Katie Jeffery and Emma Aarons
INTRODUCTION
Human virus infections may affect all ages and assume any degree of severity. They may be acute or chronic, be recurrent or elicit lifelong immunity. They are acquired through various routes via contact with humans, animals or the environment. They present as various syndromes involving fever, rash, arthralgia/myalgia, respiratory or gastrointestinal disorders and occasionally serious organ malfunction with deaths from pneumonia, cardiac, liver or kidney failure or encephalitis. They have to be rapidly distinguished from bacteriological and other infectious and non-infectious diagnoses if the appropriate clinical management is to be given.
Host factors are crucial to the outcome of virus infections. For any virus infection, age may be critical to determining outcome, those at extremes of age being more vulnerable as a consequence of lack of immunocompetence, inexperience of vaccination and waning of immunity. For some infections, gender and race may confer advantages or disadvantages, but malnutrition, pre-existing organ damage and social neglect are always potentially disadvantageous. Thus, in assessing prognosis and deciding on the investigation, management and treatment of virological infections, the individual patient must be carefully considered in their medical and social context. Any natural tendency towards spontaneous immune-mediated clearance of a virus is likely to be compromised if factors such as these are unfavourable.
A substantial part of clinical virology is taken up with the investigation and treatment of patients either constitutionally or iatrogenically immunosuppressed, or suffering from an existing immunocompromising infection, such as human immunodeficiency virus (HIV). These patients often need pre-emptive and continuing investigation if intensive treatment for other conditions is not to be nullified by overwhelming virological and other opportunistic infections. The management of these patients must be planned and rigorous, and will differ from that of other virological patients.
Because virus infections are contagious, diagnosis cannot be confined to a consideration of the individual patient. Two questions may be crucial: where did the infection originate? And who may contract it next? Each question clearly gives rise to the potential for wider investigation and possible action to protect contacts through behaviour modification, isolation or prophylaxis with drugs or vaccines. If the infection is sufficiently contagious or is life-threatening, more extensive public health measures may be required and the clinical diagnosticians must not lose sight of the possible implications of their conclusions for the wider community.
Clinical virology in the 1980s was characterized by the widespread use of enzyme-linked immunosorbent assay (ELISA) technology, and in the 1990s by the entry into routine diagnostic use of molecular methods for virus detection. During the early years of the twenty-first century real-time polymerase chain reaction (PCR) and virus quantification have come of age, alongside increasing automation of molecular diagnostics. Concurrently, the emphasis and priorities of diagnostic virology laboratories have shifted. This is in response to the availability of rapid diagnostic methods, the identification of new viruses many of which are non- or poorly cultivable, the increasing availability of effective antiviral agents, the emergence of antiviral resistance, the increasing number of immunocompromised patients in whom opportunistic viral infections are life-threatening, and the cost pressures on pathology services.
This chapter will provide, firstly, an overview of diagnostic techniques set against this background and presented in order of historical development. Secondly, it will highlight the ways in which these techniques may be applied to arrive at accurate diagnosis thereby facilitating effective management of virus infections, including prevention of their onward spread.
ELECTRON MICROSCOPY
Electron microscopy (EM) is the only technique available for directly visualizing viruses, and therefore has many applications beyond purely diagnostic ones. With the advent of alternative diagnostic methods, EM retains a limited role in the clinical setting for the diagnosis of viral gastroenteritis and examination of skin lesions for herpes and pox viruses.
Preparation of specimens for EM and the technique of negative staining are straightforward and quick, and the method is a 'catch-all' approach to detecting viruses. However, it has a limit of sensitivity of approximately 106 viral particles per millilitre of fluid, making negative results unreliable. Vast numbers of virions are present during acute skin and gastrointestinal disease and a diagnosis is easily made, but later in the course of infection viral shedding is reduced below the level of detection. Although sensitivity can be enhanced by antibody-induced clumping of virus (immune EM) or ultracentrifugation, it is unrealistic to undertake these methods routinely. The advantages and disadvantages of EM are summarized in Table 1.1.
The survival of EM within the routine clinical virology laboratory hinges on the emergence of alternative, more sensitive methods of diagnosis. Many centres now use latex agglutination for rotavirus diagnosis, and PCR is more sensitive than EM for detection of herpesviruses in vesicular fluid (Beards et al., 1998) and for the detection of noroviruses (previously called Norwalk-like viruses ) (O'Neill et al., 2001). Thus, the future of EM in clinical virology is in some doubt. However, one of the first indications for EM was for the rapid diagnosis of smallpox and, in the era of bioterrorism, EM may continue to play a role in specialist centres in the event of a bioterrorist attack.
