RNA genome.

43.28.1 Epidemiology and clinical significance

HDV, also known as Hepatitis delta virus is a defective virus which requires the HBV envelope to replicate and therefore can only be transmitted together with HBV (coinfection) or to a chronic HBV carrier (superinfection) /1/. HDV is transmitted by the parenteral route through infectious body fluids HDV superinfection causes severe disease, usually also becomes chronic and leads to late complications such as liver cirrhosis more often than HBV infection alone. Clinical studies have shown that chronic hepatitis D is the most severe and progressive form of viral hepatitis in humans. In coinfection, the clinical course can vary from asymptomatic infection to fulminant hepatitis due to the unstable coexistence of the two viruses. The coinfection usually resolves. HDV is distributed worldwide, with high prevalences in Southern and Eastern Europe, Central Africa, and Russia. There are eight genotypes (1–8). Genotype 3 is responsible for more severe disease in South American regions. In Europe, genotype 1 is prevalent.

43.28.2 Viral serology

Anti-HDV

Anti-HDV antibodies confirm the presence of active or previous HDV infection /2/.

Anti-HDV IgM

Anti-HDV IgM is detectable both in acute, probably resolving HDV coinfection and in chronic superinfection. It appears a little earlier in the course of disease than anti-HDV IgG.

43.28.3 HDV antigen test

RNA detection

The presence of HDV RNA, detected preferably by RT/PCR, confirms the positive anti-HDV IgM result and indicates active infection. In resolving infection, the viremia disappears long before the specific antibodies. Interferon therapy may be of benefit in patients with a high viral load. If such therapy is initiated, the viral load should be monitored with RT-PCR /3/.

43.28.4 Diagnostic approach

Laboratory testing for HDV is indicated in all patients with HBV infection who have not been tested for HDV as well as after every exacerbation of chronic HBV infection, especially if there is a risk of parenteral infection.

References

1. Asselah T, Rizzeto M. Hepatitis D virus infection. N Engl J Med 2023; 389 (1): 58–70.

2. Shen L, Sun L, Yang Y, Wang F, Ly Y, Bi S. Development of a hepatitis delta virus antibody assay for study of the prevalence of HDV among individuals infected with hepatitis B virus in China. J Med Virol 2012; 84: 445–9.

3. Mederacke I, Bremer B, Heidrich B, Kirschner J, Deterding K, Bock T, et al. Establishment of a novel quantitative hepatitis D virus (HDV) RNA assay using Cobas TaqMan platform to study HDV RNA kinetics. J Clin Microbiol 2010, 48: 2022–9.

43.29 Hepatitis E virus (HEV)

Family: Hepeviridae

Genus and species: Hepatitis E virus with 4 genotypes

Viral structure: naked, icosahedral capsid, 30 nm in diameter, with positive-sense RNA genome

43.29.1 Epidemiology and clinical significance

Major HEV outbreaks associated with the human specific genotypes 1 and 2 were observed in Southeast and Central Asia, the Middle East, North and West Africa, and Central America /1/. Sporadic infections in Europe and North America were associated with HEV genotype 3. Since similar viruses were identified in rodents, chicken and pigs, it is believed that in these areas the infection is transmitted from humans to other animals (zooanthroponosis) through food (raw sausages).

Like HAV, HEV is transmitted through the fecal-oral route. In contrast to HAV, direct transmission from individual to individual is rare, possibly because the concentration of HEV in stool is lower than that of HAV. In endemic regions, transmission seems to occur mainly through ingestion of contaminated drinking water, which then leads to epidemic outbreaks. Of the few cases of hepatitis E identified in the industrial nations about half were seen in travellers from endemic areas; the other half were evidently due to zoonotic transmission. It is debatable whether the possibility of HEV infection has in the past been given sufficient consideration in differential diagnoses.

The hepatitis begins 4 to 5 weeks after infection. In terms of symptoms it can barely be distinguished from HAV infection. In contrast to HAV, some outbreaks were associated with a high rate of mortality (20%) in HEV infected pregnant women, the cause of which is not clearly evident. Chronic HEV infections can occur in immunocompromized individuals /2/.

43.29.2 Viral serology

Commercial antibody assays are available which give a positive result at or shortly after the onset of disease (immunopathogenesis). However, many assays do not contain HEV capsids with conformational epitopes, which are required for sensitive testing. Moreover, the specificity, especially that of IgM assays, is often limited (monitoring is important). IgM and IgG antibodies basically show the same kinetics in HEV as in HAV infection. IgG positivity is thought to be correlated with immunity, but a proportion of the results may be false positive, especially in the absence of corresponding exposure. On the other hand, acute HEV infections are not always detected. There are no commercial antigen assays available.

43.29.3 RNA detection

In at least 90% of acute infections viral HEV RNA can be detected in stool and/or blood.

43.29.4 Diagnostic approach

Detection of HEV RNA is evidence of active HEV infection; its absence does not reliably exclude infection. HEV antibodies are diagnostic if the patient shows unequivocal symptoms and has recently spent time in an endemic area. A rise in titer in a serum sample obtained 8–10 days later can aid diagnosis (Fig. 43.29-1 – Course of an acute hepatitis E infection).

References

1. Ecevaria JM, Gonzalez JE, Lewis-Ximenez LL, dos Dantos DR, Munne MS, Pinto MA, et al. Hepatitis E virus infection in Latin America: a review. J Med Virol 2013; 85: 1037–45.

2. Aggarwal R. Diagnosis of hepatitis E. Nat Rev Gastroenterol Hepatol 2013; 10: 24–33.

43.30 Hepatitis G virus

Family: Flaviviridae

Genus: not defined

Viral structure: enveloped virus with single stranded positive-sense RNA

43.30.1 Epidemiology and clinical significance

Hepatitis G virus (HGV or GB virus C (GBV-C).Comprehensive studies by various research groups have been unable to demonstrate an association between persistent HGV infection and hepatitis or another disease. The viremia in HGV infection reaches higher levels than HCV viremia (approximately 10⁶/mL) and persists for several years. It clears with the appearance of antibodies to the E2 protein of HGV. Coinfection with HGV may mitigate the course of an HIV infection /1/.

43.30.2 Laboratory diagnosis

Testing for HGV infection is not part of the differential diagnosis for suspected viral hepatitis. However, detection of the virus by RT-PCR is useful in the testing of drugs that are made from human material and are subjected to a viral clearance process.

References

1. Reshetnyak VI, Karlovich TI, Ilchenko LU. Hepatitis G virus. World J Gastroenterl 2008; 14: 4725–34.

43.31 Herpes simplex virus (HSV) type 1

Family: Herpesviridae

Subfamily: Alphaherpesvirinae

Genus: Simplexvirus

Species/types: Human Herpes simplex virus type 1 and 2

Viral structure: see Herpesvirus (Section 43.33 – Herpesvirus).

43.31.1 Epidemiology and clinical significance

HSV type 1 causes predominantly orofacial lesions, particularly “simple” herpes in on mucous membranes (mouth and throat area), on skin-mucous membrane borders (lips), on the cornea (eye), and more lately on other extra-genital body surfaces /1/. It also plays a certain role in genital efflorescences (Tab. 43.31-1 – Herpes simplex virus diseases).

HSV-1 infection usually remains localized. However, if the skin is already damaged (e.g., due to neurodermatitis) there is risk of eczema herpeticum. Occasionally, during the primary viremia, the virus may infect the CNS, resulting in meningitis or encephalitis. The latter typically affects the temporal lobes of the cerebrum. Like all herpesvirus infections, HSV infection also remains persistent. The viruses migrate along sensory neural pathways into the spinal ganglia, especially into the Gasserian ganglion of the trigeminal nerve, where latent infection (i.e., infection without proliferation of virus particles) is established and from where reactivations to the innervation site can occur /2/. Triggers of recurrent infection include skin irritations (UV light), fever and psycho-endocrine stress factors as well as immune defects.

More than 30% of the population are infected as early as during childhood, 70–80% during adulthood. Primary infection rarely becomes clinically manifest. Irrespective of this, a considerable proportion of the population have regular recurrences of herpes simplex, usually in the form of herpes simplex labialis. A very unpleasant form of the infection, with an unfavorable long term prognosis, is corneal herpes. This slow, creeping efflorescence is what gave the disease and virus its name (Greek “herpein” = to creep).

A more common form of herpes is genital herpes caused by HSV-1 which, like HSV-2 infection, can be transmitted congenitally to the newborn (see HSV-2 – Section 43.32).

Note: neonatal infection through transmission of the virus from oral herpes efflorescences. Genital herpes caused by HSV-1 is usually associated with a more favorable course and rate of recurrence than the classic genital herpes caused by HSV-2. If herpes encephalitis is suspected, laboratory tests must be performed and the necessary therapy must be initiated immediately even if the test results are not yet available.

43.31.2 Virus detection

The easiest and most reliable way to diagnose herpes infection is by virus detection e.g., antigen test (ELISA, IIFT) using fluid or a smear of a blister base from a vesicular efflorescence /3/. The two HSV types can be distinguished by monocloncal antibodies. Virus cultivation in cell cultures (typically fibroblasts or Vero cells) also is easy to perform and allows phenotypic testing for resistance to therapy. Electron microscopic visualization of the HSV particles in vesicle fluid is another method that is rapid and easy, but does not distinguish between the different herpesvirus species.

Molecular methods (PCR) are popular, especially in situations of high clinical relevance (e.g., in prenatal care and perinatal diagnostic testing) /3/. PCR is ideally suited for early and follow-up cerebrospinal fluid (CSF) testing for CNS infection /4/. HSV cultivation from CSF is rarely successful. PCR amplicons of selected genes can be accurately characterized by restriction fragment length polymorphism electrophoresis or by DNA sequencing. This allows the construction of infection chains and the genotypic analysis of a patient’s resistance to therapy, which is usually due to a mutation in the Thymidine kinase gene or, less frequently, the viral polymerase gene.

43.31.3 Viral serology

For the detection of seroconversion of a virus specific IgM response, the serological diagnosis of an acute infection only makes sense in the course of a primary infection. EIA, IIFT and neutralization test are applied. After the acute phase there are hardly any significant changes in titer. Serum antibody tests are mainly used in exclusion diagnosis. Apart from primary infection, serum IgM is only positive in encephalitis and eczema herpeticum, although often not until days or even weeks after the onset of illness. The detection of IgA is of no relevance. A positive serum IgM test in suspected herpes encephalitis is reportable.

43.31.3.1 Serological testing in cerebrospinal fluid

Infection of the CNS usually cannot be diagnosed until 1–2 weeks later, when intrathecal antibody production begins. To differentiate intrathecally produced antibodies from contaminating immunoglobulins from the blood, it is recommended to simultaneously determine the antibody activity in serum and CSF and to calculate the antibody index Section 46.2.6 – Antibody index). If the blood-brain barrier is compromised, all the antibodies can leak across from serum into the CSF. If the barrier is intact, the pathognomonic intrathecal synthesis of antibodies by few B cells that have migrated into the CSF is directed only against the CNS bound virus. It is oligoclonal and often restricted to a single Ig class or subclass.

References

1. Arduino PG, Porter SR. Herpes simplex virus type 1 infection: overview on relevant clinico-pathological features. J Oral Pathol Med 2008; 37: 107–21.

2. Preston CM. Repression of viral transcription during herpes simplex virus latency. J Genera Virol 2000; 81: 1–19.

3. Ashley RL. Laboratory techniques in the diagnosis of herpes simplex infection. Genitourin Med 1993; 69: 174–83.

4. Aurelius E, Johansson B, Staland A, Forsgren M, Aurelies E, Skoldenberg B. Rapid diagnosis of herpes simplex encephalitis by nested polymerase chain reaction assay of cerebrospinal fluid. Lancet 1991; 337: 189–92.

43.32 Herpes simplex virus (HSV) type 2

Classification and viral structure: see HSV-1 – Section 43.31.

43.32.1 Epidemiology and clinical significance

HSV-2 is most commonly found in the genital tract, rarely in extragenital regions. HSV-2 tends to reside in the sacral ganglia. The classic manifestation is primary and recurrent genital herpes. Once viremia has commenced, HSV-2 can also spread to the CNS, where it tends to cause meningitis rather than encephalitis. However, this occurs rarely, and when it does occur, it is usually during primary infection /1/. Refer to Tab. 43.32-1 – Human herpesviruses.

In women with florid genital herpes at the end of pregnancy, the infection can be transmitted congenitally to the newborn, usually affecting all organs and the skin and thus resulting in severe and often fatal illness of the newborn. This is referred to as generalized neonatal herpes. Due to a longer period of virus production and release, congenital primary infection with HSV-2 is associated with a higher risk than recurrent infection or HSV-1 infection /2/.

Prenatal infections are considered rare. HSV-2 infection occurs after puberty and affects approximately 20% of adults /3/.

43.32.2 Virus detection

If florid herpes is suspected in the genital tract at the end of a pregnancy, then swab tests (PCR, virus isolation, antigen test) must be performed. Monoclonal antibodies can distinguish between HSV-1 antigen and HSV-2 antigen in the immunoassay. However, this has little significance for the clinical management, even though genital herpes caused by HSV-2 runs a more serious course than that caused by HSV-1. If the result is negative, a cesarian section to prevent generalized neonatal herpes is no longer recommended. The usefulness of preventive therapy with acyclovir is controversial.

43.32.3 Viral serology

There are commercial ELISA which use type specific envelope glycoprotein. They allow the differentiation of antibodies, but are slightly less sensitive than the conventional, cross reactivity assays, which employ whole virus antigen or extracts from virus infected cells.

The detection of HSV-2 specific antibodies is recommended for prenatal care and perinatal diagnostic testing, as it warns of the risk of HSV-2 recurrence.

The immunoblot is more complex and more difficult to evaluate. It is considered the gold standard in type specific HSV antibody testing.

References

1. Gupta R, Warren T, Wald A. Genital herpes. Lancet 2007; 370: 2127–37.

2. Toltzis P. Current issues in neonatal herpes simplex virus infection. Clinics in Perinatology 1991; 18: 193–207.

3. Enders G, Risse B, Zauke M, Bolley I, Knotek F. Seroprevalence study of herpes simplex virus type 2 among pregnant women in Germany using a type-specific immunoassay. Eur J Clin Microbiol Infect Dis 1998; 18: 870–2.

43.33 Herpesvirus

Family: Herpesviridae

Subfamilies: Alpha-, Beta-, Gammaherpesvirinae

Genera: Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lymphocryptovirus, Rhadinovirus

Viral structure: Herpesviruses have a complex structure and are among the largest viruses, having a diameter of 150–200 nm. The linear double stranded DNA is encased within a protein capsid (icosahedral nucleocapsid) surrounded by a membrane envelope. Between the capsid and the envelope is a layer of pleomorphic tegument proteins. The envelope is created by budding of the virus particles produced in the cell nucleus from internal and external cell membranes, which contain virus encoded proteins. The genome functions of the human specific Herpesvirus is very well understood /1/.

Gene products expressed early in the replication cycle include enzymes that contribute to the synthesis of the viral nucleic acid, such as virus specific kinases and polymerases. Their inhibition allows for specific antiviral therapy for some herpesvirus diseases with minimal side effects.

43.33.1 Epidemiology and clinical significance

Herpesviruses are species specific and widely distributed in the animal kingdom. In humans, there are eight known species divided among six genera:

• Herpes simplex virus (HSV) type 1 and type 2
• Varicella-zoster virus (VZV)
• Human cytomegalovirus (HCMV)
• Epstein-Barr virus (EBV)
• Human herpesvirus 6 (HHV-6) A and B
• Human herpesvirus 7 (HHV-7)
• Human herpesvirus 8 (HHV-8)

Besides virus specific immune responses, Herpesvirus infection also frequently triggers autoimmune processes, which can determine the disease process (see infectious mononucleosis due to by EBV infection). The prerequisite for transmission of the virus is close physical contact (exception: chickenpox), such as that between a mother and her infant or between adolescent intimate partners (two peaks of prevalence).

43.33.1.1 Latent infection

The biology of Herpesvirus infection is characterized by lifelong persistence of the virus in certain cells of the host. Latent infection occurs when replication of the virus ceases and no structural components of the virus are synthesized /2/. Latent infection is a prerequisite for virus induced oncogenesis, which has been shown unequivocally to occur for some animal Herpesviruses and can also be assumed for human Herpesviruses (EBV, HHV-8). Latent infection may (with some Herpesviruses more frequently than with others) return to productive infection with potential disease exacerbation. This is due, inter alia, to impaired cell mediated immune responses. Herpesviruses are thus opportunists of HIV infection and other immune deficiencies. Refer to Tab. 43.32-1 – Human herpesviruses.

References

1. Rajcani J, Durmanova V. Early expression of herpes simplex virus (HSV) proteins and reactivation of latent infection. Folia Microbiol 2000; 45: 7–28.

