ICP/RPD/002
LIMITED DISTRIBUTION ENGLISH ONLY
~RKING GROUP ON SEROEPIDEMIOLOGICAL TOOLS ~FOR CONTROL PROGRAMMES IN TROPICAL DISEASES
Kuala Lumpur, Malaysia 9-13 November 1987
Not for sale Printed and distributed by the Regional Office for the Western Pacific of the World Health Organization Manila, Philippines February 1988 WHO! '.Vl'!W LlRHARY
t
NOTES The views expressed in this report are those of the members of the. Working Group on Seroepidemiological Tools for Control Programmes ~n Tropical Diseases and do not necessarily reflect the policies of the Organization.
This report has been prepared by the World Health Organization Regional Office for the Western Pacific for Governments of Member States in the Region and for the members of the Working Group on Seroepidemiological Tools for Control Programmes in Tropical Diseases, held in Kuala Lumpur on 9-13 November 1987.
CONTENTS
1. 2.
INTRODUCTION SUMMARY OF DISCUSSIONS ON COUNTRY REPORTS ...••...........•••.•.•••.....•..... SUMMARY OF DISCUSSIONS ON SEROEPIDEMIOLOGY OF SPECIFIC DISEASES
1 1
3.
5 5 6 7 8 9 10 10
3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.
Dengue
.......................................... .
Haemorrhagic fever with renal syndrome •.....••.•.. Leprosy ....... ,. .................................. .
Malaria Schistosomiasis ••..••••••••••••••••••••.•...•••.•• Trichinellosis and Angiostrongyliasis •••••.•••..•• Filariasis, Amoebiasis and Toxoplasmosis ••....••.• TDR inputs
...................................... .
12 12 12 12
Biostatistical and epidemiological considerations .....•..••...•..•.••••••••.•••.••.•
SUMMARY OF RECOMMENDATIONS 4.1 General recommendations 4.2 Specific recommendations ANNEXES: ANNEX 1 - OPENING REMARKS OF DR H. NAKAJIMA, REGIONAL DIRECTOR, WHO/WPRO .................. ANNEX 2 - LIST OF TEMPORARY ADVISERS, OBSERVERS, RESOURCE PERSONS, WHO CONSULTANT AND SECRETARIAT .. . . • . . . . . • . . . . . . . • . . . . . . . . . . ANNEX 3 - AGENDA ..••...............•••.....••....•...•• ANNEX 4 - SHARING OF RESOURCES WITHIN THE WESTERN PACIFIC REGION . . . . . • . • . . • • • • . . . • • . . . . . . . . . • • ANNEX 5 - LIST OF WORKING PAPERS ISSUED
13
17
19 23 27
29
1.
INTRODUCTION
In recent years, there have been significant advances in serodiagnostic techniques for infectious diseases like malaria, filariasis, dengue, schistosomiasis and leprosy. Related developments in culture techniques, for example in malaria and filariasis, have allowed the production of the required antigens for large-scale use of these tests. The availability of large batch processing facilities, especially for sensitive serological techniques like the enzyme-linked immunosorbent assay (ELISA), has made these tests very attractive as seroepidemiological tools. In spite of the availability of these tools, their application in control programmes of infectious diseases has been minimal in countries of the Western Pacific Region. Therefore, at the 7th WHO/IMR (Kuala Lumpur) Coordination Meeting, held in the Institute for Medical Research, Kuala Lumpur, in 1986, it was recommended that the WHO Regional Centre for Research and Training in Tropical Diseases at the Institute for Medical Research (IMR) organize a meeting to discuss this very important topic. Arising from the recommendation, a Working Group on Seroepidemiological Tools for Control Programmes in Tropical Diseases was held at the Institute for Medical Research, Kuala Lumpur, from 9 to 13 November 1987. The objectives of the Working Group were: (1) to review recent advances in seroepidemiological techniques available for important endemic diseases in the Region; (2) to review the use of seroepidemiological tools in the control of these diseases; (3) to critically review the suitability, relevance and efficiency of such techniques in the monitoring of control programmes in the Region. The Working Group was opened by the Hon. Deputy Minister of Health, Malaysia, Datok N. Pathmanaban, who stressed the importance of the development of newer and more efficient tools for the monitoring and assessment of control programmes of infectious diseases endemic in the Region. Dr L. Verstuyft, on behalf of Dr H. Nakajima, Regional Director of the WHO Regional Office for the Western Pacific, also stressed the urgent need to train personnel and to test these tools in an effort to improve the efficiency of the disease control programmes (Annex 1). The list of attendees and the agenda are presented as Annexes 2 and 3 respectively. 2. SUMMARY OF DISCUSSIONS ON COUNTRY REPORTS
Dr A. Shirai opened the discussions by reviewing the reasons for the Working Group and the objectives to be addressed. Dr S.K. Lam and
- 2 -
Dr Ho Wang Lee were elected Chairman and Co-Chairman respectively and Dr Ray Sanders Rapporteur. Country reviews were then presented. Dr Ko reviewed the major parasitic diseases endemic in Hong Kong and the immunodiagnostic techniques used. The indirect fluorescent antibody assay (IFA) is used for toxoplasmosis, the counterimmunoelectrophoresis (CIE) and enzyme-linked immunosorbent assay (ELISA) for amoebiasis, and ELISA for trichinellosis, angiostrongyliasis and cysticercosis. No seroepidemiological tools are being used for control programmes in any of these parasitic diseases. Dr Sanders reported that acute respiratory infections constitute the major health problem in Papua New Guinea, malaria being the second major disease followed by childhood diarrhoea. Measles, viral hepatitis, filariasis, leprosy and dengue are also present. CIE is used for detection of pneumococcal and haemophilus antigens; IFA for the detection of adenovirus, influenza A and B, parainfluenza 1 and 3, measles, RSV and herpes simplex antigens. ELISA is used as a seroepidemiological tool for malaria and for the detection of rotavirus, adenovirus and for monitoring of measles vaccination. Some problems associated with the use of seroepidemiological tools were discussed. Dr Garcia noted that important endemic diseases in the Philippines include parasitic, bacterial, rickettsial and viral infections. Although immunoassays have been used for diagnosis, seroepidemiological tools have not been used for control programmes. However, some seroepidemiological studies have been carried out in schistosomiasis and toxoplasmosis. He cited the lack of appreciation by control programme directors of the role of seroepidemiological tools as one of the reasons for the lack of their use and suggested that training programmes in the use of these techniques as well as in basic epidemiology be carried out for control programme directors. Dr Mulkit Singh noted that seroepidemiological tools have limited application in the study of parasitiC infections in Singapore