Teste de aglutinação direta no sorodiagnóstico da leishmaniose visceral no estado do Pará

Garcez, Lourdes M.; Shaw, Jeffrey J.; Silveira, Fernando T.

doi:10.1590/S0037-86821996000200009

Resumos

Avaliou-se o teste de aglutinação direta (TAD) no sorodiagnóstico da leishmaniose visceral (LV) humana e de canídeos (cão e raposa Cerdocyon thous), sendo os resultados comparados com aqueles obtidos em imunofluorescência indireta (IFI) e ensaio imunoenzimático (ELISA). Utilizaram-se soros: humanos (303): indivíduos com LV confirmada (16), suspeitos de LV (65), em outras condições (102), controles negativos (15), indivíduos em área endêmica (105): de cães (82): de área endêmica (68), Salvaterra/Marajó/PA. (21 parasitologicamente positivos), e controles negativos (14), de Belém; de raposas (9): espécimes capturados na Ilha do Marajó. Antígenos para o TAD foram preparados a partir de promastigotas de Leishmania (Leishmania) donovani e L. (L.) chagasi. Aqueles utilizados em ELISA e IFI, respectivamente, de promastigotas (antígeno solúvel) e amastigotas deL. (L.) chagasi. Em humanos, a especificidade e sensibilidade do TAD, utilizando-se antígeno deL. (L.) donovani, foram altas (98,4% e 100%, respectivamente) e comparáveis às de IFI (97,5% e 100%). ELISA foi menos específico (84,8%), embora igualmente sensível. Em cães, o TAD foi mais específico com antígeno de L. (L.) donovani do que com aquele preparado a partir de L. (L.) chagasi. Entretanto, ELISA e TAD foram menos sensíveis (ambos 71,4%) que IFI (100%). Essa diferença foi reflelida nos resultados de cães de área endêmica, 87% dos quais foram positivos para IFI, mas somente 54% para ELISA e 49% para o TAD. Resultados similares foram observados com raposas, quando todos os 9 soros foram positivos para IFI, 7 de 9 (78% ) positivos para ELISA, enquanto que o TAD não revelou nenhum resultado positivo. Concluiu-se que o TAD, usando antígeno de L. (L.) donovani é um teste útil para LV humana; sua utilização em ampla escala é indicada, desde que monitorada por um laboratório de referência que garanta qualidade dos antígenos. Contudo, não é a melhor opção em inquéritos sorológicos caninos, já que foi menos específico que ELISA e, especialmente, IFI, na detecção de anticorpos em casos infecção canina.

Teste de aglutinação direta; Leishmaniose visceral; Humanos; Canídeos

The direct agglutination test (DAT) was evaluated for serodiagnosis of visceral leishmaniasis (VL) in human and canids (dogs and foxes Cerdocyon thous). The results were compared with those of the imnunofluorescent antibody assay (IFAT) and enzyme-linked immunosorbent assay (ELISA). The sera used were from: humans (303): confirmed VL (16), suspected VL (65), other conditions (102), negative controls (15) and individuals from an endemic area (105); dogs (82): from an endemic area (68), Salvaterra/Marajó/Pará (21 of which were parasitologically positive), and negative controls (14), from Belém;foxes (9): caught on Marajó Island. Antigens for DAT were prepared from promastigots of L. (L.) donovani, L. (L.) chagasi. Antigens used in ELISA and IFAT were prepared from promastigotes (soluble antigen) and amastigotes respectively of L. (L.) chagasi. In humans, the specificity and sensitivity of DAT using L. (L.) donovani were high (98.4% and 100% respectively) and comparable to that of IFAT (97.5% and 100%). ELISA was less specific (84.8%) although similarly sensitive (100%). In dogs, DAT was more specific using L. (L.) donovani as antigen than using L. (L.) chagasi. However, both DA Tand ELISA were less sensitive (both 71.4%) than IFAT (100%). This difference was reflected in the results from endemic dogs, 87% of which were positive by IFAT but only 54% by ELISA and 49% by DAT. Similarly, all 9 fox sera were positive by IFAT, 7 of 9 (78%) by ELISA but none by DAT. In conclusion, DAT using L. (L.) donovani antigen can provide a useful test for human VL; utilization on a large scale would be possible with a suitable reference laboratory to monitor antigen quality. However, DAT appears less useful for canine studies, as it was less sensitive than ELISA and especially IFAT in detecting canine infection.