2. Croen KD. Latency of the human herpesviruses. Annu Rev Med 1991; 42: 61–7.

43.34 Herpes B virus

Family: Herpesviridae

Subfamily: Alpha Herpesvirinae

Genus: Simplexvirus

Species: Cercopithecine herpesvirus 1

43.34.1 Epidemiology and clinical significance

Herpes B virus causes infection in monkeys. If the virus is accidentally transmitted to humans, it is highly likely to cause fatal encephalitis. Human-to-human transmission is unknown /1, 2/.

43.34.2 Laboratory diagnosis

The most diagnostic tests are virus isolation or the detection of viral genome sequences with PCR on cerebrospinal fluid.

Serologic antibody testing can be requested from special laboratories. Antibodies in monkey serum samples can be detected through cross reactivity by HSV complement fixation test.

References

1. Whitley RJ, Hilliard JK. Cercopithecine herpesvirus 1 (B Virus). In: Knipe DM, Howley PM (eds). In: Fields Virology, Knipe DM, Howley PM, eds. Philadelphia 2007; Lippincott Williams, p. 2889–2904.

2. Huff JL, Barry PA. B-virus (Cercopithecine herpesvirus 1) infections in humans and macaques: potential for zoonotic disease. Emerg Infect Dis 2003; 9 (2): 246–50.

43.35 Human herpesvirus (HHV-) 6 and 7

Family: Herpesviridae

Subfamily: Betaherpesvirinae

Genus: Roseolovirus

Species: Human herpesvirus 6 (HHV-6) with genotypes A and B; Human herpesvirus 7 (HHV-7)

Viral structure: see Herpesvirus – Section 43.33

43.35.1 Epidemiology and clinical significance

Like HSV, HHV-6A and HHV-B as well as HHV-7 are ubiquitous and highly prevalent.

Transmission occurs as a result of exposure to saliva. The main target cells are T lymphocytes, predominantly CD4⁺T cells, which undergo lytic infection, resulting in transient immunosuppression. HHV-6 can also be isolated from B cells. In addition, the virus establishes latent, persistent infection in mononuclear cells. While HHV-6A typically remains silent, HHV-6B and, less frequently, HHV-7 cause exanthema subitum (3-day fever) in young children /1/.

After the incubation period of a few days, patients develop a febrile infection with a rash that mainly affects the trunk.

The infection usually resolves without complications. The role of HHV-6 in patients with impaired immunocompetence (AIDS, organ or bone marrow transplantation) is comparable to that of Cytomegalovirus, but less significant in terms of numbers affected. Pneumonia, encephalitis and transplant rejection crises have been described. Neurologic complications, such as meningitis or encephalitis, or other consequences of HHV-6/7 infection are rarely observed.

43.35.2 Virus detection

Routine laboratory testing has so far become established only for HHV-6 and is performed by in house PCR detection of viral DNA in lymphocytes or by virus isolation with lymphocyte cultures.

43.35.3 Viral serology

Serum antibody tests that differentiate between Ig classes can be performed by IIFT and ELISA, in some cases with commercial kits. Due to the close relatedness, serologic assays also detect HHV-7 infections through cross reactivity. IgG antibodies persist for life, provided there is no reinfection with the same pathogen. A positive serum HHV IgM result indicates active or recent/reactivated infection.

References

1. Braun DK, Dominguez G, Pellett PE. Human herpesvirus 6. Clin Microbiol Rev 1997; 10: 521–67.

43.36 Human herpesvirus (HHV-) 8

Family: Herpesviridae

Subfamily: Gammaherpesvirinae

Genus: Rhadinovirus

Species: Herpesvirus 8, also known as Kaposi’s sarcoma-associated herpesvirus (KSHV)

Viral structure: see Herpesvirus – Section 43.33

43.36.1 Epidemiology and clinical significance

Unlike other human Herpesviruses, HHV-8 is not widespread in the general population. In Germany less than 3% of the population are infected. In Central Africa and Mediterranean countries there are geographic pockets with much higher prevalences. Men are affected significantly more often than women /1/. As with all herpesviruses, a significantly higher prevalence is seen in HIV carriers and HIV risk groups as well as in recipients of organ transplants who are receiving immunosuppressive therapy.

The virus is spread through close physical contact. In highly immunosuppressed patients, the virus causes proliferation of capillary endothelia, which manifests as Kaposi’s sarcoma of the skin. After immune reconstitution the tumor may disappear completely. The increased incidence of the tumor outside Central Africa in the early 1980s called attention to the emerging AIDS epidemic /2/. Multicentric Castleman’s disease and primary effusion lymphoma, which both originate in the plasma B cells, are also associated with HHV-8. In many of these lymphomas the EBV genome can be found as well.

43.36.2 Viral serology

If Kaposi’s sarcoma or another HHV-8 associated tumor is suspected, a basic serum antibody test (EIA or IIFT) without Ig class differentiation should be performed initially. A negative result excludes HHV-8 infection and the presence of a true Kaposi tumor.

A positive antibody test is associated with a high risk for the development of the tumor disease, although the latter usually only manifests in immunodeficient individuals. IgM and IgA antibody tests are of no particular relevance.

43.36.3 Virus detection

The determination of virus DNA is performed by PCR in a tumor biopsy sample. Detection of the virus in whole blood (mononuclear cells), saliva or sexual secretions is not always successful.

References

1. Kedes DH, Oerskalski E, Busch M, Kohn R, Flood J, Ganem D. The seroepidemiology of human herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus: distribution of infection in KS risk groups and evidence for sexual transmission. Nature Medicine 1996; 2: 918–24.

2. Reinheimer C, Allwinn R, Stürmer M. Do fewer cases of Kaposi’s sarcoma in HIV-infected patients reflect a decrease in HHV-8 seroprevalence? Med Microbiol Immunol 2011; 200: 161–4.

43.37 Human immunodeficiency virus

Family: Retroviridae

Genus: Lentivirus

Species: Human immunodeficiency virus type 1 (HIV-1)(groups M, N, O and P) and Human immunodeficiency type 2 (HIV-2) with many subtypes

The complete virus particle (virion) has a diameter of approximately 100 nm /1/. The diploid genome consists of two copies of single stranded RNA of 9.4 and 9.7 kbp. In addition to the three structural genes (Env for the synthesis of the viral envelope, Core for the synthesis of the conical capsid, Pol for the synthesis of the viral replication enzymes), a number of regulatory genes have been identified /2/. Refer to Fig. 43.37-1 – Structure of HIV and its genome.

HIV infection begins with adsorption of the virus to receptors and coreceptors of lymphocytes and cells of a part of the reticuloendothelial system and especially of the mononuclear phagocyte system. The main target cells are T helper lymphocytes (CD4, coreceptor CXCR4) as well as macrophages (CD4, coreceptor CRC5) and microglia (galactosylceramide). The biological function of the coreceptors consists in the binding of special cytokines (chemokines such as fusin, RANTES).

The virus enters the cell when its envelope fuses with the cell membrane /3/. The inner viral capsid (core) is enzymatically removed, exposing the diploid genome. The genome is then reversely transcribed (reverse transcriptase) into double stranded DNA by viral RNA polymerase and transported into the nucleus, where it is integrated into the genome of the cell. Provided the CD4⁺T helper cells do not develop into memory cells, the proviral DNA genome is continually transcribed and translated in terms of a productive infection. As a result, these cells appear foreign to the immune system and are initially successfully attacked by non infected components of the immune system. The memory cells and the less productive macrophages, microglia and other cells previously mentioned serve as virus reservoirs.

43.37.1 Epidemiology and clinical significance

It is accepted that HIV infection is the necessary and probably sufficient prerequisite for the pathogenesis of acquired immunodeficiency syndrome (AIDS).

Distribution

Based on genetic comparisons it is believed that HIV derives from Simian immunodeficiency virus of the chimpanzee (Section 43.61 – Retroviruses) and crossed to humans in Central Africa about 70 years ago. The first epidemic occurred in Subsaharan Africa in the 1970s. The second epidemic occurred in the Caribbean and North America, where AIDS cases were observed in outpatient clinics from 1981. From there the virus spread to Europe. Germany reported its first case of AIDS in 1983. The third epidemic was seen in Eastern Europe, Asia and South America from 1990 onward.

Subtypes

The spread of the immune weakening infection was mainly driven by HIV-1 which, over time, has diversified into numerous subtypes (A–K and many recombinants) with varying geographic prevalence /4/. Most subtypes can be combined into one main group M (major). The other groups are O (outlier), N (new) and P. HIV-1 M, O and N are believed to have been transmitted separately from chimpanzees to humans. O-like Simian immunodeficiency virus has also been found in gorillas.

HIV-1 subtype B predominates in North America and Europe, subtype C is the most common subtype worldwide. In Africa, many different subtypes coexist due to the much longer epidemic duration. HIV-2 has seven known subtypes (A–G), which separately derive from Simian immunodeficiency virus variants endemic in the sooty mangabey monkey.

Prevalence

In some Southern African countries, a quarter of the population, regardless of gender, are HIV carriers. In Thailand the rapid spread of an AE recombinant form of HIV-1 was reported. In 2010, 33.3 million people were infected. Of these, 23 million cases were reported in Subsaharan Africa and 70,000 in Germany. HIV-2 mainly remained confined to West Africa and then also spread to certain regions of India.

Transmission

Transmission occurs mainly through sexual contact and via blood, through the same routes as hepatitis B infection, although in HBV infection the viral load in blood is usually much higher and the viral content of sexual secretions much lower /5/. The following groups are at increased risk: homosexual men with frequently changing sexual partners, injection drug abusers who share needles, and individuals who have contact with people in these two major risk groups. Once a certain level of infection is reached, the virus spreads to the general population without any recognizable specific sexual behaviors and then equally affects both genders. It has been suggested that the virus is more easily transmitted through the anal than the vaginal mucosa. HIV-1-AE is associated with a higher degree of mucosal infectivity than the other HIV-1 subtypes and HIV-2. However, all HIV (sub)types can be isolated from sexual secretions in significantly larger amounts than from other bodily fluids (except blood). In countries that have implemented systematic screening, the risk of transmission via blood and blood products has been virtually eliminated.

Resistance to infection or pathogenicity

Some individuals have a special genetic resistance to infection or pathogenicity. Alleles of the CCR5 coreceptor are associated with this observation in high risk individuals. However, the resistance to pathogenicity is epidemiologically insignificant.

43.37.2 Infection and infectious disease

Infectious disease is insidious in onset after an acute phase of varying severity. Thorough monitoring has shown that the CD4⁺T cell count in blood initially declines very gradually and not noticeably. Only in the final stage is there a dramatic breakdown in the balance between CD4⁺T cell depletion and regeneration, accompanied by a burst of viremia. The infection progresses in three stages (A, B, C) (Fig. 43.37-2 – Course of HIV infection).

Acute infection

Approximately 2–4 weeks after infection a flu-like syndrome of varying severity occurs. During this phase patients typically develop high levels of viremia, in the course of which up to 50% of the CD4⁺T cells are destroyed directly by the infection or indirectly through various pathomechanisms (normal count is approximately 1,000 cells/μL blood), especially in the lymphatic tissues of the gastrointestinal tract. The body nevertheless mounts a strong, effective immune response to the infection. This is often reflected in the blood count by an infectious mononucleosis and clinically by generalized lymphadenopathy. The infected lymphocytes are filtered out in the lymph nodes, where the virus infects the dendritic cells. In systemic infection, the virus also crosses the blood-brain barrier and infects the microglial tissues. By developing cytotoxic CD8⁺T cells and neutralizing antibodies, the body manages to initially suppress the HIV infection after 4–8 weeks, but is unable to eliminate it. The virus establishes lifelong persistence in the aforementioned niche cells, which are infected less productively and immunogenically.

The unusually high genome mutation rate of the retroviruses at a replication rate as high as 10⁹ virions/day in CD4⁺T cells also significantly contributes to the viruses evading the immune system with more or less success. In actual fact the HIV does not exist as a defined molecular complex, but produces a population of variants known as quasi species.

Clinical latency

The further development of HIV infection is believed to be determined by the initial virus-host interaction. Depending on the extent of the primary viremia, HIV infection remains subclinical for a few to many years or decades. During this period, untreated carriers of the infection cannot transmit the virus during normal social contact. The virus can, however, be transmitted through invasive blood contamination or through sexual secretions in which it is present. The number of CD4⁺T helper cells and thus the efficiency of the immune system gradually decline. Some carriers of the infection may develop generalized lymphadenopathy (lymphadenopathy syndrome; LAS).

AIDS

After an average of 6–10 years (15–20 years with HIV-2), the CD4⁺T cell count falls definitely below 300/μL of blood. Now the immune system breaks down relatively quickly as a result of the interplay between various pathomechanisms. The first signs of AIDS that patients may experience include sub febrile temperatures, night sweats, skin changes, long lasting diarrhea and respiratory diseases, which are usually due to other opportunistic infections (ARC; AIDS related complex). Full blown AIDS is characterized by pneumonia or a tumor (lymphoma, Kaposi’s sarcoma) /6/. Typical virologic findings are anal herpes (caused by HSV-2), herpes zoster (caused by VZV), severe cytomegaly, often with loss of eyesight from CMV retinitis.

Microbiologic testing may reveal a multitude of additional opportunistic infections, which may also affect the CNS. Apart from this, direct HIV encephalopathy is also possible (Tab. 43.37-1 – Spectrum of important clinical manifestations of AIDS). If left untreated, the disease will lead to death within several months. From an epidemiologic point of view it is important to know that the virus cannot only be transmitted horizontally through sexual and blood contact, but also vertically. This occurs most commonly at the end of a pregnancy, when the placental barrier becomes thinner and micro blood transfusions from mother to fetus are possible.

Since the late 1990s it has been possible in the industrialized countries to suppress and successfully treat the infection over many years with a combination of different HIV anti-virals so that patients return to the latent state. In pregnant women, the viremia (viral load) can be effectively suppressed during the second and third trimester so that HIV transmission to the unborn child is rare.

Timely HIV testing persists a major public health challenge because many individuals receive HIV testing late in the course of disease and unknowingly infect others, which accounts for most new HIV infections /7, 8/.

Pregnancy

Women living with HIV become pregnant and receive antiretroviral therapy (ART) during pregnancy. In the absence of ART 15–30% of infants born of persons with HIV acquire HIV antenatally or perinatally, with additional transmission during breast-feeding. Neonatal mortality varies according to the maternal ART regimen during pregnancy (Tab. 43.37-4 – Adverse pregnancy outcomes associated with antiretroviral therapy in pregnancies with HIV). Newer ART regimens are associated with better pregnancy outcomes than the older antiviral agents /14/.

43.37.3 Screening for HIV antibodies

ELISA

This is the most important screening test for antibodies in blood. In most cases, genetically engineered antigen from different structural components of the virus is used. HIV antibodies usually appear at about 3–4 weeks after infection. They are stimulated throughout life by the persistent viral infection, even if the infection remains restricted to niche cells for years. The diagnosis can be made up to one week earlier, if the HIV antibody assay is combined with the detection of HIVp24 antigen. Antibody testing that differentiates between Ig classes has not become established. The detection of IgM and IgA antibodies is of no particular relevance, even in perinatal medicine. After infancy, the diagnostic conclusion is: HIV carrier. During infancy, transplacental antibodies from an HIV seropositive mother must be considered /9/.

Since most vertical infections occur near the end of pregnancy, no HIV specific IgM and IgA antibodies are found in most cases. The risk of vertical infection is 15–20% in HIV seropositive pregnant women, but less than 1% in cases under treatment. Vertical infection must be diagnosed by viral genome detection, because the diagnosis cannot be deferred until the maternal antibodies have been eliminated definitely from the fetal blood circulation after 1–2 years.

With regard to cross reacting non-HIV antibodies (e.g. antibodies to endogenous retroviruses), a positive screening result should always be verified by an alternative method. The following methods have proven successful.

Immunoblot

The structural proteins of the HIV are separated by electrophoresis and trans blotted onto a reaction carrier, usually a nitrocellulose or nylon strip. The next steps correspond to the ELISA procedure with defined single antigens. A minimum number of antigens producing a positive antibody binding signal (bands) excludes an unspecific cross reaction /10/. Refer to:

• Fig. 43.37-3 – HIV-1 and HIV-2 immunoblots
• Tab. 43.37-2 – Evaluation of HIV-1 immunoblot results.

Special applications of specific antibody detection with single antigens include comparative analyses of cerebrospinal fluid (CSF) samples versus serum (plasma). The detection of an additional reactive band in the CSF is indicative of intrathecal antibody production as a result of the CNS also being infected by HIV. A similar analysis can be attempted in order to differentiate neonatal antibodies from transplacental IgG.

The most important rule of HIV serology is to rule out errors in the workflow from blood collection and testing through to reporting, by cross-checking with separately obtained blood samples. Therefore, an antibody test result should only be reported as positive when it has been confirmed by another, separately obtained patient sample. The test kits are available commercially.

