6 Hunt KJ, Valentittc MD, Sobotka AK, Benton AW, Amiodio FJ, Lichtcnlstcin LM. A controlled trial of immunotherapy in insect hypersensitivity. kEngl7 fed 1978;299: 157-61. 7 Golden DBK, M1eyers DA, Kagev-Sobotka A, Valentine ID, Lichtenstein LM1. Clinical rclevance of the vcnom-specific immunoglobulin G antibody during immunotherapy. 7 Allergy C/lin Imnmunol 1982;69:489-93. 8 Ijungstrom KG, Renck H, (iruber UF, et al. Prevcntion ot dextratt-induccd anaphylactic reactions by hapten inhibition: a Scandinavian multicentre study. Acta C(hirScantd 1983;149:341-60. 9 Glcisch GJ, Zimmerman EM, Henderson LL, Yunginger JWC. Effect of immunotherapy oin immunoglobulin E and immunoglobulin G antibodies to ragweed antigens: a six year prospective study. J Allergy Clin Immunol 1982;70:261-71. 10 Rocklin RE, Sheffer AL, Greeineder DK, MNIelmott KL. (ieneration of antigen-specific sttppressor cells during allergy desensitization. NF InglJ .lIed 1980;302:1213-9. 11 Canonica GWV, Minari G, Colombatti M, Mloretta L.. Imbalance ofTF cell subpopulations in patients with atopic diseases and effects of specific immunotherapy. ] Immunol 1979;123:2669-7 1. 12 Hsieh K-H, Lue K-H, Chiang C-F. Inmunological changes after hyposensitisation in house-dustsensitive asthmatic children: a review. 7Asthma 1987;24:19-27. 13 Rak S, Hakansson L, Venge I'. Fosinophil chemotactic activity in allergic patients in the birch pollen season: the effect ot immunotherapy. Int Arch Allergv Appl Immunol 1987;82:349-50. 14 McHugh SM, Ewan PE. Reduction of inicreased serum netutrophil chemotactic actis-ity following
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effective hyposensitization in house dust mite allergy. Clini-cal andti Experimental Allergy 1989;19:327-34. Warner JO, Price JF, Soothill JF, Hey EN. Controlled trial of hyposensitisation to D)ermatophagoides pteronvssinus in children with asthma. Lancet 1978;ii:912-5. Bousquet J, Michel FB. Specific immunotherapy in asthma. In: Reed CE, ed. P'roceedings of tthe twelfth international congress of allcrgology and clinical immunology. St Louis: Mosby, 1986:397402. Norman PS. Specific therapy in allergy. Med Clin North Am 1974;58:111-25. Warner JO. Hyposensitization in asthma: a review.,7 R Soc Med 1981;74:60-5. Osterballe 0. Immunotherapy with grass pollen major allergens. Clinical results from a prospectiVe 3 year double blitsd study. Allergy 1982;37:379-88. Norman PS, Lichtenstein LM. Comparison of allum-precipitated and unprecipitated aqueous ragweed cxtracts in the treatment of hayfever.,71 AllergyClin Immunol 1978;61:384-9. Norman PS. An otverview of immunotherapy; implications for the future.] .Allergy Clin Immunol 1980;65:87-96. Bousquet J, Fontez A, Aznar R, Robinet-Lesv M, Michel F-B. Combination of passive and active immunisation in honeybee venom immunotherapy.]Y Allergy Cltn Immunol 1987;79:947-54. AMuller UR, Morris T, Bischof M, Friedli H, Skarvil F. Combined active and passive immunotherapy in honevbee-sting allergy. 7Allergy Clin Immunol 1986;78:115-22.
Regular Revieew
Rapid viral diagnosis in perspective Many viruses can now be detected routinely in small laboratories; even more will be possible by 2000 Though viral infections are the commonest cause of human disease, diagnostic services are available in only a few microbiology laboratories.' Traditionally, the procedures used for viral diagnosis have been too slow to influence patient management and too expensive and tedious to encourage widespread use. The self limiting nature of most viral diseases has encouraged clinicians not to seek a specific diagnosis, and, even when a diagnosis has been made, therapeutic options have been limited. In the past 10 years, however, the picture has changed dramatically, and viral diagnostic tests need to be more widely available. With the increasing use of immunosuppressive and cytotoxic drugs and as a consequence of AIDS severe viral infections are a common complication of modern medicine. Knowledge of the specific cause has obvious benefits, such as more accurate prognosis, reduction in clinical investigations and the use of antibiotics, and better patient management and compliance. It also leads to a better understanding of the epidemiology of viral diseases, which is important for controlling transmission. Antiviral chemotherapy has made important advances, and therapeutic options now exist for several infections.2 Acyclovir has been proved to be of value in treating established herpes simplex virus and varicella zoster virus infection in both immunocompetent and immunosuppressed patients. Serious respiratory viral disease due to influenza A virus and respiratory syncytial virus are now treatable with amantadine and tribavirin respectively. Even the newly discovered human immunodeficiency virus may be controlled at least in part by zidovudine. Recently ganciclovir and foscarnet have given promising results in immunosuppressed patients with serious cytomegalovirus disease. There are also tantalising reports suggesting that both papillomavirus infection and hepatitis B virus infection may be susceptible to immunomodulatory treatment with interferons. Though infection with these viruses is amenable to treatment, chemoprophylaxis and immunoprophylaxis may be an effective alternative for other viruses.2 Acyclovir and immunoglobulin prophylaxis have shown benefit in preventing or reducing the severity of varicella zoster virus and cytomegalovirus infections. Amantadine offers effective prophylaxis against influenza A infection and is also a useful adjunct to late immunisation in people at high risk. Although BMJ VOLUME 300
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antiviral treatment and prophylaxis are still in their infancy, they are making an impact and their influence is bound to increase in the future. As a result of these developments the demand for rapid viral diagnosis has increased substantially in recent years. Much technical progress has been made, and the laboratory can now provide reliable diagnosis of a comprehensive range of virus infections within a day or two after receiving the specimen. We review here the laboratory tests now available, emphasising those that are applicable "in the field" away from the reference laboratory.
Performance evaluation of a new test The performance measures used to evaluate the usefulness of a new test are sensitivity, specificity, and positive and negative predictive values. These terms are familiar and are defined elsewhere,' but a couple of points are worth emphasising. For culturable viruses the reference test to which these measures relate is usually isolation in tissue culture. Though specificity of virus isolation may be safely assumed, sensitivity depends on several factors that may compromise recovery of virus from clinical specimens and so generate false negative results.4 As the technology improves rapid tests are appearing that are clearly more sensitive than the reference methods. This may be a manifestation of that very problem, and other tests-for example, serological confirmation-must then be applied to determine whether the additional positive results are real or false. An example of this is the recently developed rapid culture test for cytomegalovirus, in which up to twice as many specimens give positive results than with conventional culture, which remains the reference method.I Another example is the detection of pathogen specific DNA after amplification with the polymerase chain reaction. Here a new test is so sensitive that validating the results by alternative tests may be difficult if not impossible. As described below, the only way that the results can be validated in these circumstances is to rely on stringent attention to quality control and technique by the operator. A measure that is often overlooked in evaluating a new test is its positive predictive value-that is, the proportion of true positive results. When the test has a specificity of under 100% 1413
the predictive value will show considerable variation for different populations depending on the prevalence ofinfection in the group under study because if most of the specimens examined give true negative results a small fraction of false positive results becomes important. The effect is shown in the table, which gives the sensitivity and specificity levels attainable with the current tests for detecting chlamydial antigen. Test kits that seem to have quite reasonable performance may generate a large percentage of false positive results in a condition with low prevalence (over 30% in this example). Effect of disease prevalence on positive predictive value Sensitivity (%) 85 85
Specificity ( 98 98
Prevalence 20 5
Positive predictive value (oN)) 91
69
These calculations apply to all screening tests with a specificity of less than 100%. It is therefore important to be aware of the disease prevalence in the population under study before relying too much on the result.
Respiratory viruses The viruses that infect the respiratory tract have remained difficult to identify with the rapid methods. The diagnosis of upper respiratory tract infection is rarely requested because the infection is usually trivial. Virus isolation continues to be the only reliable approach for detecting the lower respiratory tract pathogens. As with conventional serology, this may take two to three weeks to provide an answer, which in most cases is too slow to influence patient management. For a few specific viruses the rapid tests do work adequately. Respiratory syncytial virus produces regular epidemics of childhood bronchiolitis in winter, and detection of antigen by either immunofluorescence or enzyme immunoassay is reliable in these circumstances.6 Many comparisons with virus isolation have been published, and sensitivities of 95% or better and specificities of around 98% are usually reported.7 These tests tend to pick up a few additional "false" positive results, which probably represent false negative isolation results. Few commercial kits exist for other respiratory viruses. This severely limits adoption of rapid testing outside the reference laboratory. Polyclonal antisera against influenza viruses A and B, parainfluenza viruses 1 and 3, and adenovirus group antigens are available from Wellcome and are suitable for immunofluorescence testing. The influenza virus A antiserum is useful during outbreaks, but the sensitivity is only around 50% (with varying specificity) compared with isolation.6" The usefulness of the other antisera is questionable. Though specificities are very high, the sensitivities are only 20-60% and are too low to justify their use as a screening test in the absence of tissue culture back up.69 Commercial enzyme immunoassays for detecting these antigens have not been evaluated. Similarly, reports on the use of monoclonal antibodies are sporadic and commercial suppliers have not yet adopted them. An alternative approach to the respiratory viruses that has received scant attention is the fluorescent focus assay, which is similar to that developed for rapid detection of cytomegalovirus in shell phials. "' The technique entails centrifugation of the specimen on to a monolayer of permissive tissue culture cells, incubation overnight, then staining the cells with immunofluorescent antisera directed against antigens expressed early in the virus's replication cycle. In one study 1414
that used monoclonal antibodies to detect influenza viruses A and B the method achieved a sensitivity of 84% and specificity of 100l compared with culture." Conceivably, respiratory specimens could be screened for a wide range of viruses by using multiwell tissue culture trays in place of cover slips. The important unanswered question is whether the commercial antisera available can detect the early antigen with sufficient sensitivity to make the test reliable.