Direct agglutination test; Visceral leishmaniasis; Humans; Canids

ARTIGO

Teste de aglutinação direta no sorodiagnóstico da leishmaniose visceral no estado do Pará

Lourdes M. Garcez; Jeffrey J.Shaw; Fernando T. Silveira

Belém Research Projects e Instituto Evandro Chagas, Fundação Nacional de Saúde, Ministério da Saúde, Belém, PA

^{Endereço para correspondência} Endereço para correspondência: Dra. Lourdes M. Garcez.

RESUMO

Avaliou-se o teste de aglutinação direta (TAD) no sorodiagnóstico da leishmaniose visceral (LV) humana e de canídeos (cão e raposa Cerdocyon thous), sendo os resultados comparados com aqueles obtidos em imunofluorescência indireta (IFI) e ensaio imunoenzimático (ELISA). Utilizaram-se soros: humanos (303): indivíduos com LV confirmada (16), suspeitos de LV (65), em outras condições (102), controles negativos (15), indivíduos em área endêmica (105): de cães (82): de área endêmica (68), Salvaterra/Marajó/PA. (21 parasitologicamente positivos), e controles negativos (14), de Belém; de raposas (9): espécimes capturados na Ilha do Marajó. Antígenos para o TAD foram preparados a partir de promastigotas de Leishmania (Leishmania) donovani e L. (L.) chagasi. Aqueles utilizados em ELISA e IFI, respectivamente, de promastigotas (antígeno solúvel) e amastigotas deL. (L.) chagasi. Em humanos, a especificidade e sensibilidade do TAD, utilizando-se antígeno deL. (L.) donovani, foram altas (98,4% e 100%, respectivamente) e comparáveis às de IFI (97,5% e 100%). ELISA foi menos específico (84,8%), embora igualmente sensível. Em cães, o TAD foi mais específico com antígeno de L. (L.) donovani do que com aquele preparado a partir de L. (L.) chagasi. Entretanto, ELISA e TAD foram menos sensíveis (ambos 71,4%) que IFI (100%). Essa diferença foi reflelida nos resultados de cães de área endêmica, 87% dos quais foram positivos para IFI, mas somente 54% para ELISA e 49% para o TAD. Resultados similares foram observados com raposas, quando todos os 9 soros foram positivos para IFI, 7 de 9 (78% ) positivos para ELISA, enquanto que o TAD não revelou nenhum resultado positivo. Concluiu-se que o TAD, usando antígeno de L. (L.) donovani é um teste útil para LV humana; sua utilização em ampla escala é indicada, desde que monitorada por um laboratório de referência que garanta qualidade dos antígenos. Contudo, não é a melhor opção em inquéritos sorológicos caninos, já que foi menos específico que ELISA e, especialmente, IFI, na detecção de anticorpos em casos infecção canina.

Palavras-chaves: Teste de aglutinação direta. Leishmaniose visceral. Humanos. Canídeos.

SUMMARY

The direct agglutination test (DAT) was evaluated for serodiagnosis of visceral leishmaniasis (VL) in human and canids (dogs and foxes Cerdocyon thous). The results were compared with those of the imnunofluorescent antibody assay (IFAT) and enzyme-linked immunosorbent assay (ELISA). The sera used were from: humans (303): confirmed VL (16), suspected VL (65), other conditions (102), negative controls (15) and individuals from an endemic area (105); dogs (82): from an endemic area (68), Salvaterra/Marajó/Pará (21 of which were parasitologically positive), and negative controls (14), from Belém;foxes (9): caught on Marajó Island. Antigens for DAT were prepared from promastigots of L. (L.) donovani, L. (L.) chagasi. Antigens used in ELISA and IFAT were prepared from promastigotes (soluble antigen) and amastigotes respectively of L. (L.) chagasi. In humans, the specificity and sensitivity of DAT using L. (L.) donovani were high (98.4% and 100% respectively) and comparable to that of IFAT (97.5% and 100%). ELISA was less specific (84.8%) although similarly sensitive (100%). In dogs, DAT was more specific using L. (L.) donovani as antigen than using L. (L.) chagasi. However, both DA Tand ELISA were less sensitive (both 71.4%) than IFAT (100%). This difference was reflected in the results from endemic dogs, 87% of which were positive by IFAT but only 54% by ELISA and 49% by DAT. Similarly, all 9 fox sera were positive by IFAT, 7 of 9 (78%) by ELISA but none by DAT. In conclusion, DAT using L. (L.) donovani antigen can provide a useful test for human VL; utilization on a large scale would be possible with a suitable reference laboratory to monitor antigen quality. However, DAT appears less useful for canine studies, as it was less sensitive than ELISA and especially IFAT in detecting canine infection.