HISTOLOGY/CYTOLOGY
Direct microscopic examination of stained histology or cytology specimens can on occasion provide the first indication that a virus may be responsible for a pathological process, for example the intranuclear (early) or basophilic (late) inclusions seen in interstitial nephritis in renal transplant biopsies due to BK virus, changes in cervical cytology seen in association with human papilloma virus (HPV) and the nuclear inclusions seen in erythroid precursor cells in Parvovirus B19 infection. Moreover, the particular viral aetiology can be confirmed by specific antigen/genome staining using labelled antibody or in situ hybridization techniques (see below).
VIRUS ISOLATION
Many of the advances in clinical virology have come about because of the ability to grow viruses in the laboratory. Historically, viruses were propagated in laboratory animals and embryonated eggs, but most virus-isolation techniques now rely on cultured cells. With appropriate specimens and optimal cell lines, this technique can be highly sensitive and specific, with a presumptive diagnosis made on the basis of a characteristic cytopathic effect (CPE). The particular diagnosis can then be confirmed by haemadsorption (certain viruses, influenza and measles for example, cause adherence of erythrocytes to infected cells in a monolayer because the viral antigens expressed include a haemagglutinin) or by immunofluorescence (IF) using a virus-specific antibody labelled with a fluorescent dye. The judicious selection of two or three cell lines, such as a monkey kidney line, a human continuous cell line and a human fibroblast line will allow the detection of the majority of cultivable viruses of clinical importance, such as herpes simplex virus (HSV), Varicella zoster virus (VZV), cytomegalovirus (CMV), enteroviruses, respiratory syncytial virus (RSV), adenovirus, parainfluenza viruses and influenza viruses. In addition, the ability to grow virus from a clinical specimen demonstrates the presence of viable virus (albeit viable within the chosen cell line)-this is not necessarily the case with detection of a viral antigen or genome. For example, following initiation of antiviral therapy for genital herpes, HSV antigen can be detected from serial genital swabs for longer than by virus propagation in cell culture. This implies that antigen is persisting in the absence of viral replication and underlines the importance of correct interpretation of laboratory results. However, failure to isolate a virus does not guarantee that the virus is not present. Virus isolation has also been shown to be diagnostically less sensitive than molecular amplification methods such as PCR for HSV and several other viruses (see below), for example for the diagnosis of herpes simplex encephalitis.
The benefits of virus isolation (Table 1.2) include: the ability to undertake further characterization of the isolate, such as drug susceptibility (see later) or phenotyping; and the identification of previously unrecognized viruses, for example human metapneumovirus (van den Hoogen et al., 2001), severe acute respiratory syndrome (SARS)-associated coronaviruses (Drosten et al., 2003) and human enteroviruses 93 and 94, associated with acute flaccid paralysis (Junttila et al., 2007). On the other hand, routine cell culture techniques available in most laboratories will not detect a number of clinically important viruses such as gastroenteritis viruses, hepatitis viruses, Epstein-Barr virus (EBV), human herpesvirus 6, 7 and 8 (HHV-6, -7, -8) and HIV. Other than HSV and some enteroviruses, most isolates of which will grow in human fibroblast cells within three days, the time taken for CPE (or, for example, haemadsorption) to develop for most clinical viral isolates is between 5 and 21 days, which is often too long to influence clinical management. For this reason, a number of modifications to conventional cell culture have been developed to yield more rapid results. These include centrifugation of specimens on to cell monolayers, often on cover slips, and immunostaining with viral protein-specific antibodies at 48-72 hours post inoculation (shell vial assay) (e.g. Stirk and Griffiths, 1988). Such techniques can also be undertaken in microtitre plates (O'Neill et al., 1996). Certain changes, for example in haemadsorption or pH, may precede the CPE and therefore can be used to expedite detection of virus. Similarly, PCR techniques (see later) can be used to detect virus in cell culture supernatants before the appearance of CPE.
The role of conventional cell culture for routine diagnosis of viral infections has been a subject of active debate within the virology community (Carman, 2001; Ogilvie, 2001). Many laboratories are discontinuing or downgrading virus isolation methods in favour of antigen or genome detection for the rapid diagnosis of key viral infections, for example respiratory and herpes viruses. Nevertheless, it is important for certain reference and specialist laboratories to maintain the ability to employ this methodology to obtain live virus isolates and allow unexpected and emergent viruses to be grown and recognized.
SEROLOGY
This term is often used to refer to diagnostic tests for the detection of specific antibodies. More properly, the term encompasses any testing of blood serum samples for the presence of a specific antigen or antibody. However, as both antigen and antibody assays are often applied to whole blood or plasma, or indeed to body fluids other than blood (e.g. cerebrospinal fluid (CSF), oral crevicular fluid), it is helpful to use the term to span all such testing. As will be seen, most of the techniques used for viral antigen detection can also be used for detection of specific antibody, and vice versa.