Gastrointestinal viruses Viruses infecting and causing illness in the gastrointestinal tract have been surprisingly difficult to isolate in cell culture. Their discovery and diagnosis have thus depended largely on electron microscopy. Viruses such as rotavirus, adenovirus type 40/41, calcivirus, astrovirus, and the small round virus group -for example, Norwalk virus-are refractory to isolation but may be detected readily on a negatively stained electron microscopy grid. 12 For many of these viruses electron microscopy is not just a rapid method (results are available within 20 minutes); it is the only method. Direct electron microscopy, in which a clarified faecal suspension is placed directly on to a plastic coated grid and stained with a heavy metal salt solution, is insensitive. At least 106 particles/g of faeces are necessary for reliable detection, which means that substantial amounts of virus may be missed. Though ultracentrifugation or immunological aggregation may be used to concentrate the virus for improved sensitivity, the technique is unsuitable for large scale screening. Immunoelectron microscopy has an additional use in identifying viruses such as the Norwalk virus (which has no characteristic morphology), but its use is restricted to the reference laboratory. Rotavirus is the only virus in this group that occurs in regular winter epidemics and for which other tests have been developed. Several enzyme immunoassay and latex agglutination tests are available commercially, and numerous comparisons with electron microscopy have been published. The enzyme immunoassays are significantly more sensitive than electron microscopy. A recent study showed that one such kit produced over twice as many positive results as electron microscopy.'" The specificity of the test is determined by performing a confirmatory test on the positive specimens by blocking the reaction with an unlabelled rotavirus antiserum. When this is done the modern enzyme immunoassays have adequate specificity, but between 1% and 5% of apparently positive results fail the confirmatory test and are deemed false positive results. This shows the principal disadvantage of enzyme immunoassay: the need to confirm all positive findings with a blocking assay. If the laboratory has only a small paediatric load the cost may also become important because of the control samples required to validate each run. Latex agglutination is not as sensitive as the best of the enzyme immunoassays, but at least one kit is as sensitive as electron microscopy, is highly specific, and seems to be a reasonable alternative for small laboratories." 4 In summary, electron microscopy continues to be an extremely useful test as the only "catch all" method of detecting the gastrointestinal viruses. The sensitivity depends on the operator, however, and, even in competent hands, is suboptimal. Alternative methods have been developed only for rotavirus, and the choice depends to some extent on workload.
Hepatitis viruses At least five viruses that infect humans are recognised for their ability to cause hepatitis: A, B, C, D, and non-A, non-B BMJ
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hepatitis viruses. Routine tests for rapid diagnosis are available only for hepatitis A and hepatitis B viruses. Hepatitis A virus is shed in large numbers in the faeces in the two weeks preceding the overt illness and can be detected readily by immune electron microscopy. Viral excretion diminishes rapidly thereafter, however, and only half of all patients give positive results within one week and less than a tenth within three weeks of the onset of symptoms. 5 A more successful approach, which most laboratories now use, is the detection of antihepatitis A virus IgM in serum. Sensitive commercial radioimmunoassays and enzyme immunoassays are 100% sensitive for up to six months after an acute infection." Diagnosing hepatitis B virus is complicated because infection may follow various patterns, each of which is associated with a different array of viral markers. Direct detection of the virus and its antigens by electron microscopy and the various staining procedures such as immunofluorescence and immunoperoxidase has been applied to biopsy or necropsy material and is useful in diagnosing the various forms of chronic hepatitis.'8 It is not, however, suitable for rapid screening of hepatitis B virus infection in routine laboratories, which rely on the serological markers of the virus. Commercial radioimmunoassay and enzyme immunoassay kits are available for detecting antibody to the virus's surface antigen (antiHBs), core antigen (anti-HBc), and e antigen (anti-HBe) as well as for detecting the antigens themselves (HBsAg and HBeAg). In a typical case of acute self limited infection with hepatitis B virus HBsAg appears first in serum followed by HBeAg, anti-HBc, anti-HBe, and then anti-HBs.'9 In 80-90% of cases rapid diagnosis is made on the basis of a positive result for HBsAg; current third generation immunoassays can detect as little as 0 ng/ml of the antigen.'9 The remaining cases are usually detected by the presence of a high titre of anti-HBc IgM in an acute phase serum sample or seroconversion to antiHBs antibody between acute and convalescent phase serum samples. Chronic infection with hepatitis B virus occurs in 5-10% of adults who are unable to eliminate HBsAg within the usual time frame-that is, less than three months.20 Continued monitoring of concentrations of HBsAg and the other markers, including hepatitis B virus DNA, is then important to determine what form (asymptomatic, chronic persistent, or chronic active) the infection is likely to take. This need arises once hepatitis B virus infection has been diagnosed and would fall beyond the scope of most routine laboratories. Identification of the recently recognised hepatitis C virus is in its infancy at present. The only specific test is for the presence of hepatitis C virus antibodies in serum by using a cloned viral polypeptide of 363 amino acids as the capture antigen in an immunoassay. In the absence of other tests for comparison the sensitivity and specificity of the assay can be assumed to be satisfactory only on the basis of published seroprevalence studies. These show that hepatitis C virus antibodies are found in up to 85% of patients who become infected with non-A, non-B hepatitis virus through transfusion and implicated donors but in only 1% or less of people without risk factors for hepatitis.2' 22 The early serological studies were done with a home grown radioimmunoassay at the Chiron Corporation, where the polypeptide was initially cloned. Commercial kits using an enzyme immunoassay format, however, are now available from Ortho Diagnostics (Buckinghamshire) and Abbott Diagnostics (Hampshire). Though these kits will be useful in providing a diagnostic test and for screening blood donations for hepatitis C, the results should be treated with caution as confirmatory tests are not available at present. -
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Herpesvirus group Herpes simplex virus Of all of the tests for viruses that the diagnostic laboratory is called on to do, that for herpes simplex virus is likely to be the most frequently requested. The virus grows rapidly in permissive cell lines; about 80% of positive cultures are usually detectable by visual cytopathic effect within two days, and more than 99% are detectable by four days.23 Nevertheless, rapid methods of diagnosis have proliferated and include centrifugation enhanced culture, the use of DNA probes, and antigen detection by immunofluorescence, enzyme immunoassay, and latex agglutination. The success of diagnostic testing for genital herpes depends on the stage of the lesion. In one study the virus was detectable in 70% or more of vesicular lesions but in fewer than 20% when the specimen had been taken after crusting had occurred.24 Overall, immunofluorescence with a monoclonal antibody based kit had a sensitivity of 74% and specificity of 85% compared with isolation but was more reliable in specimens taken from early stage lesions than from those taken later on. Enzyme immunoassay may prove to be more sensitive than immunofluorescence as it detects both extracellular and -intracellular antigens and does not impose the demands for collection of infected cells that are required for reliable immunofluorescence testing. In one recent study a newly released enzyme immunoassay kit was shown to have a sensitivity of 95% with 100% specificity for the diagnosis of genital herpes.25 In contrast, latex agglutination seems to be insufficiently sensitive for use without culture back up, detecting only half of the cases with positive results on isolation in one series.26 Compared with the same day tests centrifugation enhanced culture in shell phials is slow, taking 24 hours to complete. The assay is, however, at least as sensitive as conventional culture and is highly specific.27 At present this is the most promising of the alternative tests to virus isolation, and for laboratories with heavy herpes simplex virus workloads it might produce savings in labour.
Vanrcella zoster virus Varicella zoster virus is notoriously labile and attempts at isolation often fail. Ironically, skin lesions caused by varicella zoster virus often contain large quantities of the virus, and diagnosis by antigen detection is the most sensitive method.2829 This approach has had limited application in the past because commercially available reagents were not available, but now a monoclonal antibody kit that uses direct immunofluorescence has been developed. In one evaluation the kit detected significantly more patients who were positive for the virus than did standard culture or centrifugation enhanced culture and had a sensitivity of 92% compared with all methods tested.29 Electron microscopy does not distinguish varicella zoster virus from other members of the herpesvirus group, but because the virus is present in high concentrations in vesicular and pustular fluid the test is often adequate to provide a rapid presumptive diagnosis.30 We have found it useful when used in conjunction with isolation to confirm the virus type.
Cytomegalovirus Rapid diagnosis of cytomegalovirus infection has made major strides since the discovery that viral antigen expressed early in the replication cycle can be detected reliably with fluorescent antisera within a day or two of inoculation into susceptible fibroblast cultures.3' When used in combination with low speed centrifugation to enhance infectivity the 1415
procedure is significantly more sensitive than conventional isolation with a variety of clinical specimens.32 This centrifugation enhanced culture has gained wide acceptance as the method of choice for diagnosing cytomegalovirus, and several commercial companies now supply monoclonal antibodies to detect early antigens. Same day diagnosis of cytomegalovirus infection by antigen detection in clinical specimens has been reported with v4rying success. One group reported a sensitivity of 93% for detecting the virus in blood leucocytes of patients with renal transplants by using a cocktail ofmonoclonal antibodies against early viral antigens.34 In another study direct detection of the virus in bronchoalveolar specimens of patients with cytomegalovirus pneumonia was less satisfactory: only 59% of culture positive specimens were positive by immunofluorescence staining.33 Epstein-Barr virus Despite the advances in direct virus detection for other members of the herpesvirus group the diagnosis of acute Epstein-Barr virus infection by routine laboratories still depends on the detection of heterophile (Paul-Bunnell) antibodies and antibodies specific for Epstein-Barr virus in serum. The Paul-Bunnell assay remains the front line screening test and can identify 85-95% of patients with infectious mononucleosis during the first month of illness.36 It has several different formats for measurement or qualitative determination, but the most common assays today are the rapid slide agglutination tests. These all depend on agglutination of horse or sheep erythrocytes by serum positive for heterophile antibodies after treatment of the serum with bovine or guinea pig kidney absorbent. Though the Paul-Bunnell test is simple to perform and inexpensive, it has several limitations -such as a false positive rate of around 7%,3 with fewer than half of children under 5 years with acute infectious mononucleosis having detectable heterophile antibodies.36 Greater sensitivity and specificity can be achieved by testing for antibody specific for EpsteinBarr virus, and commercial kits are available. These tests, which recognise antibody responses to several distinct viral antigens, are useful for resolving cases in which the infection is suspected but the Paul-Bunnell test gives a negative result and to help determine the state of the infection-that is, whether it is current, recent, past, or reactivated.39 The tests are based on immunofluorescence staining of lymphocytes infected with Epstein-Barr virus and are therefore subjective and rarely used outside the reference laboratory. Recently some enzyme immunoassay kits that use part of the EpsteinBarr virus associated nuclear antigen, EBNA-1, as capture antigen have appeared on the market. They can detect antiEBNA IgM and anti-EBNA IgG with appropriate conjugates, and the relative difference in antibody titres is used to correlate with the stage of disease. The manufacturers (Ortho Diagnostics, Buckinghamshire, and Clinical Sciences, New Jersey, United States) claim that the kits have sensitivities of 98-8-100% and specificities of 94-98-6% compared with western blot and immunofluorescence tests. The kits look promising, although independent evaluations have yet to be
published. Human immunodeficiency virus Diagnosis of infection with HIV has made great progress since the virus was discovered in 1983, and an array of commercial tests is now available for detecting viral antigen and antibody. The subject is too large to cover here but it has been reviewed by Jackson and Balfour.4' Most screening tests for HIV infection entail detecting HIV 1416
antibody by using one of the commercially licensed enzyme immunoassays. These kits use recombinant or partially purified viral antigens bound to polystyrene beads or plastic microtitre wells as the capture antigen and then use conventional or competitive binding assays to detect serum IgG and IgM antibodies to HIV. The sensitivity and specificity of these kits are excellent when compared with the western blot as the reference test; proportions above 98% were obtained for both measures in one comparison of different kits.4' Various rapid screening tests for detecting antibody have become available recently. The assays work on the principle of agglutination or dot immunobinding and give a visually readable result within 30 minutes. The tests are particularly suitable for use in developing countries, where enzyme imrnunoassays may be impracticable. In one comparative evaluation of five of these kits sensitivities ranged from 84-6% to 99 1% and specificities from 92-7% to 98-8% compared with the western blot assay.42 The western blot assay is the most sensitive of the tests for detecting antibody to viral core proteins and the envelope protein gp4l, though the radioimmunoprecipitation assay is better for detecting antibody to HIV glycoproteins.4344 Use of these tests is normally restricted to reference laboratories to help confirm positive results detected during screening tests. They are unsuitable for large scale use in regional laboratories, however, because of their technical difficulty. The principal limitation of antibody detection for diagnosis of infection with HIV is the long lag phase that may exist between infection and serocQnversion. This period is usually less than two to three months but may last for years.43 Assays to detect the virus directly either by antigen assay, virus isolation, or DNA hybridisation testing may help to clarify HIV state in high risk people during this "window" period. At this stage, however, only the antigen assay is suitable for routine use. Commercial enzyme immunoassays for detecting antigen are available from both Abbott Laboratories (Kent) and DuPont de Nemours (Wilmington, United States). The kits use polyclonal HIV antibody bound to polystyrene beads or plastic microtitre wells to capture free antigen in the patient's specimen, and the reaction is then detected with a double sandwich enzyme immunoassay to produce a coloured end product. The assay may be quantified by running it against known amounts of HIV antigen to generate a standard curve. In practice it detects mostly p24 core antigen because of the much higher affinity the specific antibody for this antigen has in relation to antibodies to the envelope proteins.46 Unfortunately the test is an insensitive indicator of infection with HIV, detecting antigen in the serum of only about a quarter to a half of seropositive people, depending on clinical state.4 The assay is therefore unlikely to be of much use for screening. In people with proved infection with HIV, however, development of p24 antigenaemia is a useful predictive marker for progression to AIDS, so the test may find some application.48 49
Chlamydia trachomatis Although Chlamydia trachomatis is a bacterial pathogen, we have included it here because its laboratory diagnosis has traditionally been made with cell culture, which has been the responsibility of the virus laboratory in most centres. Of all the microbial antigen detection tests developed in recent years none has received more attention or been more successfully adopted by testing laboratories than those now available for C trachomatis. Kellogg provides a more detailed discussion.