key-words: Direct agglutination test. Visceral leishmaniasis. Humans. Canids.

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REFERÊNCIAS BIBLIOGRÁFICAS

1. Andrade CR, Silva OA, Andrade PP, Kolk AHJ, Harith AE. A direct agglutination test discriminative toward Chagas' desease for serodiagnosis of visceral leishmaniasis in Brazil: preliminary results. Annal Institut Pasteur/ Immunology 138:457-459, 1987.

2. Andrade CR, Nascimento AE, Moura PMMF, Andrade PP. Leishmania donovani donovani e Leishmania donovani chagasi as antigens in the direct agglutination assay for the diagnosis of kala-azar. Brazilian Journal of Medical Biology Research 22:611-615, 1989.

3. Badaró R, Reed SG, Carvalho EM. Immunofluorescent antibody teste in American visceral leishmaniasis: sensitivity and especificity of different morfological forms of two Leishmania species. The American Journal of Tropical Medicine and Hygiene 32:480-484, 1983.

4. Celeste BJ, Guimarães MC. Growth curves of Leishmania braziliensis braziliensis promastigotes and surface antigen expression before and after adaptation to Schneider's drosophila medium as assessed by anti-Leishmania human sera. Revista do Instituto de Medicina Tropical de São Paulo 30:63-67, 1988.

5. Chowdhury S, Haque F, Al-masum AE, Karim E. Positive response to sodium antimony gluconate administration in visceral leishmaniasis seropositive patients. The American Journal of Tropical Medicine and Hygiene 44:390-393, 1991.

6. El Safi SH. Evans DA. A comparison of the direct agglutination test and enzyme-linked immunosorbent assay in the sero-diagnosis of leishmaniasis in the Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene 83:334-337, 1989.

7. Guimarães MCS, Celeste BJ, Castilho EA, Mineo J, Diniz JMP. Immunoenzimatic assay (ELISA) in mucocutaneous leishmaniasis, kala-azar and Chagas disease. An epimastigote Trypanosoma cruzi antigen able to distinguish between anti-Trypanosoma and anti-Leishmania antibodies. The American Journal of Tropical Medicine and Hygiene 30:942-947, 1981.

8. Guimarães MCS, Coutinho SG, Antunes CMF. Normas para sorologia de moléstias parasitárias. Revista da Sociedade Brasileira de Medicina da Sociedade Brasileira de Medicina Tropical 20:55-58, 1987.

9. Hailu A. Pre and post-treatment antibody levels in visceral leishmaniasis. Transactions of the Royal Society ofTropical Medicine and Hygiene 84:673-675, 1990.

10. Harith AE, Kolk AH, Kager PA, Leeuwenburg I. Muigai R, Kiugu S, Laarman JJ. A simple and economical direct agglutination test for serodiagnosis and sero- epidemiological studies of visceral leishmaniasis. Transactions of the Royal Society ofTropical Medicine and Hygiene 80:583-587, 1986.

11. Harith AE, Kolk AH, Kager PA, Leeuwenburg J. Faber FJ, Muigai R, Kiugu S, Laarman JJ. Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA. Transactions of the Royal Society of Tropical Medicine and Hygiene 81:603- 606, 1987.

12. Harith AE, Kolk AHJ, Leeuewenburg J. Muigai R, Huigen E, Jelsma T, Kager PA. Improvemente of a direct agglutination test for field studies of visceral leishmaniasis. Journal Clinical Microbiology 26:1321-1325, 1988.

