Evaluation of the ICT whole blood antigen card test to detect infection due to nocturnally periodic Wuchereria bancrofti in South India

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1 TMIH559 Tropical Medicine and International Health volume 5 no 5 pp may 2000 Evaluation of the ICT whole blood antigen card test to detect infection due to nocturnally periodic Wuchereria bancrofti in South India S. P. Pani, S. L. Hoti, A. Elango, J.Yuvaraj, R. Lall and K. D. Ramaiah Vector Control Research Centre, Pondicherry, India Summary The commercially available ICT Card Test for bancroftian filariasis was evaluated for its sensitivity and specificity in detecting microfilaria carriers among 189 individuals each in filariasis-endemic and nonendemic areas in South India, and compared to both conventional night blood finger prick thick blood smear examination and venous blood membrane filtration. Though the specificity of the test was 100% in comparison to both, the sensitivity was 98.5% against the finger prick thick blood smear and 71.9 compared to the membrane filtration technique. Similarly, the positive predictive value was 100% against both techniques, but the negative predictive values were 99.5% against the finger prick thick blood smear and 88.3% compared to the membrane filtration technique. The test s lower sensitivity compared to the filtration technique requires further investigation. keywords ICT card test, Wuchereria bancrofti, immunodiagnosis, filariasis correspondence Dr S.P. Pani, Vector Control Research Centre, Medical Complex, Indira Nagar, Pondicherry , India. E mail: mosquito@md2.vsnl.net.in Introduction Lymphatic filariasis caused by the nematode parasites Wuchereria bancrofti, Brugia malayi and Brugia timori results in disabling disease and is endemic in 73 countries in Africa, Asia, South-east Asia and the Pacific (Michael et al. 1996). Globally, about 1.1 billion people (constituting approximately 20% of the world population) live in areas endemic for this disease, and more than 90% of them are exposed to the risk of infection with W. bancrofti. There are about 120 million people with infection or lymphatic pathology. India alone bears 40% of the global burden of this disease (Michael et al. 1996). Clinically asymptomatic infected individuals have hidden pathology such as renal and lymphatic abnormalities (Dreyer et al. 1992; Freedman et al. 1994), and early detection and treatment of infected individuals might prevent the pathology and appearance of clinical manifestations. Rapid, sensitive and specific diagnostic tests are also required for monitoring and evaluating intervention measures. The current method of diagnosis based on a finger prick thick blood smear test for microfilaria has low sensitivity, is costly and not well accepted by either community or programme personnel (Das et al. 1995; Weil et al. 1997). Therefore, there is an urgent need to develop and validate alternative diagnostic tests which could be done on finger prick blood samples during the day and provide accurate information to permit treatment immediately. There have been several attempts to detect active infection using specific filarial antigen (Paranjape et al. 1986; More & Copeman 1990) and antibody tests (Dissanyake et al. 1994; Chandrashekar et al. 1994). Parasite-specific DNA identification by PCR techniques has also been attempted (Abbasi et al. 1996; Williams et al. 1996). A commercial ELISA test kit for detection of W. bancrofti antigen, using a monoclonal antibody, Og4C3, has also become available. Although the Og4C3 test could detect antigens in day blood samples collected on filter paper (Itoh et al. 1998), none of the tests developed so far could provide on-the-spot results. The ICT test kit (ICT Diagnostics, Balgowlah, Australia), is claimed to be specific, sensitive and instant (Weil et al. 1997). A multicentre, multicountry study was conducted under the direction of WHO/TDR to evaluate its specificity and sensitivity in detecting W. bancrofti (the major filarial parasite globally) in endemic areas of the world. This paper presents the results of the study s Indian component Blackwell Science Ltd 359

2 Materials and methods The study included 89 microfilaria carriers, 100 amicrofilaraemic persons from an endemic locality and 189 amicrofilaraemic individuals from a nonendemic locality. Chennakunam village in Villupuram District of Tamil Nadu State and Goundanplayam Locality of Pondicherry Union Territory comprised the endemic study sites (microfilaria prevalences of 11% and 10%, respectively, in 1997, VCRC unpublished data). The tribal villages of Kollimalai and Ithalar in Uthagamandalam District, Tamil Nadu State, located 1000 m above sea level in the Neelagiri hills, were the nonendemic study sites. By oral questioning we ascertained that the individuals from the nonendemic study site had not visited or lived in endemic localities before. Before the collection of blood samples, participants from both endemic and nonendemic areas were examined and found to have no clinical symptoms of filariasis or any other chronic illness such as tuberculosis, leprosy, diabetes and