Diagnosis of human herpesviruses: memorandum from a WHO meeting.

Memoranda/Memorandums Diagnosis of human herpesviruses: Memorandum from a WHO meeting* This memorandum reviews current methods for the diagnosis of human herpesvirus diseases, including appropriate techniques that can be utilized in the developing countries, and presents recommendations on procedures and reagents that should be developed and made available to laboratories. Introduction The discussions of the participants at the WHO Meeting on Diagnosis of Human Herpesvirus Infec- tions, which was held in Berlin, Germany, in August 1990,a complemented and updated a previous WHO Meeting held in 1983 (1). The herpesvirus family contains several impor- tant human pathogens. They possess a large number of genera, some of which have proved to be susceptible to antiviral chemotherapy. The out- standing property of herpesviruses is their ability to establish lifelong persistent infections in their hosts and to undergo periodic reactivation. It is their frequent reactivation in immunosuppressed patients which now particularly poses serious health compli- cations. The reactivated infection may be clinically quite different from the disease caused by the primary infection. The growing epidemic of human immunodeficiency virus (HIV) infection leading to AIDS (acquired immunodeficiency syndrome) has been associated with an increased incidence of herpesvirus diseases in these immunocompromised patients. * This memorandum is based on the report of a WHO Meeting on Diagnosis of Human Herpesvirus Infections, which was held in Berlin, Gerrnany, on 25-26 August 1990. The participants were Professor I.F. Barinsky, Moscow, USSR; Dr M. Grandien and Dr A. Linde, Stockholm, Sweden; Professor P. Griffiths and Dr H.O. Kangro, London, England; Dr S.K. Lam, Kuala Lumpur, Malaysia; Professor J.L. Melnick, Houston, TX, USA; and Dr Y. Pervikov, WHO, Geneva, Switzerland. A French translation of this article will appear in a later issue of the Bulletin. Requests for reprints should be sent to Microbiology and Immunology Support Services, Division of Communicable Diseases, World Health Organization, 1211 Geneva 27, Switzerland. Requests for copies of the working papers for this meeting should be sent to Microbiology and Immunology Support Ser- vices, Division of Communicable Diseases, World Health Organ- ization, 1211 Geneva 27, Switzerland. Reprint No. 5173 Herpesviruses of humans include herpes sim- plex virus types 1 and 2 (HSV1 and HSV2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6). Monkey B virus (cerco- pithecine herpesvirus 1) is also a human pathogen. Classification A useful division of Herpesviridae into subfamilies is based on the biological properties of the agents (Table 1). Alphaherpesviruses (HSV, VZV) are fast-growing cytolytic viruses that tend to establish latent infections in neurons. Betaherpesviruses (CMV) are slow-growing and cytomegalic (involving. massive enlargements of infected cells) and become latent in salivary glands and kidneys. Gammaher- pesviruses (EBV) infect lymphoid cells. Another herpesvirus, human B-lymphotropic virus (HBLV), has been recovered recently from patients with lymphoproliferative disorders and has been desig- nated as human herpesvirus 6 (HHV-6); its genome resembles that of cytomegalovirus. There is little antigenic relatedness among the Herpesviridae. Only herpes simplex virus types 1 and 2 share a significant number of common antigens. This is not surprising, since there is approximately 50% homology between those two viral genomes. Diseases caused by Herpesviridae Although infection is usually inapparent, a wide range of diseases are associated with the Herpesviri- dae. Disease caused by primary or reactivated infection by a given virus may involve different cell types and present different clinical pictures, as described below. (1) HSV1 and HSV2 infect epithelial cells and establish latent infections in neurons. Type 1 virus is classically associated with oropharyngeal lesions and Bulletin of the World Health Organization, 69(3): 277-283 (1991) © World Health Organization 1991 277 Memorandum Table 1: Classification of human herpesviruses (Family: Herpesviridae) Biological properties Name of virus Growth Latent Subfamily cycle Cytopathology infections Official Common Alphaherpesvirinae Short Cytolytic Neurons Human herpesvirus 1 Herpes simplex virus 1 Human herpesvirus 2 Herpes simplex virus 2 Human herpesvirus 3 Varicella-zoster virus Betaherpesvirinaea Long Cytomegalic Salivary glands, Human herpesvirus 5 Cytomegalovirus kidneys Gammaherpesvirinae Variable Lymphoproliferative Lymphoid Human herpesvirus 4 Epstein-Barr (EB) virus tissue a The HHV-6 genome resembles that of CMV more closely than it does the lymphotropic gammaherpesviruses. causes