Isolation of C trachomatis in cell culture has long been BMJ
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recognised as the reference method with the highest sensitivity and specificity. The test has, however, never been widely used because of technical and other difficulties. 5' Antigen detection kits became available from commercial sources about seven years ago and have been extensively evaluated ever since. The kits, which use either immunofluorescence or enzyme immunoassay, have been used principally for diagnosing genitourinary infections, and in most of these studies sensitivities of 70-95% and specificities of 95-99% have been reported." The variation in kit performance depends on a host of factors such as the choice of kit, population sampled, and the skill of both the specimen collector and the technician. Immunofluorescence has the advantages over enzyme immunoassay of low cost, good quality assurance of specimen, rapid testing (results available within 30-60 minutes), and marginally btter performance profiles. It is unsuitable for processing many specimens a day, however, and reliable interpretation of fluorescence microscopy requires at least six months' training in detecting chlamydial elementary bodies. At least six immunofluorescence kits are now available commercially. Most comparisons suggest that the Syva MicroTrak test (Syva Company, Palo Alto, United States) gives better results, and a recent study of the brightness, consistency, and specificity of staining against 14 different serovars of the organism confirmed its superiority.52 Of the various enzyme immunoassay kits only Chlamydiazyme (Abbott) has been thoroughly evaluated. Its principal disadvantage stems from the use of a polyclonal antiserum, which can give reduced specificity owing to cross reaction with lipopolysaccharides of various Gram negative bacteria.5' An enzyme immunoassay that uses monclonal antibodies and was released recently by Pharmacia (Middlesex) may prove to be better." Evaluation of the antigen detection tests for diagnosing extragenital infection is limited. The Syva kit seems to work well on conjunctival specimens51 but is likely to be unsatisfactory for detecting the organism from nasopharyngeal specimens from infants.' Recently the possibility that the organism shares an antigen from its major outer membrane with strains of parainfluenza 2 virus has been raised. If this observation is confirmed additional tests to check specificity would be required. Special mention should be given to the use of these tests in evaluating suspected abuse in children. Both the Chlamydiazyme and Syva tests have been found wanting,5- and given the potential medicolegal implications of such forensic test results culture is the only option at present. Finally, as an aid in the diagnosis of trachoma antigen detection tests seem to be insufficiently sensitive because of the cryptic nature of the chlamydial infection, and diagnosis by using a DNA probe may be better. New diagnostic approaches Reports on new techniques to improve sensitivity, specificity, or the speed of diagnostic tests appear constantly, and it is difficult to determine which have genuine merit and will stand the test of time. Detection of microbial antigen by using polyclonal or monoclonal antibodies in conventional enzyme immunoassays and immunofluorescence assays has been examined exhaustively, and this technology is probably now close to its ultimate performance levels in terms of sensitivity and
specifcity. A novel detection method that deserves further evaluation is that of time resolved fluoroimmunoassay. In this technique time lapse fluorometry of lanthanide chelates conjugated to antibody permits detection of minute quantities of protein antigen. The lower limit of sensitivity is about 10 pg of BMJ
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protein,i which is 100-1000 times less than that detectable with conventional conjugates."' It is not clear whether this will translate into enhanced detection rates in clinical specimens, but preliminary evidence suggests that improvements over the standard assays may be significant.i The most exciting new development, however, is detection of DNA after amplification with the polymerase chain reaction. Detection of specific DNA by conventional methods that use hybridisation of target DNA to a labelled probe has had limited success as a diagnostic tool because of insensitivity (about 106 molecules of target DNA are required).i The polymerase chain reaction is, however, theoretically capable of amplifying a DNA segment from a single gene copy to a level that is readily detectable by conventional means such as ethidium bromide staining or hybridisation. In practice, a figure of 0 15 fg of DNA, equivalent to about six gene copies, has been detected in the case of cytomegalovirus.69 Viruses that cannot be isolated by routine culture are good candidates for diagnosis by this technique: HIV, papillomavirus, hepatitis B virus, and the papovaviruses BK virus and JC virus are examples.6669 Though the polymerase chain reaction looks promising as a rapid and highly sensitive alternative to conventional methods of diagnosis, important problems have to be solved before its routine use becomes practicable. The test is so sensitive that contamination of samples with stray DNA from the laboratory may generate false positive results. The problem can be detected by running negative controls alongside the test samples but a solution may prove more difficult. (The polymerase chain reaction assay may even need to be run in a building remote from the one in which the reagents are generated-a luxury that few laboratories would have available.) Another difficulty is that of determining the importance of a positive result. Distinction between DNA levels in health and in disease has not been made, and though for most acute viral diseases this is not an issue, the same is not true in chronic disease. Papillomavirus type 16 provides a good example: the polymerase chain reaction assay has shown it to occur in the cervix of over four fifths of women with normal cervical cytology and in every biopsy specimen of women with cervical cancer.6 The dilemma here seems to have been resolved by the recent discovery of subtypes of papillomavirus type 16-a and b -one of which was strongly associated with samples from women with carcinoma of the cervix. In the absence of such discoveries, however, the importance of a positive polymerase chain reaction result may remain uncertain. Selection of the specific segment of DNA to be amplified is clearly going to be critical. Finally, the potential application of the polymerase chain reaction in mass screening is limited because the procedures used for detecting the reaction products are laborious. Recently a colorimetric assay has been described for this, and it should be adaptable for use in a microtitre tray.' Whether such a format will control the problem of contamination remains to be seen. Conclusion Diagnosis of viral infection has come far since the days when isolation of the agent in cell culture and retrospective serology were the only options. For many of the commonly encountered viruses simple and reliable tests are now available in kit form from commercial sources and are suitable for use by smaller laboratories that may lack the equipment or skill required hitherto. As a group the respiratory viruses have remained refractory to detection by this approach, and the most prevalent causes of the common cold (rhinovirus and 1417
coronavirus) cannot be diagnosed at all in this way. Nevertheless, for viruses such as respiratory syncytial virus and rotavirus, which regularly cause sizable epidemics in winter, the antigen detection tests have become essential for both infection control and epidemiological purposes. Similarly, for the herpesviruses and C trachomatis, for which effective treatments are available, rapid diagnosis by these tests is making an important contribution to improved management and reduced morbidity of patients. More sensitive methods of detecting both antigen and DNA are being developed. They hold much promise for the future, especially for those viruses for which present methods
are unsatisfactory or unwieldy. Though predicting future trends is always hazardous, we think that DNA detection assays after amplification by the polymerase chain reaction will become commonplace once the current glitches have been sorted out. Given the pace of modern technology, that should occur well before 2000.
I Fenner FJ, White DO. Medical virology. 2nd ed. New York: Academic Press, 1976. 2 Reines ED, Gross PA. Update on antibiotics II: antiviral agenits. Med Clin North Am 1988;72:691715. 3 Galen RS, Gambino SR. Beyond normalisy-the predictive value and efficiencs of medical diagnosis. New York: John Wiley and Sons, 1975. 4 Lee IC. Quality control in clinical virology. In: Specter S, Lancz GJ, eds. Clinical virology mtanual. New York: Elsevier, 1986:3-14. 5 Paya CV, Wold AD, Smith TF. Detection of cytomegalovirus from blood leukocytes separated by sepracell-MN and ficoll-paque/macrodex method. 7 Clin Microbiol 1988;26:2031-3, 6 Ray CG, Minnich LL. Efficiency of immunofluorescence for rapid detection of common respiratory viruses. ] Clin Microbiol 1987;25:355-7. 7 Kumar ML, Super DMI, Lembo RM, Thomas PC, Prokay SL. Diagnostic efficacy of two rapid tests for detection of respiratory syncytial virus antigen.] Clin Microbiol 1987;25:873-5. 8 Shalit I, McKee PA, Beauchamp H, Waner JL. Comparison of polyclonal antiserum versus monoclonal antibodies for the rapid diagnosis of influenza A virus infections by immunofluorescence in clinical specimens.] Clin Microbiol 1985;22:877-9. 9 Hallsworth PG, McDonald PJ. Rapid diagnosis of viral infections with fluorescent antiscra.