hypertension. Between 2000 h and 2200 h we collected 1 ml of venous blood in a heparin coated tube and 60 l of finger prick thick blood smear on a slide from each individual recruited for the study. The blood smears were stained with JSB stain using standard procedures (Sasa 1976) and the venous blood samples were processed by membrane filtration (Chularerk & Desowitz 1970). The stained smears and the membranes were examined for microfilaria (mf) and mf were counted and recorded. The ICT card test was carried out on the the next day between 1000 h and 1600 h. Briefly, 100 l of capillary blood was collected from an individual by finger prick method, applied to the test card immediately and the reading was taken following manufacturer s guidelines. A single observer, who had no knowledge about the infection status of the individual (microfilaria results), read all cards. Similarly, the technician examining the blood smear had no knowledge about the ICT card test results. The ICT cards, preserved at room temperature, were reread by three independent observers from different organizations within 6 months of the original reading. Of 378 cards tested, 371 were available for this exercise to study interobserver variation. All microfilaraemic or ICT card-positive individuals were treated with DEC 6 mg/kg/day for 12 days. Results Night blood parasitaemia 189 individuals from endemic localities who were asymptomatic for filariasis were tested for night blood parasitaemia both by finger prick thick smear method and venous blood membrane filtration technique. By thick smear examination technique, 65 were mf-positive and 124 mf-negative (Table 1). All individuals diagnosed positive by this technique were also positive by membrane filtration technique. Parasite density ranged between 1 and 1060/60 l of blood. By the membrane filtration technique, 89 persons were mf-positive and 100 mfnegative (Table 2), and parasite density ranged between 1 and 1000/ml. All 189 individuals from nonendemic localities were mf-negative both by finger prick thick smear method and membrane filtration technique and were also asymptomatic for filariasis. Day blood antigenaemia by the ICT card test Of the 65 mf carriers detected as positive by thick smear examination, the ICT card test identified 64 as positive ICT test results Groups No. Positive (%) No. negative (%) Total Table 1 Microfilaria status as determined by 60 l finger prick thick blood smear examination and ICT card test Microfilaria-positive 64 (98.5) 001 (1.5) 065 Microfilaria-negative (endemic site) 17 (13.7) 107 (86.3) 124 Microfilaria-negative (nonendemic site) 00 (0.0) 189 (100.0) 189 ICT card test Groups No. positive (%) No. negative (%) Total Table 2 Microfilaria status as determined by membrane filtration technique and the ICT card test Microfilaria positive 64 (71.9) 025 (28.1) 089 Microfilaria-negative (endemic sites) 17 (17.0) 083 (83.0) 100 Microfilaria-negative (nonendemic site) 00 (0.0) 189 (100.0) Blackwell Science Ltd

3 Table 3 Results of ICT test in relation to microfilarial count determined by the membrane filtration Mf count No. No. ICT No. ICT (membrane filtration) tested positive (%) negative (%) (31.8) 15 (68.2) (37.5) 05 (62.5) (100.0) 00 (0.0) (100.0) 00 (0.0) (88.9) 01 (11.1) (84.6) 02 (15.4) (85.7) 02 (14.3) (100.0) 00 (0.0) Total (71.9) 25 (28.1) Table 4 Sensitivity, specificity, positive and negative predictive value of the ICT card test compared to thick smear examination and membrane filtration technique Thick smear examination Sensitivity 098.5% 071.9% Specificity 100.0% 100.0% Positive predictive value 100.0% 100.0% Negative predictive value 099.5% 088.3% Membrane filtration technique value in comparison with membrane filtration was only 88.3% (Table 4). (98.5%), and of 124 mf-negative individuals by the thick smear examination, 17 (13.7%) (Table 1). 64 of the 89 mf carriers detected by the membrane filtration technique tested positive with the ICT card test. The relationship between density of microfilaraemia by membrane filtration technique and positivity in the ICT card test is described in Table 3. The ICT card test failed to detect 20 of the 30 (67%) mf carriers whose microfilaraemic count was 10/ml. Seventeen of 100 individuals diagnosed as mf-negative by membrane filtration technique were positive by the ICT card test. All 189 mfnegative individuals from nonendemic localities were negative by the day blood ICT card test. Sensitivity and specificity The sensitivity, specificity, negative and positive predictive values obtained by the ICT card test compared to nonendemic normals and microfilaria carriers in comparison with the night blood parasite tests were calculated using the EPIINFO 6 software. Compared to the finger prick thick smear examination, the sensitivity and specificity of the ICT card test were 98.5% and 100%, respectively, and against the membrane filtration test, 71.9% and 100% (Table 4). The positive and negative predictive values of the ICT card test in comparison with finger prick thick smear examination were 100% and 99.5%, respectively, while the negative predictive Interobserver variation To study