but that serological techniques have been used for immunodiagnosis of malaria, toxoplasmosis and filariasis. Antigens for all these tests are being produced. The indirect haemagglutination (IHA) technique was used to detect toxoplasma antibodies; 17.2% apparently healthy blood donors were positive while antibody prevalence rates of 4.5% in chicken, 20.7% in cats, 27.2% in pigs and 35.7% in cattle were found. IFA and IgMELISA are also used currently. IFA using microfilarial antigens is used as a diagnostic tool in filariasis. Dr Chan noted that seroepidemiology is playing an increasing role in public health planning and evaluation of immunization programmes for the control of infectious diseases in Singapore. A seroepidemiological study using the flocculation technique was carried out in 1982-1984 to determine the herd immunity to diphtheria and to identify high-risk groups in the population. Although 94.6% of those surveyed had a protective antitoxic level of ~ 0.01 IV/ml, 14.3% of adults above 40 years of age had no protective antitoxic immunity. However, it was
- 3 -
concluded that the existing immunization programme is effective and adequate in the control of diphtheria. A seroepidemiological survey of 50 persons showed 10% having antibodies to Legionella at a titre of ~1: 32. In 1976, rubella immunization was introduced to girls aged 11-12 years and subsequently extended to school boys and national servicemen in 1982 and later to females of the reproductive age group. A preliminary serological survey in 1986 among ante-natal mothers showed a haemagglutination inhibition (HI) antibody rate of 69%. A later survey in 1986, in a public housing community among females aged 15-49 years, showed a positive rate of 76.7% by the latex agglutination (LA) test. A seroepidemiological survey to assess the immunization programme and the immune status of the population to poliomyelitis showed a high herd immunity to the three types of poliovirus, the neutralizing antibodies to poliovirus types 1, 2 and 3 being detected in 93.9% 96.7% of the population aged 6 months to 40 years and above. A serological study in 1982-1984 to determine the immune status of the population to dengue following the intensive control of Aedes mosquitos showed 54.4% of the population having no haemagglutination inhibition (HI) antibodies to dengue type 2 virus. Of great concern was the fact that 96.6% of children below age 10 years were seronegative. Serological studies were carried out for rotavirus antibodies and 73.6% of 425 healthy persons aged 6 months to 40 years had antibodies by the immune adherence haemagglutination test, thus suggesting that rotavirus may be responsible for the large numbers of nonbacterial diarrhoeal diseases in adults. Using the IgG-ELISA, 31.8% of 1,630 persons were found to be seropositive for hepatitis A virus antibodies in 1984-1985. Several seroepidemiological studies of hepatitis B virus infection have been carried out. HBsAg carrier rates of 13.6%, 7.3% and 6.0% were found among Chinese, Malay and Indian male blood donors respectively. Similarly, 6.2%, 2.3% and 0.6% of Chinese, Malay and Indian pregnant women were positive. In 1985-1986 serological surveys among urban rats showed that 28% of Rattus norvegicus had IFA antibodies to Hantaan virus. A survey among laboratory rats in the National University showed 40% had antibodies. Among hospitalized patients in 1984-1985, 1.6% of those with nephritis, 2.5% clinically diagnosed as leptospirosis, 4.8% clinically diagnosed as dengue and 19.4% clinically diagnosed as non-A, non-B hepatitis had significant IFA titres against Hantaan virus. Dr Lee noted that soil-transmitted helminthic infections are still common and important in the Republic of Korea. Clonorchis sinensis, Paragonimus westermani, Metagonimus yokogawai and Diphyllobotrium latum
- 4 -
infections are focally endemic. In 1987, stool examinations showed a C. sinensis average prevalence rate of 21.5% in inhabitants in the Nakdong, Yeongsan, Seomjin, South Han, Tanjin, Kum and Mangyong River basins, which had a population of 4 million. Several outbreaks of haemorrhagic fever of unknown cause with about 20% mortality have occurred in rural areas since 1981. Recently, some of these were diagnosed as leptospirosis or scrub typhus. In fact, many cases of leptospirosis and scrub typhus have been misdiagnosed clinically as haemorrhagic fever with renal syndrome (HFRS) in the Republic of Korea. Dr Cho noted that seroepidemiological tools are not employed for control programmes for malaria, filariasis or leprosy but that specific serodiagnostic test for leprosy may be implemented in a few clinics in the future in the Republic of Korea. Filariasis has been reduced from 14.7% in 1970 to 0.9% in 1985 in Chegu Province due to the intensive control programme there. Only two indigenous cases of malaria were seen in the Republic of Korea between 1980 and 1984. Leprosy is still a major infectious disease with 25 000 registered cases (prevalence 0.61 per 1000). However, the number of new cases declined from 832 in 1977 to 215 in 1985. A couple of on-going studies in the use of the phenolic glycolipid-I Mycobacterium leprae-specific antigen based serology are being carried out to evaluate the feasibility of using this test for leprosy control. Dr Lam reviewed the various serological tools applied to epidemiological studies of infectious diseases, especially childhood infections in tropical countries. Blood specimen collection techniques were discussed. He favoured the use of heparinized capillary tubes over filter paper collection of blood samples for seroepidemiological studies. Seroepidemiological techniques used for studies were described and their advantages, disadvantages and applications discussed. Techniques discussed were the complement fixation test (CFT), haemagglutination-inhibition (HI), neutralization test (NT), the single radial haemolysis (SRH), ELISA, the latex agglutination (LA), the passive and reverse passive haemagglutination (PHA & RPHA) and IFA tests. The criteria for the selection of these tools were also discussed. Dr Mak reviewed the important parasitic diseases in Malaysia, these being malaria, filariasis, toxoplasmosis, amoebiasis, soi1transmitted helminthiasis and schistosomiasis. Malaria is still the most important of these diseases, there being 44,145 cases with 67 deaths in 1986. Seroepidemiologica1 techniques used include IFA and ELISA for malaria and filariasis; IFA, IHA and ELISA for toxoplasmosis; ELISA for toxocariasis; circumova1 precipitin test (COPT) and ELISA for schistosomiasis. The results of IFA and ELISA for malaria reflect closely the level of malaria endemicity in the areas studied.