43.37.4 Screening for HIV antigen

HIV infected cells release viruses and viral structure components. The main component of the inner viral capsid (core) is a protein with a molecular weight of 24 kDa (p24). During acute HIV infection and later during full blown AIDS, sufficient viral (sub)particles are released so that the p24 antigen can be detected by ELISA. By immunoadsorption, the titer of anti-p24 antibodies, which can be measured, e.g., by immunoblot, is often decreased. Using an acid or alkaline dissociation, the p24 antigen can be dissociated from immune complexes and thus be detected in serum or plasma with even more sensitivity /11/.

An HIV p24 antigen test can detect HIV infection up to a week before the appearance of HIV antibodies. Modern assays therefore offer combined antigen/antibody testing. The p24 antigen test is of limited use as a marker of viremia.

43.37.5 Molecular based methods

Analog to the hepatitis B and hepatitis C viruses, in HIV infection the viral load is also quantified by plasma HIV RNA testing. The basic idea here is that the copies of the viral genome must be derived from virus particles (virions), since free nucleic acid in blood is cleaved by nucleases. Thus the number of copies is correlated with the amount of virus (one HIV particle contains two genome copies). This method of viral load quantification has become established and is clearly superior to quantitative HIV antigen detection. The detection of HIV-2 and group N or group O HIV-1 requires special primers or gene probes, which are not present in commercial HIV-1 RNA assays.

PCR with an in-house genomic standard

The viral load is quantified by comparing the amplification of the in-house standard to that of the test nucleic acid, preferably using a real time assay. This eliminates the need to assess the specificity of the amplicon by molecular weight determination with electrophoresis or by hybridization with a gene probe. The result is available after 1–2 hours. Due to its high detection limit, PCR is susceptible to contamination (false positive result). At the same time, the polymerase activity may be reduced by inhibitors in the specimen (urine) (false negative result) /12/. Refer to Section 52.3 – Amplification techniques.

Nucleic acid-based amplification (NASBA) or TMA

Unlike PCR, nucleic acid sequence based amplification (NASBA) and transcription mediated amplification (TMA) do not require artificial separation of the double stranded DNA produced from the HIV RNA by reverse transcription. Instead, the more physiologic technique of DNA-RNA transcription by T7 RNA polymerase is employed. NASBA is an isothermal method.

PCR and NASBA are essentially well suited for assessing HI viremia in adults with good reproducibility. PCR has become established, and HIV RNA may be detectable in plasma as early as 1–2 weeks after infection.

A number > 15,000 IU/mL (90,000 genome copies/mL) is associated with a poor prognosis in terms of progression and treatment outcome in HIV infection. Refer to Tab. 43.37-3 – Evaluation criteria for HIV-1 viral load testing.

In follow-up tests, which must be performed by the same laboratory since testing has as yet not been standardized across regions /13/. A decrease or increase in the number of copies (IU/mL) by a factor of 4 or more is considered clinically significant. For copy numbers < 500 IU/mL a factor of 8 applies.

Opportunistic infections or vaccinations can interfere with the tests as they can stimulate the viremia transiently.

In children, spontaneous viral load fluctuations are very common, so they are of limited use as markers of pathogenicity and treatment outcome.

The detection of HIV RNA by PCR is the most conclusive marker of prenatal or perinatal vertical infection from a mother to her child.

43.37.6 Genotypic resistance testing

More than a dozen antiviral drugs targeting different step in the viral life cycle are used in anti-retroviral therapy /13/. The combination of at least three substances from two different drug classes, also referred to as “highly active anti-retroviral therapy” (HAART), has proven to be a successful strategy. Nevertheless, the rapid development of drug resistant HIV variants poses a major challenge. These variants can be identified by comparing selected and PCR amplified nucleic acid sequences (genotyping) (Fig. 43.37-4 – HIV-1 gene sequencing and mutation analysis). For this, commercial analytical systems are available, which also offer interpretation systems. The interpretation is made on the basis of databases in which clinical follow-up tests and in-vitro tests with HIV infected cell cultures (phenotyping) using specific antiviral drugs are stored. They must be continuously updated.

43.37.7 Virus isolation in cell cultures

Isolation of the HIV from EDTA blood, plasma or isolated lymphocytes and mononuclear cells can provide valuable information for the prognosis of HIV infection. The infectious titer is determined indirectly by measuring the p24 antigen released from the cultured cells. For cell culture, T cell lines or fresh umbilical cord lymphocytes, which are more sensitive, are used.

The cells must be stimulated with mitogens and interleukin-2. The prognosis is considered to be unfavorable if the virus isolate induces the formation of syncytia in the indicator cells (SI strain). Cell culture replication of HIV is time consuming, labor intensive, costly, and subject to stringent safety requirements (S3 laboratory). It is therefore only used for scientific purposes. The isolate can be tested phenotypically in culture for sensitivity to chemotherapeutic agents (virogram). However, there is a risk of in-vitro alteration of the quasi species, and for this reason, genes from the patient’s virus are cloned into a defined test virus.

References

1. Gelderblom HR. Assembly and morpholpgy of HIV: potential effect of structure on viral function. AIDA 1991; 5: 617–38.

2. Gallo RC. Human retroviruses: a decade of discovery and link with human disease. J infect Dis 1991; 164: 235–43.

3. Sattentau QJ. CD4 activation of HIV fusion. Int J Cell Cloning 1992; 10: 323–332.

4. Alaeus A. Significance of HIV-1 genetic subtypes. Scand J Infect Dis 2000; 32: 455–63.

5. Yerly S, Vora S, Rizzardi P, Chave JP, Vernazza PL, Flepp M, Telenti A, et al. Acute HIV infection: impact on the spread of HIV and transmission of drug resistance. AIDS 2001; 15: 2287–92.

6. Middleton GW, Lau RKW. AIDS lymphomas. Int J STD&AIDS 1992; 3: 173–81.

7. Mahajan AP, Stemple L, Shapiro MF, King JB, Cunningham WE. Consistency of state statutes with the CEnters for Disease Control and Prevention HIV testing recommendations for health care settings. Ann Intern Med 2009; 150: 263–9.

8. Quaseem A, Snow V, Shekelle P, Hopkins Jr. R, Owens D. Screening for HIV in health care settings: A guidance statement from the American College of Physicians and HIV Medicine Association. Ann Intern Med 2009; 150: 125–31.

9. Maskill WJ, Croft N, Waldman E, Healey S, Howard TS, Silvester C, et al. An evaluation of competitive and second generation ELISA screening tests for antibody to HIV. J Virol Methods 1988; 22: 61–73.

10. Jackson JB, Parsons JS, Nichols LS, Knoble N, Kennedy S, Piwowar EM. Detection of human immunodeficiency virus type 1 (HIV-1) antibody by western blotting and HIV-1 DNA by PCR in patients with AIDS. J Clin Microbiol 1997; 35: 1118–21.

11. Polywka S, Feldner J, Duttmann H, Laufs R. Diagnostic evaluation of a new combined HIV p24 antigen and anti-HIVI/2/O screening assay. Clin Lab 2001; 47: 351–6.

12. Cao Y, Ho DD, Todd J, Kokka R, Urdea M, Lifson JD, et al. Clinical evaluation of branched DNA signal amplification for quantifying HIV type 1 in human plasma. AIDS Res and Human Retroviruses 1995; 3: 353–61.

13. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 1995; 373: 123–6.

14. Eke AC, Mirochnick MM, Lockman S. Antiretroviral therapy and adverse pregnancy outcomes in people living with HIV. N Engl J Med 2023; 388 (4): 344–54.

43.38 Human T cell leukemia virus (HTLV)

Family: Retroviridae

Genus: Deltaretrovirus

Species: HTLV type 1 and type 2

Viral structure: see Retrovirus – Section 43.61

43.38.1 Epidemiology and clinical significance

Prevalence

HTLV is present throughout the world. It generally has a very low prevalence (affecting less than 0.5% of the world’s population), except in the Caribbean countries and Central and South America, where the prevalence is 2–7%, and in Southern Japan, where the prevalence of HTLV-1 is approaching a similar level in some regions. In Central Africa, 1–5% of the population are seropositive for both types of the virus. In Europe, HTLV is most prevalent in France and Portugal, presumably due to the countries’ special colonial relationship with Africa (prevalence of approximately 0.5%).

In the other European and North American countries, HTLV infection is extremely rare, except in i.v. drug abusers.

Transmission

HTLV is infectious through cell bound virus and spread via blood and sexual contact. It establishes persistent infection in CD4⁺T cells. The infection spreads from cell to cell through direct contact only. The viral genome integrates into the infected cells.

Diseases

After a latency period of 30–45 years, rarely after 5 or more than 60 years, the cell is transformed and proliferates by cloning in 0.1% of carriers of the infection, resulting in acute T cell leukemia, which becomes chronic in 30% of cases. Adult T cell leukemia/lymphoma (ATL) is rare, but when it does occur, it has a very poor prognosis. CD8⁺T cell and B cell lymphomas are also seen. Occasionally, HTVL infection is associated with Sézary syndrome and with mycosis fungoides /1/. In Europe, less than 10% of patients with ATL are seropositive for HTLV.

Approximately 1% of HTLV carriers develop chronic progressive myelopathy due to lymphocytic infiltration of the spinal cord. HTLV-1 associated myelopathy presents with spasticity, muscle weakness (tropical spastic paraparesis), hyperreflexia of the lower extremities, bladder dysfunction, and cerebellar ataxia. Uveitis has also been observed in HTLV carriers.

The role of HTLV-2 in human disease remains poorly understood, although it has been implicated in isolated cases of leukemia and neurological disease.

43.38.2 Laboratory diagnosis

Screening

The method of choice for diagnosis by detection or exclusion is the ELISA antibody test in serum with considerable cross reaction between the two types of virus. Virus detection by PCR in EDTA or citrate whole blood is not always successful in seropositive individuals due to the lack of circulating lymphocytes that carry the viral genome. In Central European blood donors, low positive ELISA results are usually unspecific. A Western blot cannot always provide clarity either.

References

1. Saito M, Jain P, Tsukasaki K, Bangham CRM. HTLV-1 infection and its associated diseases. Leukemia Research and Treatment 2012. doi: 10.1155/2012/123637.

43.39 Influenza viruses

Family: Orthomyxoviridae

Genus: Influenzavirus A, B, C

Viral structure: the virus particle is approximately 100 nm in diameter. The genome consists of 8 helical segments (7 in C genus) of single stranded negative sense RNA (viral chromosomes), which are enclosed in tubular capsids. These viral capsids are surrounded by a membrane (envelope). Their structural elements are the matrix proteins, which form a layer underneath the envelope, and the hemagglutinin and neuraminidase spikes on the outer surface of the envelope.

43.39.1 Epidemiology and clinical significance

Distribution

Influenza A and B viruses are present throughout the world and regularly cause major and minor seasonal outbreaks of influenza in countries with cold winters (influenza C viruses are of no epidemiologic significance, and influenza due to the type C species is very rare in Europe). Influenza viruses circulate between the northern and southern hemispheres. Global pandemics are always due to a new influenza A virus, which previously mainly occurred in Southern China, possibly due to very close contact with pigs, poultry and other domestic animals. Species specific A subtypes are widely distributed in the animal kingdom, especially among (water) birds, which are the natural reservoirs of influenza A virus. While avian Influenza A viruses usually do not cause any harm in water birds (ducks, geese, gulls), they can become highly pathogenic if spread to poultry farms. In rare cases, selected highly pathogenic avian viruses (pathogens of avian flu) can be spread directly to humans, in the case of the H5N1 subtype often with a fatal outcome /1/.

The Influenza A virus subtypes and strains are classified based on the hemagglutinin and neuraminidase variants and the place of initial isolation (Tab. 43.39-1 – Distribution of influenza virus subtypes by location and time period). There are 16 known hemagglutinin (H) and 9 known neuraminidase (N) serosubtypes (i.e., a total of 16 × 9 = 144 combinations are possible). Of these, 105 have been described in water birds to date. Three subtypes have become established in humans (H1N1, H2N2, H3N2) and have caused major pandemics: H1N1 was responsible for the Spanish flu, H2N2 for the Asian flu, and H3N2 for the Hong Kong flu pandemic. Minor, less dangerous pandemics began in Vladivostok (Russian flu of 1978/79 caused by H1N1). In April 2009, an outbreak caused by a variant of H1N1 virus in Mexican swine (swine flu) spread to humans and developed into a global pandemic that occurred in multiple waves. At present, influenza B virus as well as influenza A virus subtypes H1N1v and H3N2 are in circulation.

Infection of the cell

Hemagglutinin is the viral protein responsible for binding the virus to the host cell receptor (N-acetylneuraminic acid; a sialic acid), thus initiating infection. There are various sialic acid modifications in birds and mammals to which the hemagglutinin of the respective types is adapted. Once inside the endosome, the hemagglutinin is cleaved by a host cell protease. Due to the low endosomal pH, the protein then undergoes conformational changes that allow its hydrophobic fusion peptide to insert into the endosomal membrane, resulting in fusion of the viral envelope with the internalized cell membrane /2, 3/.

In addition, the matrix protein M2, which acts as an ion channel, lowers the pH inside the virus, resulting in dissociation of the ribonucleoprotein (RNP). The RNP contains the viral RNA polymerase, which is required for the transcription and replication of the viral genome. However, the RNA polymerase transcription error rate is higher than that of DNA polymerases (1 error/10,000 nucleotides) due to the lack of repair mechanisms. As a result, influenza viruses have a relatively high mutation rate, which leads to antigenic drift, enabling the virus to partly evade the immune response. Furthermore, the antigenic variability is dramatically increased through re assortment of the 8 genome segments of each virus during coinfection with two different virus variants (antigenic shift). Such re assortment of genome segments can have serious consequences, if a cell (e.g. cell of a pig as mixing vessel) is simultaneously infected with both a human and an avian influenza virus.

Pigs are especially suitable for re assortant formation, because they have both sialic acid modifications on their respiratory cells. Although statistically very rare, genetic re assortment can result in a hybrid virus that can infect humans and against which there is as yet no partial immunity in the population. After replicating in the nucleus, the viruses are assembled by budding at the outer cell membrane, and released. The neuraminidase in the viral envelope cleaves the sialic acid to allow the newly produced viruses to be released and to prevent the binding to desquamated cell debris in the mucous of the respiratory tract.

Immunity to infection depends on the formation of antibodies to the individual H subtypes and variants, and also partly to the N spike. Antigenic shift is only known to occur with Influenza A virus, since types B and C are not endemic in the animal kingdom, apart from single type C isolates in swine.

Clinical presentation

Classic influenza is an Influenza virus infection of the respiratory tract. After an incubation period of just a few days, the disease begins acutely with chills, sore throat and body aches, high fever, fatigue, and poor circulation. After 1–2 weeks, gradual recovery occurs.

Convalescence can last several weeks or even months, if relapses occur. Influenza is associated with more severe objective signs and subjective symptoms of malaise than a common cold, which is caused by many different viruses and bacteria. Transmission occurs via droplets of throat secretions and is more likely in a cold and humid environment or in overheated rooms, where the dry air can lead to drying out of the mucous membranes /2/.

True influenza virus pneumonia is rare. By contrast, bacterial superinfections descending to the lungs are more common and also dangerous, as they cause leukocytosis. A classic complication of influenza is superinfection with Hemophilus influenzae, which gave the disease its name. This pathogen promotes virus infectivity by secreting proteases that cleave hemagglutinin /3/.

43.39.2 Virus detection

Early diagnosis of the infection is best accomplished by detecting the pathogen from nasopharyngeal swab or gargle samples. Special viral transport media, possibly commercial ones, should be used. The most sensitive method is culture of the virus in permanent dog kidney cell lines. The optimum temperature for this is around 33 °C. As early as 24–48 hours after inoculation, viral antigen can be detected immunohistologically with monoclonal antibodies. RT-PCR testing for viral RNA genome sequences only takes between 2 and 3 hours. This method is less susceptible to interference due to suboptimal transport of samples, but slightly more costly than virus culture. It can, however, be combined with screening for other viruses (influenza A, B, parainfluenza 1, 2, 3) as multiplex PCR. The molecular and cell biological methods of virus detection allow fine typing of the virus isolate, which is of critical importance for assessing the epidemiologic situation and for the composition of the vaccine.

For subtyping cultured viruses, there are commercial monoclonal antibodies available. Subtyping by PCR uses specific H gene probes, which also are available commercially. Direct antigen testing can be performed with ELISA and IIFT. An immunochromatographic variant of the method can be used as a rapid bedside test. The methods are suitable for group diagnosis, not so much for individual cases, because the diagnostic sensitivity is low at 60–70%. Individual diagnoses or virus exclusion are best accomplished by RT-PCR.