13 Harith AE, Splappendel RJ, Reiter 1, Knapen F, Korte P. Huigen E, Kolk AHJ. Application of a direct agglutination test for detection of especific anti-Leishmania antibodies in canine reservior. Journal Clinical Microbiology 27:2252- 2257, 1989.

14. Jahn A, Diesfield HJ. Evaluation of a visually read ELISA for serodiagnosis and sero-epidemiological studies of kala-azar in the Beringo District, Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 77:451-454, 1983.

15. Korte PM, Harith AE, Dereure J, Huigen E, Faucherre V, Kaay HJ. Introduction of an improved direct agglutination test (DAT) is assessed for the detection of Leishmania infantum infection in southern France. Parasitology Research 76:526-530, 1990.

16. Lemestre JL, Rizvi FS, Afehain D, Sadigursky M, Capron A, Santoro F. Subspecies-specifes surface antigens of promastigotes of the Leishmania donovani complex. Infect. Immunol. 50:136-141, 1985. apud Andrade CR, Silva OA, Andrade PP, Kolk AHJ, Harith AE. A direct agglutination test discriminative toward Chagas disease for the diagnoses of visceral leishmaniasis in Brazil: preliminary results. Annal Institut Pasteur/Immunology 138:457-459.

17. Mengistu G. Kiessling R, Akuffo H. The value of a direct agglutination test in the diagnoses of cutaneous and visceral leishmaniasis. Transactions o f the Royal Society ofTropical Medicine and Hygiene 84:359-362, 1990.

18. Mengistu G, Akuffo H, Fehniger TE, Negese Y, Nilsen R. Comparison of parasitological and immunological methods in the diagnosis of leishmaniasis in Ethiopia. Transactions of the Royal Society of Tropical Medicine and Hygiene 86:154-157, 1992.

19. Neogy AB, VouldoukisI, Silva OA, TselenSis Y, Lascomhe JC, Thierry S, Rzerpa D, Monjour L. 1992. Serodiagnosis and screening of canine visceral leishmaniasis in a endemic area of Corsica: applicability of a direct agglutination test and immunoblot analysis. The American Journal of Tropical Medicine and Hygiene 47:772-777.

20. Pappas MG, McGreevy Hajkowski R, Hendricks LD, Oster CN, Hockmeyer WT. Evaluation of promastigote and amastigote antigens in the indirect fluorescent antibody test for American cutaneous leishmaniasis. The American Journal o f Tropical Medicine and Hygiene 32:1260-1267, 1983.

21. Shaw JJ, Ishkawa EAY, Lainson R. A rapid and sensitive method for the identification of Leishmania with monoclonal antibodies using fluorescein-labelled avidin. Transactions of the Royal Society o f Tropical Medicine and Hygiene 83:783-784, 1989.

22. Shaw JJ, Lainson R, McMahon PD, David JR. Serodemes of the Leishmania braziliensis complex. Leishmania. Taxonomie et phylogenese. Applications eco- epidemiologiques. (Coll. int. CNRS/INSERM, 1984). IMEEE, Montpellier p. 179-183, 1986.

23. Voller A, Bartlett A, Bidwell DE. Enzyme immunoassays with especial reference to ELISA techniques. Reprinted from the from the Journal Clinical Pathology 31:507, 1978.

24. Voller A, Bidwell DE, Bartlett A. Enzyme immunoassay in diagnostic medicine. Bulletin of the World Health Organization 53:55-65, 1976.

26. Zijlstra EE, Siddig Ali M, El-Hassan AM, Eltoum IA, Satti M, Ghalib HW, Kager PA. Direct agglutination test for diagnosis and serological and sero-epidemiological survey of kala-azar in the Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene 85:474-476, 1991.

Recebido para publicação em 08/01/96.