- 5 -
Dr Shirai gave a review of some important infectious diseases present in Viet Nam. A total of 172,904 cases with 772 deaths due to dengue haemorrhagic fever were seen between May-August 1987. For the first half of 1987, there were 52 cholera, 74 plague, 13,175 pertussis, 479 tetanus, 433 diphtheria, 11,800 measles and 691 poliomyelitis cases. The total number of leprosy cases as at the end of 1986 was 36,198. For the first six months of 1987, there were 45,823 malaria cases of which 14,982 were Plasmodium falciparum, 30,740 P. vivax and 101 mixed infections. As Dr Liu could not attend the meeting due to some unforeseen circumstances, Dr Mulkit Singh presented his review paper. The important parasitic diseases in China are malaria, filariasis and schistosomiasis. The number of malaria cases in 1986 were 364,000. SChistosomiasis was seen in 94 counties in 1986 with 463,000 cases reported while filariasis due to Wuchereria bancrofti and Brugia malayi in 1985 were 4.01 and 0.84 million respectively. The microfilarial rate was < 1% in 113 counties, 1-5% in 61 counties and 5.1-20% in 30 counties. IFA was used as an epidemiological tool for malaria in Shandong Province where a positive correlation was seen in the positive IFA and incidence rates. IFA has also been used for malaria surveillance and for monitoring the effectiveness of the control programme. The skin-test, COPT, IHA and LA are used for detecting antibodies against Schistosoma japonicum. ELISA and IFA are used as serological tests for filariasis.
3. SUMMARY OF DISCUSSIONS ON SEROEPIDEMIOLOGY OF SPECIFIC DISEASES 3.1 Dengue
Dr Lam reviewed the seroepidemiology of dengue with special reference to an ongoing longitudinal study in Malaysia. Baseline data using the HI test on 5,138 subjects aged 3 months to 19 years showed 19.4% of children under 1 year of age had antibody to dengue and this declined to 8.6% within a year. It then increased by an average of 5% every year until the age of 18 years when all subjects tested were positive. A cohort study on children 6-7 years of age was carried out at six monthly intervals. The antibody prevalence was shown to increase progressively with time. The serological tests employed were HI, haemadsorption immunosorbent technique (HIT) and IgM-ELISA. Blood specimens were collected on Nobutu filter paper. HI is the 'gold standard' test for arbovirus serology. Sera were extracted with kaolin and treated with goose-red blood cells to remove non-specific haemagglutinins. HI is very useful not only for epidemiological surveillance but also for the diagnosis of clinical infection. It is sensitive and economical but the sera have to be extracted with kaolin or acetone. In addition non-specific agglutinins must be removed by adsorption with red blood cells. Furthermore, in endemic countries, single serum samples often give uninterpretable results unless the antibody levels are exceptionally high (~ 1:1280). HIT was developed for the detection of dengue IgM. This is useful for
- 6 -
diagnosing primary dengue infection as well as secondary infection where high-titred dengue IgG does not appear to interfere with the test. Rheumatoid factors also do not appear to interfere with the test. The test is relatively simple to perform and does not require any major instrumentation but it is not as sensitive as IgM-·ELISA. The antibody-capture IgM-ELISA assay for the diagnosis of clinical infection is particularly useful for testing of single specimen as the presence of IgM is diagnostic of a recent infection. There is 100~ correlation between this test and the HI test in properly spaced paired sera. In addition, about 60% of the acute sera of such pairs already have detectable IgM, thus making this a potentially powerful tool for rapid diagnosis. One drawback of the IgM-ELISA is that blood collected on filter paper is not suitable for the assay as the IgM does not easily elute off the filter papers. In this case, blood collected in heparinized capillary tubes will be useful. Virus isolation is necessary in any epidemiological study of dengue as it is important to determine the sequence of infection and correlate this with serotypes and disease severity. In recent years, sensitive methods such as the use of mosquito cell lines (AP 61 and C6/36) and macrophage cell line (P388Dl) and the inoculation of mosquito adults and larvae have improved this significantly. The virus isolated is identified by IFA using type-specific monoclonal antibodies. The HI test is recommended for epidemiological study, while for laboratory diagnosis, other than HI, HIT and ELISA should also be considered. Another test which may have promise as a seroepidemiological tool for dengue is the single radial haemolysis (SRH), which has been shown to be useful for the diagnosis of dengue and Japanese encephalitis infections. Dr Chan stated that HI and NT, which measure long lasting antibodies, are well established methods for seroepidemiological studies of dengue virus infection. HI is more widely used because of its simpliCity, rapidity and suitability for testing large numbers of sera. Dengue type 2 virus is commonly used in the HI test as it is the most broadly reactive antigen. ELISA has recently been introduced in dengue seroepidemiological studies but the interpretation of results is difficult. 3.2 Haemorrhagic fever with renal syndrome
Dr Lee reviewed the clinical presentation of haemorrhagic fever with renal syndrome (HFRS). There are four serotypes of virus in the Genus Hantavirus under Bunyaviridae, these being Hantaan virus, Puumala virus, Seoul virus and Prospect Hill virus. Hantaan virus is distributed in Euro-Asia continent, Puumala virus in Europe and Seoul virus is world-wide. There are evidences of additional serotypes of Hantavirus, which can cause HFRS in the Euro-Asia continent and in other parts of the world. Seroepidemiological studies show that Hantaan and Hantaan-related agents are widely distributed; the severe form is common in Asia but the majority of European cases are mild.