43.39.3 Viral serology

Antibodies often do not appear in serum until 1–2 weeks after the onset of disease. The best established test methods are ELISA and IIFT /4/. The antigens used are whole virus or matrix and nucleoproteins, which allow the detection of type specific antibodies. IgG antibodies are not a clinically reliably marker of immune protection. Elevated titers of IgM, or better IgA antibodies, are indicative of relatively recent infection. Only a significant (at least four-fold) rise in titer is confirmatory of infection. Immunity can be tested with a cell culture virus neutralization or hemagglutination inhibition assay. The results of these assays are largely virus subtype and strain specific despite cross reactions. The same applies to the test for antibody activity against neuraminidase with an enzyme inhibition assay.

References

1. Monto AS. Vaccines and antiviral drugs in pandemic preparedness. Emerging Infectious Diseases 2006; 12 (1): 55–60.

2. Wong S S Y, Yuen KY. Avian influenza virus infections in humans. Chest 2006; 129: 156–68.

3. Liu J, Xiao H, Lei F, Zhu Q, Qin K, Zhang XW, et al. Highly pathogenetic H5N1 influenza virus infection in migratory birds. Science 2005; 309. doi: 10.1126/science.1115273.

4. Allwinn R, Geiler J, Berger A, Cinatl j M, Doerr HW. Determination of serum antibodies against swine-origin influenza A virus H1N1/09 by immunofluorescence, haemagglutination inhibition, and by neutralisation tests. How is the prevalence rate of protecting antibodies in humans? Med Microbiol Immunol 2010: 199: 117–121.

43.40 Measles virus

Family: Paramyxoviridae

Subfamily: Paramyxovirinae

Genus: Morbillivirus

Species: Measles virus with 22 genotypes, but only one serotype

Viral structure: see Paramyxoviridae – Section 43.51

43.40.1 Epidemiology and clinical significance

Prevalence

Humans are the only hosts for measles virus. To spread efficiently, the infection requires a large cohesive, susceptible population. Its prevalence therefore varies significantly throughout the world. In regions with established vaccination programs, it now only occurs as an introduced infection or in areas where vaccination rates are low. In many developing countries, measles is one of the leading causes of death among children besides malaria and diarrhea. The WHO’s goal of eliminating measles by 2010 has not been achieved.

Transmission

Measles is highly contagious, typically from one week before the appearance of the rash to 4 days after its onset. Transmission occurs via droplets, which can infect hosts several meters away. Almost all non immune individuals contract measles if exposed to infection. The portals of entry of infection are the nasopharynx and the conjunctiva.

Clinical presentation

The virus first reproduces in the upper respiratory tract and in the conjunctiva. This is followed by two viremic phases (Fig. 43.40-1 – Course and clinical manifestations of measles infection). Prodromal symptoms typically begin 8–12 days after exposure and include fever, lack of appetite, cough, runny nose, sore throat, and conjunctivitis with light sensitivity. During this early stage, the characteristic Koplik spots can be seen on the buccal mucosa. Approximately 4–5 days after the prodromal symptoms, an erythematous macular or maculopapular rash appears on the face and head and then spreads to the trunk and extremities, including the palms and soles, before resolving after approximately 5 days in the same order /1/. The rash results from damage to the endothelial cells of major capillaries caused by infection and cellular immune response (cytotoxic T cells) and may be absent in immunodeficient patients. These “white measles” often lead to encephalitis with a poor prognosis. Measles virus also targets leukocytes and especially lymphocytes. The resulting leukopenia and lymphopenia is associated with marked immunosuppression, which gives rise to superinfections /2/. In most cases, however, the immunosuppression resolves.

Possible complications include:

(i) Otitis media in up to 10% of cases, with otosclerosis as a sequela

(ii) Croup (with bouts of respiratory distress) as a result of epiglottitis

(iii) Pneumonia in 1–5% of cases, either in the form of measles giant cell pneumonia or as a bacterial superinfection

(iv) Encephalitis, which may take different forms:

• Acute post infectious encephalitis due to an autoimmune response, therefore no virus detection; with a mortality rate of 15–25% in 0.1% of cases
• Acute progressive infectious form of measles encephalitis in immunodeficient patients
• Subacute sclerosing pan encephalitis as a late sequela of persistent infection with measles virus mutants. It has an incidence of approximately 1 : 100,000 and usually occurs 7–8 years after measles infection. Infants, in particular, are at risk due to their immature immune system.

(v) Negative impact on other diseases such as tuberculosis, since the immune defence is weakened by T-cell infection with the measles virus.

43.40.2 Viral serology

Serodiagnosis can be made with all the relevant methods. Complement fixation test titers of 1 : 40 and higher indicate relatively recent infection. The antibody activities measured by hemagglutination inhibition assay (HIA) remain detectable for life at unchanged high titers /3/. Both assays have been replaced by immunoassays, with IgM positivity indicating (relatively) recent infection. Most ELISA are sufficiently sensitive to detect IgG antibodies shortly after the IgM test, 1–3 days after the incubation period of approximately 2 weeks /4/. After immunization against measles, high positive titers (serum dilutions) should be reached to prove immunity (i.e., > 10 with HIA and > 80 with IgG ELISA) /5/. The antibody test also is the preferred method for cerebrospinal fluid diagnosis in the workup of encephalitis.

43.40.3 Virus detection

Isolation of the pathogen from nasal, conjunctival and throat swabs in cell cultures is easy, since the virus shows marked syncytia formation as a cytopathic effect. The test duration can be limited to 24–48 hours by antigen detection with labeled antibody probes. However, the test is hardly ever successful in cerebrospinal fluid. This also applies to RT-PCR, which therefore is also only performed in special laboratories. Genotyping of the virus (sequencing of selected cDNA regions) is used epidemiologically (construction of infection chains). For CSF diagnosis, the antibody test is the method of choice. This applies even more to the diagnosis of measles associated subacute sclerosing panencephalitis (SSPE), in which high antibody activities can be detected in serum and cerebrospinal fluid.

Laboratory confirmation of measles infection is a critical component of the surveillance required to support measles control and elimination programs. Though determination of measles specific IgM antibodies (ELISA) is the most widely used method to confirm measles infection, suspected measles cases in highly vaccinated populations may require additional testing. Inconclusive results obtained by IgM testing can be confirmed by detection of measles RNA by RT-PCR or the finding of high concentration of measles neutralizing antibodies ( higher than 40,000 mIU/mL (WHO Second International Standard) /6/.

References

1. Orenstein WA, Perry RT, Halsey NA. The clinical significance of measles: a review. J Infect Dis 2004; 189 suppl 1: S4–S16.

2. Mina MM, Metcalf CJE, de Swart RL, Osterhaus ADME, Grenfell BT. Long-term measles induced immunomodulation increases overall childhood infectious disease mortality. Science 2015; 348: 694–9.

3. Chen RT, Markowitz LE, Albrect P, Stewart JA, Mofenson LM, Preblud SR, et al. Measles antibodies. J Infect Dis 1990; 162: 1036–42.

4. Arisra S, Ferraro D, Cascio A, Vizzi E, di Stefano R. Detection of IgM antibodies specific for measles virus by capture and indirect enzyme immunoassays. Res Virol 1995; 146: 225–32.

5. Ozanne G, D’Halewyn MA. Secondary immune response in a vaccinated population during a large measles epidemic. J Clin Microbiol 1992; 30: 1778–82.

6. Sowers SB, Rota JS, Hickman CJ, Mercader S, Redd S, McNall RJ, et al. High concentrations of measles neutralizing antibodies and high avidity measles IgG accurately identify measles reinfection cases. Clin Vaccine Immunol 2016. doi: 10.1128/CVI.00268-16.

43.41 Milker’s nodule virus

The milker’s nodule virus belongs to the Parapoxvirus genus (see Poxvirus – Section 43.55). It causes skin lesions on the udder of dairy cattle. Individuals exposed to the virus through their occupation (previously milkers) develop Poxvirus associated local lesions on the hands and lower arms. The illness is benign; usually no laboratory diagnosis is performed /1/.

References

1. Kaviarasan PK, Yamini M, Prasad PVS, Viswanathan P. Milker’s nodule. Indian J Dermatology 2009; 54: 78–9.

43.42 Merkel cell carcinoma virus

See Polyomavirus – Section 43.57.

43.43 Metapneumovirus

Family: Paramyxoviridae

Subfamily: Pneumovirinae

Genus: Metapneumovirus

Species: Human metapneumovirus (subtypes A and B)

Viral structure: see Paramyxoviridae – Section 43.51

43.43.1 Epidemiology and clinical significance

Respiratory infection with this virus is ubiquitous and has a high early prevalence in the population /1, 2/. After an incubation period of just a few days, the infection manifests clinically in a similar way to respiratory syncytial virus infection.

Refer to Section 43.60 – Respiratory syncytial virus (RSV).

43.43.2 Laboratory diagnosis

The test method of choice is virus detection by RT-PCR in nasopharyngeal swabs, sputum or broncho alveolar lavage fluid. Antibody testing is not commonly performed.

References

1. Viazov S, Ratjen F, Scheidhauer R, Fiedler M, Roggendorf M. Hifg prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the virus isolates. J Clin Microbiol 2003; 41: 3043–5.

2. Bastien N, Ward D, Caeseele PV, Brandt K, Lee SHS, McNaabb G, et al. Human metapneumovirus infection in the Canadian population. J Clin Microbiol 2003; 41: 4642–6.

43.44 Molluscum contagiosum virus

Family: Poxviridae

Subfamily: Chordopoxvirinae

Genus: Molluscipoxvirus

Species: Molluscum contagiosum virus

Viral structure: see Poxvirus – Section 43.55

43.44.1 Epidemiology and clinical significance

Molluscum contagiosum infection is transmitted through direct contact, including sexual contact. It mainly affects children and is especially common in individuals infected with HIV. In immunocompetent patients it is usually self limiting. The infection causes wart-like bumps, very characteristic, vesicular papules with an indented center (mollusca) /1/.

43.44.2 Laboratory diagnosis

The diagnosis is made visually based on clinical appearance. Poxvirus can be detected in vesicle fluid by electron microscopy or as cytoplasmic inclusion bodies in a biopsy sample by light microscopy. The most sensitive test for virus detection is PCR. Serology is of little value.

References

1. Chen X, Anstey AV, Burgert JJ. Molluscum contagiosum virus infection. The Lancet infectious diseases 2013; 13: 877–88.

43.45 Mumps virus

Family: Paramyxoviridae

Subfamily: Paramyxovirinae

Genus: Rubulavirus

Species: Mumps virus

Viral structure: see Paramyxovirus – Section 43.51

43.45.1 Epidemiology and clinical significance

Prevalence

Mumps viruses are distributed worldwide. Their only host are humans. Since the introduction of mumps vaccination, the incidence has been very low.

Transmission

Transmission occurs via droplets. The virus first replicates in the nasopharyngeal epithelium.

Clinical presentation

After an incubation period of 2–4 weeks (on average 18 days), 50–70% of patients develop the characteristic parotitis with swelling of the parotid gland (bilateral in 90% of cases) /1/. Regardless of whether or not parotitis is present, the virus is excreted in saliva from 1 week prior to until 9 days after the onset of symptoms. Although involvement of the CNS is common, meningitis develops in only 5–10% and encephalitis in only 0.1% of cases. A dreaded late complication is deafness. The risk of CNS symptoms increases with increasing age.

Up to a quarter of patients with postpubertal infection develop (unilateral) orchitis, which can lead to pressure necrosis and testicular sterility. An ovaritis usually remains undetected due to the lack of a thick fibrous capsule of the ovaries. In addition to the parotid gland(s), the pancreas may also be affected (pancreatitis). Although it has been shown that the virus can replicate in the pancreatic β cells (animal experiment), a causal relationship between Mumps virus infection and diabetes mellitus has yet to be conclusively demonstrated.

Mumps virus infection during the first trimester of pregnancy can lead to miscarriage. However, since congenital malformations have not been described, termination of pregnancy is not indicated /2/.

43.45.2 Viral serology

IgM antibodies are detectable in serum by ELISA as early as 2–3 days after the onset of symptoms and persist for 2–3 months /3/.

IgG antibodies appear in the serum (plasma) 7–10 days after the onset of symptoms and then practically persist for life. They usually indicate protection from secondary infection.

Serology shows cross reactivity with acute parainfluenza type 2 infection (case history).

43.45.3 Virus detection

The Mumps virus is not stable. Its isolation can only be successful if the sample is cooled to 4 °C. The length of culture can be shortened by antigen detection in the infected cell culture with labeled antibodies. The virus is detectable:

• In saliva: from 5 days prior to until approximately 7 days after the onset of symptoms
• In urine: for a longer period
• In cerebrospinal fluid: only shortly after the onset of illness.

RT-PCR is performed on the same samples as virus isolation, which it is increasingly replacing. Sequencing of a suitable cDNA amplicon allows to distinguish between vaccine-strain and wild-type Mumps virus infection.

Virus or genome detection is indicated if other viral causes of the parotitis, such as infections with Parainfluenza 3 virus, Coxsackievirus or Influenza A virus, can be excluded. Other reasons are absent or recurrent parotitis, and aseptic meningitis /4/.

References

1. Kutty PK, Kyae MH, Dayan GH, Brady MT, Bocchini Jr, JA, Reef SE, et al. Guidance for isolation precautions for mumps in the United States: a review of the scientific basis for policy change. Clin Infect Dis 2010; 50: 1619–28.

2. Aase JM, Noren GR, Reddy DV, St Germe, Jr J. Mumps-virus infection in pregnant women and the immunology response of their offspring. N Engl J Med 1972; 286: 1379–82.

3. Allwinn R, Zeidler B, Steinhagen K, Rohwäder E, Wicker S, Doerr HW. Assessment of mumpsvirus-specific antibodies by different serologic assays: Which test correlates best with mumps immunity? Eur. J. Clin. Microbiol. Infect. Dis. 2011; 30: 1223–8.

4. Carr MJ, Moss E, Waters A, Dean J, Jin L, Coughlan S, et al. Molecular epidemiological evaluation of the recent resurgence in mumps virus infection in Ireland. J Clin Microbiol 2010; 48: 3288–94.

43.46 Newcastle disease virus

This virus is the cause of atypical avian influenza. The infection may occasionally cross from poultry to humans who are exposed through farming, and can manifest as conjunctivitis /1/.

The laboratory diagnosis of this Paramyxovirus infection in animals is made by isolating the pathogen in cell cultures.

References

1. Nelson NJ. Scientific interest in Newcastle disease virus is reviving. J Natl Cancer Inst 1999; 91: 1708–10.

43.47 Norovirus

See Calicivirus – Section 43.11.

Human noroviruses are a group of viral agents that afflict people of all age groups. The viruses are the most causative common agent of nonbacterial acute gastroenteritis and foodborne viral illness worldwide. Molecular methods for the detection and characterization of norovirus are described in Ref. /1/.

References

1. Che H, Hu Y. Molecular diagnostic methods for detection and characterization of human noroviruses. Open Microbiol J 2016;10: 78–89.

43.48 Papillomavirus

Family: Papillomaviridae

Genus: Papillomavirus in many vertebrate species

Types: more than 100 in humans, plus numerous types in various mammals such as cattle and rabbit. Unlike other viruses, Papillomavirus is classified based on molecular (genotypes) rather than serologic typing.

Viral structure: icosahedral, stable, non enveloped virus measuring 55 nm in diameter. The genome consists of a double stranded circular DNA of 8000 base pairs, of which only one strand is transcribed. In the early region of the genome, two genes were identified whose protein products (E6 and E7) inactivate the tumor suppressor proteins p53 and pRb.

43.48.1 Epidemiology and clinical significance

Prevalence

Human Papillomaviruses (HPV) are ubiquitous dermatotropic pathogens that cause infection in humans. Apart from the classic wart, a multitude of other, more serious skin efflorescences, which proliferate as benign tumors, are associated with HPV /1/.

Clinical presentation

The most intensive workup with regard to the role of HPV has been performed for cervical carcinoma /2/. The different clinical manifestations are preferentially caused by certain of the 90 HPV genotypes. The infection is considered one of the necessary, although insufficient, conditions for malignant degeneration of the infectious lesion /3/. If additional nutritional and environmental cofactors are present, HPV infection may progress to cancer years or decades later.

Laboratory diagnosis of the characteristic warts is usually unnecessary, as it is of no additional clinical relevance. However, if malignant degeneration of the skin efflorescences is suspected, the identification or exclusion of an oncogenic type of HPV is of additional prognostic value.

Refer to: The oncogenic types are listed in Tab. 43.48-1 – HPV types and lesions.

43.48.2 Virus detection

The virus is identified by molecular diagnostic methods using in situ hybridization or PCR /4/:

• In situ hybridization in deparaffinized tissue sections or dot blot hybridization with DNA extract from biopsy material can be performed easily with labeled gene probes of commercial kits. The kits also allow the selective detection of a number of more or less oncogenic genotypes. Predominantly enzymes are used as marker molecules. The detection limit of the commercial kits is limited.
• PCR is considerably more sensitive and specific. There are no commercial tests, but suitable type specific primers are available for purchase. Genotyping is performed by sequencing selected DNA amplicons.