38 Fleisher G, Lennette ET, Henle G, Henle W. Incidenice of heterophile antibody responses in children with infectious mononucleosis. 7 Pediatr 1979;94:723-8. 39 Lennette ET. Epstein-Barr virus. In: Lennette EH, Balows A, Hausler WVJ, Shadomy HIJ, eds. Manual of clinical microbiology. 4th ed. Washington, DC: American Socicty for Microbiology,
Pathology 1985;17:629-32. 10 Gleaves CA, Smith TP, Shuster EA, Pearson GR. Comparison of standard tube and shell vial cell culture technique for the detection of cytomegalovirus in clinical specimens. ] Clin Microbiol 1985 ;21:2 17-2 1. 11 Stokes CE, Bernstein JMNl, Kyger SA, Hayden FG. Rapid diagnosis of influenza A and B by 24-h fluorescent focus assays. ] Clin Microbiol 1988;26:1263-6. 12 Madeley CR. Viruses in the stools. .7 Clin Pathol 1979;32:1-10. 13 Kok TW, Burrell CJ. Comparison of five enzyme immunoassays, electron microscopy and latex 1989I27:364-6. agglutination for detection of rotavirus in fecal specimens.] Clin Microbiol 14 Brandt CD, Arndt CW, Evans GL, et al. Evaluation of a latex test for rotavirus detection. ] Clin Microbiol 1987;25: 1800-2. 15 Feinstone SiM, Kapikien AZ, Purcell RH. Hepatitis A: detectioni by immune electron microscopy of virus like antigen associated with acute illness. Science 1973;182:1026-8. 16 Flehmig G, Ranke M, Berthold H, Gerth H-J. A solid-phase radioimmunioassay for detection of IgM antibodies of hepatitis A virus. ] Infect Dis 1979;140:169-75. 17 Duermayer W, Wielaard F, van der Veen J. A new principle for the detection of specific IgM antibodies applied in an ELISA for hepatitis A. ] Med Iirol 1979;4:25-32. 18 Huang SN, Neurath AR. Immunohistologic demonstration of hepatitis B viral antigens in liver with reference to ist significances in liver in'jury. Lab Invest 1979;40:1-17. 19 Locarnini SA, Gust ID. Hepadnaviridae: hepatitis B sirus and the delta virus. In: Balows A, Hausler WJ, Lennette EH, eds. Laboratory diagnosis of infectious diseases. New York: SpringerVerlag, 1988:750-96. 20 Nielsen JO, Dietrichson 0, Elling P. Incidence and meaning of persisteltce of Australia antigen in patients with acute viral hepatitis: development of chronic hepatitis. N Engl.7 Med 1981;285: 1157-60. 21 Esteban JI, Esteban R, ViladomiL L, etal. Hepatitis C virus antibodies among risk groups in Spain. Lancet 1989;ii:294-6. 22 Van der Poel CL, Reesink HW, I.elie PN, et al. Anti-hepatitis C antibodies and non-A, non-B posttransfusion hepatitis in The Netherlands. Lancet 1989;ii:297-8. 23 Callihan DR, Menegus MA. Rapid detection of herpes simplex virus in clinical specimens with human embryonic lung fibroblast and primary rabbit kidnev cell culture. ] Clin Microbiol 1984;19:563-5. 24 Lafferty WIE, Krofft S, Remington M, et al. Diagnosis of herpes simplex virus by direct immunofluorescence and viral isolation from samples of external genital lesions in a highprevalence population. ] Clin Microbiol 1987;25:323-6. 25 Dascal A, Chan-Thim J, Morahan M, IPortnov J, Mendelson J. Diagnosis of herpes simplex virus infection in a clinical setting by a direct antigen detection enzyme immunoassav kit. J Clin
Microbiol 1989;27:700-4. 26 Halstead DC, Beckwith DG, Sautter RL, Plosila L, Schneck K. Evaluation of a rapid latex slide aggltttination test for herpes simplex sirus as a specimen screen and culture identification method. ] Clin Microbiol 1987;25:936-7.
27 Espy MJ, Smith rP. Detectiott of hcrpes sinmplex sirus in conventional tube cell ctultures and in shell vials with a DNA probe kit and monoclonal antibodies. ] Clin Microbiol 1988;26:22-4. 28 Olding-Stenkirst E, Grandien M. Early diagnosis of virus-caused sesicular rashes by immunofluorescence on skin biopsies 1. Varicella zoster and herpes simplex. Scand.7 Infect Dis 1976;8: 27-35.
29 Gleaves CA, Lee CF, Bustamante Cl, Meyers JD. Use of murine monoclonal antibodies for laboratory diagnosis of varicella-zoster sirus itnfection. 7 Clin .icrobiol 1988;26: 1623-5. 30 D)oane FW. Electron microscopy and immitnoelectron microscopy. In: Specter S, Lancz GJ, eds. Clinicall virology manual. New York: Elsevier, 1986:71-88. 31 Griffiths PD, Stirk PR, Ganczakowski M, st al. Rapid diagnosis of cvtomegalovirtts infection in immunocompromised patients by detection of early antigen fluorescent foci. Lancet 1984;ii: 1242-5.
32 Swenson PD, Kaplan MH. Comparison of two rapid culture methods for detcction of cytomegalovirtus in clinical specimens.] Clin Microbiol 1987 25:2445-6. 33 Randazzo DN, Michalski PJ. Comparison of antibodies for rapid dctection of cytomegalovirus. ] Clin Microbiol 1988;26:369-70. 34 van de Bij W, Torensma R, van Son WJ, et al. Rapid immttnodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leucocytes. .7 Med Virol 1988;25:179-88. 35 Crawford SW, Bowdeni RA, Hackman RC, et al. Rapid detection of cytomegalovirus pulmonary infection by bronchoalveolar lavage and centrifuge culture. Ann Intern Mled 1988;108:180-5. 36 Davidson I, Lee CL. The clinical serology of infectious mononucleosis, In: Carter RL, Penmnan HG, eds. Intfectious mononucleosis. Oxford: Blackwell, 1969:177-200. 37 Evans AS, Hiederman JC, Cenabre LC, West B, Richards VA. A prospective evaluation of heterophile and Epstein-Barr sirus-specific IgM1 antibody tests in clinical and subclinical infcctious mononucleosis: specificity and sensitivity of the tests and persistence of antibody. J Infect Dis 1975;132:546-54.
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Staff Specialist in Clinical M:crobiology PETER HALLSWORTH Senior NVirologist Department of Clinical Microbiology, Flinders Medical Centre, South Australia 5042.
1985:728-32. 40 Jackson BJ, Balfour HH. Practical diagnostic testing for human immunodeficiency virus. Clinical Microbiology Reviews 1988;1:124-38. 41 Reesink HW, Lelie PN, Huisman JG, et al. Evaluation of six enzyme imnmunoassays for antibody against human immunodeficiency virus. Lancet 1986;ii:483-6. 42 Spielberg F, Kabeya CM, Ryder RW, et al. Field testing and comparative esaluation of rapid, visually read screening assays for antibody to human immunodeficienc sirus. Lanzcet 1989;i:
580-4. 43 Chiodi F, Bredberg-Raden U, Biberfeld G, et al. Radioimtnunoprecipitation and western blottitsg with sera of human immunodeficiency virus infected patients: a comparative study. AIDS Res 1987;3: 165-76. 44 Gaines H, Sonnerborg A, Czajkowski J, et al. Antibody response in primary human immuLnodeficiency virus infection. Lancet 1987;i: 1249-53. 45 Ranki A, Valle S, Krohn M, et al. Long latency precedes osert seroconversion in sexuallv transmitted human immunodeficiencv virus infection. ILancet 1987;ii:589-93. 46 Goudsmit J, Lange JMA, Paul DA, Dawson GJ. Antigenemia and antibody titers to core anid envelope antigens in AIDS, AIDS-related complex and subclinical human immunodeficiencv virus infection. 7 InJect Dis 1987;155:558-60. 47 Re MC, Furlini G, La Placa M. Patterns of antigenaemia and antibody response in patients infected with human immunodeficiency virus (HIV) according to clinical state..7 Clin Pathol 1989;42: 282-3. 48 Kenny C, Parkin J, Undershill G, et al. HIV antigen testing. I.ancet 1987;i:565-6. 49 Eyster ME, Ballard JO, Gail MH, Drummond JE, Goedert jj. Predictive markers for the acquired immunodeficiencv syndrome (AIDS) in hemophiliacs: persistence of p24 antigen and low T4 cell count. Ann Intern Med 1989;110:963-9. 50 Kellogg JA. Clinical and laboratory considerations of culture vs antigen assays for detection of Chlamycdia trachaomatis from genital specimens. Arch Pathol Lab Med 1989;113:453-60. 51 Stamm WE. Diagnosis of Chlamydia trachomatis genitourinarv infections. Ann Intern Med
1988;108:710-7. 52 Cles LD, Bruch K, Stamm WlE. Staining characteristics of six commercially available monoclonal immunofluorescence reagents for direct diagnosis of Chlamvdia trachomatis infection. ] Clin Mtlcrobtol 1988;26:1735-7. 53 Mumtaz G, Ridgewav GL, Nayagam A, Oricl JD. Enzyme immunoassay compared with cell culture and immunofluorescence for detecting genital chlamydia. ] Clin Pathol 1989;42:658-60. 54 Potts MI, Paul ID, Rooma AP, Caul EO. Rapid diagnosis of Chlamydia trachomatis infection in patients attending an ophthalmic casualty department. Br] Ophthalmol 1986;70:677-80. 55 Roblin PM, Hammerschlag MR, Cttmmings C, Williams TH, Worku M. Comparison of two rapid microscopic methods and culture for detection of Chlamydia trachomatis in ocular aitd nasopharyngeal specimens from infants. ] Chlin icrobiol 1989;27:968-70. 56 Fox AS, Saxon EMI, Doveikis S, Beem 10. Chlamvdia trachomatis and parainfluenza 2 virus: a shared antigenic determinant?] Clin Mficrobiol 1989;27:1407-8. 57 Hammerschlag MR, Rettig PJ, Shields MIE. False positive results with the use of chlamrdial antigen detection tests in the esaluation of suspected sexual abuse in children. Pediatr InfeJct l)isj 1988;7: 11-4. 58 Hauger SB, Brown J, Agre F, et al. Failure of direct fluorescent antibody staining to detect Chlamydia trachomatis from genital tract sites of prepubertal children at risk for sexual abusc. Pediatr Infect D)isj 1988;7:660-1. 59 Dean D, Palmer L, Pant CR, et al. Use of a Chlamydia trachomatis DNA probe for detectiots of' ocular chlamydiae. J Clin Microbiol 1989;27: 1062-7. 60 Walls HH, Johanssott KH, Harmon MW, Halonen PE, Kendal AP. Time-resolved fluoroimmtunoassay with monoclonal antibodies for rapid diagnosis of influenza infections. ] Clin Microhiol
1986;24:907-12. 61 Grandien M, Petterson CA, Gardner PS, Linde A, Stanton A. Rapid viral diagnosis of acute respiratory inf'ections: comparison of enzyme-linked immunosorbent assay and the immunofluorescence technique for detcction of viral antigen in nasopharyngeal secretions. ] f,in Microbiol 1985;22:757-60. 62 Hierholzer JC, Johansson KH, Anderson LJ, 'I'sou CJ, Halonen PE. Comparison of monoclolnal time-resolved fluoroimmunoassav with monoclonal capture-biotin'ylated detector enzyme immunoassav for adenovirus antigen detection.]I Clin Microbiol 1987;25:1662-7. 63 Hierholzer JC, Binham PG, Coombs RA, et al. Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection. 7 Clin Microbiol 1989;27: 1243-9. 64 Chou S, Roark L, Merigan TC. DNA of 21 clinical c-tomegalovirus strains detected by hybridisation to cloned DNA fragments of laboratory strain AD-169. 3Med Virol 1984;14:263-8. 65 Olive DM, Simsek M, Al-Mufti S. Polymerase chain reaction assay for detectiots of' hbumats cVtomegalovirus. 7 Clin Microbiol 1989;27:1238-42. 66 Rogers MF, Oui MID, Rayfield M, et al. Use of the polyimerase chain reaction for early dctectoiot of the proviral sequences of human immunodeficiencv sirus in infants born to seropositive motlers. N Engl]Med 1989;320:1649-54. 67 'Tidy JA, Parrv GCN, 'Vard P!'et al. High rate of human papillomavirus type 16 ittfectioll in cVtologically normal cervices. Lancet 1989;i:434. 68 Sumazaki R, NMotz M, Wolf H, et al. Detection of hepatitis B sirus in serum tisiitg amplificatioit oif viral DNA by meatis of the polymerase chain reaction.]7 Med l'irol 1989;27:304-8. 69 Arthur RR, Dagostin S, Shah KV. Detection of BK virus and JC sirus in urine and braiti tissuc by the polymerase chain reaction. ] Clin Microbiol 1989;27:1174-9. 70 Tidy JA, VoLusden KH, Farrell PJ. Relation between infection with a subtype of HPV' 16 and cervical neoplasia. Lancet 1989;i: 1225-7. 71 Kemp DJ, Smith DB, Foote SJ, et al. Colorimetric detection of specific DNA scgmelts amplified by polymerase chain reaction. Proc NatlAcadSci USA 1989;86:2423-7.