- 7 -
It is impossible to diagnose Hantavirus infection on clinical grounds alone in the individual case with moderate or mild symptoms. A correct diagnosis was made only in 50% of seropositive patients in the Republic of Korea where HFRS has been endemic for 35 years. A rise in specific IFA titre in paired sera collected a week apart is diagnostic. ELISA and neutralizing antibodies appear in the first week of symptoms, peak at 2 weeks and persist for as long as 34 years. IFA antibodies are group-specific but neutralizing antibodies are type-specific. The IgM-ELISA test is a rapid and reliable early diagnostic test but cannot differentiate between Hantaan and Seoul virus infections. HI is cheap and can differentiate between Hantaan and Seoul virus but is not as sensitive as IFA and ELISA. The plaque reduction neutralizing test (PRNT) in Vero E6 cells is expensive and time-consuming and cannot be used for routine serological diagnosis of HFRS. IFA, ELISA and PRNT antibodies persists for 5-17 years after infection. PRNT is the most sensitive and specific in differentiating the original serotype virus infection among Hantavirus. ELISA is the most sensitive serological test for seroepidemiological study and IFA can be used as a routine test for diagnosis while PRNT is used for serotyping of Hantavirus infection. It is recommended that seroepidemiological surveillance of the distribution of Hantaan and related viruses in the world, including surveys of urban and laboratory rats,be carried out. Dr Chan agreed that the IFA test used for detecting anti-Hantaan antibodies is broadly reactive and cannot distinguish infections caused by the different members of the Hantavirus genus. Furthermore, it is known to exhibit false positive reactions, especially at a low antibody titre of 1: 32.
<
The neutralizing antibody is the most specific indicator of Hantavirus infection. He recommended that antibodies detected by the IFA test be tested by NT. The immune adherence haemagglutination (IAHA) test employing infected Vero E6 cells as antigen was found to be a useful method for detecting antibody to hantaviruses in human and rodent sera. It can discriminate between antibodies to the different types of hantaviruses and can be used for titration of a large number of sera, making it suitable for seroepidemiological studies. The indirect immunoperoxidase antibody (IPA) test has recently been described for the detection of anti-Hantaan IgG antibody. It uses protein A-peroxidase to detect antigen-antibody binding and results are read with a light-microscope. It has been shown to be as sensitive as the IFA test and is suitable for large-scale antibody screening of a variety of animal species using only one conjugate. 3.3 Leprosy
Dr Cho reviewed the seroepidemiological techniques for leprosy. With the availability of leprosy bacillus in relatively large quantities since the late 1970s, considerable efforts have been made to isolate and characterize :1ycobacterium leprae-specific antigens. Two
- 8 -
serodiagnostic techniques are currently used. One uses the phenolic glycolipid I (PGL-I) in indirect ELISA and the other uses monoclonal antibodies (McAbs) directed against protein antigens of ~ leprae in the competitive inhibition assay. PGL-I is a M.leprae-specific antigen and has been characterized as a triglycosylphenolic diacylphthiocerol (MW, about 2000). Using this antigen in indirect ELISA, the seropositive rate among lepromatous leprosy patients was high (80-90%) and among tuberculoid patients low (35-45%). Seropositivity was significantly higher in active patients with less than two years of treatment (95-100%) than in inactive patients (67-76%). However, 2-5% of healthy controls, tuberculosis and other mycobacteriosis patients were seropositive. The test is therefore useful in detecting untreated lepromatous patients. Synthetic neoglycoprotein antigens have been shown to have comparable or greater seroreactivities than the native glycolipid in the test. As an alternative tool to ELISA, LA or gelatin agglutination (GL) tests using neoglycoprotein antigens for sensitization have been shown to be comparable to ELISA in detecting anti-PGL-I antibodies. However, further evaluation is necessary. Enzyme or radio-labelled McAbs to M. leprae have been used in competitive inhibition ELISA or radioimmunoasssay (RIA). In one study using McAb ML04 in inhibition RIA, all lepromatous and about 50% of tuberculoid patients were seropositive. Specificity was close to 100%. Detection of PGL-I antigens from the urine and serum, though laborious, provides evidence of current M.leprae infection. It was detected in all untreated lepromatous patients but not from tuberculoid patients. The PGL-I ELISA results correlated with the bacterial index in untreated leprosy patients. Serological tools can be used for the early detection of ~ leprae infection and for monitoring the effectiveness of chemotherapy. However, these tools need to be simplified and made less expensive before they can be implemented in the field. For detecting anti-PGL-I antibodies, the agglutination tests will be the choice but ELISA should be available on a regional basis to serve as a back-up to the simpler tests. More convincing results of the effectiveness of serological tests for early detection of cases and for monitoring effectiveness of chemotherapy are needed. Furthermore, serological tests at the moment are not suitable for detection of antibodies in tuberculoid leprosy patients. 3.4 Malaria
Dr Sanders reported that the ELISA technique was used to study the transmission dynamics of a serotype of P. falciparum designated FC27 in Papua New Guinea. Sera from residents in Madang were screened for the
- 9 -
presence of circulating S-antigen. The specificity of ELISA was ensured by serotyping with rabbit antibodies raised against a fusion polypeptide (produced in Escherichia coli), containing 23 repeats of an 11-amino acid sequence characterizing the S-antigen of FC27. Dr Liu's paper on serological techniques for detecting malaria antigens using McAbs was discussed. Serological techniques were needed for antigen detection in areas with low endemicity and as a tool for rapid screening of malaria carriers during mass population movements. He noted that, although IFA has been used for the detection of malarial antibodies, it cannot be used for case detection. Two McAbs against P. falciparum designated No. 32 and 3F9 (both IgM) and reacting with different erythrocytic stages including ring and gametocytes were used in the competitive ELISA test. These McAbs cross-reacted with these stages of P. vivax, P.ovale, P.cynomolgi, P.knowlesi and P.yoelii. The test could detect 20 out of 22 blood smear P.vivax positive samples as positive. However, 3 out of 32 samples from blood negative subjects in a non-endemic area were also positive while all 8 samples from slide negatives from an endemic area were negative. The McAbs were also used in the Avidin-biotin complexantigen spot test. The McAbs 32 and 3F9 were coupled with biotin. Serum samples were dropped into nitrocellulose paper and allowed to react with the coupled McAbs. Avidin-HRP was added for colour visualization. The sensitivity was found to be not high enough as it could only detect parasitaemias of 1 per 10 red blood cells. 3.5 Schistosomiasis
Dr Garcia reviewed the seroepidemiological tools for Schistosoma japonicum infection. Parasitological techniques for the demonstration of Schistosoma eggs are rather insensitive. To achieve a sensitivity of 95% at least three 1-gram stool samples should be examined. Furthermore, in light infections, eggs are not regularly passed out while in chronic infections with hepato-splenic disease, eggs are entrapped in fibrous tissue in the mucosa of the intestine. In immunodiagnostic assays, egg antigens have been observed to be more specific than adult worm antigens. Among the available immunodiagnostic tests only the intradermal test (lOT), COPT and ELISA using soluble egg antigens have been used for seroepidemiological studies. IDT using adult worm antigen is relatively easy to perform and 99.9% of infected individuals react positively. The results can be read in 15 minutes. lOT is of value as a rapid screening method in the field for schistosomiasis. COPT is highly sensitive and specific. It is easy to perform and lyophilized eggs are used in the test. However, its main drawback is the 24-48 hours incubation time needed for the test.