43.48.3 Viral serology

Serum antibody screening is of no value due to the high prevalence of HPV infection in the population. For some HP viruses such as 16 and 18, the oncogenes are well studied. The E6 and E7 proteins are used as antigens scientifically for seroepidemiologically relevant antibody diagnosis of oncogenic HPV infections, but are of no value for individual diagnosis.

References

1. Fernandes JV, De Araujo JMG, de Medeiros Fernandes TA. Biology and natural history of human papillomavirus. Dovepress 2013; 5: 1–12. doi: 10.2147/OAJCT.S37741.

2. Bosch FX, Lorincz A, Meijer CLM, Shah KV. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002; 55: 244–65.

3. Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. British Journal of Cancer 2003; 88: 63–73.

4. Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutlee F, Hildesheim A, et al. Improved amplification of genital human papillomaviruses. J Clin Microbiol 2000; 38: 357–61.

43.49 Pappataci (sandfly) fever virus

Family: Bunyaviridae

Genus: Phlebovirus

Species: Pappatacivirus

Types: Toscana virus, Sicilian virus, Naples virus.

43.49.1 Epidemiology and clinical significance

Toscana virus is found in the Mediterranean region, especially Italy, Spain and Cyprus, while Sicilian virus and Naples virus are present in the Eastern Mediterranean area and in the Middle East. The infection is transmitted by sand flies. Sand fly bite often causes severe itching. Most infections are clinically inapparent. Symptomatic disease presents similar to influenza, in severe cases with high fever, headache, and meningism /1/. The disease usually resolves completely. The virus is not known to persist.

Note: sand flies can also transmit leishmaniosis.

43.49.2 Laboratory diagnosis

The diagnosis can be made by antibody detection. Cross reactivity between the individual types is low. The level of immunity is determined by IIFT or immunoblot. In encephalitis, RT-PCR in cerebrospinal fluid provides an early positive result.

References

1. Silvas JA, Aguilar PV.The emergence of severe fever with thrombocytopenia syndrome virus. Am J Trop Med Hyg. 2017 (4): 992–996.

43.50 Parainfluenza virus

Family: Paramyxoviridae

Subfamily: Paramyxovirinae

Genus: Paramyxovirus

Types: Parainfluenza virus 1, 3 and 4; type 2 belongs to the Rubulavirus genus.

Viral structure: see Paramyxoviruses – Section 43.51.

43.50.1 Epidemiology and clinical significance

Parainfluenza viruses have 3 serotypes and are highly prevalent in human populations. They cause common colds all year round, especially in the winter /1, 2/. Type 4 is of no epidemiologic significance in Central Europe.

43.50.2 Viral serology

Complement fixation test remains the standard method for type specific serology testing. Antibody titers of 1 : 40 or higher are indicative of relatively recent infection. Cave: cross reaction between Parainfluenza 2 virus and Mumps virus. Only an early, significant rise in titer is confirmatory of infection.

43.50.3 Virus detection

For viral culture, cell cultures are available. The cytopathic effect is demonstrated by hemagglutination of added erythrocytes. Serologic typing is performed by hemagglutination inhibition using immune sera. An alternative, more rapid method is antigen detection in cell culture with labeled monoclonal antibodies.

The antigen test may also be performed directly using swabs on slides or in a micro vessel, but has to be set up and established in house. The limit of detection is low.

RT-PCR is considered the best method for direct virus detection from samples (nasopharyngeal swab, sputum, broncho alveolar lavage fluid).

References

1. Burke CW, Mason JN, Surman SL, Jones BG, Dalloneau E, Hurwitz JL. Illumination of parainfluenza virus infection and transmission in living animals reveals a tissue-specific dichotomy. PLOS Pathogens 2011. doi: 10.1371/journal.ppat.1002134.

2. Fiore AE, Iverson C, Messmer T, Erdman D, Lett SM, Talkington D, et al. Outbreak of pneumonia in a long-term care facility: antecedent human parainfluenza virus 1 infection may predispose to bacterial pneumonia. J Amer Geriatric Society 1998; 46: 1112–7.

43.51 Paramyxovirus

Family: Paramyxoviridae

Subfamilies: Paramyxovirinae, Pneumovirinae

Genera: Paramyxovirus, Rubulavirus, Morbillivirus; Pneumovirus and Metapneumovirus with many species and types

Viral structure: the virions are > 150 nm in size and are very pleomorphic. The negative stranded RNA is surrounded by a helical nucleoprotein coat, which in turn is surrounded by an envelope /1, 2/.

43.51.1 Epidemiology and clinical significance

Paramyxovirus is species specific and comprises a group of human and animal pathogens. The following species and types occur in humans: Morbillivirus with Measles virus, Paramyxovirus with Parainfluenza virus and Rubulavirus with Mumps virus (Parainfluenza 2 virus belongs to the Rubulavirus genus), Pneumovirus with Respiratory syncytial virus.

In 2001, another widely distributed Paramyxovirus was identified in humans as the cause of respiratory illness: Metapneumovirus. Important Paramyxoviruses in animals include Rinderpest virus, Canine distemper virus and, in poultry, Newcastle disease virus, which is occasionally spread to humans who are exposed through occupation.

References

1. Thibault PA, Watkinson RE, Moreira-Soto A, Drexler JF, Lee B. Zoonotic potential of emerging paramyxoviruses: knowns and unknowns. Adv Virus Res 2017; 98: 1–55.

2. Battisti AJ, Meng G, Winkler DC, McGinnes LW, Plevka P, Steven AC, et al. Structure and assembly of a paramyxovirus matrix protein. PNAS 2012; 109: 13996–14000.

43.52 Parvovirus

Family: Parvoviridae

Subfamily: Parvovirinae (Parvovirus of vertebrates)

Genera: Parvovirus, Dependovirus, Erythrovirus, Bocavirus, Amdovirus

Viral structure: measuring just 18–26 nm in diameter, Parvovirus is the smallest viral pathogen of infection in animals. The virus has a single stranded DNA of positive or negative sense, which is enclosed by an icosahedral protein capsid.

43.52.1 Epidemiology and clinical significance

The Parvovirus genus is widely distributed in the animal kingdom. In cats Parvovirus causes violent diarrheas and panleukocytopenias (Parvovirus genus).

The pathogen crossed over to dogs about 35 years ago and spread to epidemic proportions in this animal until it was contained by vaccination. In humans, Parvovirus is a rare incidental finding in stool without any pathological significance. These are Adenovirus associated Parvoviruses whose replication in cell culture depends on the presence of Herpesvirus or Adenovirus. They are therefore classified as belonging to the Dependovirus genus.

The pathogen of fifth disease (erythema infectiosum), Parvovirus B19, is a human specific Parvovirus. Parvovirus B19 infection must be considered in the differential diagnosis of hypo regenerative anemias. The target cells of this viral infection are erythroblasts. The pathogens constitute their own genus (Erythrovirus) which, as of late, also includes the PARV4 virus, whose clinical role is not well understood /1/.

In addition to canine and bovine Bocaviruses, a human pathogen of the Bocavirus genus has also been described. See Section 43.8 – Bocavirus.

References

1. Kailasan S, Agbandje-McKenna M, Parrish RC. Parvovirus family conundrum: what makes a killer?. Annu Rev Virol 2015; Nov 2 (1): 425–50.

43.53 Parvovirus B19

Family: Parvoviridae

Subfamily: Chordoparvovirinae

Genus: Erythrovirus

Species: Parvovirus B19

Viral structure: see Parvovirus – Section 43.52

Epidemiology and clinical significance

43.53.1 Epidemiology and clinical significance

Prevalence

Parvovirus B19 can be found worldwide. In Europe, infection is most common in late winter and spring and follows a cyclic pattern, with increased rates of infection occurring every 4–6 years. The prevalence of IgG antibodies as a sign of past infection increases dramatically with increasing age and is 50–80% in adults.

Transmission

Transmission is via droplets and occurs mainly during the incubation period. The virus targets erythropoietic progenitor cells in the bone marrow, where it causes a temporary decrease in erythrocyte production which, in the setting of a shortened erythrocyte life span, can manifest as acute anemia or possibly as an aplastic crisis. The incubation period is 5–10 days. Meanwhile, the viremia reaches levels of up to 10¹²/mL. The virus is excreted in nasopharyngeal secretions and urine from day 5 after infection.

Due to the high levels of viremia and antigenemia, blood plasma donated during the incubation period can potentially contaminate an entire plasma pool despite the presence of anti-B19 antibodies, and lead to infectious plasma derivatives.

Clinical presentation

Prodromal symptoms during the incubation period are similar to those of influenza. At least 20% of infections are asymptomatic /1/.

Clinical findings:

• The classic erythema infectiosum begins after a symptom free phase of up to one week and manifests in school children as a bright red rash on the cheeks ("slapped-cheek rash"). Within a few days, a characteristic maculopapular, garland shaped rash develops, which varies in intensity and can last from a few days up to several weeks before disappearing completely. This syndrome is also known as fifth disease.
• Arthralgia is usually symmetric and affects several joints, especially the small ones. The prevalence increases with patient age, and women are more often affected than men.
• Hypo regenerative anemia and reduced reticulocyte count. The infection persisting in the erythroid precursor cells can lead to aplastic anemia.
• Pregnancy: during a Parvovirus B19 infection, the virus can spread to the fetus via the placenta at any stage during the pregnancy. The possible consequences are most severe during the second trimester. In about 10% of cases, hydrops fetalis and miscarriage may occur. Malformations have not been observed. However, in most cases, women exposed to Parvovirus B19 during pregnancy deliver a healthy baby /2, 3/.
• Persistent infections are predominantly seen in immunodeficient patients. Chronic B19 virus infection may also occasionally occur in immunocompetent patients. In some patients with previous infection, the virus may persist in the bone marrow for years /4/.
• In the past few years, Parvovirus B19 infections have also been associated with a number of other diseases, such as myocarditis, but a causal relationship has not been firmly established.

Patients presenting with typical erythema infectiosum do not need laboratory testing to confirm Parvovirus B19 infection, especially if there is an epidemic.

43.53.2 Viral serology

Acute Parvovirus infection is demonstrated by seroconversion or the presence of anti-B19 IgM antibodies. The most common assays used are ELISA and IIFT.

The presence of IgG antibodies in the absence of IgM antibodies is indicative of immunity after past infection. IgM antibodies can persist for weeks.

Cave: immune complexes due to long term viremia.

The following must be observed during antenatal testing or if clinical symptoms are present:

• IgM antibodies may only be detectable for a few days
• The tests are not sufficiently sensitive to exclude acute infection.

43.53.3 Virus detection

For pregnant women who have come into contact with Parvovirus B19, genome detection by PCR in plasma or serum is recommended, in addition to serologic testing. The viremia can persist for weeks despite antibodies being produced.

Since most children are born healthy, even if the mother is found to be infected with Parvovirus B19, the developing child should be monitored with serial ultrasonography by an experienced examiner. If, due to an abnormal ultrasound finding (hydrops/anemia), an intrauterine transfusion is indicated, fetal blood can be collected for PCR testing prior to the transfusion. Generally, the more pronounced the symptoms, the higher the rate of detection of B19 by PCR. With hydrops, 3/4 of the tests are positive, with an asymptomatic fetus only 1/3.

Newborns to mothers with B19 infections are generally asymptomatic. If there are existing abnormalities (e.g. anemia), a PCR from blood or bone marrow should be performed, if possible.

The detection of DNA and IgM antibodies in the fetal blood is useful only if performed after the first week of life, due to the risk of contamination by maternal blood. Virus culture is successful only in special hematopoietic cell cultures and is performed solely for scientific purposes.

In transfusion medicine, highly viremic plasma donations are identified and excluded.

References

1. Adamson-Small LA, Ignatovich IV, Laemmerhirt MG, Hobbs JA. Persistent parvovirus B19 infection in non-erythroid tissues: possible role in the inflammatory and disease process. Virus Research 2014; 190: 8–16.

2. Jordan JA. Identification of human parvovirus B19 infection in idiopathic nonimmune hydrops fetalis. AJOG 1996; 174: 37–42.

3. Miller E, Fairley CK, Cohen BJ, Claude S. Immediate and long-term outcome of human parvovirus B19 infection in pregnancy. British J Obstet and Gynaecol 1998; 105: 174–8.

4. Curraturo A, Catalani V, Ottaviani D, Menichelli P, Rossini M, Terella D, et al. Parvovirus B19 infection and severe anemia in renal transplant recipients. The Scientific World J 2012; article ID 102829.

43.54 Picornavirus

Family: Picornaviridae

Genera: Enterovirus, Rhinovirus (humans), Aphthovirus (artiodactyla), Cardiovirus (mice), Hepatovirus (humans)

Viral structure: Picornavirus (derived from Greek “pico” = small) are among the smallest pathogens of infection, having a diameter of just 30 nm. Their genome consists of a linear, positive stranded RNA, which is enclosed by an icosahedral protein capsid composed of a small number of polypeptides.

43.54.1 Epidemiology and clinical significance

Picornavirus is widely distributed with type specific genera among humans and animals /1/. The animal viruses do not cross to humans. In veterinary medicine, Aphthovirus is of major importance as a causative agent of foot-and-mouth disease in cattle and sheep. Since Enterovirus and Rhinovirus are subject to relatively rapid variation (antigenic drift), new virus types can be expected to emerge. Some of the pathogens are very stable, so that they are transmitted via aerosols, via the fecal-oral route, and via contaminated food and drinks. Hepatitis A virus in shellfish harvested from sewage polluted water can also act as a pathogen of infection. Enterovirus and the Hepatitis A virus are transmitted via the fecal-oral route, although in most cases they do not cause any gastrointestinal symptoms. Detection of foot-and-mouth disease is based on RT-PCR test /2/.

References

1. Whitton C, Cornell CT, Feuer R. Host and virus determinants of picornavirus pathogenesis and tropism. Nature Rev Microbiology 2005; 3: 765–76.

2. Gorna K, Relmy A, Romey A, Zientara S, Blaise-Boisseau S, Bakkali-Kassimi L. Establishment and validation of two duplec one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease. J Virol Methods 2016; pii: SO 166-0934(15)30074-4.

43.55 Poxviruses

Family: Poxviridae

Subfamily: Chordopoxvirinae

Genera: Orthopoxvirus (human and primate Poxviruses), Molluscipoxvirus (molluscum contagiosum), Parapoxvirus (milker’s nodule virus), and numerous other types in various mammals.

Viral structure: the virus particles are brick shaped or ovoid, with external dimensions of 220–450 nm (length) × 140–260 nm (width/depth). The outer membrane contains lipids and globular proteins. In total, more than 100 different polypeptides have been identified in the Poxvirus. The dsDNA has a molecular weight of (86–250) × 10⁶ kDa with 130–375 kbp. This largest known animal virus can just be seen with a light microscope.

43.55.1 Epidemiology and clinical significance

Prevalence

The once feared human specific pathogen of true pox is considered eradicated as a result of a systematic global immunization program. New fears have emerged over the risk of bioterrorist activities using existing laboratory strains. The risk assessment of this virus in terms of the likelihood of an outbreak is controversial. Since infected individuals are not contagious during most of the incubation period (1–3 weeks, on average 12 days), quarantine measures and post exposure immunization can be used.

Clinical presentation

The disease (variola major/smallpox) begins with high fever, head and body aches, vomiting, and severe fatigue. Petechial rashes in the inguinal and axillary area are pathognomonic. Approximately 3–5 days after the onset of illness, the true smallpox efflorescences appear as a uniform, focal rash on the skin and mucosa, with lesions evolving from macules to papules and then to virus containing vesicles, from where the pathogen spreads. The main risk of infection is transmission via saliva droplets from vesicles of the respiratory tract mucosa. There also is a risk of developing hemorrhagic pneumonia. Approximately 12 days after the rash appears, the pustules dry up and crust and scab formation begins, if the disease does not end fatally (approximately 25%). The differential diagnosis is varicella (chickenpox), in which the efflorescences are multiform /1/.

Chemotherapy may be attempted with the phosphono- nucleoside cidofovir. Due to relatively common and serious complications, vaccination with the classic vaccinia virus should occur only during active disease and primarily as post exposure immunization to halt infection chains.

Numerous other Orthopoxviruses circulate widely among various animal species. They can occasionally spread to humans and cause a smallpox-like illness. This includes in particular monkeypox and camel pox. Monkey pox occurs primarily in West Africa. Contrary to its name, the natural reservoir hosts of the virus are not monkeys, but rodents, from which the infection occasionally spreads to monkeys and humans via the fecal-oral route, through inhalation of aerosolized excretions and feces.

Monkeypox infections first occurred outside Africa in 2003, when several individuals in Wisconsin and Illinois, USA, contracted a milder form of smallpox and had to be given in-patient treatment. The outbreak was traced to prairie dogs that were sold as pets by a dealer where the dogs were housed with an infected Gambian giant rat imported from West Africa. No human-to-human transmission was found.