interobserver variation, results of the original card readings were compared with those of three independent observers. The overall agreement was 87% (330/371): 93% for mf carriers, 83% for amicrofilaraemic endemic individuals and 88% for amicrofilaraemic nonendemic individuals. Sensitivity, specificity, positive and negative predictive values in the ICT card test, as obtained by the original reading and independent observers, are presented in Table 5. The results suggest that overall there is good agreement between observers. Discussion Assessment of the filariasis burden in a community is a prerequisite for planning, implementation and evaluation of control strategies (Pani et al. 1997). We found the performance of the ICT card test to be comparable to that of the conventional finger prick night blood thick smear examination. It has several advantages over the conventional technique in that it can be performed on the whole blood sample collected by finger prick at daytime and that results are instant. Interobserver variation was minimal even after storage of the cards for up to 6 months. Therefore, the test will be acceptable to both community and programme personnel for screening bancroftian microfilaria carriers. Table 5 Comparison of sensitivity, specificity, positive and negative predictive values of ICT test card between original readings and those of three independent observers Sensitivity Specificity Positive predictive Negative predictive ICT card readers (%) (%) value (%) value (%) Original reading Independent observer Independent observer Independent observer Blackwell Science Ltd 361

4 The ICT card test diagnosed several amicrofilaraemic individuals as positives. Possibly the test detects antigens of other stages of W. bancrofti also, such as L4, L5 or single sex adult worms. The test is based on (Weil et al. 1996) and detects adult parasite antigen (Itoh et al. 1998). Dreyer et al. (1996) reported that a significant proportion of amicrofilaraemic individuals had live adult worms in their scrotal area when examined by the ultrasound technique. However, the test was found to have lower sensitivity than membrane filtration in this study. A significant proportion of microfilaraemics, especially those with low-level parasitaemia detected by the membrane filtration technique, were ICTnegative. These results differ from earlier ones, where the sensitivity was estimated to be more than 90% in endemic regions of other countries (Weil et al. 1997). The reasons for these variations are unclear. In the reported studies, the test was carried out on serum, whereas we used whole blood. Multi-centre studies in different parts of India must validate this observation. Large-scale field use of the ICT card test in control programmes in endemic countries will require assessment of its costs and suitability. The cost of a conventional night thick blood examination is US $0.5 (Rs 20.6) inclusive of overhead cost (personnel, canvassing, blood collection and examination, data compilation and logistics including vehicles and fuel, etc.). The cost of an ICT card alone is US $1.05 (Rs 45), and US $1.29 (Rs 54) inclusive of shipping expenses (US $120 for 500 tests). However, for larger orders, as for national programmes, shipping cost would be less per test. The card needs to be stored at 2 C to 8 C until use, which may not be feasible in many rural areas. To make the ICT card test a practical field tool to screen populations in endemic countries, it needs to be more sensitive, suitable for storage at tropical ambient weather conditions and cheaper. If these problems are overcome, the technique will find wider applications. Acknowledgements This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Disease (TDR). We are grateful to Dr P.K. Das, Director of the Vector Control Research Centre, for his critical review of the study s design and results, and thank Dr Q.B. Saxena, Deputy Director General, ICMR, New Delhi; Dr P.R. Narayanan, Director, Tuberculosis Research Centre, Madras and Dr N.B.L. Saxena, Deputy Director, National Malaria Eradication Programme, New Delhi for reading of the ICT cards as independent observers. We acknowledge the help of Dr N. Balakrishnan, Deputy Assistant Director, National Institute of Communicable Diseases, Coonoor, for testing the ICT cards in a nonendemic area. References Abbasi I, Hamburger J, Githure J et al. (1996) Detection of Wuchereria bancrofti DNA in patient sputum by the polymerase chain reaction. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, Chandrashekar R, Curtis KC, Ramzy RM, Liftis F, Li BW & Weil GJ (1994) Molecular cloning of Brugia malayi antigens for diagnosis of lymphatic filariasis. Molecular and Biochemical Parasitology 64, Chularerk P & Desowitz RS (1970) A simplified membrane filtration technique for diagnosis of microfilaraemia. Journal of Parasitogy 56, Das PK, Manoharan A, Ramaiah KD, Balarajan K & Dhanda V (1995) Cost analysis of blood surveys for the detection of microfilaria carriers in rural areas. National Medical Journal of India 8, Dissanyake S, Zheng H, Dreyer G et al. 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