Laboratory diagnosis of measles infection and monitoring of measles immunization: memorandum from a WHO meeting.

Laboratory diagnosis of measles infection and monitoring of measles immunization: Memorandum from a WHO meeting* Measles infection continues to be a major global health problem, and in many countries the disease is frequently diagnosed on clinical grounds alone, although it is easily confused with other conditions. In order to discuss approaches to improving this situation, a WHO Consultation on Laboratory Diagnosis of Measles Infection and Monitoring of Measles Immunization was held in Glasgow on 7-8 August 1993. The discussions and recommendations made by the participants are summarized in this Memo- randum. Introduction Measles continues to be a major health problem in developing countries, and results in the deaths of approximately 1.13 million infants and children, with 45 million cases worldwide per annum. Also, some industrialized countries have reported an increase in the number of measles cases over the last 3-5 years. The disease is characterized by a prodromal stage with fever and respiratory symptoms, and 4-5 days after the onset of the respiratory symptoms a macu- lopapular rash usually occurs. The WHO case defini- tion calls for the presence of fever and rash with one or more of the following symptoms: cough, coryza, or conjunctivitis. Owing to its highly infectious na- ture, measles affects virtually all children who are not immunized, and in some countries the disease causes over 20% of all infant deaths. In many countries, measles infection is frequent- ly diagnosed on clinical grounds alone, although the signs and symptoms of the disease are easily con- fused with those of other conditions that are associat- ed with a rash. Furthermore, several recent studies have indicated that measles virus may circulate in vaccinated populations, causing mild symptoms or * This Memorandum is based on the report of a WHO Consulta- tion on Laboratory Diagnosis of Measles Infection and Monitoring of Measles Immunization held in Glasgow on 7-8 August 1993. The participants at the Meeting were: Dr M. Grandien, Stock- holm, Sweden; Dr A.D.M.E. Osterhaus, Bilthoven, Netherlands; Dr P.A. Rota, Atlanta, GA, USA; Dr M.F. Smaron, Chicago, IL, USA; Dr T.F. Wild, Lyon, France; Observer: Dr J. Nascimento, Rio de Janeiro, Brazil; WHO Secretariat: Dr M.M. El-Nageh, Dr J. LeDuc, Dr Y. Pervikov (Secretary), Dr R.M. Scott. Requests for reprints should be sent to Division of Commu- nicable Diseases, Microbiology and Immunology Support Ser- vices, World Health Organization, 1211 Geneva 27, Switzerland. A French translation of this article will appear in a later issue of the Bulletin. Reprint No. 5469 even asymptomatic infections. Laboratory diagnosis of measles infection is therefore recommended, both to detect it and to differentiate it from other condi- tions, including many other infectious diseases (den- gue and other arboviral infections, roseola infantum, rubella, varicella, tick-bome rickettsioses, meningo- coccaemia, and scarlet fever) as well as noninfec- tious conditions. The lack of measles diagnostic laboratories in many countries and absence of techniques suitable for use at the primary health care level often result in misdiagnosis of the disease. In many developing countries measles surveil- lance is incomplete and does not reflect the inci- dence or the age distribution of cases. For effective control and the eventual eradication of the disease, a sensitive surveillance system is indispensable, and the ability to diagnose measles unequivocally is therefore essential. The development of measles diagnostic methods that are sensitive, specific, affordable and practicable for screening under field conditions has been recently identified as a high pri- ority by the WHO Expanded Programme on Immu- nization (EPI). Also, in view of the efforts to devel- op new generations of vaccines that should facilitate the eradication of measles, diagnostic tools to deter- mine antibody levels and protective immunity are required. Most of the current laboratory techniques for the diagnosis and surveillance of measles are based on classic biological assay systems, which are both labour-intensive and time-consuming. The advent of molecular biology and the better understanding of immunological principles of protection have created the opportunity to develop more appropriate diagnos- tic tools for measles. Under the auspices of WHO, a network of regional and national laboratories for the diagnosis of viral diseases has been established for the labora- tory confirmation, epidemiological surveillance, and Bulletin of the World Health Organization, 1994, 72 (2): 207-211 © World Health Organization 1994 207 Memorandum reporting of diseases caused by human immuno- deficiency virus (HIV), enteroviruses, arboviruses, and respiratory viruses. It is now proposed to use this network or establish a similar network for measles. The present article describes the proposed laboratory network, reviews available laboratory tests, and recommends at what level the tests should be implemented. The laboratory network The capability to confirm the clinical diagnosis of measles is required, especially with increased immu- nization coverage and the diverse clinical presenta- tion. As global control of measles is approached, the contribution of the diagnostic capabilities at all lev- els will become increasingly important. Experience has shown that a tiered system of