43.55.2 Laboratory diagnosis

If (variola major) smallpox /2/ is strongly suspected, it must be ensured that the infection cannot be spread by the patient or through the sample.

The virus can be detected rapidly from vesicles or scabs with electron microscopy or PCR. Using PCR, Orthopoxvirus can be reliably distinguished from Parapoxvirus. Further information can be obtained from the Robert Koch Institute in Berlin and the Bernhard Nocht Institute for Tropical Medicine in Hamburg.

43.55.3 Virus detection

Stool sample

If poliomyelitis is suspected (reportable), the diagnostic method of first choice is a stool sample test. The enterovirus continues to be replicated and excreted in the feces for several weeks after the onset of infection. Due to the stability of the virus, conservation of the stool sample is not required.

CSF sample

This usually only contains small amounts of virus. For transportation of the sample to the laboratory, a continuous refrigeration chain (4 °C) must be guaranteed.

Throat swab/gargle

During the early stage of infection, the virus may also be detected in the pharynx.

Blood, serum sample

During the acute stage of illness, the viral genome can be detected by RT-PCR /3/.

For virus detection in the stool sample (which is usually not diarrheic, as gastrointestinal problems are rare), electron microscopy is the easiest, most rapid and least costly method. A much more sensitive method is culture of the virus in cell cultures (monkey kidney, amnion), which can be accomplished easily, but takes several days to give a positive result. The RNA viral genome can be detected by RT/PCR within a couple of hours. RT/PCR generally uses primers which target all human pathogenic Enteroviruses. Enterovirus PCR is also commercially available. It is the preferred method of CSF enterovirus testing. Isolation of the infecting virus can be even more sensitive, provided that optimal transport of the material is ensured.

The isolated virus can be typed with type specific immune sera or monoclonal antibodies in the neutralization assay. It takes one week for the result to be available (almost two weeks from receipt of the material). By contrast, the sequencing of a PCR product allows typing within 2 days.

Before oral (live) polio vaccination was abandoned, the vaccination history always had to be obtained, since the attenuated vaccine viruses also can be excreted for weeks.

Wild-type and vaccine viruses can be typed and differentiated most accurately by nucleotide sequencing of a cDNA PCR amplicon in special laboratories. Failure to detect the virus in three separately obtained stool samples indicates that no (relatively recent) Poliovirus infection is present.

43.55.4 Viral serology

The diagnosis of exclusion of Poliovirus infection can be made by serology (negative antibody test) /4/. The most reliable method is the demonstration of neutralizing antibodies with a cell culture virus neutralization assay, also with regard to determining the type specific immune status. However, readings cannot be made until after 5–7 days’ incubation.

A rapid method of screening for Enterovirus is the detection of IgM. Due to the lack of sensitivity and specificity, enterovirus PCR on a stool or serum sample is preferable.

43.55.5 Monkey pox

Monkey pox virus belongs to the family poxviridae subfamily chordopox virinae, and genus orthopoxvirus. The genus encompasses many other poxviruses as well as more recently isolated poxviruses. Monkey pox affects both children and adults and has typically the phases incubation 13 days (range 3 to 34), prodrome (1 to 4 days with high temperature headache, fatique and often lymphadenopathy, especially in the cervical and axillary region), and the eruptive state (lasts 14 to 28 days with clinical skin signs, in centrifugal distribution are macules, papules, vesicles, and finally pustules). The most frequent complications are cutaneous bacterial infections /3/.

In Africa both animal-to-person and person to person transmission have been documented. However, monkey pox virus should be considered a sexually transmitted infection. The virus can be isolated from semen and rectal and oral fluids and most patients have been presented with anogenital or oropharyngeal lesions , frequently in association with other sexual transmitted infections /4/.

Vaccination against smallpox generally provides some protection against monkeypox.

References

1. McFadden G. Poxvirus tropism. Nature Rev Microbiology 2005; 3: 201–13.

2. Breman JG, Henderson DA. Diagnosis and management of smallpox. N Engl J Med 2002; 346: 1300–8.

3. Gessain A,Nakoune E.Yazdan Y. Monkeypox. N Engl J Med 2022; 387: 1783–93.

4. Golden MR, Wasserheit JN. Monkeypox – a sobering sentinel for pandemic preparedness and sexual health system capacity N Engl J Med 2022; 387: 1826–7.

43.56 Poliovirus

Family: Picornaviridae

Genus: Enterovirus

Type: Poliovirus 1, 2 and 3, see also Enterovirus – Section 43.18

Viral structure: see Picornavirus – Section 43.54

43.56.1 Epidemiology and clinical significance

Poliovirus is the causative agents of poliomyelitis (infectious infantile paralysis).

Prevalence

As a result of a systematic global vaccination campaign, poliomyelitis has been largely eradicated. In 2010, poliomyelitis cases caused by Poliovirus types 1 and 3 were reported in the tropical and subtropical regions of Nigeria through to India. Poliovirus 2 has been eradicated. The WHO is currently undertaking great efforts to eradicate the other two poliovirus types, too /1/.

Transmission

The virus spreads through the fecal-oral route and saliva droplets. It has an incubation period of only a few days.

Clinical presentation

In most cases, the enteral infection runs a subclinical course or presents as a minor illness with fever and a sore throat /2/. If the disease progresses to major illness, the virus spreads from the intestinal tract, where it replicates well in the epithelial cells, via the bloodstream to the brain or to a myelomere. The spinal cord infection affects the motor ganglion cells of the anterior horn. If the irreversible damage to the nerve cells cannot be compensated, persistent paralyses and atrophies develop in the skeletal muscles innervated by this myelomere. Particularly feared are the failure of the auxiliary respiratory muscles of the chest or diaphragm, and the often fatal polioencephalitis. Before vaccination was introduced, there was a considerable increase in the sickness rate, although the neurotropic infection has a manifestation index of only 1–2%. To differentiate poliovirus from other enterovirus infections, see Tab. 43.14-1 – Clinical spectrum of enterovirus infections.

In the polio free countries, the inactivated vaccine has replaced the three attenuated poliovirus vaccine serotypes (oral poliovirus vaccine). Although the latter builds up immunity in the population better and easier, it carries the risk of vaccine associated poliomyelitis due to reversion of the mutation to the wild-type virus (incidence of 1 : 1 million to 1 : 5 million doses of vaccine).

43.56.2 Virus detection

Stool sample

If poliomyelitis is suspected (reportable), the diagnostic method of first choice is a stool sample test. The enterovirus continues to be replicated and excreted in the feces for several weeks after the onset of infection. Due to the stability of the virus, conservation of the stool sample is not required.

CSF sample

This usually only contains small amounts of virus. For transportation of the sample to the laboratory, a continuous refrigeration chain (4 °C) must be guaranteed.

Throat swab/gargle

During the early stage of infection, the virus may also be detected in the pharynx.

Blood, serum sample

During the acute stage of illness, the viral genome can be detected by RT-PCR /3/.

For virus detection in the stool sample (which is usually not diarrheic, as gastrointestinal problems are rare), electron microscopy is the easiest, most rapid and least costly method. A much more sensitive method is culture of the virus in cell cultures (monkey kidney, amnion), which can be accomplished easily, but takes several days to give a positive result. The RNA viral genome can be detected by RT/PCR within a couple of hours. RT/PCR generally uses primers which target all human pathogenic Enteroviruses. Enterovirus PCR is also commercially available. It is the preferred method of CSF enterovirus testing. Isolation of the infecting virus can be even more sensitive, provided that optimal transport of the material is ensured.

The isolated virus can be typed with type specific immune sera or monoclonal antibodies in the neutralization assay. It takes one week for the result to be available (almost two weeks from receipt of the material). By contrast, the sequencing of a PCR product allows typing within 2 days.

Before oral (live) polio vaccination was abandoned, the vaccination history always had to be obtained, since the attenuated vaccine viruses also can be excreted for weeks.

Wild-type and vaccine viruses can be typed and differentiated most accurately by nucleotide sequencing of a cDNA PCR amplicon in special laboratories. Failure to detect the virus in three separately obtained stool samples indicates that no (relatively recent) Poliovirus infection is present.

43.56.3 Viral serology

The diagnosis of exclusion of Poliovirus infection can be made by serology (negative antibody test) /4/. The most reliable method is the demonstration of neutralizing antibodies with a cell culture virus neutralization assay, also with regard to determining the type specific immune status. However, readings cannot be made until after 5–7 days’ incubation.

A rapid method of screening for Enterovirus is the detection of IgM. Due to the lack of sensitivity and specificity, enterovirus PCR on a stool or serum sample is preferable.

References

1. Hovi T, Shulman LM, van der Avoort H, Deshpande J, Roivainen M, de Gourville EM. Role of environmental poliovirus surveillance in global polio eradication and beyond. Epidemiol Infect 2011; pages 1–13. doi: 10.1017/s095026881000316x.

2. Tebbens RJ, Pallansch MA, Chumakov KM, Halsey NA, Hovi T, Minor PD, et al. Expert review on poliovirus immunity and transmission. Risk Anal 2013; 33: 544–605.

3. Rossomando RF, White L, Ulfelder KJ. Capillary electrophoresis: separation and quantitation of reverse transcriptase polymerase chain reaction products from polio virus. J Chromatography B: Biochemical Sciences and Applications 1994; 656: 159–68.

4. Buxbaum S, Berger A, Preiser W, Rabenau H, Doerr HW: Enterovirus infections in Germany: Comparative evaluation of different laboratory diagnostic methods. Infection 2001; 29: 138–42.

43.57 Polyomaviruses

Family: Polyomaviridae

Genus: Polyomavirus in various mammals

Species: Human polyomavirus

Types: BK, JC, Ki and Wu, Merkel cell polymomavirus (MCV)

Viral structure: polyomaviruses are DNA viruses with a circular double-stranded genome consisting of 5300 base pairs. Both DNA strands are transcribed. The genome is contained within an icosahedral naked capsid measuring 40–45 nm.

43.57.1 Epidemiology and clinical significance

Polyomaviruses comprise five serotypes that are highly prevalent in humans. Infection with the JC, BK and MCV types is generally apathogenic. In immunosuppressed or immunocompromised individuals, the JC virus can cause progressive multi focal encephalopathy /1/, which is usually fatal unless the immune system can be reconstituted in time. The BK virus has been described as the cause of hemorrhagic cystitis in such patients, although the extent of viruria does not correlate with the symptoms. The BK virus can cause tubular nephritis with persistence of the virus and lead to problems in kidney transplant recipients. Two other polyomavirus types (Ki and Wu) cause upper respiratory tract infections in children and must be considered in the differential diagnosis (Coronavirus, Metapneumovirus, Parainfluenzavirus, RS virus) of common colds.

A primate Polyomavirus (simian virus 40) has attained great importance in experimental tumor research due to the viral tumor antigen /2/. In humans, only the MCV causes Merkel cell carcinoma of the skin.

43.57.2 Laboratory diagnosis

The detection of antibodies in serum is of no value. The JC virus is detected in cerebrospinal fluid using a non-commercial PCR assay. A positive result is pathognomonic.

The BK virus can also be detected by an in-house PCR assay /3/. A positive qualitative result is of little informative value, because the pathogen is also found in patients without hemorrhagic cystitis. By contrast, quantitative monitoring of the viral load in urine with reduction of immunosuppression at repeated levels > 10⁴ GEq/mL is recommended. The detection of viruria by electron microscopy is less sensitive, but usually pathognomonic. The presence of decoy cells in urine is also relevant to diagnosis.

Ki and Wu viruses are detected by PCR assay on a throat swab, sputum, and broncho alveolar lavage fluid. Primers and gene probes can be ordered online. The tests must be established and validated in-house.

The Merkel cell polymomavirus is detected by PCR on skin swabs of healthy individuals and individuals with Merkel cell carcinoma, where it is found in higher concentrations and integrated within the DNA of the cancer cells.

References

1. Gordon J, Khalili K. The human polyomavirus, JVC, and neurological diseases Int J Mol Med 1998; 1: 647–55.

2. Dalianis T, Hirsch HH. Human polyomaviruses in disease and cancer. Virology 2013; 437: 63–72.

3. Elfaitouri A, Hammarin Al, Blomberg J. Quantitative real-time PCR assay for detection of human polyomavirus infection. J Virol Methods 2006; 135: 207–13.

43.58 Prions

Classification: unconventional subcellular agents

Structure: amyloid fibrils on nerves and other cells

43.58.1 Epidemiology and clinical significance

Prions (proteinaceous infectious organisms) are special subcellular infectious agents that lack a nucleic acid genome. They are composed entirely of a conformational variant of a physiological asialoglycoprotein, which has been shown, by animal experiments of no essential importance, to be present on the membrane of many cells of the body and in particular of the nervous system /1/. Homozygous mutations of the cell gene PrP on chromosome 20 cause, and heterogenous mutations promote, a conformational change, which inhibits the metabolic process (Fig. 43.58-1 – Mutations in the human PrP gene as the cause of familial spongiform encephalopathies).

This results in fibrillar amyloidosis and spongy degeneration of the brain tissue (spongiform encephalopathy), which are clearly distinguishable from other cerebral amyloidoses such as Alzheimer’s disease. By coming into contact with the infectious conformation of a prion, the physiological protein also undergoes the pathologic conformational change. A genetic metabolic disease thus becomes infectious.

Creutzfeldt Jakob disease (CJD) /2/ has long been known as a typical human prion disease affecting the CNS. It has a reported incidence of approximately 1 : 1 million population per year. In the animal kingdom, sheep and goats (scrapie) have been known for centuries to be carriers of similar pathogens. All prion diseases are characterized by a very long incubation period (years to decades) and an irreversibly fatal course once symptoms have begun.

Refer to Tab. 43.58-1 – Spongiform encephalopathies in humans and animals.

In 1986, a spongiform encephalopathy found in cattle in Great Britain spread to epidemic proportions /3/. This bovine spongiform encephalopathy (BSE), commonly known as mad cow disease, has so far led to the death of more than 180,000 cattle. The number of infected animals is estimated at 1 million, half of which ended up as meat for human consumption. Outside Great Britain and Ireland the rates of infection are lower by several powers of ten. In Germany, approximately 300 infected cattle have to date been identified by laboratory testing. It is believed that the infectious agent of BSE either derives from a mutated prion strain of sheep [PrP sc(rapie)] or was selected out as a previously undetected agent through factory farming and non biologic feeding practices. In Great Britain, a special type of cattle feed in the form of meat and bone meal rendered from the carcasses of slaughtered sheep had been developed and used. Due to energy saving measures, this meat and bone meal had not been adequately heat sterilized since 1982. At the end of 1987, the production and use of meat and bone meal from animal carcasses was prohibited. Since then the BSE epidemic has decreased dramatically.

In 1996, several cases of a new form of human CJD were reported in Great Britain. This form of the disease, known as v(ariant) CJD, differs from classic CJD in that it causes changes in the brain tissue that are more similar to BSE. Analysis of this PrPcjd from biopsied brain tissue of deceased individuals shows the same glycosylation pattern as that of PrPbse of cattle. To date, there have been about 220 fatal cases of vCJD, most of them in Great Britain. The latest projections indicate that a CJD epidemic is unlikely, even in Great Britain, since the incidence of vCJD is decreasing, although there have recently been several cases of transmission by blood donors who later became ill.

43.58.2 Laboratory diagnosis

At present, no diagnostic test exists for the detection of specific prion pathogens in live animals or humans. The amyloid fibrils are demonstrated by post mortem histological detection. Animal species specific prions can be visualized by a special immunoblot assay. This method helped confirm the relationship between the pathogen of BSE and the causative agent of vCJD. The prion can also be analyzed and characterized genetically (mutations in the amino acid sequence)using experimental animals (i.e., hamsters experimentally infected with prions). All tests are performed by special veterinary medical institutes.

Refer to Fig. 43.58-1 – Mutations in the human PrP gene as the cause of familial spongiform encephalopathies.

In human medicine, surrogate markers of the disease have been described for CSF diagnosis. These are neuronal proteins released during the disease process, such as enolase, p130/131, and especially p14-3-3. The tests are performed with two dimensional gel electrophoresis and ELISA. These methods are not suitable for diagnosing infection, but should only be used for differentiating prion diseases from other CNS disorders.

Anti-JCV antibody status is used for progressive multi focal leukoencephalopathy (PML) risk stratification in multiple sclerosis (MS) patients before and during natalizumab therapy. JCV antibodies are detected in approximately 60% of MS patients, however, only a small proportion actually develops PML. The JCV antibody response in MS patients seems to be largely independent of any anti-viral immunity /4/.

References

1. Aguzzi A, Nivolone M, Zhu C. The immunobiology of prion diseases. Nature Review Immunology 2013; 13: 888–902.

2. Ironside JW. Creutzfeldt-Jacob disease. Brain Pathol 1996; Oct 6 (4): 379–88.

3. Lee J, Kim SY, Hwang KJ, Yu JR, Woo HJ. Prion diseases as transmissible zoonotic disease. Osong Public Health and Research Perspectives 2013; 4: 57–64.