1. Andrade CR, Silva OA, Andrade PP, Kolk AHJ, Harith AE. A direct agglutination test discriminative toward Chagas' desease for serodiagnosis of visceral leishmaniasis in Brazil: preliminary results. Annal Institut Pasteur/ Immunology 138:457-459, 1987.
2. Andrade CR, Nascimento AE, Moura PMMF, Andrade PP. Leishmania donovani donovani e Leishmania donovani chagasi as antigens in the direct agglutination assay for the diagnosis of kala-azar. Brazilian Journal of Medical Biology Research 22:611-615, 1989.
3. Badaró R, Reed SG, Carvalho EM. Immunofluorescent antibody teste in American visceral leishmaniasis: sensitivity and especificity of different morfological forms of two Leishmania species. The American Journal of Tropical Medicine and Hygiene 32:480-484, 1983.
4. Celeste BJ, Guimarães MC. Growth curves of Leishmania braziliensis braziliensis promastigotes and surface antigen expression before and after adaptation to Schneider's drosophila medium as assessed by anti-Leishmania human sera. Revista do Instituto de Medicina Tropical de São Paulo 30:63-67, 1988.
5. Chowdhury S, Haque F, Al-masum AE, Karim E. Positive response to sodium antimony gluconate administration in visceral leishmaniasis seropositive patients. The American Journal of Tropical Medicine and Hygiene 44:390-393, 1991.
6. El Safi SH. Evans DA. A comparison of the direct agglutination test and enzyme-linked immunosorbent assay in the sero-diagnosis of leishmaniasis in the Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene 83:334-337, 1989.
7. Guimarães MCS, Celeste BJ, Castilho EA, Mineo J, Diniz JMP. Immunoenzimatic assay (ELISA) in mucocutaneous leishmaniasis, kala-azar and Chagas disease. An epimastigote Trypanosoma cruzi antigen able to distinguish between anti-Trypanosoma and anti-Leishmania antibodies. The American Journal of Tropical Medicine and Hygiene 30:942-947, 1981.
9. Hailu A. Pre and post-treatment antibody levels in visceral leishmaniasis. Transactions of the Royal Society ofTropical Medicine and Hygiene 84:673-675, 1990.
10. Harith AE, Kolk AH, Kager PA, Leeuwenburg I. Muigai R, Kiugu S, Laarman JJ. A simple and economical direct agglutination test for serodiagnosis and sero- epidemiological studies of visceral leishmaniasis. Transactions of the Royal Society ofTropical Medicine and Hygiene 80:583-587, 1986.
11. Harith AE, Kolk AH, Kager PA, Leeuwenburg J. Faber FJ, Muigai R, Kiugu S, Laarman JJ. Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA. Transactions of the Royal Society of Tropical Medicine and Hygiene 81:603- 606, 1987.
12. Harith AE, Kolk AHJ, Leeuewenburg J. Muigai R, Huigen E, Jelsma T, Kager PA. Improvemente of a direct agglutination test for field studies of visceral leishmaniasis. Journal Clinical Microbiology 26:1321-1325, 1988.
13 Harith AE, Splappendel RJ, Reiter 1, Knapen F, Korte P. Huigen E, Kolk AHJ. Application of a direct agglutination test for detection of especific anti-Leishmania antibodies in canine reservior. Journal Clinical Microbiology 27:2252- 2257, 1989.
14. Jahn A, Diesfield HJ. Evaluation of a visually read ELISA for serodiagnosis and sero-epidemiological studies of kala-azar in the Beringo District, Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 77:451-454, 1983.
15. Korte PM, Harith AE, Dereure J, Huigen E, Faucherre V, Kaay HJ. Introduction of an improved direct agglutination test (DAT) is assessed for the detection of Leishmania infantum infection in southern France. Parasitology Research 76:526-530, 1990.
16. Lemestre JL, Rizvi FS, Afehain D, Sadigursky M, Capron A, Santoro F. Subspecies-specifes surface antigens of promastigotes of the Leishmania donovani complex. Infect. Immunol. 50:136-141, 1985. apud Andrade CR, Silva OA, Andrade PP, Kolk AHJ, Harith AE. A direct agglutination test discriminative toward Chagas disease for the diagnoses of visceral leishmaniasis in Brazil: preliminary results. Annal Institut Pasteur/Immunology 138:457-459.
17. Mengistu G. Kiessling R, Akuffo H. The value of a direct agglutination test in the diagnoses of cutaneous and visceral leishmaniasis. Transactions o f the Royal Society ofTropical Medicine and Hygiene 84:359-362, 1990.
18. Mengistu G, Akuffo H, Fehniger TE, Negese Y, Nilsen R. Comparison of parasitological and immunological methods in the diagnosis of leishmaniasis in Ethiopia. Transactions of the Royal Society of Tropical Medicine and Hygiene 86:154-157, 1992.
20. Pappas MG, McGreevy Hajkowski R, Hendricks LD, Oster CN, Hockmeyer WT. Evaluation of promastigote and amastigote antigens in the indirect fluorescent antibody test for American cutaneous leishmaniasis. The American Journal o f Tropical Medicine and Hygiene 32:1260-1267, 1983.
21. Shaw JJ, Ishkawa EAY, Lainson R. A rapid and sensitive method for the identification of Leishmania with monoclonal antibodies using fluorescein-labelled avidin. Transactions of the Royal Society o f Tropical Medicine and Hygiene 83:783-784, 1989.
22. Shaw JJ, Lainson R, McMahon PD, David JR. Serodemes of the Leishmania braziliensis complex. Leishmania. Taxonomie et phylogenese. Applications eco- epidemiologiques. (Coll. int. CNRS/INSERM, 1984). IMEEE, Montpellier p. 179-183, 1986.
23. Voller A, Bartlett A, Bidwell DE. Enzyme immunoassays with especial reference to ELISA techniques. Reprinted from the from the Journal Clinical Pathology 31:507, 1978.
24. Voller A, Bidwell DE, Bartlett A. Enzyme immunoassay in diagnostic medicine. Bulletin of the World Health Organization 53:55-65, 1976.
26. Zijlstra EE, Siddig Ali M, El-Hassan AM, Eltoum IA, Satti M, Ghalib HW, Kager PA. Direct agglutination test for diagnosis and serological and sero-epidemiological survey of kala-azar in the Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene 85:474-476, 1991.