referral labora- tories is required, with essential capabilities avail- able at the local level, supported by more sophistica- ted capacities in regional laboratories and intemation- al reference centres. Such a network has the inher- ent benefit of offering a framework for efficient and effective technology transfer, surveillance reporting, and training. Field testing. Self-contained diagnostic tests that can be performed under field conditions to screen for potential measles cases are not currently available; the development, testing, and implementation of such tests are, however, currently under way. When these tests do become available, their use should be rapidly integrated into the primary health care system. Outbreaks identified by this method will have to be confirmed in the laboratory. Local laboratories. The local laboratory is typically the first available clinical laboratory to offer specific diagnostic support at, for example, district or region- al hospitals. It should have the capability and resources to provide laboratory confirmation of clini- cal diagnosis, obtain, store, and transfer clinical specimens, and monitor the immune status of the local population. To achieve these objectives, the laboratory should be staffed by appropriately trained personnel and be equipped to conduct routine sero- logical assays and antigen detection. Since many of the technical requirements are similar for different viral diseases, every effort should be made to devel- op and employ assays and procedures that use com- mon reagents and equipment. National/intercountry laboratories. These should serve as referral laboratory facilities, with all the technical capabilities of local laboratories, plus more sophisticated assay systems, e.g., virus isolation and culture. Intercountry laboratories provide necessary support for countries where national laboratories are not available. These laboratories should offer the fol- lowing: training; new technologies at the local level; quality control and proficiency testing; and distribu- tion of specialized reagents. They should also receive and summarize surveillance information and periodi- cally report through the appropriate channels to WHO. International reference centres. Such centres should obtain and distribute relevant standards, reference materials and panels for proficiency testing; further- more, they should act as a resource for the develop- ment and use of training materials and provide train- ing sites. These centres are responsible for the following: definitive identification, comparison and banking of viral isolates; development of new test methods; evaluation of proposed standards and refer- ence materials; and answers to specific research questions. Laboratory diagnosis for measles in clinical materials Serological assays Demonstration of total specific immunoglobulins. Several techniques have been developed for demon- strating measles-virus-specific immunoglobulins in serum samples. These assays can be used to diagnose measles if paired serum samples are available; and they can also be used to assess the immune status of individuals and for epidemiological purposes. The following are the most frequently used assays for diagnostic purposes: virus neutralization (VN) tests; haemagglutination inhibition (HAI) tests; and enzy- me immunoassays (EIA). Of these, the EIA are avail- able as commercial diagnostic test kits, but compo- nents or ingredients for the other assays can be obtained also commercially. In general, the VN and HAI tests are relatively time-consuming and labour- intensive-the VN requiring tissue culture facilities and the HAI a source of monkey red blood cells. Despite these disadvantages, both these tests are being used in many national and local laboratories, and have proved to be useful in epidemiological sur- veys and vaccine efficacy assessment studies. There are several other assays used for the detection and measurement of measles-virus-specific antibodies. Some of them, such as the complement fixation test and the haemolysis inhibition test, are less suitable for routine diagnostic purposes; others, e.g., single radial haemolysis and the indirect immunofluores- cence test, are only used in a limited number of lab- oratories. Demonstration of specific IgM antibody. Immuno- assays for the detection of measles-virus-specific WHO Bulletin OMS. Vol 72 1994208 Laboratory diagnosis of measles and monitoring measles immunization immunoglobulins, which are associated with ongoing or recent infections, are based on the detection of IgM. Such techniques include two enzyme-linked immunosorbent assays (ELISAs) - the IgM capture ELISA (CEIA) and indirect IgM ELISA (IEIA) and the indirect IgM immunofluorescence assay (IFA). Among other assays that have been developed are the IgM capture haemadsorption assay (CHA), the line-immunoblot assay (LIBA), and a radio- immunoassay (RIA) for the detection of IgM in saliva. The capture assays are generally superior to the indi- rect assays, but more studies are needed to evaluate their relative sensitivities and specificities. Both the CEIA and IEIA are available commer- cially, although their current versions may be too expensive for some laboratories. However, these tests should be made available to laboratories that are equipped to perform ELISAs. In addition, the development of simplified versions of these tech- niques should be encouraged. Although IgM is the best characterized of the immunoglobulin subclasses that are useful for