4. Auer M, Borena W, Laer DH, Deisenhammer F. Correlation between anti-JCV-virus and anti-cytomegalovirus, -Epstein-Barr-virus and -measles/-rubella/-varicella-zoster-virus antibodies. J Med Virol 2016 Jun 2. doi: 10.1002/jmv.24590.

43.59 Reoviruses

Family: Reoviridae

Genus: Orthoreovirus. There are 14 additional genera in animals and humans, including Rotavirus (see Section 43.64 – Rotavirus).

Viral structure: Reovirus is composed of a special tree-layered icosahedral capsid without an envelope. It contains a dsRNA, which consists of ten segments. The virus particle is 70 nm in diameter.

43.59.1 Epidemiology and clinical significance

Reoviruses are a large group of pathogens that cause infection in the respiratory and enteric tracts /1/. They often cannot be associated with a disease (orphan). The Orthoreovirus is very stable outside the cell and is transmitted via the fecal-oral route and via droplets. Its clinical relevance in immunocompetent individuals is debatable.

43.59.2 Laboratory diagnosis

The pathogen is detected by electron microscopy of infected samples (stool, gargle). Virus isolation in cell cultures or RT-PCR are performed only for scientific purposes. The same applies to antibody testing.

References

1. Danthi P, Guglielmi KM, Kirchner E, Mainou B, Stehle T, Dermody TS. From touchdown to transcription: the reovirus cell entry pathway. Curr Top Microbiol Immunol 2010; 343: 91–119.

43.60 Respiratory syncytial virus (RSV)

Family: Paramyxoviridae

Subfamily: Pneumovirinae

Genus: Pneumovirus

Types: A and B

Viral structure: see Paramyxovirus – Section 43.51.

43.60.1 Epidemiology and clinical significance

Most people have been infected with the virus by age 2. There is no animal reservoir for the virus. RSV spreads through droplets. It causes annual winter outbreaks of respiratory tract illness and usually remains subclinical.

A high risk group of severe RSV disease are individuals of extreme age with progressively reduced immune immunity /1/. Lower respiratory tract infections with RSV are especially associated with high morbidity and mortality in allogenic hematopoietic cell transplant recipients. In lung transplant recipients RSV infections are associated with the bronchiolitis obliterans syndrome /2/.

Although most RSV infections remain restricted to the upper respiratory tract and are associated with relatively mild clinical signs, approximately 0.5–2% of primary infections in infants below six months of age result in severe bronchiolitis or pneumonia requiring hospitalization /3/. Young infants with a history of premature birth and those with congenital heart or lung disease have an increased risk of developing severe disease upon primary RSV infection

The virus has an incubation period of only a few days. Type A strains are more aggressive than those of type B. Immunity after RSV infection does not protect against all types of the virus and only lasts for 1–2 years. Therefore, reinfections are common. It has been hypothesized that an allergic component may play a role in the pathogenesis.

43.60.2 Virus detection

The virus can be detected in cell culture with the eponymous cytopathic effect or, more rapidly and routinely, from a nasal or throat swab or gargle with the commercial antigen test (ELISA).

RT-PCR, in comparison, is considerably more laborious, but allows the identification of infection chains by subsequent cDNA sequencing. This is important with regard to nosocomial infections, which are relatively common.

43.60.3 Viral serology

RSV infection can also be diagnosed by the detection of serum antibody, which remains detectable for 1–2 weeks after the onset of illness.

References

1. Falsey AR, McElhanney JE, Beran J, van Essen GA, Duval X, Esen M, et al. Respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness. J infect Dis 2014; 209: 1873–81.

2. Kim YJ, Guthrie KA, Waghmare A, Walsh EE, Falsey AR, Kuypers J, et al. Respiratory syncytial virus in hematopoietic cell transplant recipients: factors determining progression to lower respiratory tract disease. J Infect Dis 2014; 209: 1195–1204.

3. Stittelaaar KJ, de Waal L, van Amerongen G, Veldhuis Kroeze EJB, Fraaij PLA, van Baalen CA, et al. Ferrets as a novel animal model for studying human respiratory syncytial virus infections in immunocompetent and immunocompromized hosts. Virus 2016; 8: 168. doi: 10.3390/v8060168.

43.61 Retroviruses

Family: Retroviridae

Genus: Lentivirus in humans (HIV), primates (SIV), sheep/goats (Visna/Maedi), cats (FIV). Deltaretrovirus with HTLV/BLV in humans and cattle. Five additional general in the animal kingdom, including highly and weakly oncogenic retroviruses.

Viral structure: enveloped, spherical particles with a conical (Lentivirus) or round core (nucleocapsid), which contains two copies of a single stranded positive sense RNA as a diploid genome and a viral polymerase (reverse transcriptase). The virus measures 80–100 nm in diameter.

43.61.1 Epidemiology and clinical significance

Retroviruses are a family of viruses with a diploid RNA genome that are widely distributed in animals. The genome is transcribed into a double stranded DNA by a viral RNA dependent DNA polymerase (reverse transcriptase/pol) and an RNase H, and integrated into the host cell genome, catalyzed by the viral integrase. The genome is surrounded by a protein capsid (gag) and an envelope (env). After budding from the cell membrane, the virus particles mature into virions, mediated by the viral protease.

Several genera exist, which are either oncogenic (Onco-cornavirus, human HTLV) or cause lytic cell infection (human HIV). The latter, through interplay with the proviral latency that is characteristic of retroviruses, can lead to a slow virus disease. These pathogens belong to the Lentivirus group of.

Sheep, goats and horses have long been known as animal carriers of the virus, with the infection preferably manifesting in the CNS (encephalitis). In humans, we know the retrovirus HIV (human immunodeficiency virus) with two types. Subtypes have been described, especially for type 1, which are numbered alphabetically and vary in their geographic distribution /1/. The analog of HIV is Simian immunodeficiency virus (SIV) in primates.

SIV infection in the natural host species is usually subclinical, which indicates a long coevolution. Phylogenetic research suggests that HIV-1 crossed to humans from chimpanzees and HIV-2 from sooty mangabeys during the first half of the last century. The known human Oncocornaviruses are human T-cell leukemia viruses 1 and 2.

References

1. Stoye J. Studies of endogenous retroviruses reveal a continuing evolutionary saga. Nature Rev Microbiology 2012; 10: 395–406

43.62 Rhinovirus

Family: Picornaviridae

Genus: Rhinovirus

Species: Rhinovirus A, B and interim species with numerous types.

Viral structure: see Picornavirus – Section 43.54

43.62.1 Epidemiology and clinical significance

Rhinoviruses have 101 serotypes (i.e. there is no cross immunity) and are strictly host specific to humans (although bovine Rhinoviruses also exist) /1/. The Rhinovirus accounts for more than half of the upper respiratory tract infections.

43.62.1.1 Common cold

The clinical symptoms of Rhinovirus infection are known as the common cold. Cold symptoms usually develop 1–3 days after being exposed to the virus and resolve within 5–7 days. The infection remains localized in the mucosa and confers limited IgA mediated humoral immunity. Symptoms include nasal stuffiness sneezing, coughing and a sore throat. About 12–32% of Rhinovirus infections in children of less than 4 years are asymptomatic. Rhinovirus infection on top of chronic obstructive pulmonary disease (COPD), asthma, or cystic fibrosis might become life threatening /2/.

43.62.2 Laboratory diagnosis

The virus can easily be cultured from nasal swabs in cell cultures at 32 °C, but usually no laboratory investigation is required. Electron microscopy cannot distinguish between Rhinovirus and other Picornaviruses.

References

1. Jacobs SE, Lamson DM, George KSt, Walsh TJ. Human rhinoviruses. Clin Microbiol Rev 2013; 26: 135–62.

2. Blaas D, Fuchs R. Mechanism of human rhinovirus infections. Molecular and Cellular Pediatrics 2016; 3: 21. doi: 10.1186/s40348-016-0049-3.

43.63 Rubella virus

Family: Togaviridae

Genus: Rubivirus

Species: Rubella virus

Viral structure: see Togavirus – Section 43.65

43.63.1 Epidemiology and clinical significance

This infectious agent causes a characteristic viral disease in children, which is usually benign.

Clinical presentation

Approximately 2–3 weeks after infection through droplets, a flu-like syndrome develops, which presents with nuchal lymphadenopathy and occasionally with a characteristic rash which, like measles, has an immunopathogenic etiology. The differential diagnosis of rubella should include EBV, adenovirus and enterovirus infection. In some cases, Rubella infection may go unnoticed /1/. Unlike measles, neurologic sequelae are rare (subacute sclerosing rubella pan encephalopathy, SSRPE). Approximately 80% of the population have been infected by wild type or vaccine strain Rubella virus by young adulthood.

Pregnancy

While Rubella infection is generally rare in adults, seronegative women of childbearing age can contract primary Rubella infection by disproportionately frequent contact with infants /2, 3/.

The virus can be transmitted vertically to the fetus at any time during the pregnancy. During embryogenesis, the infection can cause malformations by arrest of development, leading to Rubella embryopathy. Rubella was first described in 1942 by the Australian ophthalmologist Norman Gregg as a triad of cataracts (blindness), sensorineural hearing loss and heart defects (Gregg’s triad). In addition, numerous other CNS and organ related defects have been described.

Fetopathies are, however, much less common than embryopathies. The rather low cytotoxicity of the virus can lead to miscarriage only during early pregnancy. The likelihood of the mother giving birth to a live, deformed child was estimated based on the evaluation of two major epidemics (1962/63 in the USA, 1978/79 in England) with the following results:

• In the 1st month of embryonic development (gravidity): 60–80%
• In the 2nd month: 30–40%
• In the 3rd month: 15–20%
• During fetal age: 10–15%.

Rubella infection during or after the 17th week of pregnancy is no longer considered an eugenic indication for abortion. According to a 16-year review of seroprevalence studies on Rubella the seroprevalence ranged from 53 to 99.3%. The review highlighted that infants lost maternally acquired immunity within 9 months of birth and were unprotected until vaccination /4/.

Subclinical infections in pregnant women

According to the evaluation of the epidemic in England, subclinical infections are rarely transmitted to the fetus with pathogenic consequences. This is consistent with the observation that secondary Rubella infections, which are usually asymptomatic, are harmless to the fetus. However, a few cases have been reported in which the immune protection that had been built up by vaccination with live attenuated vaccine viruses was overcome. Accidental infections with attenuated Rubella vaccine virus during pregnancy have not been associated with defects in the newborn. Nevertheless, Rubella vaccine is contraindicated during pregnancy. If Rubella is detected during routine prenatal testing, it must always be treated the same, regardless of whether there are symptoms or not.

43.63.2 Viral serology

Several well established and standardized test methods exist for antibody detection in serum or other body fluids (e.g., cerebrospinal fluid) to investigate an acute infection or assess the immune status /5, 6/.

The most commonly used test is the ELISA, which quantifies the individual immunoglobulin classes (IgG, IgM and IgA). The first antibodies to appear after the incubation period are those of the IgM class, which may be present with or without pronounced symptoms. The antibodies usually disappear 3 to 8 weeks after the onset of illness or fall to low levels (below 2 × threshold), at which they can persist for months. This is observed especially after vaccination. Only elevated test results (> 3 × threshold) indicate acute primary infection. Low levels of IgM can result from recent infection or vaccination. They are, however, also seen in reinfections (which are usually subclinical and harmless in terms of perinatal risk).

The indication for pregnancy termination should be made based on double testing, ideally using two different test modifications (sandwich and anti-μ techniques). Low IgM levels in the presence of indefinite symptoms should be further investigated with paired IgG serological testing or with IgG avidity testing. The presence of reinfection versus primary infection is best confirmed with a serum sample obtained well before the pregnancy.

The specific detection of IgM antibodies in venous cord blood or serum of the newborn confirms prenatal infection, while IgG antibodies detected may be maternal antibodies transferred through the placenta. Cord serum IgM levels usually exceed the upper reference interval of 0.2 g/L.

Depending on the amount or quality of the antigen used (whole virus, core protein, E2 glycoprotein), IgG can be detected shortly after or not until 3–6 weeks after IgM antibodies. Commercial test kits must include instructions as to whether all antibodies or only the high avidity antibodies that appear later are to be detected. Acute primary infection can be detected and a positive IgM test can be confirmed by a significant rise in Rubella IgG antibody titer between acute and convalescent phase sera. The significant (i.e., at least 4-fold) rise in antibody titer within 2–3 weeks cannot distinguish between primary and reinfection, if Rubella antibodies were already detected in the first blood sample.

A high positive IgG indicates prior infection or immunization (at some time in the past), and immunity. Depending on the test kit, the threshold is usually 10 or 15 IU/mL. As a precaution, immunity should only be assumed if Rubella IgG levels are at least 2-fold the threshold level (20 or 30 IU/mL). Serial testing shows that titers range from 10 to > 500 IU/mL. A single antibody result is therefore insufficient to determine an individual’s infectious status. A very high positive IgG in the absence of IgM is highly suggestive of recent reinfection.

Fig. 43.63-1 – Antibody titers and virus detection during the course of a rubella infection shows the kinetics of antibody production and antibody persistence. Because immunity to Rubella is very important for pregnancy, the blood samples of women of childbearing age should be retained for an extended period of time.

Hemagglutination inhibition (HI) assay

Antibody detection with the HI assay is still used for diagnostic serology, to determine the immune status and to assess the protective effect of antibody preparations.

Test kits and individual reagents are both commercially available. The antigen is the viral hemagglutinin from the envelope, which is responsible for adsorption of the virus to the target cell of the infection. The HI assay thus serves as an approximate neutralization method. By contrast, the ELISA IgG antibodies can only indicate past infection or vaccination.

The HI assay detects all acute and convalescent phase Rubella antibodies. Thus, the humoral immune response can be detected immediately after the onset of illness or after the incubation period. A significant (at least 4-fold) rise in titer between two serum samples collected a few days apart is confirmatory of acute infection, if both serum samples are tested in parallel. The rise in titer can, however, not distinguish a secondary reinfection if the first serum sample is already positive. Low antibody activities also indicate a current or past (vaccine) virus infection which, based on clinical experience, leads to long term immunity. However, lipoproteins in serum of a lower dilution (less than 1 : 32) can also inhibit the test virus hemagglutinin and simulate antibodies.

Low antibody activities therefore need to be verified by another method, e.g. ELISA or hemolysis-in-gel test (HIG).

Overall, the HI titers must be interpreted as follows:

• A titer of 1 : 32 or a threshold titer specified by the manufacturer indicates humoral immunity, provided there are definitely no clinical signs of recent infection. If acute infection cannot be definitely ruled out, an IgM test is indicated. For this, the IgM fraction must be isolated from the serum sample. As an alternative, special IgM HI assays were developed. The IgM HI assay is usually only positive in acute primary infection (rarely in secondary infection).
• When determining the immune status, a low HI titer must be confirmed with a second IgG test, generally an ELISA (or HiG), by demonstrating Rubella IgG levels of at least 20 IU/mL (observe manufacturer’s instructions). If levels are less than 10 IU/mL, the overall result is negative. In between lies an indeterminate range, in which retesting after 6 months can bring clarity, provided that no immediate decisions have to be made.

Hemolysis-in-gel (HiG) test

Like most IgG specific ELISA, this test uses relatively small amounts of viral antigen and detects only higher-avidity antibodies, which often appear only 3–6 weeks after the onset of disease or after the incubation period. The HiG test cannot be automated and is more difficult to standardize. The advantage of this test is that it only becomes positive a long time after the infection and then unequivocally indicates immunity. Conversely, a still negative HiG test in the presence of a positive HI assay and/or ELISA is highly suggestive of an acute or recent infection.

Immunoblot and antibody avidity test

In the immunoblot, the detection of antibodies to the E2 antigen correlates with immunity, while the absence of anti-E2 antibodies in the presence of a positive HI assay and/or ELISA correlates with recent infection. Similarly, low avidity antibodies correlate with recent infection, high avidity antibodies with remote or secondary infection. The measurement of antibody avidity is performed by ELISA.

43.63.3 Virus detection

The pathogen can easily be detected in the urine of newborns, who excrete high levels of the virus for months. By contrast, virus detection in throat secretions or EDTA blood samples often is of little value. The non cytopathic virus must be demonstrated in the infected culture cells with an antigen test, usually using the immune peroxidase technique. This test takes 24–48 hours from material receipt. RT-PCR can also be used for highly sensitive detection of Rubella virus RNA. The test takes only 2–3 hours. During early pregnancy, both virus isolation and genome detection are possible from amniotic fluid and chorionic villus samples. The material to be used is selected based on pregnancy status and length of time since symptoms presented. During late pregnancy, IgM antibody detection from fetal blood can also be performed.