- 10 -
The ELISA test using egg antigens can detect 97-100% as positive in patients with parasitological evidence of S. japonicum infection. 3.6 Trichinellosis and angiostrongyliasis
Dr Ko reviewed antigen preparations of Trichinella and the serological methods available for the diagnosis of trichinellosis. Among the available tests are the immunoradiometric assay (IRMA) using excretory-secretory (ES) antigens, the radioimmunoassay (RIA) using crude saline extracts of trichinella larvae, IHA using extract of muscle larvae, IFA using sections of muscle larvae or sonicated larvae, the flocculation tests (FT) using various carrier particles e.g. bentonite for antigenic extracts, ELISA using various antigenic extracts, and eIE using muscle larvae extracts. The methods used for the diagnosis of porcine trichinellosis are basically similar to those for the human infection. ELISA is the best method for seroepidemiological purposes in terms of practicability, sensitivity and specificity. The crude worm extract used may give some false positive reaction and a more specific antigen is needed. While the indirect ELISA method is satisfactory for the detection of IgG, IgM or IgA antibodies, the 'sandwich' method should be used for the detection of IgE antibodies present in acute infections. The various antigenic preparations of Angiostrongylus and the serological methods available for detection of angiostrongliasis were reviewed. Crude worm extracts, purified fractions and ES antigens of adults have been used. Other tests used are the immunodiffusion (10), the immunoelectrophoresis (IE), ELISA using antigenic extracts of larval stages from the brain of infected rats and purified adult worm fractions, complement fixation test (CFT) with somatic antigens, intradermal test using saline extracts of adult worms and IFA using frozen sections of 1st and 3rd stage larvae, adult worms or purified antigen coupled to cyanogen bromide-activated Sepharose 4B beads. As the parasitological diagnosis of angiostrongyliasis is seldom possible, serological methods are extremely important. Due to the cross-reactiveness of crude antigenic extracts, serological techniques have limited application. A more specific antigen or novel methods to demonstrate circulating antigen are needed. The micro-ELISA (or its variation) appears to be the best method available at present for seroepidemiological studies of angiostrongyliasis. Serological methods are not really necessary for epizoological studies as parasitological methods are sufficient. 3.7 Filariasis, amoebiasis and toxoplasmosis
Dr Mulkit Singh noted that serological methods are useful for detecting filarial infections where microfilaria is not demonstrable. Antigens from different stages have been used for these assays. Various crude and purified extracts of human and animal filariae are used for the intradermal test, which has low sensitivity and
- 11 -
specificity. ELISA using various antigens including ES, microfilaria, infective larva and adult of homologous and heterologous filarial parasites has been used. ELISA using microfilaria ES antigens appears promising but needs further evaluation as a seroepidemiological tool. IFA using whole parasites, fragments, sections or soluble antigens coated on inert carrier particles has been used. Using papainised or sonicated microfilariae as antigens in IFA, antibodies were detected in all microfilaraemics and those with clinical manifestations. IRA using crude extracts of adult worms gives fairly reliable results that correlate well with those of IFA. IRA using ES antigens from homologous species deserves further evaluation as a seroepidemiological tool. Detection of circulating antigen is the best alternative to a parasitological confirmation of filariasis. However, such tests can only detect 82-95~ of microfilaraemics. IRMA for antigen detection in filariasis has also been developed. Serological tests are useful for detecting extra intestinal amoebiasis where a strong immune response is present. These tests are less useful for the diagnosis of intestinal infections and asymptomatic cyst passers. Intact or extracts of Entamoeba histolytica trophozoites from axenic cultures are used in IFA, CIE, IHA, LA and ELISA. CIE is rapid, highly specific and detects 95~ of positives in hepatic amoebiasis and 90~ of intestinal amoebiasis. lHA is highly sensitive and specific, detecting 81~ of intestinal amoebiasis and 92~ of invasive cases. However, haemagglutinating antibodies persist for a long time. IFA is sensitive and detects 100% of hepatic amoebiasis but only 15% of intestinal amoebiasis. ELISA and its variant, the Dot-ELISA, are sensitive tests and can detect 100% of amoebic liver abscess but only 87% of intestinal cases. Serology is generally used for the diagnosis of toxoplasmosis. The Sabin-Feldman dye test, though reliable and giving very few false positive, is cumbersome and needs live organisms for the test. CFT is difficult to standardize between laboratories. The IFA test using fixed tachyzoites as antigens is convenient, specific and sensitive. The IgM-IFA test allows acute infections to be detected. The lHA test has some pitfalls. It has been found to be negative in some cases of congenital toxoplasmosis. lHA titres are slow to appear but tend to remain at high levels longer than those in the dye test. The direct agglutination test has been used for routine screening and diagnosis of congenital infection in France. It is convenient, specific, sensitive and reliable for the diagnosis of IgG and for IgM in its modified form. The LA test is similar to the direct agglutination test except that antigen-coated beads are used. IgG or IgM-ELISA is easy to perform and allows large-scale screening of sera. The IgM capture ELISA is an improvement over the IgM-ELISA. Most ELISAs detect specific antibodies but can be adapted
- 12 -
for detection of antigenemia, which would be useful if other serological tests are inconclusive and when antibody responses are suppressed. The ELISA and IFA test results generally correlate well. 3.8 TDR inputs
Dr Bergquist reviewed the objectives and inputs of the Special Programme for Research and Training in Tropical Diseases (TDR) in research on the development of sensitive, specific, simple and inexpensive immunodiagnostic techniques. The appropriate use of seroepidemiological tools was discussed. The status of such tests used in filariasis, schistosomiasis, malaria and toxoplasmosis was also reviewed. A recent TDR initiative of importance for seroepidemiology is the establishment of a registry of monoclonal antibodies recognizing schistosome and malaria antigen epitopes.