recurrent attacks of "fever blisters". Type 2 virus primarily infects the genital mucosa and is mainly responsible for genital herpes. Both viruses also cause neurological disease; HSV1 is one of the main causes of sporadic encephalitis in temperate climates. (2) VZV causes chickenpox (varicella) on prim- ary infection and establishes latent infection in neu- rons. Upon reactivation, the virus causes "shingles" (zoster). Adults infected for the first time with VZV often develop serious pneumonia. Encephalitis has also been reported as a complication of VZV infection. (3) CMV replicates in the epithelial cells of the respiratory tract, salivary glands, kidneys and in lymphocytes. CMV may cause infectious mononu- cleosis and cytomegalic inclusion disease (in new- borns); it is an important cause of congenital defects and mental retardation. (4) EBV replicates in epithelial cells of the oropharynx and parotid gland, and establishes latent infections in lymphocytes. It causes infectious mono- nucleosis and appears to be the cause of or closely associated with two human cancers, one a lymph- oma and the other a carcinoma. (5) Human herpesvirus 6 (HHV-6) infects lymphocytes. It is typically acquired in early infancy and causes exanthem subitum (roseola infantum). Target cells for latent infections and the conse- quences of reactivations are not known. (6) Monkey B virus (cercopithecine herpesvirus 1) can infect humans. Such infections are rare, but those that occur result in severe neurological disease and are usually fatal. (7) Human herpesviruses are frequently reactiv- ated in immunocompromised patients (e.g., trans- plant recipients, cancer patients) and may cause severe disease, such as pneumonia or lymphomas. (8) Herpesviruses have been linked with malig- nant diseases in humans and lower animals, EBV both with Burkitt's lymphoma of African children and with nasopharyngeal carcinoma, Luck' virus with renal adenocarcinomas of the frog, Marek's disease virus (MDV) with a lymphoma of chickens, and a number of primate herpesviruses with reti- culum cell sarcomas and lymphomas in monkeys. MDV also induces atherosclerosis in chickens and current investigations have linked CMV with atherogenesis in humans. Diagnosis Each of the viruses presents its own problems and requires its own diagnostic procedures. The main procedures used for the different herpesviruses are summarized in Tables 2 to 5. Detailed methods are not given in this paper but can be found in standard laboratory manuals (2-4). Some general features have emerged; these are described below. (1) Laboratory diagnosis of herpesvirus infec- tions is becoming an increasingly important part of patient management as more effective means of treatment and prevention become available. The emphasis of these investigations is now directed towards diagnosis for intervention and screening for prevention. These considerations play an important role in deciding which techniques to deploy in diagnostic laboratories. (2) The main effort in recent years has been to develop methods for rapid diagnosis, thus providing information that is useful for patient management and disease control. Methods using monoclonal antibodies for detection of viral antigen(s) directly in clinical specimens, by fluorescence microscopy or chromogenic systems, have proved highly successful and are within the scope of general diagnostic laboratories. The same methods have also been used for detection of virus in cell cultures within 24-48 hours after inoculation with clinical specimens. This is a considerable improvement in the utility of virus WHO Bulletin OMS. Vol 69 1991.278 Diagnosis of human herpesviruses isolation, traditionally regarded as the most defini- tive diagnostic method for all the herpesviruses (except EBV) but, because of the long incubation period often required, of limited clinical value. It will, however, in the future be important to have access to virus isolation at least in reference labora- tories, specifically to aid the monitoring of drug resistance amongst the herpesviruses. (3) Detection methods for viral DNA in clinical specimens are being advanced. It is expected that the PCR (polymerase chain reaction) method may ultimately be used for routine diagnostic purposes. (4) Serology continues to be used for diagnosis, often in conjunction with other methods, but its role is diminishing. The reasons for this are the change in diagnostic approaches generally towards methods which do not rely on paired serum samples to demonstrate a significant rise in antibody level and doubts about the value of serological investigations for the herpesviruses in immunosuppressed patients. Concomitant antibody responses to more than one herpesvirus are also often encountered in these patients, making the