diag- nosis of measles, tests designed to determine IgA or different IgG subclasses should be developed. The relative sensitivity and specificity of these new tests should then be compared to the "gold standard" IgM assays by the national laboratories. Virus isolation Virus isolation can be used to confirm the diagnosis of measles but, more importantly, it provides samples for epidemiological assessment and phylo- genetic comparison. In immunocompetent individuals, measles virus may be isolated from blood and nasopharyngeal secretions (NPS) for a short period of time, usually during the first few days after the appearance of the rash; however, in urine the virus is often present for a week or longer. Correct collection, storage, and shipment of samples are important for successful virus isolation. Aspirates of NPS must contain suffi- cient numbers of epithelial cells and all samples must be kept cold. Samples should preferably be inoculated into tissue culture within 2 hours. If sam- ples must be stored, they should be kept for not more than 48 hours at +4 °C in viral transport medium or frozen at -70 "C for longer periods. Isolation can be accomplished in a number of cell substrates, such as primary rhesus monkey kidney cells and human or monkey B-lymphoblastoid cell lines. Although mea- sles virus infection usually produces characteristic cytopathic effects, a confirmatory procedure, such as IFA, peroxidase staining, haemadsorption inhibition, or RNA detection by the polymerase chain reaction (PCR), is required. Isolation is an arduous procedure, being labour- intensive, time-consuming, requires tissue culture capability, and is expensive. It may take up to 6 weeks to complete the procedure. Isolation facilities should be available in intemational reference centres and in selected national/intemational laboratories when required epidemiologically. Virus isolates should be made available for fur- ther characterization in reference laboratories. Direct detection of measles virus antigens in clinical specimens The detection of viral antigens in clinical specimens offers direct proof of virus infection, and IFA is cur- rently the most frequently used technique for detec- ting measles virus antigens in such specimens. Anti- gen can be detected in NPS aspirates during the prodromal stage and up to 1-2 days after the onset of the rash in the majority of cases. The reliability of IFA depends largely on the quality of the reagents used and on the sampling and preparation procedures, but has the advantage of a rapid response (3 hours) and the possibility of testing for several viruses simultaneously if antibodies against different viruses are used. Since all the reagents are available either commercially or through WHO, IFA can be rapidly disseminated. The need for a fluores- cence microscope unfortunately restricts the tech- nique to the reference or specialized local laboratory level at present; however, the development of a tech- nique involving enzyme conjugates could alleviate this problem. Detection of specific nucleic acids Assays that directly demonstrate measles virus RNA in clinical specimens are the PCR and direct nucleic acid hybridization tests. PCR can be used for diag- nostic purposes in local outbreak or epidemic set- tings where large numbers of samples are being ex- amined, and may provide a confirmatory test for oth- er assays that demonstrate directly the presence of measles virus or viral antigen. In addition PCR may be especially useful for diagnosing measles virus infections with an aberrant clinical course, such as subacute sclerosing panencephalitis (SSPE), measles inclusion body encephalitis (MIBE), and giant cell pneumonia. It may also serve for the diagnosis of asymptomatic or subclinical cases of measles virus infection. The most important application of PCR will be as a research tool for molecular epidemiolog- ical studies, when used in conjunction with sequence analysis of appropriate regions of the genome. In this way, the extent of genetic variation between different WHO Bulletin OMS. Vol 72 1994 209 Memorandum wild-type measles virus strains and, ultimately, the transmission pathways of these strains could be assessed. A future application may be the identifica- tion of molecular markers for virulence and changes in host cell tropism. Collection and primary handling of specimens for PCR analysis may be carried out locally. These samples include NPS aspirates, urine, peripheral blood mononuclear cells, and biopsy materials. Sam- ples should be shipped frozen as rapidly as possible to the appropriate laboratories. To ensure sensitivity and consistency, a standard protocol should be developed that can detect the spectrum of genetically diverse virus strains. This should cover all technical aspects, including sample collection, availability of appropriate positive and negative controls, RNA extraction, cDNA synthesis, PCR, and analysis of the PCR products. The protocol should also define the specimen of choice, preferably one on which virus or antigen detection can also be carried out. All the reagents needed are currently available at reasonable cost, and specific reagents such as intemal standards and oligonucleotide prim- ers should preferably be provided through the net- work of reference laboratories. The costs of PCR analysis and the technical skills needed for diagnostic and epidemiological studies are higher than those associated with the use of antibody and antigen detection systems. There- fore, it is