Endereço para correspondência:

Dra. Lourdes M. Garcez.

Datas de Publicação

Publicação nesta coleção
09 Abr 2013
Data do Fascículo
Abr 1996

Histórico

Aceito
08 Jan 1996
Recebido
08 Jan 1996

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. Andrade CR, Silva OA, Andrade PP, Kolk AHJ, Harith AE. A direct agglutination test discriminative toward Chagas' desease for serodiagnosis of visceral leishmaniasis in Brazil: preliminary results. Annal Institut Pasteur/ Immunology 138:457-459, 1987.

[2] 2. Andrade CR, Nascimento AE, Moura PMMF, Andrade PP. Leishmania donovani donovani e Leishmania donovani chagasi as antigens in the direct agglutination assay for the diagnosis of kala-azar. Brazilian Journal of Medical Biology Research 22:611-615, 1989.

[3] 3. Badaró R, Reed SG, Carvalho EM. Immunofluorescent antibody teste in American visceral leishmaniasis: sensitivity and especificity of different morfological forms of two Leishmania species. The American Journal of Tropical Medicine and Hygiene 32:480-484, 1983.

[4] 4. Celeste BJ, Guimarães MC. Growth curves of Leishmania braziliensis braziliensis promastigotes and surface antigen expression before and after adaptation to Schneider's drosophila medium as assessed by anti-Leishmania human sera. Revista do Instituto de Medicina Tropical de São Paulo 30:63-67, 1988.

[5] 5. Chowdhury S, Haque F, Al-masum AE, Karim E. Positive response to sodium antimony gluconate administration in visceral leishmaniasis seropositive patients. The American Journal of Tropical Medicine and Hygiene 44:390-393, 1991.

[6] 6. El Safi SH. Evans DA. A comparison of the direct agglutination test and enzyme-linked immunosorbent assay in the sero-diagnosis of leishmaniasis in the Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene 83:334-337, 1989.

[7] 7. Guimarães MCS, Celeste BJ, Castilho EA, Mineo J, Diniz JMP. Immunoenzimatic assay (ELISA) in mucocutaneous leishmaniasis, kala-azar and Chagas disease. An epimastigote Trypanosoma cruzi antigen able to distinguish between anti-Trypanosoma and anti-Leishmania antibodies. The American Journal of Tropical Medicine and Hygiene 30:942-947, 1981.