3.9 Biostatistical and epidemiological considerations Dr Patwary reviewed the biostatistical and epidemiological requirements in seroepidemiology. Survey techniques, analyses of results and epidemiological modelling techniques were discussed. 4. 4.1 SUMMARY OF RECOMMENDATIONS
General recommendations
(1) Seroepidemiology should be defined as "the systematic collection and testing of blood and other biological samples from a target population or a representative sample thereof to identify current and past infections by means of immunological tests". (i) Since antibodies can now be detected in biological fluids other than blood, the term "immuno-epidemiology" is recommended. (ii) A "target population" would include an animal population. (2) Use of the term "tropical diseases" should be replaced by "important endemic diseases of the Western Pacific Region". (3) Any test used as a seroepidemiological tool should be sensitive, specific, inexpensive, objective and standardized, and should have reagents readily available. (4) Finger-prick blood collection is relatively rapid and inexpensive and should be used for seroepidemiological studies. This is in preference to venous blood collection, which requires skilled personnel and is expensive. Venous blood collections do, however, have the advantage of allowing large volumes of blood to be taken. Fingerprick blood can be collected by:
- 13 -
(i) (ii) (iii)
vial/microtainers which hold up to 1 mI. (approximate cost: USSO.20 each); capillary tubes (approximate cost: US$O.06 each); filter papers (approximate cost: USSO.10 each).
It should be noted that collections using capillary tubes and filter papers have inherent problems for some studies. (5) Appropriate situations for the use of seroepidemiological tools would be the determination of: disease distribution incidence prevalence severity/intensity contact patterns prognosis effectiveness of control, including immunization new agents or diseases risk factors early indication of disease outbreaks (6) WHO should continue to conduct workshops and individual training programmes on seroepidemiological tools. In addition, control programme managers should be included in future workshops in order to familiarize them with the use of such tools. (7) WHO should provide funding for laboratories, both WHOdesignated and others, offering to contribute reagents and services to the Resources Sharing Scheme (see Annex 4). (8) A catalogue of currently available reagents, including monoclonal antibodies, and their sources should be prepared by the WHO Regional Office. Information on these from other regions should also be made available, through the Regional Office for the Western Pacific. (9) Standard reagents and panels of sera should be made available through the Regional Office for the Western Pacific for evaluation and standardization of seroepidemiological tools. 4.2 Specific recommendations (1) Malaria
The recommended test is ELISA. IFA is also currently used and may be considered for diagnostic purposes. An indirect immunoperoxidase test has been applied for antibody testing in malaria. It has been shown to be as sensitive as IFA but does not require a fluorescent microscope. This test needs further evaluation before it can be recommended.
- 14 -
(2)
Filariasis
At present, serological tests have not been found to be useful for epidemiological studies although some tests (e.g. IFA) are used for diagnosis. (3) Toxoplasmosis
IRA and LA are recommended as general screening tools. IgM-ELISA is useful for the detection of acute infections. IFA is also currently used. (4) Schistosomiasis
The recommended test is IRA. COPT and ELISA are currently used.
(5)
Trichinellosis
The recommended test is ELISA. Currently used tests employ crude antigens prepared from infective larvae. There is a need for more specific antigens (e.g. ES antigens). (6) Angiostrongyliasis
The recommended test is ELISA. Purified antigens prepared by preparative isoelectric focusing or affinity chromatography are used. ES antigens from in vitro culture of 3rd stage larvae have also been used with some success. (7) Amoebiasis
At present, serological tests have not been found to be useful for epidemiological studies although some tests (e.g. CIE, ELISA, IRA) are used for diagnosis. (8) Leprosy
The following serological tests for the detection of Mycobacterium leprae infection have been developed: indirect ELISA using PGL-I antigen, agglutination (latex or gelatin particle) test also using PGLI, and competitive inhibition assay (ELISA or RIA) using monoclonal antibodies against specific epitopes of protein antigens. The application of these serological tests in seroepidemiological surveys is being evaluated. I
I
J
- 15/16 -
(9)
Haemorrhagic fever with renal syndrome
IFAT is group-specific and is the recommended test for detecting all four serotypes of Hantavirus. PRNT is recommended for the typing of hantavirus infection. ELISA has also been developed as a seroepidemiological tool but it requires further standardization and evaluation. (10) Dengue
The recommended test is HI. PRNT remains the method of choice for the serotyping of dengue virus infection. The use of ELISA as a seroepidemiological tool requires further evaluation. SRH shows promise as a seroepidemiological tool and requires evaluation.
- 17/18 ANNEX 1
OPENING REMARKS OF DR HIROSHI NAKAJIMA, REGIONAL DIRECTOR, WHO REGIONAL OFFICE FOR THE WESTERN PACIFIC AT THE OPENING CEREMONY OF THE WORKING GROUP ON SEROEPIDEMIOLOGICAL TOOLS FOR CONTROL PROGRAMMES IN TROPICAL DISEASES, Kuala Lumpur, 9-13 November 1987 On behalf of Dr Hiroshi Nakajima, Regional Director of the WHO Regional Office for the Western Pacific, allow me to extend a warm welcome to all members participating in this Working Group on Seroepidemiological Tools for Control Programmes in Tropical Diseases. I would like to express my gratitude to the Government of Malaysia for agreeing to host this meeting and to thank Dr Lim Teong Wah, the past Director of this Institute for Medical Research, and Dr Khalid Hassan, Deputy Director, for making available the facilities. I also wish to thank Dr Mak Joon Wah and his colleagues for all the hard work which went into the preparation of this meeting. In 1978, an agreement was signed which established the Institute for Medical Research as a WHO Regional Centre for Research and Training in Tropical Diseases. This meeting is an activity of this Centre, in line with one of its objectives to improve control methods of major communicable diseases which are prevalent in the Western Pacific Region as a whole. Tropical diseases such as malaria, dengue fever and dengue haemorrhagic fever, leprosy, filariasis and schistosomiasis, to mention a few, are major causes of morbidity and mortality in many countries of the Region. It is therefore of the utmost importance to develop a control programme which will include strengthening of surveillance and the upgrading of laboratory diagnostic capacities. The WHO Regional Office for the Western Pacific is deeply involved in collaborating with the national control programmes in developments in these fields, and has recognized the urgent need to train personnel and to test the tools for detecting and monitoring infections in an effort to improve the efficiency of the disease control programmes. Great advances have been made in serological methods over the past ten years. There is therefore a need to review the various serological tools available today and the applicability of such techniques in the control of the various diseases. Nor should we overlook the important issues of standardization and quality control. I am confident that, with your active participation and exchange of ideas and information, this meeting will be a great success. I wish you all a very pleasant and fruitful stay in Kuala Lumpur. Thank you.
