Field evaluation of ELISA using Wuchereria bancrofti mf ES antigen for bancroftian filariasis

Bulletin of the World Health Organization, 62 (6): 941 - 944 (1984) (3 World Health Organization 1984 Field evaluation of ELISA using Wuchereria bancrofti mf ES antigen for bancroftian filariasis B. C. HARINATH,' ASHOK MALHOTRA,2 S. N. GHIRNIKAR,3 S. D. ANNADATE,4 V. P. ISAACS,5 & M. S. BHARTI6 An enzyme-linked immunosorbent assay using Wuchereria bancrofti microfilarial excretory-secretory antigen was used in field studies to screen blood samples collected on filter-paper from persons residing in areas endemic for bancroftian filariasis. This assay system, when compared with examination ofnight wet bloodsmearsfor microfilariae, gave a relative sensitivity of98% and a relative specificity of86%. Daytime blood samples can also be used in this test, which can thus replace tedious examination ofnight blood samples in field surveys in endemic areas. The only definitive method currently available for diagnosis of lymphatic filarial infection is the examination of blood films (20 11) for microfilariae. In the case of bancroftian filariasis, the blood films must be collected at night. To increase the sensitivity of the test, use of an increased volume of blood (50-60 Al) for thick film preparation, or concen- tration and membrane-filtration techniques using as much as 1.0 ml of venous blood, have been explored (3). Considerable loss of microfilariae is observed during processing and staining of blood films (2, 10). The time of collection of blood at night also affects the test results (A. Malhotra & B. C. Harinath, unpublished observations, 1983). Membrane- filtration techniques are costly, time-consuming, and not convenient for large-scale field studies in endemic areas. The parasitological examination techniques are also not useful where there is a low level of micro- filaraemia, or occult or clinical filariasis. Hence, there is a need for a sensitive and specific immuno- diagnostic test, which would avoid the need for tedious night blood surveys in nocturnally periodic Wuchereria bancrofti infections. Gidel et al. (4) con- ducted a trial of two immunological tests (intradermal and complement fixation) using purified Dirofilaria immitis antigens without success. W. bancrofti micro- filarial excretory-secretory (mf ES) antigen has been found to be useful in detection of filarial antibody (5, X Professor and Head, Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha 442 102, India. 2 Research Officer, Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, India. 3 Medical Officer-in-Charge, Research cum Training Centre, Wardha, India. 4 Filaria Officer, Survey Unit, Nagpur, India. 5 Filaria Officer, Filaria Control Unit, Nagpur, India. 6 Research Officer, Filaria Control Unit, Nagpur, India. 7). This communication reports the field evaluation of an enzyme-linked immunosorbent assay (ELISA) using W. bancrofti mf ES antigen for detection of bancroftian filariasis in an endemic area. MATERIALS AND METHODS Collection and processing of blood samples Social service camps were organized by the Mahatma Gandhi Institute of Medical Sciences in Pipri and Sukalibai villages, which are endemic for bancroftian filariasis. At the time of these camps, blood was collected from 462 individuals by finger- prick between 20 h 00 and 24 h 00 on to Whatman No. 3 filter-paper in such a way that it covered a circle of more than 0.9-cm diameter, and on to glass slides (20-mm3 thick smear). A wet blood smear was examined at the same time. The filter-paper blood samples were dried at room temperature in the laboratory and a circle of 0.9-cm diameter was cut for elution. The filter paper with the dried blood sample was cut into small pieces and eluted into 0.75 ml of 0.05 mol/litre phosphate-buffered saline (PBS), pH 7.2, in a tube by shaking at 37 IC for 2 hours. After centrifugation at 600 g for 10 minutes, the supernatant was separated and stored at - 10 IC. Sodium azide (1 ml/litre) was used as preservative. The blood dilution of the undiluted filter-paper eluate was assumed to be 