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effective hyposensitization in house dust mite allergy. Clini-cal andti Experimental Allergy 1989;19:327-34. Warner JO, Price JF, Soothill JF, Hey EN. Controlled trial of hyposensitisation to D)ermatophagoides pteronvssinus in children with asthma. Lancet 1978;ii:912-5. Bousquet J, Michel FB. Specific immunotherapy in asthma. In: Reed CE, ed. P'roceedings of tthe twelfth international congress of allcrgology and clinical immunology. St Louis: Mosby, 1986:397402. Norman PS. Specific therapy in allergy. Med Clin North Am 1974;58:111-25. Warner JO. Hyposensitization in asthma: a review.,7 R Soc Med 1981;74:60-5. Osterballe 0. Immunotherapy with grass pollen major allergens. Clinical results from a prospectiVe 3 year double blitsd study. Allergy 1982;37:379-88. Norman PS, Lichtenstein LM. Comparison of allum-precipitated and unprecipitated aqueous ragweed cxtracts in the treatment of hayfever.,71 AllergyClin Immunol 1978;61:384-9. Norman PS. An otverview of immunotherapy; implications for the future.] .Allergy Clin Immunol 1980;65:87-96. Bousquet J, Fontez A, Aznar R, Robinet-Lesv M, Michel F-B. Combination of passive and active immunisation in honeybee venom immunotherapy.]Y Allergy Cltn Immunol 1987;79:947-54. AMuller UR, Morris T, Bischof M, Friedli H, Skarvil F. Combined active and passive immunotherapy in honevbee-sting allergy. 7Allergy Clin Immunol 1986;78:115-22.
Regular Revieew
Rapid viral diagnosis in perspective Many viruses can now be detected routinely in small laboratories; even more will be possible by 2000 Though viral infections are the commonest cause of human disease, diagnostic services are available in only a few microbiology laboratories.' Traditionally, the procedures used for viral diagnosis have been too slow to influence patient management and too expensive and tedious to encourage widespread use. The self limiting nature of most viral diseases has encouraged clinicians not to seek a specific diagnosis, and, even when a diagnosis has been made, therapeutic options have been limited. In the past 10 years, however, the picture has changed dramatically, and viral diagnostic tests need to be more widely available. With the increasing use of immunosuppressive and cytotoxic drugs and as a consequence of AIDS severe viral infections are a common complication of modern medicine. Knowledge of the specific cause has obvious benefits, such as more accurate prognosis, reduction in clinical investigations and the use of antibiotics, and better patient management and compliance. It also leads to a better understanding of the epidemiology of viral diseases, which is important for controlling transmission. Antiviral chemotherapy has made important advances, and therapeutic options now exist for several infections.2 Acyclovir has been proved to be of value in treating established herpes simplex virus and varicella zoster virus infection in both immunocompetent and immunosuppressed patients. Serious respiratory viral disease due to influenza A virus and respiratory syncytial virus are now treatable with amantadine and tribavirin respectively. Even the newly discovered human immunodeficiency virus may be controlled at least in part by zidovudine. Recently ganciclovir and foscarnet have given promising results in immunosuppressed patients with serious cytomegalovirus disease. There are also tantalising reports suggesting that both papillomavirus infection and hepatitis B virus infection may be susceptible to immunomodulatory treatment with interferons. Though infection with these viruses is amenable to treatment, chemoprophylaxis and immunoprophylaxis may be an effective alternative for other viruses.2 Acyclovir and immunoglobulin prophylaxis have shown benefit in preventing or reducing the severity of varicella zoster virus and cytomegalovirus infections. Amantadine offers effective prophylaxis against influenza A infection and is also a useful adjunct to late immunisation in people at high risk. Although BMJ VOLUME 300
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antiviral treatment and prophylaxis are still in their infancy, they are making an impact and their influence is bound to increase in the future. As a result of these developments the demand for rapid viral diagnosis has increased substantially in recent years. Much technical progress has been made, and the laboratory can now provide reliable diagnosis of a comprehensive range of virus infections within a day or two after receiving the specimen. We review here the laboratory tests now available, emphasising those that are applicable "in the field" away from the reference laboratory.
Performance evaluation of a new test The performance measures used to evaluate the usefulness of a new test are sensitivity, specificity, and positive and negative predictive values. These terms are familiar and are defined elsewhere,' but a couple of points are worth emphasising. For culturable viruses the reference test to which these measures relate is usually isolation in tissue culture. Though specificity of virus isolation may be safely assumed, sensitivity depends on several factors that may compromise recovery of virus from clinical specimens and so generate false negative results.4 As the technology improves rapid tests are appearing that are clearly more sensitive than the reference methods. This may be a manifestation of that very problem, and other tests-for example, serological confirmation-must then be applied to determine whether the additional positive results are real or false. An example of this is the recently developed rapid culture test for cytomegalovirus, in which up to twice as many specimens give positive results than with conventional culture, which remains the reference method.I Another example is the detection of pathogen specific DNA after amplification with the polymerase chain reaction. Here a new test is so sensitive that validating the results by alternative tests may be difficult if not impossible. As described below, the only way that the results can be validated in these circumstances is to rely on stringent attention to quality control and technique by the operator. A measure that is often overlooked in evaluating a new test is its positive predictive value-that is, the proportion of true positive results. When the test has a specificity of under 100% 1413
the predictive value will show considerable variation for different populations depending on the prevalence ofinfection in the group under study because if most of the specimens examined give true negative results a small fraction of false positive results becomes important. The effect is shown in the table, which gives the sensitivity and specificity levels attainable with the current tests for detecting chlamydial antigen. Test kits that seem to have quite reasonable performance may generate a large percentage of false positive results in a condition with low prevalence (over 30% in this example). Effect of disease prevalence on positive predictive value Sensitivity (%) 85 85
Specificity ( 98 98
Prevalence 20 5
Positive predictive value (oN)) 91
69
These calculations apply to all screening tests with a specificity of less than 100%. It is therefore important to be aware of the disease prevalence in the population under study before relying too much on the result.
Respiratory viruses The viruses that infect the respiratory tract have remained difficult to identify with the rapid methods. The diagnosis of upper respiratory tract infection is rarely requested because the infection is usually trivial. Virus isolation continues to be the only reliable approach for detecting the lower respiratory tract pathogens. As with conventional serology, this may take two to three weeks to provide an answer, which in most cases is too slow to influence patient management. For a few specific viruses the rapid tests do work adequately. Respiratory syncytial virus produces regular epidemics of childhood bronchiolitis in winter, and detection of antigen by either immunofluorescence or enzyme immunoassay is reliable in these circumstances.6 Many comparisons with virus isolation have been published, and sensitivities of 95% or better and specificities of around 98% are usually reported.7 These tests tend to pick up a few additional "false" positive results, which probably represent false negative isolation results. Few commercial kits exist for other respiratory viruses. This severely limits adoption of rapid testing outside the reference laboratory. Polyclonal antisera against influenza viruses A and B, parainfluenza viruses 1 and 3, and adenovirus group antigens are available from Wellcome and are suitable for immunofluorescence testing. The influenza virus A antiserum is useful during outbreaks, but the sensitivity is only around 50% (with varying specificity) compared with isolation.6" The usefulness of the other antisera is questionable. Though specificities are very high, the sensitivities are only 20-60% and are too low to justify their use as a screening test in the absence of tissue culture back up.69 Commercial enzyme immunoassays for detecting these antigens have not been evaluated. Similarly, reports on the use of monoclonal antibodies are sporadic and commercial suppliers have not yet adopted them. An alternative approach to the respiratory viruses that has received scant attention is the fluorescent focus assay, which is similar to that developed for rapid detection of cytomegalovirus in shell phials. "' The technique entails centrifugation of the specimen on to a monolayer of permissive tissue culture cells, incubation overnight, then staining the cells with immunofluorescent antisera directed against antigens expressed early in the virus's replication cycle. In one study 1414
that used monoclonal antibodies to detect influenza viruses A and B the method achieved a sensitivity of 84% and specificity of 100l compared with culture." Conceivably, respiratory specimens could be screened for a wide range of viruses by using multiwell tissue culture trays in place of cover slips. The important unanswered question is whether the commercial antisera available can detect the early antigen with sufficient sensitivity to make the test reliable.
Gastrointestinal viruses Viruses infecting and causing illness in the gastrointestinal tract have been surprisingly difficult to isolate in cell culture. Their discovery and diagnosis have thus depended largely on electron microscopy. Viruses such as rotavirus, adenovirus type 40/41, calcivirus, astrovirus, and the small round virus group -for example, Norwalk virus-are refractory to isolation but may be detected readily on a negatively stained electron microscopy grid. 12 For many of these viruses electron microscopy is not just a rapid method (results are available within 20 minutes); it is the only method. Direct electron microscopy, in which a clarified faecal suspension is placed directly on to a plastic coated grid and stained with a heavy metal salt solution, is insensitive. At least 106 particles/g of faeces are necessary for reliable detection, which means that substantial amounts of virus may be missed. Though ultracentrifugation or immunological aggregation may be used to concentrate the virus for improved sensitivity, the technique is unsuitable for large scale screening. Immunoelectron microscopy has an additional use in identifying viruses such as the Norwalk virus (which has no characteristic morphology), but its use is restricted to the reference laboratory. Rotavirus is the only virus in this group that occurs in regular winter epidemics and for which other tests have been developed. Several enzyme immunoassay and latex agglutination tests are available commercially, and numerous comparisons with electron microscopy have been published. The enzyme immunoassays are significantly more sensitive than electron microscopy. A recent study showed that one such kit produced over twice as many positive results as electron microscopy.'" The specificity of the test is determined by performing a confirmatory test on the positive specimens by blocking the reaction with an unlabelled rotavirus antiserum. When this is done the modern enzyme immunoassays have adequate specificity, but between 1% and 5% of apparently positive results fail the confirmatory test and are deemed false positive results. This shows the principal disadvantage of enzyme immunoassay: the need to confirm all positive findings with a blocking assay. If the laboratory has only a small paediatric load the cost may also become important because of the control samples required to validate each run. Latex agglutination is not as sensitive as the best of the enzyme immunoassays, but at least one kit is as sensitive as electron microscopy, is highly specific, and seems to be a reasonable alternative for small laboratories." 4 In summary, electron microscopy continues to be an extremely useful test as the only "catch all" method of detecting the gastrointestinal viruses. The sensitivity depends on the operator, however, and, even in competent hands, is suboptimal. Alternative methods have been developed only for rotavirus, and the choice depends to some extent on workload.