- 19 -
ANNEX 2 LIST OF TEMPORARY ADVISERS, OBSERVERS, RESOURCE PERSONS, WHO CONSULTANT AND SECRETARIAT TEMPORARY ADVISERS Dr Liu Er-Xiang Department of Parasitology Institute of Basic Medical Sciences Beijing People's Republic of China Dr Ronald C. Ko Department of Zoology Science Faculty Hong Kong Dr R. Sanders Institute of Medical Research Goroka Papua New Guinea Dr E. Garcia College of Public Health University of the Philippines Manila
Philippines Dr Mulkit Singh Department of Microbiology Faculty of Medicine National University of Singapore Singapore Republic of Singapore Dr Chan Yow Chong Department of Microbiology Faculty of Medicine National University of Singapore Singapore Republic of Singapore Dr Ho Wang Lee Institute of Viral Diseases Korea University Seoul Republic of Korea
- 20 Annex 2
Dr Sang-Nae Cho Department of Microbiology College of Medicine Yonsei University Seoul Republic of Korea Dr Lam Sai Kit Department of Microbiology Medical Faculty University of Malaya Kuala Lumpur Malaysia OBSERVERS Professor Mohd. Roslani Abdul Majid Dean School of Medical Sciences Universiti Sains Malaysia Penang Malaysia Dr Chan Boon Tek Parasitology Department Medical Faculty Universiti Kebangsaan Malaysia Kuala Lumpur Malaysia
Mr Gan Seng Chiew Serology Division Institute for Medical Research Kuala Lumpur Malaysia Dr Norilla bt PDPD Hj Abdul Rahim Principal Epidemiologist Brunei Dr Kan Nyunt Medical Officer of Health Brunei
- 21 Annex 2
RESOURCE PERSONS Dr Lye Munn Sann Community Health Division Institute for Medical Research Kuala Lumpur Dr M. Palanicavaban National Leprosy Control Centre Sungei Buloh Dr Lim Kuan J 00 Public Health Institute Kuala Lumpur Dr Yong Hoi Sen Department of Zoology Faculty of Science University of Malaya Kuala Lumpur Dr Chua Sau Peng Department of Parasitology Faculty of Medicine University of Malaya Kuala Lumpur CONSULTANT Dr Mak Joon Wah Head Division of Filariasis/Malaria Institute for Medical Research Kuala Lumpur Malaysia SECRETARIAT Dr A. Shirai Research Promotion and Development WHO Regional Office for the Western Pacific Manila
Dr N.R. Bergquist Special Programme on Research and Training in Tropical Diseases WHO Headquarters Geneva Dr K.M. Patwary WHO Regional Centre for Research and Training in Tropical Diseases Institute for Medical Research Kuala Lumpur
- 22 Annex 2
Mr J. Storey WHO Regional Anti-Malaria Team Kuala Lumpur Dr Khalid Hassan Deputy Director Institute for Medical Research Kuala Lumpur
Mr Cheong Weng Hooi Entomology Division Institute for Medical Research Kuala Lumpur Dr C.K. Ow-Yang Parasitology Division Institute for Medical Research Kuala Lumpur Dr K.D. Sukumaran Serology Division Institute for Medical Research Kuala Lumpur
- 23 -
ANNEX 3 AGENDA Monday. 9 November 1987 (Day 1) 8:30 a.m. 10:30 a.m. 11:00 a.m. Registration Opening ceremony - Lecture Hall II Group photograph Tea Introductory remarks by Dr A. Shirai, WPRO Introduction of participants Election of chairman, co-chairman Adoption of agenda Presentation of country reports on "Seroepidemiological tools for control programmes on tropical diseases - current status" Dr Liu Er-Xiang (People's Republic of China) (Working paper no. 1) Dr Ronald C. Ko (Hong Kong) (Working paper no. 2) Lunch Break Dr R. Sanders (Papua New Guinea) (Working paper no. 3) Dr E. Garcia (Philippines) (Working paper no. 4) Dr Mulkit Singh (Singapore) (Working paper no. 5) Dr Chan Yow Cheong (Singapore) (Working paper no. 6) 3:30 p.m. Tea Dr Ho Wang Lee (Republic of Korea) (Working paper no. 7) Dr Sang-Nae Cho (Republic of Korea) (Working paper no. 8)
11:30 a.m. 12:00 noon 12:30-2:00 p.m. 2:00 p.m. 2:30 p.m. 3:00 p.m.
3:45 p.m.
4:15 p.m.
Dr Lam Sai Kit (Malaysia) (Working paper no. 9)
- 24 Annex
3
4:45 p.m.
Dr Mak Joon Wah (Malaysia) (Working paper no. 10)
Tuesday. 10 November 1987 (Day 2) 8:30 a.m. 10:15 a.m. 10:30 a.m. 11:00 a.m. 11: 30 a.m. 12:00 noon General discussions on country reports Tea Seroepidemiology of dengue Dr Lam Sai Kit (Working paper no. 11) Haemorrhagic fever with renal syndrome (Dr Ho Wang Lee) (Working paper no. 12) Dr Chan Yow Cheong (Working paper no. 13) Seroepidemiological techniques for leprosy control (Dr Sang Nae Cho) (Working paper no. 14) Lunch break Dr N.R. Bergquist (Working paper no. 15) Dr Ray Sanders (Working paper no. 16) Seroepidemiology in the control of Schistosoma japonica (Dr E. Garcia) (Working paper no. 17) Tea
12:30-2:00 p.m. 2:00 p.m. 2:30 p.m. 3:00 p.m.