References

1. Drutz JE (ed). Rubella. Pediatrics in Review. 2010; 31: issue 3.

2. Simons EA, Reef SE, Cooper LZ, Zimmerman L, Thompson KM. Systematic review on the manifestations of congenital rubella syndrome in infants and characterization of disability-adjusted life years. Risk Analysis 2014. doi: 10.1111/risa.12263.

3. Dewan P, Gupta P. Burden of congenital rubella syndrome (CRS) in India: a systematic review. Indian Pediatr 2012; 49: 377–99.

4. Dimech W, Mulders MN. A 16-year review seroprevalence studies on measles and rubella. Vaccine 2016; June 20. doi: 10.1016/j.vaccine.2016.06.002.

5. Enders G. Serologic test combinations for safe detection of rubella infections. Rev Infect Dis 1985, suppl 1; 113–22.

6. Mendelson E, Aboudy Y, Smetana Z, Tepperberg M, Grossman Z. Laboratory assessment and diagnosis of congenital viral infections: rubella, cytomegalovirus (CMV), varicella-zoster virus (VZV), herpes simplex virus (HSV), parvovirus B19 and human immunodeficiency virus (HIV). Reproductive Toxicology 2006; 21: 350–82.

43.64 Rotavirus

Family: Reoviridae

Genus: Rotavirus

Species: Rotavirus

Groups: A, B and C in humans and animals; D–G to date only in animals.

Types: the many serotypes are grouped according to antigenic relationships. Similar to Influenza A virus, serotyping is performed based on two different combined classification characteristics: there are 19 G types (defined by glycoprotein VP 7) and 30 P types (defined by protein VP 4). For example, a Rotavirus strain of Group A is called G1P1A(8). Within the P types, the genotypes differ from the serotypes and indicated by a number in brackets after the serotype number.

Viral structure: see Reovirus – Section 43.59. The genome consists of 11 segments, and the enveloped icosahedral capsid, which is approximately 75 nm in diameter, of three protein layers. The structural proteins VP 7, VP 4 (and VP 6) define the serotypes.

43.64.1 Epidemiology and clinical significance

Rotaviruses, especially those of group A, are ubiquitous in humans worldwide. They are the most common viral cause of acute gastroenteritis in infants and young children. The incubation period is 1–3 days. In the arid regions of the subtropics, they contribute considerably to infant mortality. Because Rotavirus is extremely stable and resistant to the environment, it is the number one pathogen causing nosocomial infections /1/. Transmission occurs via the fecal-oral route. The virus replicates in the villi of the small intestine and is then excreted in stool for approximately one week. In temperate climates, Rotavirus infection has a winter seasonal pattern, and minor nosocomial outbreaks on pediatric wards are common.

After an incubation period of 1–3 days, Rotavirus infection typically manifests with fever, vomiting, and diarrhea. Children up to 2 years of age can quickly become critically ill from dehydration. Beyond this age, Rotavirus infection has a prevalence of over 90% and runs a milder or asymptomatic course. The pathogen can nevertheless spread effectively /2/.

Reinfections are common due to the multitude of serotypes resulting from genetic re-assortment, including with animal adapted viruses, within a Rotavirus group. Reinfections often remain subclinical. The virus is not known to persist /3/. IgA antibodies confer clinical immunity on the intestinal mucosa.

43.64.2 Virus detection

Rotavirus screening should be performed predominantly in infants and young children with severe gastroenteritis. In adults, gastroenteritis is more likely to be caused by Norovirus. Rotavirus is excreted in high concentrations in stool, where it can be detected by electron microscopy or antigen ELISA. The ELISA usually detects group A antigen and may be unsuccessful in less common, usually imported infections with group B and C viruses. The great advantage of electron microscopy is that it allows screening for several viruses in parallel.

Molecular-based methods

Because the virus is excreted in high concentrations, the dsRNA segments of the viral genome can be demonstrated by gel electrophoresis of nucleic acid extracted directly from stool samples. The antigentic drift can often easily be detected by the different patterns of the RNA segments in the electrophoretic gel.

Typing by RNA electrophoresis detects infection chains, which is important when investigating nosocomial infections (Fig. 43.2-2 – Analysis of the segmentation/restriction fragment length polymorphism using the example of RNA segments of rotavirus and DNA PCR amplicons of HSV-1 from different patient isolates).

The fine typing and phylogenetic analysis are performed by sequencing a cDNA amplicon. Due to the good quality of the antigen test, RT-PCR is not yet as well established for the detection of rotavirus as it is for the detection of norovirus /4/.

Cell culture

Rotavirus can be isolated in cell cultures pretreated with trypsin. This method is useful for the testing of disinfectants.

43.64.3 Viral serology

Compared to the methods of direct virus detection, serology is of no value for routine diagnosis. Neutralizing antibodies can be determined with a cell culture virus neutralization assay (e.g., after vaccination). For scientific purposes, an immunoblot assay may be used.

References

1. Cox E, Christenson JC. Rotavirus. Pediatrics in Review 2012; vol 33, issue 10.

2. Maldonado YA, Yolken RH. Rotavirus. Baillieres Clin Gastroenterol 1990; 4 (3): 609–25.

3. Surendran S. Rotavirus infection: molecular changes and pathphysiology. EXCLI Journal 2008; 7: 154–62.

4. Tate JE, Mijatovic-Rustempasic S, Tam KI, Lyde FC, Payne DC, Szilagyi P, Edwards K, et al. Comparison of 2 assays for diagnosing rotavirus and evaluating vaccine effectiveness in children with gastroenterits. Emerging Infectious Disease Journal 2013, 19: No 8.

43.65 Togavirus

Family: Togaviridae

Genera: Rubivirus, Alphavirus

Viral structure: Togaviruses are spherical, enveloped particles containing an icosahedral nucleocapsid /1/. The genome consists of a single stranded positive sense RNA. The particles have a diameter of 60–70 nm.

Significance: this virus family includes the human rubivirus genus (rubella). Exotic zoonotic Togavirus infections spread by insects: see Alphavirus – Section 43.4.

References

1. Westerway EG, Brinton MA, Gaidamovic SY, Horzinec MC, Igarashi A, Kääriäinen L, et al. Togaviridae. Intervirology 1985, 24: 125–39.

43.66 Rabies virus

Family: Rhabdoviridae

Genus/species: Lyssavirus, Rabies virus

Types: genotypes 1–7. GT 1 (Rabies virus) is predominant.

Viral structure: the Rabies virus has a bullet like shape with a length of about 180 nm and a diameter of 75 nm. The nucleocapsid is helical and contains a single stranded negative sense RNA. It is surrounded by an envelope studded with spikes.

43.66.1 Epidemiology and clinical significance

Genotype 1 of the virus is widely distributed in carnivorous mammals /1, 2, 3/. In Europe, rabies mainly affects foxes, badgers and occasionally dogs and cats. In America it is also seen in racoons and skunks. In Asia and Africa, every dog bite carries the threat of rabies. Globally, bats may also carry different genotypes of the virus. Cases of transmission through organ transplantation have been reported.

The virus travels along the nerves from the bite site up to the brain, where it causes fatal encephalitis. From the brain, the multiplied virus is transported centripetally to the salivary glands and visceral organs. The incubation period in dogs ranges from 7 to 10 days. In Germany, the last reported autochthonous rabies case occurred in 1986. Untreated, the virus usually has an incubation period of 10–30 days. In rare cases, this period may be more than 3 months, or sometimes as long as several years.

The disease typically runs through a prodromal phase (persistent pain and severe itching at the bite site), followed by an excitation phase (raging fury) and a paralytic phase (with paralyses), and usually leads to death.

All cases of suspected exposure to rabies are immediately treated by both active and passive immunization. Immunization performed within a week of infection is usually successful.

43.66.2 Laboratory diagnosis

Bite history and observation of the biting animal. Involvement of the relevant public health authority. If there is reasonable suspicion of infection, the animal must be killed and examined histopathologically in special laboratories approved for this purpose.

In humans, diagnosis can be attempted by the detection of rabies antigen using direct immunofluorescence or by genomic detection using RT-PCR on skin biopsies, saliva, or throat secretions. Serum antibodies generally become detectable only after the onset of illness. Antibody detection by neutralization assay using a defined laboratory virus and neuroblastoma cells predominantly serves to determine the patient’s immune status after vaccination and can only be performed by special laboratories.

References

1. Fishbein DB, Robinson L. Rabies. N Engl J Med 1993; 329: 1632–8.

2. Vitasek J. A review of rabies elimination in Europe Vet Med Czech 2004; 49: 171–85.

3. Consales CA, Bolzan VL. Rabies Review: immunpathology, clinical aspects andtreatment. J Venom Anim Toxins incl Trop Dis 2007. doi: 10.1590/S1678-91992007000100002

43.67 Varicella-zoster virus (VZV)

Family: Herpesviridae

Subfamily: Alphaherpesvirinae

Genus: Varicellovirus

Species: Varicella-zoster virus with 5 geographically distinct genotypes

Viral structure: see Herpesviruses – Section 43.33.

43.67.1 Epidemiology and clinical significance

VZV causes chickenpox (incubation time is approximately 2 weeks) and herpes zoster.

Prevalence

The infection is ubiquitous. It is transmitted primarily through droplet or airborne spread of respiratory secretions of infected hosts, but also through contact with discharges from the characteristic vesicles, which contain high viral titers. Most people have been infected by age 20, with clinically manifest disease occurring in 30–60% of cases.

Clinical presentation

In immunocompetent individuals VZV infection is harmless /1, 2/. Complications of the infection (pneumonia, meningitis, encephalitis) are more likely to occur in older patients. In contrast to other Herpesviruses, prior infection with VZV confers demonstrable clinical immunity to exogenous reinfection for decades. VZV also persists in the sensory ganglia of the spinal cord, probably not only in the nervous cells but also in satellite Schwann cells /3/. From about the age of 40, sometimes earlier, VZV infection may reactivate in the form of Herpes zoster (shingles), which can be very painful, especially when the virus has affected the facial area (trigeminal neuralgia, zoster ophthalmicus or oticus). Often, the entire dorsal root ganglion is inflamed and meningitis develops. Reactivation causes strong immune stimulation which, in contrast to Herpes simplex, can easily be diagnosed by serology. From age 60 onward, the incidence of herpes zoster is 20%.

Note: while HSV recurrences are common, most people typically experience only one episode of Herpes zoster in their lifetime. Herpes zoster duplex or multiplex is indicative of a T cell immune deficiency: AIDS opportunist (Tab. 43.37-1 – Spectrum of important clinical manifestations of AIDS). With increasing age, there is an increased risk of post herpetic neuralgia which, by definition, persists for more than six weeks after the onset of Herpes zoster. Although rare, there are documented cases of secondary chickenpox as a result of exogenous reinfection 30–40 years after primary infection.

Pregnancy

Like HSV infection, perinatal varicella infection (but not reactivated Herpes zoster infection) can also be transmitted vertically to the child at the end of the pregnancy and cause life threatening generalized neonatal herpes (Section 43.32 – Herpes simplex virus (HSV) type 2). In this case, the newborn must immediately be administered hyper immunoglobulin, if it is unlikely that sufficient maternal antibodies will be transferred through the placenta /4, 5/. If necessary, antiviral Herpes medication must be given.

The risk of congenital varicella syndrome resulting from prenatal primary varicella infection is 2%, with most of the reported cases relating to the first 21 weeks of pregnancy. If hyper immunoglobulin is not administered to the seronegative woman early enough i.e., within a few days after infection, the otherwise contraindicated use of antiviral herpes medication must be considered, especially if the infection becomes life-threatening to the expectant mother /5/. In the individual cases reported to date, the fetus was not damaged. Occasionally, prenatal maternal infection may result in mild neonatal herpes zoster, which usually has a good prognosis.

43.67.2 Viral serology

Unlike HSV serology, VZV serology is well suited for detecting primary varicella infection and subsequently reactivated Herpes zoster infection. Complement fixation test for the detection of IgM, IgG and IgA antibodies has been replaced by ELISA /6/. In varicella, the IgM assay is always positive, while in Herpes zoster it may occasionally remain negative. The IgA assay is more sensitive. Since there are no international units in antibody testing, the test kit units should be adapted to titer levels.

Interpretation of results:

• A titer > 1280 is indicative of Herpes zoster
• Acute infection and exacerbation can often be reliably diagnosed based on the rise in titer (IgG, IgM, IgA)
• An IgG titer of at least 160 confers immunity to varicella
• When determining the immune status during pregnancy, the differential diagnosis of acute infection must take into account that, during primary infection, IgG antibodies may be detectable before IgM antibodies.

43.67.3 Virus detection

Virus detection from throat swabs or skin efflorescences can be done easily by isolating the virus in cell cultures, but is more time-consuming than in the case of HSV infection. Rapid diagnosis is usually performed by PCR and rarely by antigen testing. One week after all the blisters have scabbed over, the infection is no longer contagious. Genotyping differentiates between wild-type virus and vaccine virus and allows the construction of infection chains.

Increased amount of VZV DNA in serum of patients with VZV central nervous disease infections seems associated with encephalitis and ongoing rush /7/.

References

1. Cohen JI. Herpes zoster. N Engl J Med 2013; 369. 255–63.

2. Zerbini L, Sen N, Oliver Sl, Arvin AM. Molecular mechanisms of varicella zoster virus pathogenesis. Nature Rev Microbiology 2014; 12: 197–2010.

3. Gilden D, Nagel M, Cohrs R, Mahalingam R, Baird N. Varicella zoster virus in the nervous system. F1000Research 2015. doi: 10.12688/f1000research.7153.1.

4. Royal College of Obstetrics &Gynaecologists. Chickenpox in pregnancy. RCOG Green-tpo Guideline No 13; 2015.

5. Enders G.. Consequences of varicella and herpes zoster in pregnancy: prospective study of 1739 cases. The Lancet 1994; 343: 1548–51.

6. Cohen JI. Recent advances in varicella-zoster virus infection. Ann Intern Med 1999; 130 922–32.

7. Grahn A, Bergstroem T, Runesson J, Studahl M. Varicella-zoster virus (VZV) DNA in serum of patients with VZV central nervous system infections. J Infect 2016 Jun 15. doi: 10.1016/j.jinf.2016.04.035.

43.68 Community-acquired pneumonia

Lothar Thomas

Community-acquired pneumonia is an acute infection of the pulmonary parenchyma in a patient who has acquired the infection in the community (as distinguished from an infection acquired in a hospital) /1/. In the United States community-acquired pneumonia is one of the leading causes of hospitalization and death /2/. The annual incidence is about 650 adults per 100,000 population.

Clinical findings: Slightly increased temperature (< 39 °C) normal blood pressure and normal respiratory rate of 20 breaths per minute. Auscultation of lungs reveales coarse bronchi over the right midlung field. Chest radiography reveals right upper-lobe consolidation /1/.

Laboratory findings: White cell count slightly elevated, serum CRP concentration about 30 mg/L, procalcitonin about 5 ng/mL (normal range 0–0.05 mg/L), oxygen saturation 92%, creatinine normal, serology is positive for Respiratory syncytial virus /1/.

Numerous microorganisms can cause community acquired pneumonia. Refer to Tab. 43.68-1 – Respiratory pathogens in community-acquired pneumonia.

43.68.1 Immune dysregulation to respiratory viruses

Respiratory viruses, e.g. Covid 19 virus, Influenza virus, and Respiratory Syncytial virus cause infections of varying severity depending on factors of the host and the virus /3/. The pathogenesis of immune dysregulation is triggered by largely inexplicable host factors. The conditions that render the host susceptible to severe viral infections will lead to preventative therapeutic strategies.

Comments to Covid 19 virus infection:

• Sars-COV-2S protein suppresses inflammatory responses by decreasing the expression and secretion of IL-8
• the infection with SARS-Cov-2 is considered as a trigger for autoimmune diseases
• diversity in response to SARS-Cov-2 exposure may be related to differences in their innate immune responses.

Comments to Influenza virus infection:

• cigarette smoke leads to severe disease in Influenza A virus infection
• cigarette smoking affect the course and severity of Influenza B virus infection and reduces the Influenza B virus specific antibodies.

Comments to Respiratory syncytial virus (RSV) infection:

• Besides Influenza virus, RSV represents a major contributor of infection-related hospitalization with significant morbidity rates among children (especially infants) and adults (elderly and immunocompromized patients).

References

1. File M, Jr, DB, Ramirez JA. Community-acquired pneumonia. N Engl J Med 2023; 389 (7): 632–41.

2. Agency for Healthcare research and quality, Community-acquired pneumonia clinical decision support implementation toolkit. January 2018 (www.ahrq.gov/hai/tools/ambulatory-care/cap-toolkit/index.html).

3. Fragkou PC, Dimopoulou D, De Angelis G, Menchinelli G, Chemaly RF, Skevaki C, et al. Editorial: Immune response to respiratory viruses and respiratory viral infections in susceptible populations. Front Med 2023. doi: 10.3389/fmed. 2023.1330265.