interpretation of results dif- ficult without further supportive findings. However, serology is still the mainstay of diagnosis of EBV infections. IgM antibody tests can be useful as confirmatory tests and for the diagnosis of con- genital infections but are generally less useful in herpesvirus than in some other acute virus infec- tions. Serological testing continues to be important for screening for past infection. (5) Diagnostic reagents are now marketed commercially. The performance of commercial test kits and reagents can vary enormously and it is, therefore, advisable that laboratories using such products for virus diagnosis or antibody screening should ensure that the test performance, in terms of sensitivity and specificity, is adequate. Diagnosis of herpes simplex virus infections The laboratory diagnosis of infections caused by herpes simplex viruses (HSV-1 and HSV-2) can be made by isolation of the virus, demonstration of the presence of viral antigen or virus-specific nucleic acid sequences in appropriate specimens from patients with serious clinical forms of the infection, or serologically by demonstrating a rise in the titre of virus-specific IgG or detection of virus-specific IgM in serum samples from the patient (Table 2). For detection of viral antigen the immuno- fluorescence test (IF) and enzyme-linked im- munosorbent assay (EIA or ELISA) are the sim- plest and most reliable methods currently available, directly demonstrating the virus in clinical speci- mens. Detection of virus by electron microscopy is also useful, and isolation and typing of HSV may be valuable in some instances, for example for monitor- ing drug resistance. Serological testing to detect IgM antibodies or a rise in IgG antibodies can be used for confirmation of primary infection or in cases with serious compli- cations. The established methods for diagnosing cases of encephalitis are antigen detection in brain biopsies or virus isolation. Direct diagnosis may be possible with PCR on cerebrospinal fluid, but until the method is fully evaluated the diagnosis should be confirmed by demonstration of intrathecal antibody production in paired serum and CSF samples. In neonatal infections, the demonstration of virus excretion by isolation or detection of antigen by IF or EIA is most important to make the diagnosis. Table 2: Diagnosis of herpes simplex virus infections Demonstration of virus or Disease Specimens viral products Serologya Lesions of skin, genitalia, Vesicle fluid, swab or scrape Immunofluorescence test, eyes material enzyme immunoassay, and electron microscopyb Sera Complement-fixation test, enzyme immunoassay Herpes encephalitis Brain biopsy Polymerase chain reactionc Paired sera Enzyme immunoassay, complement-fixation test Neonatal herpes Vesicle fluid, swab or scrape Immunofluorescence test, material (post-mortem enzyme immunoassay, and material) electron microscopyb a Using HSV1 and HSV2 specific assays for epidemiological studies. ° Can be complemented with virus isolation. c Not yet used for routine diagnosis. WHO Bulletin OMS. Vol 69 1991. 279 Memorandum Monoclonal antibodies are preferred for use in IF and EIA tests. New techniques are being introduced for the diagnosis of diseases caused by herpes simplex virus, such as the detection of virus-specific nucleotide sequences by DNA hybridization or by PCR. These may in the future be used for routine diagnosis. Diagnosis of varicella-zoster virus (VZV) infections A variety of techniques are available for the diagnosis of VZV infections. The main uses of these techniques are summarized in Table 3. Electron microscopy (EM) is simple to perform and gives results within 1-2 hours but it requires expensive equipment and cannot distinguish be- tween HSV and VZV. Techniques (such as IF or the immunoperoxidase test (IP)) using monoclonal anti- bodies to probe for VZV antigen directly in clinical specimans can be used for rapid diagnosis (2-3 hours) and are more sensitive and specific than conventional cytology or histology Virus isolation is the definitive means of diagno- sis but can take 3 weeks to perform. The "shell vial technique" (see below) can be used with monoclonal antibodies to allow identification of VZV in cell cultures within two days after inoculation. Serology is commonly used for the diagnosis of VZV infections and currently is the main method for diagnosing the disease in the central nervous system. However, serological diagnosis can be complicated by apparent cross-reactivity with HSV. Testing for VZV IgM antibodies can provide additional confir- mation of recent VZV infection. The most impor- tant use of serology, however, is for the determina- tion of antibody status of at-risk patients and health care personnel to prevent VZV infection. Sensitive ETAs and fluorescent