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"There_is_no_longer_a_need_for_diagnostic_laboratories_to_attempt isolation_of_viruses_in_tissue_culture"_Discuss_this_statement

 

Isolation of viruses in tissue culture has been for many years the mainstay of diagnosis of many virus infections. However, in recent years, rapid diagnostic tests based on the direct examination of the clinical material, and serological tests have made inroads into what was once the preserve of tissue culture alone.

Uses_of_tissue_culture

Cell cultures are divided into 3 types: (1) primary cells, which are prepared from adult animals eg. monkey kidney, and can be subcultured only once or twice. (2) semi-continuous cells, which are derived from human fetal tissues eg. human embryo lung fibroblasts, and can be subcultured 20 to 50 times. (3) Continuous cell-lines, which are established from human or animal tumours eg. vero, hep-2, and can be subcultured indefinitely. Cell cultures vary greatly in their susceptibility to different viruses and it is important to use the most sensitive cell cultures available for the isolation of suspected viruses in the clinical specimen.

The clinical specimen is inoculated into test-tubes or tissue culture plates containing confluent layers of cell-culture and incubated for up to 4 weeks depending on the laboratory policy. The cell-culture is examined periodically for the presence of viral-induced changes such as cytopathic effect (CPE) or the ability to haemadsorb. The identity of the isolated virus can be confirmed, or if necessarily typed by several different methods, such as neutralization, fluorescent antibody, haemadsorption-inhibition tests.

The isolation of a virus from the site of disease eg. an enteroviruses from the CSF, or HSV from a genital lesion is highly diagnostic for the infection. The diagnosis is more tenuous if the virus is isolated from another site eg. from the faeces in a case of suspected enterovirus meningitis.

Disadvantages of cell culture: The greatest disadvantage of cell culture is the length of time required for the viral induced changes to appear which usually takes a few days to a few weeks. The exceptions are herpes simplex viruses and sometimes enteroviruses where CPE may appear within one day of inoculation. Cell cultures are very susceptible to bacterial contamination and requires experienced personnel to perform. Another problem with virus culture is that the sensitivity is often low and depends on a large extent the quality of the clinical specimen received. Cell culture is not applicable to viruses which grow poorly or not at all in cell culture eg. diarrhoeal viruses, hepatitis B and C, parvovirus.
 
 

Other_diagnostic_techniques

Serology

Serology remains the bulk of the work carried out by a routine diagnostic virus laboratory. A large variety of serological tests are available eg. complement-fixation (CFT) , haemagglutination-inhibition (HAI), enzyme-linked immunoassay (EIA), radioimmunoassay (RIA), particle agglutination, immunofluorescence etc. The sensitivity and specificity varies greatly between different techniques. Most techniques will detect all classes of antibody whereas some assays eg. RIA, EIA and IF can be made to detect one specific class only ie. IgM, IgG or IgA.

A serological diagnosis of a primary infection can be made by (1) detection of virus-specific IgM, (2) 4 fold increase in titre of specific total antibody or IgG. (3) demonstration of seroconversion, or (4) a very high level of total antibody or IgG. Serological diagnosis of reinfection or reactivation may be difficult but is generally made on the finding of rising titres of antibody. Specific IgM should either be undetectable or at a low titre.

How useful a serological result is depends on the individual virus. For example, for viruses such as rubella and hepatitis A, the onset of clinical symptoms coincide with the development of antibodies and thus a diagnosis can be made on the detection of specific IgM from a single specimen. However, many viruses often produce clinical disease before the appearance of antibodies, such as those responsible for gastroenteritis and respiratory infections. So in this case, any serological diagnosis would be retrospective and therefore its use would be limited. There are also other circumstances when serology is of limited value. For example ;in case of local infections such as herpes genitalis when a systemic humoral immune response may not be induced: in immunocompromized patients, who may produce abnormal humoral immune response: infectious mononucleosis, when a large number of antibodies may be produced: following transfusion with blood or blood products: serology is also of limited use in the case of latent viruses which may reactivate from time to time such as viruses of the herpesvirus family.

Direct_examination

In direct examination, the clinical specimen is examined directly for the presence of virus particles, virus antigens, viral nucleic acids and virus-induced histological changes. Direct examination methods include :detection of viral antigens by fluorescent antibody or ELISA: virus particles by electron microscopy: viral nucleic acids by polymerase chain reaction or hybridization: viral induced changes by histological examination of lesions. The main advantage of direct examination methods is the short length of time required for a result, often within the same day. With the advent of specific antiviral chemotherapy, this is becoming an increasingly important consideration. On the debit, these tests are usually labor intensive and require experienced personnel. However, this is likely to change with the arrival to simple automated molecular biology techniques. Current commercial examples include the PCR-based Roche Amplicor system, Abbott LCR system, and the Chiron branched DNA system. Indeed the Roche Amplicor and the Chiron branched DNA system are now widely used for the determination of HIV viral load. The major drawback of these tests is that they are very expensive. However, when the costs come down, they should gain widespread acceptance in the virology laboratory. Looking further ahead, DNA chip technology may one day be used to detect viruses from clinical specimens. Besides detecting the presence of the virus, it may be able to tell the viral load, and to which antiviral agent that the virus is sensitive to at the same time. Therefore, molecular techniques are likely to pose the strongest challenge to tissue culture.
 

The advent of reliable serological and direct examination techniques has diminished the role of tissue culture as a means of diagnosis of virus infection. However, for some viruses, tissue culture is still the method of choice for diagnosis. The following virus infections are commonly diagnosed by tissue culture;-

HSV 1 and 2 - tissue remains the best and easiest way for diagnosing local primary or recurrent HSV infection. Serology is usually unreliable and direct examination techniques eg. electron microscopy, immunofluorescence and PCR, are tedious and are only indicated for severe cases.

Enteroviruses (polio, coxsackie B, echoviruses) - many enteroviruses can be easily cultured from faecal and throat swab specimens during the acute phase of the illness. Serological tests for enteroviruses are usually complicated and may be unreliable. In any case, a serological diagnosis would be retrospective as paired acute and convalescent sera are required.

Respiratory viruses - RSV, influenza A and B, parainfluenza 1-3, non-enteric adenoviruses and rhinoviruses can often be isolated from respiratory specimens of patients with respiratory disease. However, direct examination of nasopharyngeal aspirates for adenoviral antigens is reported to be a highly sensitive method for some respiratory viruses, in particular RSV, influenza A and B, and adenoviruses. However this technique is not as sensitive for parainfluenza viruses. Serological diagnosis is unreliable and is usually retrospective.

mumps - mumps virus can be isolated from saliva and urine specimens. However, a serological diagnosis can be easily made by CFT or by ELISA techniques.

CMV - virus culture, whether conventional or rapid (DEAFF test) remains the gold standard for the diagnosis of active CMV infection. Newer techniques such as the CMV antigenaemia test and PCR are yet to be universally accepted as reliable means of diagnosing CMV infection

Other viruses;- rubella, VZV, measles, and some arboviruses can seldomly be isolated by tissue culture. The diagnosis of these infections are usually made by serology.

In conclusion, although tissue culture is likely to remain the predominant means of diagnosis for some virus infections such as HSV, CMV and enteroviruses. Its role is likely to diminish with time as simpler and more reliable direct examination techniques, especially molecular te become available. Tissue culture is unlikely to be completely dispensed with as a means of diagnosis in the near future.
 

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