1:37.5 (7). These eluates were analysed for filarial antibody by indirect ELISA using W. bancrofti mf ES antigen and anti-human IgG + IgM + IgA penicillinase conjugate. A further 1144 night blood samples were collected during a filaria field survey of Wardha district by the Filaria Survey Unit and Control Unit of Nagpur; these samples were also dried, eluted, and screened 4494 -941- 942 B. C. HARINATH ET AL. in an indirect ELISA. The staff of the Filaria Control Unit also examined 20-mm3 stained blood smears from each of these subjects, to check for micro- filaraemia. Further blood samples were collected on to filter- paper from 35 of these cases between 12 h 00 and 16 h 00 for screening by ELISA. Of 146 apparently false positive cases (ELISA- positive, microfilariae-negative by stained smear examination), 71 were recontacted and examined for filarial infection by inspection of a 20-mm3 wet blood smear, and examination for signs and symptoms of clinical filariasis. In addition, peripheral blood films from 25 of these apparently false positive cases were examined for eosinophilia. Filter-paper eluate from 10 persons who had microfilaraemia, confirmed by wet blood smear examination, were used as positive controls. Filter-paper blood sample eluates from students coming to the Mahatma Gandhi Institute from non-endemic areas were used as negative controls. Also 7 serum samples from subjects from non-endemic areas, who had non-filarial helminth infections were examined (4 samples with Ascaris lumbricoides, 1 with Enterobius vermicularis and 2 with hookworm). Microfilarial excretory-secretory antigen Culture fluid containing knf ES antigen was obtained by in vitro maintenance of about 3000 W. bancrofti microfilariae per ml of medium 199 supplemented with organic acids and sugars of Grace's medium for 14 days (5). The medium was changed every 24 hours. The antigenic protein content of the medium, estimated by Lowry's method (6), was found to be about 140 ug/ml. As the optimum mf ES antigen required per test in ELISA was found to be 0.35 ng, maintenance of 3000 micro- filariae per ml per day provided sufficient antigen for 400 000 tests. Enzyme-linked immunosorbent assay (ELISA) The substrate for the ELISA consisted of soluble starch (150 mg) in 27.5 ml of 0.2 mol/litre phosphate-buffered saline, pH 7.0, containing 10.64 mg of penicillin V0 and 65 1l of 0.08 mol/litre iodine in 3.2 mol/litre potassium iodide solution. The substrate was prepared fresh before use. Conjugation of antihuman IgG + IgM + IgAb and penicillinase° was achieved by the method of Avrameas (1) using glutaraldehyde. Bovine serum albumina and polystyrene or poly(vinyl chloride) microtitration platesC were used in the assay. ' Sigma Chemical Co., USA. b Immunodiagnostics, New Delhi, India. c Dynatech Laboratories, Singapore. Indirect ELISA The indirect ELISA was carried out as described by Malhotra et al. (7). Optimum working dilutions of W. bancrofti mf ES antigen (3.5 ng of protein/ml) and penicillinase-labelled antihuman IgG + IgM + IgA conjugate (1: 1600) were determined by chequer- board titration. Volumes of 100 Al of mf ES antigen, blood sample (1:150), penicillinase-labelled antihuman IgG + IgM + IgA conjugate, and starch iodine penicillin substrate were used in the assay. After addition of substrate, the plate was incubated at 37 IC for 30 min and the colour changes were read visually. Complete decolorization or decolorization with a slight tinge of substrate colour was taken as a positive reaction. Suitable positive and negative controls were included in each set. As some of the negative controls gave a positive reaction at a blood sample dilution of 1: 75, we considered a titre of 1:150 as positive for filarial antibody. RESULTS Of 462 individuals from Pipri and Sukalibai villages, screened by night wet blood smear examination, 188 were found to be positive for microfilariae. When the filter-paper blood samples of these individuals were analysed by ELISA using W. bancrofti mf ES antigen and antihuman IgG +IgM + IgA-penicillinase conjugate, 184 of the 188 microfilariae-positive