antibody to membrane antigen tests (FAMA) have been developed for this pilr- pose. To provide a full service a laboratory offering routine diagnosis will require a direct demonstration method for rapid diagnosis of VZV disease, and a sensitive and specific test for VZV antibody status. An antigen detection system with monoclonal anti- bodies is most suitable for rapid diagnosis with the optional addition of electron microscopy or virus isolation if the facilities and expertise are available.. An EIA is the most suitable serological test for screening for antibody status. It may be possible to provide a screening programme based on question- ing for past history of varicella plus a well-optimized complement-fixation test (CFT), but only if a back-up service is available for testing CFT-negative or equivocal sera by a sensitive EIA. A regional or national reference laboratory needs to provide support and confirmatory testing for the routine laboratories including virus isolation and antigen detection. It should provide serological tests for diagnosis, such as the CFT and preferably an IgM test, and it should provide a sensitive reference test Table 3: Diagnosis of varicella-zoster virus infections Demonstration of virus or Disease Specimens viral products Serology Rash Vesicle fluid, scrape or swab Immunofluorescence test or material immunoperoxidase test, electron microscopya Serum Complement-fixation test, enzyme immunoassayl Pneumonia Biopsy, lavage, aspirate Immunofluorescence test or immunoperoxidase testa Serumc Complement-fixation test, enzyme immunoassay' CNS disease Paired serum and CSF Complement-fixation test, enzyme immunoassay (Biopsy) Immunofluorescence test or immunoperoxidase test Screening for antibody status Serum Enzyme immunoassay, fluorescent antibody to membrane antigen test a Can be complemented with isolation (conventional or rapid identification with monoclonal antibodies). IgM for confirmatory testing. c Serology is of limited use for the diagnosis of pneumonia. 280 WHO Bulletin OMS. Vol 69 1991. Diagnosis of human herpesviruses for antibody screening. Blood transfusion labora- tories do not need to screen for VZV antibody status since the virus is not transmitted by transfusion. However, a specific quantitative EIA or latex test is useful for selecting units of blood with high titre VZV antibody for the preparation of VZV immune globulin. Fully evaluated tests or reference tests are not yet available, so more developmental work and evaluation will be required to establish a VZV service. Diagnosis of cytomegalovirus (CMV) infections CMV infection is common. The virus rarely causes disease unless the host's immune response is im- mature (fetus) or compromised (organ transplant or AIDS patients). Two antiviral drugs (ganciclovir and foscarnet sodium) are available for treatment but are toxic and should only be used after CMV infection has definitely been diagnosed. The methods currently used for diagnosis are summar- ized in Table 4. Rapid methods of virus detection DEAFF test. Cell cultures inoculated with clinical specimens can be fixed after overnight incubation, reacted with monoclonal antibodies against CMV a or /3 proteins and then with an immunofluorescent conjugate. This technique is called DEAFF (detec- tion of early antigen fluorescent foci) or shell vial assay (because the cells may be prepared on coverslips in shell vials). Leukocyte antigen detection (LAD). Peripheral blood mononuclear cells can be separated by dex- tran sedimentation, centrifuged, and stained as above with monoclonal antibodies and immuno- peroxidase or immunofluorescent conjugates. Both the above techniques require monoclonal antibodies but DEAFF requires cell culture in addition. LAD may, therefore, be more practicable for laboratories which are not familiar with cell cultures whereas those already using cell cultures may prefer DEAFF because it can process samples other than blood (e.g., urine from neonates or bronchial lavage fluid). CMV lung infection does not of itself progress to disease until a T-cell immunopathological process becomes involved. Since AIDS patients appear not to mount this response, they do not get CMV pneumonitis and, therefore, treatment of CMV infections of the lung in these patients is not required, unlike the situation in transplant recipients. Serology CMV-IgG antibodies should be detected by EIA (or the latex agglutination test) in donors of organs or blood, in order to exclude those who are seroposit- ive, where possible, or to match seronega- tive recipients with only seronegative donors. Transplant patients who are seronegative can be monitored separately to detect seroconversion using the same IgG methods. However, this is not generally recommended because the diagnosis of active infection is