Hepatitis viruses At least five viruses that infect humans are recognised for their ability to cause hepatitis: A, B, C, D, and non-A, non-B BMJ
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hepatitis viruses. Routine tests for rapid diagnosis are available only for hepatitis A and hepatitis B viruses. Hepatitis A virus is shed in large numbers in the faeces in the two weeks preceding the overt illness and can be detected readily by immune electron microscopy. Viral excretion diminishes rapidly thereafter, however, and only half of all patients give positive results within one week and less than a tenth within three weeks of the onset of symptoms. 5 A more successful approach, which most laboratories now use, is the detection of antihepatitis A virus IgM in serum. Sensitive commercial radioimmunoassays and enzyme immunoassays are 100% sensitive for up to six months after an acute infection." Diagnosing hepatitis B virus is complicated because infection may follow various patterns, each of which is associated with a different array of viral markers. Direct detection of the virus and its antigens by electron microscopy and the various staining procedures such as immunofluorescence and immunoperoxidase has been applied to biopsy or necropsy material and is useful in diagnosing the various forms of chronic hepatitis.'8 It is not, however, suitable for rapid screening of hepatitis B virus infection in routine laboratories, which rely on the serological markers of the virus. Commercial radioimmunoassay and enzyme immunoassay kits are available for detecting antibody to the virus's surface antigen (antiHBs), core antigen (anti-HBc), and e antigen (anti-HBe) as well as for detecting the antigens themselves (HBsAg and HBeAg). In a typical case of acute self limited infection with hepatitis B virus HBsAg appears first in serum followed by HBeAg, anti-HBc, anti-HBe, and then anti-HBs.'9 In 80-90% of cases rapid diagnosis is made on the basis of a positive result for HBsAg; current third generation immunoassays can detect as little as 0 ng/ml of the antigen.'9 The remaining cases are usually detected by the presence of a high titre of anti-HBc IgM in an acute phase serum sample or seroconversion to antiHBs antibody between acute and convalescent phase serum samples. Chronic infection with hepatitis B virus occurs in 5-10% of adults who are unable to eliminate HBsAg within the usual time frame-that is, less than three months.20 Continued monitoring of concentrations of HBsAg and the other markers, including hepatitis B virus DNA, is then important to determine what form (asymptomatic, chronic persistent, or chronic active) the infection is likely to take. This need arises once hepatitis B virus infection has been diagnosed and would fall beyond the scope of most routine laboratories. Identification of the recently recognised hepatitis C virus is in its infancy at present. The only specific test is for the presence of hepatitis C virus antibodies in serum by using a cloned viral polypeptide of 363 amino acids as the capture antigen in an immunoassay. In the absence of other tests for comparison the sensitivity and specificity of the assay can be assumed to be satisfactory only on the basis of published seroprevalence studies. These show that hepatitis C virus antibodies are found in up to 85% of patients who become infected with non-A, non-B hepatitis virus through transfusion and implicated donors but in only 1% or less of people without risk factors for hepatitis.2' 22 The early serological studies were done with a home grown radioimmunoassay at the Chiron Corporation, where the polypeptide was initially cloned. Commercial kits using an enzyme immunoassay format, however, are now available from Ortho Diagnostics (Buckinghamshire) and Abbott Diagnostics (Hampshire). Though these kits will be useful in providing a diagnostic test and for screening blood donations for hepatitis C, the results should be treated with caution as confirmatory tests are not available at present. -
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Herpesvirus group Herpes simplex virus Of all of the tests for viruses that the diagnostic laboratory is called on to do, that for herpes simplex virus is likely to be the most frequently requested. The virus grows rapidly in permissive cell lines; about 80% of positive cultures are usually detectable by visual cytopathic effect within two days, and more than 99% are detectable by four days.23 Nevertheless, rapid methods of diagnosis have proliferated and include centrifugation enhanced culture, the use of DNA probes, and antigen detection by immunofluorescence, enzyme immunoassay, and latex agglutination. The success of diagnostic testing for genital herpes depends on the stage of the lesion. In one study the virus was detectable in 70% or more of vesicular lesions but in fewer than 20% when the specimen had been taken after crusting had occurred.24 Overall, immunofluorescence with a monoclonal antibody based kit had a sensitivity of 74% and specificity of 85% compared with isolation but was more reliable in specimens taken from early stage lesions than from those taken later on. Enzyme immunoassay may prove to be more sensitive than immunofluorescence as it detects both extracellular and -intracellular antigens and does not impose the demands for collection of infected cells that are required for reliable immunofluorescence testing. In one recent study a newly released enzyme immunoassay kit was shown to have a sensitivity of 95% with 100% specificity for the diagnosis of genital herpes.25 In contrast, latex agglutination seems to be insufficiently sensitive for use without culture back up, detecting only half of the cases with positive results on isolation in one series.26 Compared with the same day tests centrifugation enhanced culture in shell phials is slow, taking 24 hours to complete. The assay is, however, at least as sensitive as conventional culture and is highly specific.27 At present this is the most promising of the alternative tests to virus isolation, and for laboratories with heavy herpes simplex virus workloads it might produce savings in labour.
Vanrcella zoster virus Varicella zoster virus is notoriously labile and attempts at isolation often fail. Ironically, skin lesions caused by varicella zoster virus often contain large quantities of the virus, and diagnosis by antigen detection is the most sensitive method.2829 This approach has had limited application in the past because commercially available reagents were not available, but now a monoclonal antibody kit that uses direct immunofluorescence has been developed. In one evaluation the kit detected significantly more patients who were positive for the virus than did standard culture or centrifugation enhanced culture and had a sensitivity of 92% compared with all methods tested.29 Electron microscopy does not distinguish varicella zoster virus from other members of the herpesvirus group, but because the virus is present in high concentrations in vesicular and pustular fluid the test is often adequate to provide a rapid presumptive diagnosis.30 We have found it useful when used in conjunction with isolation to confirm the virus type.
Cytomegalovirus Rapid diagnosis of cytomegalovirus infection has made major strides since the discovery that viral antigen expressed early in the replication cycle can be detected reliably with fluorescent antisera within a day or two of inoculation into susceptible fibroblast cultures.3' When used in combination with low speed centrifugation to enhance infectivity the 1415
procedure is significantly more sensitive than conventional isolation with a variety of clinical specimens.32 This centrifugation enhanced culture has gained wide acceptance as the method of choice for diagnosing cytomegalovirus, and several commercial companies now supply monoclonal antibodies to detect early antigens. Same day diagnosis of cytomegalovirus infection by antigen detection in clinical specimens has been reported with v4rying success. One group reported a sensitivity of 93% for detecting the virus in blood leucocytes of patients with renal transplants by using a cocktail ofmonoclonal antibodies against early viral antigens.34 In another study direct detection of the virus in bronchoalveolar specimens of patients with cytomegalovirus pneumonia was less satisfactory: only 59% of culture positive specimens were positive by immunofluorescence staining.33 Epstein-Barr virus Despite the advances in direct virus detection for other members of the herpesvirus group the diagnosis of acute Epstein-Barr virus infection by routine laboratories still depends on the detection of heterophile (Paul-Bunnell) antibodies and antibodies specific for Epstein-Barr virus in serum. The Paul-Bunnell assay remains the front line screening test and can identify 85-95% of patients with infectious mononucleosis during the first month of illness.36 It has several different formats for measurement or qualitative determination, but the most common assays today are the rapid slide agglutination tests. These all depend on agglutination of horse or sheep erythrocytes by serum positive for heterophile antibodies after treatment of the serum with bovine or guinea pig kidney absorbent. Though the Paul-Bunnell test is simple to perform and inexpensive, it has several limitations -such as a false positive rate of around 7%,3 with fewer than half of children under 5 years with acute infectious mononucleosis having detectable heterophile antibodies.36 Greater sensitivity and specificity can be achieved by testing for antibody specific for EpsteinBarr virus, and commercial kits are available. These tests, which recognise antibody responses to several distinct viral antigens, are useful for resolving cases in which the infection is suspected but the Paul-Bunnell test gives a negative result and to help determine the state of the infection-that is, whether it is current, recent, past, or reactivated.39 The tests are based on immunofluorescence staining of lymphocytes infected with Epstein-Barr virus and are therefore subjective and rarely used outside the reference laboratory. Recently some enzyme immunoassay kits that use part of the EpsteinBarr virus associated nuclear antigen, EBNA-1, as capture antigen have appeared on the market. They can detect antiEBNA IgM and anti-EBNA IgG with appropriate conjugates, and the relative difference in antibody titres is used to correlate with the stage of disease. The manufacturers (Ortho Diagnostics, Buckinghamshire, and Clinical Sciences, New Jersey, United States) claim that the kits have sensitivities of 98-8-100% and specificities of 94-98-6% compared with western blot and immunofluorescence tests. The kits look promising, although independent evaluations have yet to be
published. Human immunodeficiency virus Diagnosis of infection with HIV has made great progress since the virus was discovered in 1983, and an array of commercial tests is now available for detecting viral antigen and antibody. The subject is too large to cover here but it has been reviewed by Jackson and Balfour.4' Most screening tests for HIV infection entail detecting HIV 1416
antibody by using one of the commercially licensed enzyme immunoassays. These kits use recombinant or partially purified viral antigens bound to polystyrene beads or plastic microtitre wells as the capture antigen and then use conventional or competitive binding assays to detect serum IgG and IgM antibodies to HIV. The sensitivity and specificity of these kits are excellent when compared with the western blot as the reference test; proportions above 98% were obtained for both measures in one comparison of different kits.4' Various rapid screening tests for detecting antibody have become available recently. The assays work on the principle of agglutination or dot immunobinding and give a visually readable result within 30 minutes. The tests are particularly suitable for use in developing countries, where enzyme imrnunoassays may be impracticable. In one comparative evaluation of five of these kits sensitivities ranged from 84-6% to 99 1% and specificities from 92-7% to 98-8% compared with the western blot assay.42 The western blot assay is the most sensitive of the tests for detecting antibody to viral core proteins and the envelope protein gp4l, though the radioimmunoprecipitation assay is better for detecting antibody to HIV glycoproteins.4344 Use of these tests is normally restricted to reference laboratories to help confirm positive results detected during screening tests. They are unsuitable for large scale use in regional laboratories, however, because of their technical difficulty. The principal limitation of antibody detection for diagnosis of infection with HIV is the long lag phase that may exist between infection and serocQnversion. This period is usually less than two to three months but may last for years.43 Assays to detect the virus directly either by antigen assay, virus isolation, or DNA hybridisation testing may help to clarify HIV state in high risk people during this "window" period. At this stage, however, only the antigen assay is suitable for routine use. Commercial enzyme immunoassays for detecting antigen are available from both Abbott Laboratories (Kent) and DuPont de Nemours (Wilmington, United States). The kits use polyclonal HIV antibody bound to polystyrene beads or plastic microtitre wells to capture free antigen in the patient's specimen, and the reaction is then detected with a double sandwich enzyme immunoassay to produce a coloured end product. The assay may be quantified by running it against known amounts of HIV antigen to generate a standard curve. In practice it detects mostly p24 core antigen because of the much higher affinity the specific antibody for this antigen has in relation to antibodies to the envelope proteins.46 Unfortunately the test is an insensitive indicator of infection with HIV, detecting antigen in the serum of only about a quarter to a half of seropositive people, depending on clinical state.4 The assay is therefore unlikely to be of much use for screening. In people with proved infection with HIV, however, development of p24 antigenaemia is a useful predictive marker for progression to AIDS, so the test may find some application.48 49
Chlamydia trachomatis Although Chlamydia trachomatis is a bacterial pathogen, we have included it here because its laboratory diagnosis has traditionally been made with cell culture, which has been the responsibility of the virus laboratory in most centres. Of all the microbial antigen detection tests developed in recent years none has received more attention or been more successfully adopted by testing laboratories than those now available for C trachomatis. Kellogg provides a more detailed discussion.