cases and 38 of the 274 microfilariae-negative cases showed a positive reaction for filarial antibody, giving this assay system a relative sensitivity of 980/ and a relative specificity of 86% compared with night wet blood smear examination. On the basis of these encouraging results, further field evaluation of this assay system was carried out, in comparison with stained blood smear examination, which is routinely employed in large-scale field surveys. Of a total of 1144 persons examined, stained blood smear examination showed the presence of microfilariae in 70 individuals, whereas ELISA showed the presence of antibody in 207 cases. Of the 70 smear-positive cases, 61 were also positive by ELISA. There were 146 cases that showed the presence of filarial antibody by ELISA but in whom no microfilaraemia was observed by stained smear examination. Of these 146 false positive cases, 71 were reexamined clinically and for microfilariae by night wet blood smear examination; 7 were found to be positive for microfilariae, 1 had clinical filariasis, while another 7 gave a history suggestive of filariasis. Of 25 false positive cases for whom peripheral blood EVALUATION OF ELISA FOR BANCROFTIAN FILARIASIS 943 films were examined, 19 had an eosinophil count > 10%. All the 7 non-filarial helminthic sera screened gave a negative reaction in the ELISA. The daytime blood samples collected between 12 h 00 and 16 h 00 from 32 carriers and 3 normal persons from endemic areas were analysed for antibody by ELISA; all the carrier samples showed the presence of antibody. DISCUSSION Epidemiological studies of bancroftian filariasis in endemic areas are beset by problems: the need for examination of night blood samples, the problems of processing and staining blood films, and the insensitivity of parasitological examination tech- niques are just a few. A precise and reliable diagnostic test is required for studies investigating the level of endemicity of the disease, transmission, and the effect of control measures, as well as for surveillance. In India alone, 304 million people living in areas endemic for bancroftian filariasis are at risk of infection (9), which emphasizes the urgent need for such a test. Though there have been a number of studies on the detection of filarial antibody using different immunological tests,d field evaluations of the tests are few. The study described here used W. bancrofti mf ES antigen in an ELISA; the test was relatively sensitive (98%) and relatively specific (86%) compared with wet blood smear examination. In further studies, of 70 people who showed microfilariae in stained blood smears, only 61 showed filarial antibody in the ELISA. Mitchell et al. (8), using a hybridoma antibody-based competitive d KAGAN, I. G. The immunodiagnosis of filariasis. In: Report of the sixth meeting of the Scientjflc Working Group on Filariasis: lymphatic filariasis-diagnosis of infection and evaluation of control. WHO unpublished document, TDR/FIL/SWG(6)/81, 1981. ELISA for the diagnosis of Schistosoma japonicum infection, observed a false negativity rate of about 10%. This may be due to excess antigen or immune complexes or immunosuppression. Analysis of 5 false negative blood samples by sandwich ELISA showed the presence of antigen in two cases (Reddy et al., unpublished observations, 1982); this subject needs further exploration. A test for the detection of antigen and immune complexes, in association with the ELISA, may decrease the proportion of false negatives. By indirect ELISA, the presence of antibody was detected in an additional 146 subjects. Re- examination of 71 of these false positive cases revealed that 10% had microfilariae and a further 11 %o had either clinical filariasis or a history suggestive of filariasis. It has also been observed that 43% of eosinophilia cases show the presence of filarial antibody by indirect ELISA (A. Malhotra & B. C. Harinath, unpublished observations, 1983). In the present study, 17 out of 25 false positive cases were found to have eosinophilia. Pipri and Sukalibai and other surrounding villages have a 20%/ endemicity rate, whereas Wardha town has about 6% endemicity for bancroftian filariasis. The predictive value of a positive result in the ELISA falls from 63.81%o when the prevalence is 20% to 31.040/ when the prevalence is 6%. However, the predictive value of a negative result is quite high (> 98%o). Thus the test may be used in seroepidemio- logical studies. The specificity of the test may be increased by the use of additional tests for detection of antigen or immune complexes. It was also observed that daytime blood samples, as well as night samples, from microfilariae carriers showed the presence of antibody by ELISA; thus, the use of this test will obviate the need for tedious collection of night blood samples. The ELISA test using W. bancrofti mf ES antigen should thus facilitate the coverage of large populations in field surveys of bancroftian filariasis in endemic areas. ACKNOWLEDGEMENTS We are grateful to the Departments of Community Medicine and Pathology of the Mahatma Gandhi Institute of Medical Sciences, Wardha, India, for help and cooperation in the collection of blood samples. We thank Dr Sushila Nayar, Director and Dr K. S. Sachdeva, Dean of the Institute for their keen interest and encouragement. Thanks are also due to Mr Sharad Tekade and Miss Sulabha Ranade for their technical assistance. This study was supported in part by a grant from the Indian Council of Medical Research and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. 944 B. C. HARINATH ET AL. RtSUMt EVALUATION SUR LE TERRAIN D'UNE TECHNIQUE ELISA UTILISANT L'ANTIGtNE ESmf DE WUCHERERIA BANCROFTI POUR LE DIAGNOSTIC DE LA BANCROFTOSE Une technique immuno-enzymatique du type ELISA, fond6e sur l'utilisation de l'antigene excr6toire-s6cr6toire de la microfilaire de Wuchereria bancrofti (ESmf) et d'une anti-IgG + IgM + IgA humaine conjugu6e a la p6nicillinase, a et6 appliquee A l'analyse d'6chantillons de sang recueillis sur papier filtre en vue de poser le diagnostic de filariose A W. bancrofti. Au d6part, on a proc6de a l'6tude de 462 6chantillons et constat6 que le syst6me d'6preuve avait une sensibilit6 relative de 98% et une sp6cificit6 relative de 86%, par comparaison avec l'examen d'un frottis sanguin a l'6tat frais (20 mm'). L'6tude a ensuite 6t6 6largie de fa$on a englober 1144 autres cas et le systeme d'6preuve a ete compar6 a l'examen d'un frottis sanguin (20 mm') aprcs coloration. Sur les 70 cas pour lesquels on a observ6 la pr6sence de microfilaires dans le sang periph6rique, 61 ont 6t6 jug6s positifs par la technique ELISA. Cinq des 9 faux n6gatifs ont alors 6t6 6tudi6s a l'aide d'une technique ELISA "en sandwich", en vue de rechercher la pr6sence de I'antigene filarien circulant; 2 se sont r6v6lks positifs. Sur les 146 faux positifs apparents, 71 ont et6 soumis A un nouvel examen clinique tandis qu'on recherchait la presence de microfilaires A l'ctat frais, dans un frottis de sang pr6levc la nuit; 7 ont 6tt trouv6s positifs (pr6sence de microfilaires), I etait atteint de filariose clinique et 7 autres avaient des antec6dents faisant cvoquer cette parasitose. L'application de ce systeme d'epreuve a 35 6chantillons de sang recueillis pendant le jour a donn6 les memes r6sultats que l'analyse des echantillons preleves la nuit. Cela 6tant, il est possible de substituer cette technique ELISA (fond6e sur l'utilisation de l'antigene ES mf de W. bancrofti et d'6chantillons de sang recueilli sur papier filtre) a la methode astreignante que constitise l'examen d'6chantillons de sang prelev6s la nuit et d'am6liorer la couverture des populations importantes lors des enquEtes effectuees sur le terrain en r6gions d'end6mie. REFERENCES 1. AVRAMEAS, S. Coupling of enzymes to protein with glutaraldehyde-use of conjugates for the detection of antigens and antibodies. Immunochemistry, 6: 43-52 (1969). 2. DENHAM, D. A. ET AL. Comparison of a counting chamber and thick smear methods of counting micro- filariae. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65: 521-526 (1971). 3. DESwWITZ, R. S. & HITCHCOCK, J. C. 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