Antigen Detection
Immunofluorescence
IF is one of the most effective rapid diagnostic tests. Direct IF involves the use of indicator-labelled virus-specific antibody to visualize cell-associated viral antigens in clinical specimens. The indirect method utilizes a combination of virus-specific antibody (of a nonhuman species) and labelled anti-species antibody. Usually, the label used is fluorescein. The indirect method is more sensitive, since more label can be bound to an infected cell. Results can be available within 1-2 hours of specimen receipt. The success of the technique depends on adequate collection of cells in the particular sample to be examined, for example epithelial cells for respiratory virus antigens or peripheral blood mononuclear cells (PBMCs) for CMV. An advantage of IF is that microscopic examination of the fixed cells can determine the presence of adequate cell numbers for analysis (Table 1.3). Whenever it is employed, however, a trained microscopist is required to interpret results, which remain subjective.
The most common use of this technique is for the diagnosis of respiratory viral infections. A panel of reagents can be used to detect RSV, parainfluenza viruses, influenza A and B, metapneumovirus and adenovirus in multiple wells of a microscope slide. Compared to cell culture this technique is rapid and sensitive, especially for detection of RSV. The ideal specimen is a nasopharyngeal aspirate or a well-taken throat/nasal swab, most usually obtained from infants with suspected bronchiolitis, in whom a rapid result is invaluable for correct clinical management and implementation of infection control measures. There is increasing evidence that community- or nosocomial-acquired respiratory viruses lead to severe disease in immunocompromised patients (reviewed in Ison and Hayden, 2002), and it is important that bronchoalveolar lavage specimens from such patients with respiratory disease are also tested for these viruses, in addition to bacterial and fungal pathogens.
IF has been used widely for the direct detection of HSV and VZV in vesicle fluid, and has advantages over EM in both sensitivity and specificity. Detection and semi-quantification of CMV antigen-containing cells in blood can also be undertaken by direct IF (CMV/pp65 antigenaemia assay). This technique involves separating PBMCs and fixing them on a slide, followed by staining with a labelled monoclonal antibody directed against the matrix protein pp65. The frequency of positive cells can predict CMV disease in the immunocompromised patient (van der Bij et al., 1989) and has been used quite extensively, though it is labour intensive. It needs large numbers of PBMCs, making it unsuitable for some patients. In addition, it requires a rapid processing of blood specimens if a reduction in sensitivity of detection is to be avoided (Boeckh et al., 1994). PCR has therefore become the method of choice for qualitative and quantitative detection of CMV as well as several other viruses.
Enzyme-linked Immunoassay (EIA), Chemiluminescent and Fluorescence-based Immunoassay
Solid-phase systems for antigen detection are still used widely. EIA is based on the capture of antigen in a clinical specimen to a solid phase (such as the base and walls of a well in a microtitre plate, or multiple magnetic microparticles/beads) via a capture antibody, and subsequent detection uses an enzyme-linked specific antibody that produces a colour change in the presence of a suitable substrate. EIA is also readily used for the detection of specific antibody as well as antigen (see later).
Elaboration of capture and detector antibody species has increased the sensitivity of EIA antigen-detection assays, which are widely used for hepatitis B virus (HBV) surface antigen (HBsAg) and 'e' antigen (HBeAg) detection and for HIV p24 antigen detection. Neutralization of the antigen reactivity by the appropriate immune serum can be used to confirm the specificity of the antigen reactivity. In primary HIV infection, HIV p24 antigen is present in the blood prior to the development of antibodies. Therefore, assays which detect this antigen in addition to anti-HIV antibodies reduce the diagnostic 'window period', that is the time from acquisition of infection to its first becoming detectable (Hashida et al., 1996). Similar assays that detect hepatitis C core antigen in addition to anti-HCV (hepatitis C virus) antibodies have been proposed for testing donated blood.
Molecules with chemiluminescent properties, for example acridinium ester, which produces chemiluminescence in the presence of hydrogen peroxide, can be conjugated to antibodies/antigens and used instead of enzymes for immunoassay detection. Fluorescent labels are another alternative. The fluorescence emissions of chelates of certain rare earth metals-lanthanides, for example Europium-are relatively long-lived. Thus, the presence of an antigen or antibody labelled with a lanthanide chelate can be detected by measuring fluorescence intensity at a delayed time point after excitation, background fluorescence having completely died away. This is the principle of the time-resolved fluorescence assay (TRFA). Both chemiluminescent and TRF methods are very sensitive and highly amenable to automation in commercial systems.
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