recommended that standardized PCR assays be developed initially and carried out by the reference laboratories and that they be implemented gradually in selected national laboratories. The use of direct nucleotide hybridization tests for the diagnosis of measles infection is currently being studied, but these tests are not yet suitable for routine diagnostic purposes. Recommendations * Taking into account that only limited efforts have been made in recent years to develop tools for the diagnosis and surveillance of measles virus infection, the main focus should be on techniques for the rapid diagnosis of measles using inexpensive techniques and technologies currently employed in other immun- ological and molecular diagnostic methods. Priority should be given to assay systems that determine parameters associated with current and recent infec- tion, such as certain classes or subclasses of specific immunoglobulin and the detection of viral antigens and nucleic acids. The evaluation and use of non- invasive techniques of sample collection should be encouraged. * An intemational network of laboratories involved in the diagnosis and surveillance of measles should be established, if possible taking advantage of simi- lar networks developed for other viral diseases. Initially, attention should be given to reference laboratories that will carry out the following: - develop reagents and simple techniques for diag- nostic and epidemiological purposes and provide facilities for quality control and training in diag- nostic techniques; - distribute to other laboratories standardized reagents for diagnosis and surveillance, including monoclonal antibodies, (recombinant) viruses, recombinant proteins, peptides and oligonucleo- tides; - develop measles virus strain banks; and study fundamental questions conceming corre- lates between protection and laboratory test results, also taking into account the importance of cell- mediated immunity in the protection against measles. Table 1: Recommended activities for the different level laboratories in the measles diagnostic network Laboratory level National/ Referencea intercountrya Locala Total antibodyb VN + + +/- HAI + + EIA + + + IgM antibodyc CEIA + + + IEIA + + + IFA + + +/- Virus isolation + +/- - Antigen detection IFA + + +/- EIA + + +/- Polymerase chain reaction + +/- - Banking viruses/ antibodies/cells primers + Quality control internal proficiency testing + +-- Reagents + + Training courses + + Surveillance information + + a Test or activity: + = recommended; - = not recommended; and +/- = recommended if facilities are available. b VN = virus neutralization tests; HAI = haemagglutination inhibi- tion tests; EIA = enzyme immunoassay tests. c CEIA = capture ELISA; IEIA = indirect ELISA; IFA = immuno- fluorescence assay. WHO Bulletin OMS. Vol 72 1994210 Laboratory diagnosis of measles and monitoring measles immunization * For the evaluation of new generations of diagnos- tic tools and the establishment of criteria for their use, panels of serum samples should be established from individuals with a well-defined history of vac- cination and/or wild-type measles virus infection. For the same purpose, a bank of clinical specimens should be established from monkeys experimentally infected or vaccinated with different measles viruses within the framework of the WHO monkey model. * Since the measles eradication programme will largely depend on the combined efforts both in the laboratory and in the field, a sound financial structure for the continuing support at the respective levels should be established. A summary of the recommend activities for the different level laboratories in the measles diagnostic network is shown in Table 1. Annex Current WHO activities in standardization of laboratory diagnosis of measles infection To improve standardization of diagnostic tests and locally produced reagents, WHO has developed the following types of reference reagents. * International standard and reference reagents (ISRR), calibrated in international units of activity: - international standard for anti-measles human serum; - international standard for FITC-conjugated sheep anti-human IgM; - international standard for FITC-conjugated sheep anti-human IgG (anti-y-chain); and - international standard for horseradish-peroxi- dase-conjugated sheep anti-human IgG (H and L chains). * Reference reagents (RR) that are not calibrated in international units of activity: - monoclonal antibodies against the measles virus H and F proteins; - strains of measles virus; and - cell lines for cultivation of measles virus. Development of each new reference reagent is the joint activity of several WHO collaborating insti- tutions. The collection of RR has been expanded to include a number of new monoclonal antibodies, measles virus antigens, and measles virus-vaccinia virus recombinants expressing one of the following measles virus proteins: F, H, N, and P. ISRR and RR are distributed upon request, free- of-charge, through the network of WHO Collaborat- ing Centres or Collaborating Laboratories. However, a charge may be levied for shipment. Only a small quantity of reference reagents may be supplied to each interested laboratory. More detailed information on reference reagents can be obtained for ISRR, from Chief, Biologicals, World Health Organization, 1211 Geneva 27, Switzerland; and for RR, from Chief, Microbiology and Immunology Support Ser- vices, World Health Organization, 1211 Geneva 27, Switzerland. WHO Bulletin OMS. Vol 72 1994 211