made more rapidly by DEAFF or LAD, and because many transplant patients with serious CMV disease have delayed serological re- sponses. Testing for IgM antibodies can be used for the diagnosis of CMV infections retrospectively and when intervention is not immediately indicated. Generally, however, IgG or IgM serological testing for CMV is not recommended in immunocomprom- ised patients for the above reasons and because they cannot reliably detect reactivations of latent infec- tion or reinfection (which both occur in AIDS and transplant patients). Congenital infection can be diagnosed using CMV-IgM but sensitive and specific assays are not readily available. Diagnosis of Epstein-Barr virus (EBV) infections The method of choice for the diagnosis of diseases induced by or associated with EBV differ, depend- ing on the disease. The diseases of main importance Table 4: Diagnosis of cytomegalovirus infections Demonstration of virus, or Patient Specimen viral productsa Serology Immunocompromised Blood DEAFF or LAD Transplant recipient Bronchial lavage DEAFF Neonate Urine DEAFF Serum IgM by enzyme immunoassay Organ donors Serum IgG by enzyme immunoassay (or latex) a DEAFF (detection of early antigen fluorescent foci); LAD (leukocyte antigen detection). WHO Bulletin OMS. Vol 69 1991. 281 Memorandum to the diagnostic laboratory are infectious mono- nucleosis (IM), Burkitt's lymphoma, and naso- pharyngeal carcinoma (NPC) in endemic areas and severe disease in the immunocompromised host. Shown in Table 5 are the minimal laboratory tests required for diagnosis of these diseases. Past EBV infection is demonstrated by the presence of EBV viral capsid antigen (VCA) IgG antibodies. Pres- ence of IgM to VCA is a sensitive and relatively specific marker of infectious mononucleosis. In teenagers and adults, IM can be diagnosed by an immediate test for heterophile antibodies. Com- plementary tests with high cost-benefit in IM are examinations for VCA IgG and Epstein-Barr nu- clear antigen (EBNA) antibodies. Assays for EBNA antibodies and EBV DNA in biopsies should be used for diagnosis of EBV-associated malignancies. In the reference laboratory all mentioned tests and further assays for antibodies to the various EBV antigens (EBNA 1-2, early antigen (EA)) should be available. The established antigens for the serologi- cal investigations are derived from Burkitt's lymph- oma cell lines. IF assays are the most widely used for EBV serology, but commercial EIAs are also available. A high priority should be given to the develop- ment of cheap and specific serological assays for predicting nasopharyngeal carcinoma and for the detection of IgM to viral capsid antigen. Diagnosis of human herpes virus type 6 (HHV-6) infections Since human herpesvirus type 6 (HHV-6) was only recently discovered, very little is known about its natural history and full clinical significance. Cur- rently, there are two means of diagnosing HHV-6 infections: virus isolation and serological testing. However, neither can be recommended for routine use and they are only for consideration in reference laboratories. DNA hybridization and PCR have been employed for research purposes but have not been evaluated as diagnostic tests. Serological tests. Most of the serological investiga- tions of HHV-6 infections to date have relied on IF for IgG and IgM antibodies using acetone-fixed infected lymphocytes. EIAs have successfully been developed which are suitable as diagnostic tests but their usefulness, particulary the IgM tests, has yet to be determined. It will be important to investigate the relationships between HHV-6, CMV and EBV, since simultaneous antibody rises are often encoun- tered with two or more of these viruses. Parallel investigations for these viruses may become routine practice in the future. For this reason it would be valuable if at least the reference laboratories ac- quired IF and probably EIAs for HHV-6 antibodies as investigative tools. Diagnosis of herpesviruses in developing countries The laboratory diagnosis of herpesvirus infections in developing countries has not been given sufficient prominence or priority in the past owing to the many other health problems faced by these countries. With the increasing problems of AIDS, the use of immunosuppressive therapy and the possibility of treatment, there is now a greater awareness of herpesvirus diseases and the need to introduce appropriate diagnostic services in countries where these do not yet exist. However, in deciding which tests should be introduced both practical and eco- nomic factors have to be considered. For example, since tissue culture technology is technically difficult as well as labour intensive and expensive to provide, WHO has recommended that developing countries, Table 5: Diagnosis of Epstein-Barr