Isolation of C trachomatis in cell culture has long been BMJ
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recognised as the reference method with the highest sensitivity and specificity. The test has, however, never been widely used because of technical and other difficulties. 5' Antigen detection kits became available from commercial sources about seven years ago and have been extensively evaluated ever since. The kits, which use either immunofluorescence or enzyme immunoassay, have been used principally for diagnosing genitourinary infections, and in most of these studies sensitivities of 70-95% and specificities of 95-99% have been reported." The variation in kit performance depends on a host of factors such as the choice of kit, population sampled, and the skill of both the specimen collector and the technician. Immunofluorescence has the advantages over enzyme immunoassay of low cost, good quality assurance of specimen, rapid testing (results available within 30-60 minutes), and marginally btter performance profiles. It is unsuitable for processing many specimens a day, however, and reliable interpretation of fluorescence microscopy requires at least six months' training in detecting chlamydial elementary bodies. At least six immunofluorescence kits are now available commercially. Most comparisons suggest that the Syva MicroTrak test (Syva Company, Palo Alto, United States) gives better results, and a recent study of the brightness, consistency, and specificity of staining against 14 different serovars of the organism confirmed its superiority.52 Of the various enzyme immunoassay kits only Chlamydiazyme (Abbott) has been thoroughly evaluated. Its principal disadvantage stems from the use of a polyclonal antiserum, which can give reduced specificity owing to cross reaction with lipopolysaccharides of various Gram negative bacteria.5' An enzyme immunoassay that uses monclonal antibodies and was released recently by Pharmacia (Middlesex) may prove to be better." Evaluation of the antigen detection tests for diagnosing extragenital infection is limited. The Syva kit seems to work well on conjunctival specimens51 but is likely to be unsatisfactory for detecting the organism from nasopharyngeal specimens from infants.' Recently the possibility that the organism shares an antigen from its major outer membrane with strains of parainfluenza 2 virus has been raised. If this observation is confirmed additional tests to check specificity would be required. Special mention should be given to the use of these tests in evaluating suspected abuse in children. Both the Chlamydiazyme and Syva tests have been found wanting,5- and given the potential medicolegal implications of such forensic test results culture is the only option at present. Finally, as an aid in the diagnosis of trachoma antigen detection tests seem to be insufficiently sensitive because of the cryptic nature of the chlamydial infection, and diagnosis by using a DNA probe may be better. New diagnostic approaches Reports on new techniques to improve sensitivity, specificity, or the speed of diagnostic tests appear constantly, and it is difficult to determine which have genuine merit and will stand the test of time. Detection of microbial antigen by using polyclonal or monoclonal antibodies in conventional enzyme immunoassays and immunofluorescence assays has been examined exhaustively, and this technology is probably now close to its ultimate performance levels in terms of sensitivity and
specifcity. A novel detection method that deserves further evaluation is that of time resolved fluoroimmunoassay. In this technique time lapse fluorometry of lanthanide chelates conjugated to antibody permits detection of minute quantities of protein antigen. The lower limit of sensitivity is about 10 pg of BMJ
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protein,i which is 100-1000 times less than that detectable with conventional conjugates."' It is not clear whether this will translate into enhanced detection rates in clinical specimens, but preliminary evidence suggests that improvements over the standard assays may be significant.i The most exciting new development, however, is detection of DNA after amplification with the polymerase chain reaction. Detection of specific DNA by conventional methods that use hybridisation of target DNA to a labelled probe has had limited success as a diagnostic tool because of insensitivity (about 106 molecules of target DNA are required).i The polymerase chain reaction is, however, theoretically capable of amplifying a DNA segment from a single gene copy to a level that is readily detectable by conventional means such as ethidium bromide staining or hybridisation. In practice, a figure of 0 15 fg of DNA, equivalent to about six gene copies, has been detected in the case of cytomegalovirus.69 Viruses that cannot be isolated by routine culture are good candidates for diagnosis by this technique: HIV, papillomavirus, hepatitis B virus, and the papovaviruses BK virus and JC virus are examples.6669 Though the polymerase chain reaction looks promising as a rapid and highly sensitive alternative to conventional methods of diagnosis, important problems have to be solved before its routine use becomes practicable. The test is so sensitive that contamination of samples with stray DNA from the laboratory may generate false positive results. The problem can be detected by running negative controls alongside the test samples but a solution may prove more difficult. (The polymerase chain reaction assay may even need to be run in a building remote from the one in which the reagents are generated-a luxury that few laboratories would have available.) Another difficulty is that of determining the importance of a positive result. Distinction between DNA levels in health and in disease has not been made, and though for most acute viral diseases this is not an issue, the same is not true in chronic disease. Papillomavirus type 16 provides a good example: the polymerase chain reaction assay has shown it to occur in the cervix of over four fifths of women with normal cervical cytology and in every biopsy specimen of women with cervical cancer.6 The dilemma here seems to have been resolved by the recent discovery of subtypes of papillomavirus type 16-a and b -one of which was strongly associated with samples from women with carcinoma of the cervix. In the absence of such discoveries, however, the importance of a positive polymerase chain reaction result may remain uncertain. Selection of the specific segment of DNA to be amplified is clearly going to be critical. Finally, the potential application of the polymerase chain reaction in mass screening is limited because the procedures used for detecting the reaction products are laborious. Recently a colorimetric assay has been described for this, and it should be adaptable for use in a microtitre tray.' Whether such a format will control the problem of contamination remains to be seen. Conclusion Diagnosis of viral infection has come far since the days when isolation of the agent in cell culture and retrospective serology were the only options. For many of the commonly encountered viruses simple and reliable tests are now available in kit form from commercial sources and are suitable for use by smaller laboratories that may lack the equipment or skill required hitherto. As a group the respiratory viruses have remained refractory to detection by this approach, and the most prevalent causes of the common cold (rhinovirus and 1417
coronavirus) cannot be diagnosed at all in this way. Nevertheless, for viruses such as respiratory syncytial virus and rotavirus, which regularly cause sizable epidemics in winter, the antigen detection tests have become essential for both infection control and epidemiological purposes. Similarly, for the herpesviruses and C trachomatis, for which effective treatments are available, rapid diagnosis by these tests is making an important contribution to improved management and reduced morbidity of patients. More sensitive methods of detecting both antigen and DNA are being developed. They hold much promise for the future, especially for those viruses for which present methods
are unsatisfactory or unwieldy. Though predicting future trends is always hazardous, we think that DNA detection assays after amplification by the polymerase chain reaction will become commonplace once the current glitches have been sorted out. Given the pace of modern technology, that should occur well before 2000.
I Fenner FJ, White DO. Medical virology. 2nd ed. New York: Academic Press, 1976. 2 Reines ED, Gross PA. Update on antibiotics II: antiviral agenits. Med Clin North Am 1988;72:691715. 3 Galen RS, Gambino SR. Beyond normalisy-the predictive value and efficiencs of medical diagnosis. New York: John Wiley and Sons, 1975. 4 Lee IC. Quality control in clinical virology. In: Specter S, Lancz GJ, eds. Clinical virology mtanual. New York: Elsevier, 1986:3-14. 5 Paya CV, Wold AD, Smith TF. Detection of cytomegalovirus from blood leukocytes separated by sepracell-MN and ficoll-paque/macrodex method. 7 Clin Microbiol 1988;26:2031-3, 6 Ray CG, Minnich LL. Efficiency of immunofluorescence for rapid detection of common respiratory viruses. ] Clin Microbiol 1987;25:355-7. 7 Kumar ML, Super DMI, Lembo RM, Thomas PC, Prokay SL. Diagnostic efficacy of two rapid tests for detection of respiratory syncytial virus antigen.] Clin Microbiol 1987;25:873-5. 8 Shalit I, McKee PA, Beauchamp H, Waner JL. Comparison of polyclonal antiserum versus monoclonal antibodies for the rapid diagnosis of influenza A virus infections by immunofluorescence in clinical specimens.] Clin Microbiol 1985;22:877-9. 9 Hallsworth PG, McDonald PJ. Rapid diagnosis of viral infections with fluorescent antiscra.
38 Fleisher G, Lennette ET, Henle G, Henle W. Incidenice of heterophile antibody responses in children with infectious mononucleosis. 7 Pediatr 1979;94:723-8. 39 Lennette ET. Epstein-Barr virus. In: Lennette EH, Balows A, Hausler WVJ, Shadomy HIJ, eds. Manual of clinical microbiology. 4th ed. Washington, DC: American Socicty for Microbiology,
Pathology 1985;17:629-32. 10 Gleaves CA, Smith TP, Shuster EA, Pearson GR. Comparison of standard tube and shell vial cell culture technique for the detection of cytomegalovirus in clinical specimens. ] Clin Microbiol 1985 ;21:2 17-2 1. 11 Stokes CE, Bernstein JMNl, Kyger SA, Hayden FG. Rapid diagnosis of influenza A and B by 24-h fluorescent focus assays. ] Clin Microbiol 1988;26:1263-6. 12 Madeley CR. Viruses in the stools. .7 Clin Pathol 1979;32:1-10. 13 Kok TW, Burrell CJ. Comparison of five enzyme immunoassays, electron microscopy and latex 1989I27:364-6. agglutination for detection of rotavirus in fecal specimens.] Clin Microbiol 14 Brandt CD, Arndt CW, Evans GL, et al. Evaluation of a latex test for rotavirus detection. ] Clin Microbiol 1987;25: 1800-2. 15 Feinstone SiM, Kapikien AZ, Purcell RH. Hepatitis A: detectioni by immune electron microscopy of virus like antigen associated with acute illness. Science 1973;182:1026-8. 16 Flehmig G, Ranke M, Berthold H, Gerth H-J. A solid-phase radioimmunioassay for detection of IgM antibodies of hepatitis A virus. ] Infect Dis 1979;140:169-75. 17 Duermayer W, Wielaard F, van der Veen J. A new principle for the detection of specific IgM antibodies applied in an ELISA for hepatitis A. ] Med Iirol 1979;4:25-32. 18 Huang SN, Neurath AR. Immunohistologic demonstration of hepatitis B viral antigens in liver with reference to ist significances in liver in'jury. Lab Invest 1979;40:1-17. 19 Locarnini SA, Gust ID. Hepadnaviridae: hepatitis B sirus and the delta virus. In: Balows A, Hausler WJ, Lennette EH, eds. Laboratory diagnosis of infectious diseases. New York: SpringerVerlag, 1988:750-96. 20 Nielsen JO, Dietrichson 0, Elling P. Incidence and meaning of persisteltce of Australia antigen in patients with acute viral hepatitis: development of chronic hepatitis. N Engl.7 Med 1981;285: 1157-60. 21 Esteban JI, Esteban R, ViladomiL L, etal. Hepatitis C virus antibodies among risk groups in Spain. Lancet 1989;ii:294-6. 22 Van der Poel CL, Reesink HW, I.elie PN, et al. Anti-hepatitis C antibodies and non-A, non-B posttransfusion hepatitis in The Netherlands. Lancet 1989;ii:297-8. 23 Callihan DR, Menegus MA. Rapid detection of herpes simplex virus in clinical specimens with human embryonic lung fibroblast and primary rabbit kidnev cell culture. ] Clin Microbiol 1984;19:563-5. 24 Lafferty WIE, Krofft S, Remington M, et al. Diagnosis of herpes simplex virus by direct immunofluorescence and viral isolation from samples of external genital lesions in a highprevalence population. ] Clin Microbiol 1987;25:323-6. 25 Dascal A, Chan-Thim J, Morahan M, IPortnov J, Mendelson J. Diagnosis of herpes simplex virus infection in a clinical setting by a direct antigen detection enzyme immunoassav kit. J Clin
Microbiol 1989;27:700-4. 26 Halstead DC, Beckwith DG, Sautter RL, Plosila L, Schneck K. Evaluation of a rapid latex slide aggltttination test for herpes simplex sirus as a specimen screen and culture identification method. ] Clin Microbiol 1987;25:936-7.