3:30 p.m.
-
3:45 p.m.
A review of the seroepidemiological techniques in trichinellosis (Dr Ronald Ko) (Working paper no. 18)
-
A review of the seroepidemiological techniques in angiostrongyliasis (Dr Ronald Ko) (Working paper no. 19)
4:45 p.m.
-
Malaria (Dr Liu Er-Xiang) (Working paper no. 20)
Wednesday. 11 November 1987 (Day 3) 8:30 a.m. Filariasis. amoebiasis and other parasitic diseases (Dr Mulkit Singh) (Working paper no. 21)
- 25/26 Annex
3
9:30 a.m. 10: 15 a.m. 10:30 a.m. 12:30-2:00 p.m. 2:00 p.m. 3:30 p.m. 3:45 p.m.
Discussions on working papers Tea Plenary session on general use of seroepidemiological tools Lunch break Continuation of plenary session Tea Group discussion on specific use of seroepidemiological tools in: (i) bacterial and viral diseases (Group I - Bilik Baca) (ii) parasitic diseases (Group II Bilik Mesyuarat Kecil)
Thursday. 12 November 1987 (Day 4)
8:30 a.m. 10:15 a.m. 10:30 a.m. 12:30-2:00 p.m. 2:00 p.m.
Continuation of group discussions on specific use of seroepidemiological tools Tea Continuation of group discussion Lunch break Plenary session on specific use of seroepidemiological tools: Reports by Groups I and II Tea Continuation of plenary session
3:30 p.m. 3:45 p.m.
Friday. 13 November 1987 (Day 5)
9:00 a.m. 10:30 a.m. 10:45 a.m. 12:00 noon
Presentation and adoption of final guidelines Tea Continuation on final guidelines Closing ceremony and official lunch
- 27/28 -
ANNEX 4
Sharing of resource within the Western Pacific Region The Institutions listed below have indicated their willingness to participate in the following activities: 1. Institute for Medical Research, Malaysia Supply of antigens from Brugia malayi, Plasmodium falciparum, Toxoplasma gondii (for IFAT and ELISA), Entamoeba histolytica, Schistosoma (Malaysian strain) and Toxocara spp. 2. Department of Zoology, University of Hong Kong Crude extracts of: Trichinella spiralis, Parangiostrongylus (=Angiostrongylus) cantonensis, Cysticercus cellulosae, and Gnathostoma spp.
3.
College of Public Health, University of the Philippines Crude extracts and intact eggs of Schistosoma japonicum
4.
Department of Microbiology, National University of Singapore Antigens of Toxoplasma gondii, Entamoeba histolytica, Giardia lamblia; and sonicated microfilaria of Brugia malayi (for IFAT) and Trichomonas vaginalis.
5.
Institute of Viral Diseases, Korea University All reagents, with the exception of conjugates, for work on HFRS. Serological testing on human and wild rodent sera.
6.
Department of Microbiology, Yonsei University, Korea Monoclonal and polyclonal antibodies to PGL-I (leprosy).
7.
Department of Medical Microbiology, University of Malaya, Malaysia The WHO Collaborating Centre for Dengue (DF/DHF) will provide information on the availability of reagents and reference panel sera for evaluation of techniques.
Reagents provided by the abovementioned laboratories are intended for initiation of control programmes only. They are also prepared to provide training facilities on individual basis on appropriate techniques.
- 29 ANNEX 5
LIST OF WORKING PAPERS ISSUED 1. 2. 3. Country review of the utilization of seroepidemiological techniques in the control programme, by Dr Liu Er Xiang Use of seroepidemiological tools in control programmes of tropical diseases in Hong Kong, by Dr Ronald C. Ko Use of seroepidemiological tools in the control of malaria and viral diseases in Papua New Guinea, by Dr R.C. Sanders and Dr M.P. Alpers Seroepidemiological tools for control programs of tropical diseases in the Philippines, by Dr Edito G. Garcia Use of seroepidemiology in the control of infectious diseases in Singapore: Parasitic Diseases, by Dr M. Singh and Dr Y.C. Chan Use of seroepidemiology in the control of infectious diseases in Singapore: Bacterial and Viral Diseases, by Dr Y.C. Chan and Dr M. Singh Brief report on important tropical diseases in Korea, by Dr H.W. Lee Tropical diseases in the Republic of Korea, by Dr Sang-Nae Cho Use of seroepidemiological tools in the control of tropical diseases, by Dr S.K. Lam Review of seroepidemiological tools for control programmes of parasitic diseases in Malaysia, by Dr Mak Joon Wah Seroepidemiology of dengue, by Dr S.K. Lam Haemorrhagic fever with renal syndrome, by Dr H.W. Lee Seroepidemiological tools in the control of dengue and hantavirus infections, by Dr Y.C. Chan and Dr M. Singh Seroepidemiological techniques for leprosy control, by Dr Sang-Nae Cho Seroepidemiological aspects of parasitic diseases in the Western Pacific Region - Summary of research carried out under the auspices of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) , by Dr N.R. Bergquist
4.
5.
6.
7. 8. 9. 10. 11. 12. 13. 14. 15.
- 30 Annex 5
16. 17. 18. 19. 20. 21.
Use of seroepidemiological techniques in Papua New Guinea, by Dr R.C. Sanders and Dr M.P. Alpers Seroepidemiology in the control of Schistosoma japonica, by Dr Edito G. Garcia A review of seroepidemiological techniques in trichinellosis, by Dr Ronald Ko A review of the seroepidemiological techniques in Angiostrongyliasis, by Dr Ronald Ko Serological techniques for detecting malaria antigens using monoclonal antibodies, by Dr Liu Er Xiang Seroepidemiological tools in the control of filariasis, amoebiasis and toxoplasmosis, by Dr M. Singh and Dr Y.C. Chan