virus infections Demonstration of virus or Disease Specimen viral products Serology Infectious mononucleosis Serum IgM viral capsid antigen (immunofluorescence test, enzyme immunoassay) Heterophile antibodya Nasopharyngeal carcinoma Serum IgA viral capsid antigen (immunofluorescence test)b Severe disease in immuno- Material EBNA (ACIF)C compromised patients from or DNA lesions 8 Rarely present below the age of 3 years. For identification of persons at high risk of developing nasopharyngeal carcinoma and for monitoring treatment. I EBNA (Epstein-Barr nuclear antigen); ACIF (anti-complement immunofluorescence test). WHO Bulletin OMS. Vol 69 1991.282 Diagnosis of human herpesviruses where possible, should use rapid techniques which do not require virus replication. Techniques such as IF and EIA are widely practised for antigen detec- tion and the detection of IgM class antibodies should also be considered in relevant diseases. In countries where information is scanty on the impact of herpesvirus diseases, serological diagnosis may have an important role for disease surveillance of the herpesviruses in different groups of patients. Such investigations may play an important role in defining national or local needs and, thus, help to target the diagnostic services provided. In order to promote herpesvirus diagnosis in developing countries, there is a need to provide technical expertise by way of workshops with a follow-up programme to enable laboratories to acquire and practise the new skills. This priority should be given to strengthening and upgrading certain laboratories in developing coun- tries to produce working reagents for the herpes- viruses of interest to that country. These reagents could then be shared on an exchange basis. Since monoclonal antibodies will probably be the most useful reagents for rapid antigen detection, work- shops on hybridoma technology should be con- ducted in the WHO Regions. Recommendations Laboratory investigations for diagnosis and screen- ing purposes are now important for the prevention and treatment of herpesvirus diseases. Demand for these services is likely to increase in the future in line with improved health care provisions generally and because of a growing population at risk of severe disease caused by these viruses. In order to provide such services the following are recommended: (1) A core of diagnostic procedures should be introduced in laboratories providing routine virolo- gical or microbiological services. (2) Regional or national reference laboratories should establish techniques for confirmatory testing and more specialized investigations to support the routine laboratories. (3) International standard preparations and ref- erence reagents, including virus strains and human sera containing IgG and IgM antibodies to the herpesviruses, should be developed and quality controlled for distribution to national reference laboratories. (4) A list of monoclonal antibodies available for collaborative diagnostic studies on the human herpesviruses should be compiled. (5) Studies on the rapid diagnosis of encephali- tides caused by herpesviruses should be promoted. Such studies should emphasize the use of PCR for viral DNA detection in the cerebrospinal fluid. References 1. WHO Meeting. Prevention and control of herpesvirus diseases. Part 1. Clinical and laboratory diagnosis and chemotherapy. Part 2. Epidemiology and immunology. Bulletin of the World Health Organization, 63: 185-201, 427-444 (1985). 2. Fields, B.N. et al. Virology, 2nd edition. New York, Raven Press, 1990. 3. Hsiung, G.D. Diagnostic virology, 3rd edition. New Haven and London, Yale University Press, 1982. 4. Schmidt, N.J. & Emmons, R.W. Diagnostic procedures for viral, rickettsial and chlamydial infections, 6th edition. Washington, American Public Health Association, 1988. Annex Suggested quality control laboratories The numbers refer to laboratories (see below) for diagnosis of infections caused by: - HSV: 1,5,6 - CMV: 3, 4, 6 - VZV: 2,4 - EBV: 2, 5 - HHV-6: 2,4 1. WHO Collaborating Centre for Herpes Virus Reference and Research, The D. I. Ivanovsky Institute of Virology, Moscow, USSR. 2. WHO Collaborating Centre for Virus Reference and Research, Department of Virology, The National Bacteriological Laboratory, Stockholm, Sweden. 3. WHO Collaborating Centre for Reference on and Research on Viral Diseases, Department of Virology, The Royal Free Hospital Medical School, London, England. 4. Virology Department, St Bartholomew's Hos- pital Medical College, London, England. 5. Department of Medical Microbiology, University of Malaya, Kuala Lumpur, Malaysia. 6. WHO Collaborating Centre for Virus Reference and Research, Department of Virology and Epidemiology, Baylor College of Medicine, Houston, TX, USA. WHO Bulletin OMS. Vol 69 1991. 283