27 Espy MJ, Smith rP. Detectiott of hcrpes sinmplex sirus in conventional tube cell ctultures and in shell vials with a DNA probe kit and monoclonal antibodies. ] Clin Microbiol 1988;26:22-4. 28 Olding-Stenkirst E, Grandien M. Early diagnosis of virus-caused sesicular rashes by immunofluorescence on skin biopsies 1. Varicella zoster and herpes simplex. Scand.7 Infect Dis 1976;8: 27-35.
29 Gleaves CA, Lee CF, Bustamante Cl, Meyers JD. Use of murine monoclonal antibodies for laboratory diagnosis of varicella-zoster sirus itnfection. 7 Clin .icrobiol 1988;26: 1623-5. 30 D)oane FW. Electron microscopy and immitnoelectron microscopy. In: Specter S, Lancz GJ, eds. Clinicall virology manual. New York: Elsevier, 1986:71-88. 31 Griffiths PD, Stirk PR, Ganczakowski M, st al. Rapid diagnosis of cvtomegalovirtts infection in immunocompromised patients by detection of early antigen fluorescent foci. Lancet 1984;ii: 1242-5.
32 Swenson PD, Kaplan MH. Comparison of two rapid culture methods for detcction of cytomegalovirtus in clinical specimens.] Clin Microbiol 1987 25:2445-6. 33 Randazzo DN, Michalski PJ. Comparison of antibodies for rapid dctection of cytomegalovirus. ] Clin Microbiol 1988;26:369-70. 34 van de Bij W, Torensma R, van Son WJ, et al. Rapid immttnodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leucocytes. .7 Med Virol 1988;25:179-88. 35 Crawford SW, Bowdeni RA, Hackman RC, et al. Rapid detection of cytomegalovirus pulmonary infection by bronchoalveolar lavage and centrifuge culture. Ann Intern Mled 1988;108:180-5. 36 Davidson I, Lee CL. The clinical serology of infectious mononucleosis, In: Carter RL, Penmnan HG, eds. Intfectious mononucleosis. Oxford: Blackwell, 1969:177-200. 37 Evans AS, Hiederman JC, Cenabre LC, West B, Richards VA. A prospective evaluation of heterophile and Epstein-Barr sirus-specific IgM1 antibody tests in clinical and subclinical infcctious mononucleosis: specificity and sensitivity of the tests and persistence of antibody. J Infect Dis 1975;132:546-54.
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Staff Specialist in Clinical M:crobiology PETER HALLSWORTH Senior NVirologist Department of Clinical Microbiology, Flinders Medical Centre, South Australia 5042.
1985:728-32. 40 Jackson BJ, Balfour HH. Practical diagnostic testing for human immunodeficiency virus. Clinical Microbiology Reviews 1988;1:124-38. 41 Reesink HW, Lelie PN, Huisman JG, et al. Evaluation of six enzyme imnmunoassays for antibody against human immunodeficiency virus. Lancet 1986;ii:483-6. 42 Spielberg F, Kabeya CM, Ryder RW, et al. Field testing and comparative esaluation of rapid, visually read screening assays for antibody to human immunodeficienc sirus. Lanzcet 1989;i:
580-4. 43 Chiodi F, Bredberg-Raden U, Biberfeld G, et al. Radioimtnunoprecipitation and western blottitsg with sera of human immunodeficiency virus infected patients: a comparative study. AIDS Res 1987;3: 165-76. 44 Gaines H, Sonnerborg A, Czajkowski J, et al. Antibody response in primary human immuLnodeficiency virus infection. Lancet 1987;i: 1249-53. 45 Ranki A, Valle S, Krohn M, et al. Long latency precedes osert seroconversion in sexuallv transmitted human immunodeficiencv virus infection. ILancet 1987;ii:589-93. 46 Goudsmit J, Lange JMA, Paul DA, Dawson GJ. Antigenemia and antibody titers to core anid envelope antigens in AIDS, AIDS-related complex and subclinical human immunodeficiencv virus infection. 7 InJect Dis 1987;155:558-60. 47 Re MC, Furlini G, La Placa M. Patterns of antigenaemia and antibody response in patients infected with human immunodeficiency virus (HIV) according to clinical state..7 Clin Pathol 1989;42: 282-3. 48 Kenny C, Parkin J, Undershill G, et al. HIV antigen testing. I.ancet 1987;i:565-6. 49 Eyster ME, Ballard JO, Gail MH, Drummond JE, Goedert jj. Predictive markers for the acquired immunodeficiencv syndrome (AIDS) in hemophiliacs: persistence of p24 antigen and low T4 cell count. Ann Intern Med 1989;110:963-9. 50 Kellogg JA. Clinical and laboratory considerations of culture vs antigen assays for detection of Chlamycdia trachaomatis from genital specimens. Arch Pathol Lab Med 1989;113:453-60. 51 Stamm WE. Diagnosis of Chlamydia trachomatis genitourinarv infections. Ann Intern Med
1988;108:710-7. 52 Cles LD, Bruch K, Stamm WlE. Staining characteristics of six commercially available monoclonal immunofluorescence reagents for direct diagnosis of Chlamvdia trachomatis infection. ] Clin Mtlcrobtol 1988;26:1735-7. 53 Mumtaz G, Ridgewav GL, Nayagam A, Oricl JD. Enzyme immunoassay compared with cell culture and immunofluorescence for detecting genital chlamydia. ] Clin Pathol 1989;42:658-60. 54 Potts MI, Paul ID, Rooma AP, Caul EO. Rapid diagnosis of Chlamydia trachomatis infection in patients attending an ophthalmic casualty department. Br] Ophthalmol 1986;70:677-80. 55 Roblin PM, Hammerschlag MR, Cttmmings C, Williams TH, Worku M. Comparison of two rapid microscopic methods and culture for detection of Chlamydia trachomatis in ocular aitd nasopharyngeal specimens from infants. ] Chlin icrobiol 1989;27:968-70. 56 Fox AS, Saxon EMI, Doveikis S, Beem 10. Chlamvdia trachomatis and parainfluenza 2 virus: a shared antigenic determinant?] Clin Mficrobiol 1989;27:1407-8. 57 Hammerschlag MR, Rettig PJ, Shields MIE. False positive results with the use of chlamrdial antigen detection tests in the esaluation of suspected sexual abuse in children. Pediatr InfeJct l)isj 1988;7: 11-4. 58 Hauger SB, Brown J, Agre F, et al. Failure of direct fluorescent antibody staining to detect Chlamydia trachomatis from genital tract sites of prepubertal children at risk for sexual abusc. Pediatr Infect D)isj 1988;7:660-1. 59 Dean D, Palmer L, Pant CR, et al. Use of a Chlamydia trachomatis DNA probe for detectiots of' ocular chlamydiae. J Clin Microbiol 1989;27: 1062-7. 60 Walls HH, Johanssott KH, Harmon MW, Halonen PE, Kendal AP. Time-resolved fluoroimmtunoassay with monoclonal antibodies for rapid diagnosis of influenza infections. ] Clin Microhiol
1986;24:907-12. 61 Grandien M, Petterson CA, Gardner PS, Linde A, Stanton A. Rapid viral diagnosis of acute respiratory inf'ections: comparison of enzyme-linked immunosorbent assay and the immunofluorescence technique for detcction of viral antigen in nasopharyngeal secretions. ] f,in Microbiol 1985;22:757-60. 62 Hierholzer JC, Johansson KH, Anderson LJ, 'I'sou CJ, Halonen PE. Comparison of monoclolnal time-resolved fluoroimmunoassav with monoclonal capture-biotin'ylated detector enzyme immunoassav for adenovirus antigen detection.]I Clin Microbiol 1987;25:1662-7. 63 Hierholzer JC, Binham PG, Coombs RA, et al. Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection. 7 Clin Microbiol 1989;27: 1243-9. 64 Chou S, Roark L, Merigan TC. DNA of 21 clinical c-tomegalovirus strains detected by hybridisation to cloned DNA fragments of laboratory strain AD-169. 3Med Virol 1984;14:263-8. 65 Olive DM, Simsek M, Al-Mufti S. Polymerase chain reaction assay for detectiots of' hbumats cVtomegalovirus. 7 Clin Microbiol 1989;27:1238-42. 66 Rogers MF, Oui MID, Rayfield M, et al. Use of the polyimerase chain reaction for early dctectoiot of the proviral sequences of human immunodeficiencv sirus in infants born to seropositive motlers. N Engl]Med 1989;320:1649-54. 67 'Tidy JA, Parrv GCN, 'Vard P!'et al. High rate of human papillomavirus type 16 ittfectioll in cVtologically normal cervices. Lancet 1989;i:434. 68 Sumazaki R, NMotz M, Wolf H, et al. Detection of hepatitis B sirus in serum tisiitg amplificatioit oif viral DNA by meatis of the polymerase chain reaction.]7 Med l'irol 1989;27:304-8. 69 Arthur RR, Dagostin S, Shah KV. Detection of BK virus and JC sirus in urine and braiti tissuc by the polymerase chain reaction. ] Clin Microbiol 1989;27:1174-9. 70 Tidy JA, VoLusden KH, Farrell PJ. Relation between infection with a subtype of HPV' 16 and cervical neoplasia. Lancet 1989;i: 1225-7. 71 Kemp DJ, Smith DB, Foote SJ, et al. Colorimetric detection of specific DNA scgmelts amplified by polymerase chain reaction. Proc NatlAcadSci USA 1989;86:2423-7.
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