[9] 9. Hailu A. Pre and post-treatment antibody levels in visceral leishmaniasis. Transactions of the Royal Society ofTropical Medicine and Hygiene 84:673-675, 1990.

[10] 10. Harith AE, Kolk AH, Kager PA, Leeuwenburg I. Muigai R, Kiugu S, Laarman JJ. A simple and economical direct agglutination test for serodiagnosis and sero- epidemiological studies of visceral leishmaniasis. Transactions of the Royal Society ofTropical Medicine and Hygiene 80:583-587, 1986.

[11] 11. Harith AE, Kolk AH, Kager PA, Leeuwenburg J. Faber FJ, Muigai R, Kiugu S, Laarman JJ. Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA. Transactions of the Royal Society of Tropical Medicine and Hygiene 81:603- 606, 1987.

[12] 12. Harith AE, Kolk AHJ, Leeuewenburg J. Muigai R, Huigen E, Jelsma T, Kager PA. Improvemente of a direct agglutination test for field studies of visceral leishmaniasis. Journal Clinical Microbiology 26:1321-1325, 1988.

[13] 13 Harith AE, Splappendel RJ, Reiter 1, Knapen F, Korte P. Huigen E, Kolk AHJ. Application of a direct agglutination test for detection of especific anti-Leishmania antibodies in canine reservior. Journal Clinical Microbiology 27:2252- 2257, 1989.

[14] 14. Jahn A, Diesfield HJ. Evaluation of a visually read ELISA for serodiagnosis and sero-epidemiological studies of kala-azar in the Beringo District, Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 77:451-454, 1983.

[15] 15. Korte PM, Harith AE, Dereure J, Huigen E, Faucherre V, Kaay HJ. Introduction of an improved direct agglutination test (DAT) is assessed for the detection of Leishmania infantum infection in southern France. Parasitology Research 76:526-530, 1990.

[16] 16. Lemestre JL, Rizvi FS, Afehain D, Sadigursky M, Capron A, Santoro F. Subspecies-specifes surface antigens of promastigotes of the Leishmania donovani complex. Infect. Immunol. 50:136-141, 1985. apud Andrade CR, Silva OA, Andrade PP, Kolk AHJ, Harith AE. A direct agglutination test discriminative toward Chagas disease for the diagnoses of visceral leishmaniasis in Brazil: preliminary results. Annal Institut Pasteur/Immunology 138:457-459.

[17] 17. Mengistu G. Kiessling R, Akuffo H. The value of a direct agglutination test in the diagnoses of cutaneous and visceral leishmaniasis. Transactions o f the Royal Society ofTropical Medicine and Hygiene 84:359-362, 1990.

[18] 18. Mengistu G, Akuffo H, Fehniger TE, Negese Y, Nilsen R. Comparison of parasitological and immunological methods in the diagnosis of leishmaniasis in Ethiopia. Transactions of the Royal Society of Tropical Medicine and Hygiene 86:154-157, 1992.

[20] 20. Pappas MG, McGreevy Hajkowski R, Hendricks LD, Oster CN, Hockmeyer WT. Evaluation of promastigote and amastigote antigens in the indirect fluorescent antibody test for American cutaneous leishmaniasis. The American Journal o f Tropical Medicine and Hygiene 32:1260-1267, 1983.

[21] 21. Shaw JJ, Ishkawa EAY, Lainson R. A rapid and sensitive method for the identification of Leishmania with monoclonal antibodies using fluorescein-labelled avidin. Transactions of the Royal Society o f Tropical Medicine and Hygiene 83:783-784, 1989.

[22] 22. Shaw JJ, Lainson R, McMahon PD, David JR. Serodemes of the Leishmania braziliensis complex. Leishmania. Taxonomie et phylogenese. Applications eco- epidemiologiques. (Coll. int. CNRS/INSERM, 1984). IMEEE, Montpellier p. 179-183, 1986.

[23] 23. Voller A, Bartlett A, Bidwell DE. Enzyme immunoassays with especial reference to ELISA techniques. Reprinted from the from the Journal Clinical Pathology 31:507, 1978.

[24] 24. Voller A, Bidwell DE, Bartlett A. Enzyme immunoassay in diagnostic medicine. Bulletin of the World Health Organization 53:55-65, 1976.

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