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A direct agglutination test discriminative toward Chagas' disease for the diagnosis of visceral Leishmaniasis in Brazil: Preliminary results
©
Ann. Inst. PasteurlImmunol. 1987, 138,457-459
ELSEVIER
Paris
1987
PRELIMINARY NOTE
A DIRECT AGGWTINATION TEST DISCRIMINATIVE TOWARD CHAGAS' DISEASE FOR THE DIAGNOSIS OF VISCERAL LEISHMANIASIS IN BRAZIL: PRELIMINARY RESULTS by C.R. Andrade (I), O.A. Silva (2), P.P. Andrade (I), A.H.J. Kolk (3) and A.E. Harith (3) (*) (1) Department of Biophysics and Radiobiology, Federal University of Pernambuco,
Av. Moraes Rego, sin, Recife (Brazil),
(2) Centro de Pesquisas Aggeu Magalhaes (FIOCR UZ), Campus Universitario,
UFPE, A v. Moraes Rego, sin, Recife (Brazil), and
(3) Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam (The Netherlands)
z-o
SUMMARY
Two-hundred and sixty-five human sera from Brazil were tested in a direct agglutination assay using trypsinated Leishmania donovani donovani antigen. The assay proved to be of value for the diagnosis of American visceral leishmaniasis in that it was discriminative toward Chagas' disease. KEY-WORDS:
Diagnosis.
Leishmaniasis, Chagas' disease, Direct agglutination;
INTRODUCTION
The immunological diagnosis of visceral leishmaniasis in Brazil is difficult since, in currently employed assays (IFAT and ELISA), cross-reactions with Chagas' disease, endemic in South America, are frequent. The clinical diagnosis is also rendered difficult when patients harbouring Schistosoma mansoni and suffering from prolonged septicaemic salmonellosis (a common finding in
Submitted November 4, 1986, accepted February 14, 1987. (*) Address for correspondence.
458
C.R. ANDRADE AND COLL.
endemic regions) have clinical signs and symptoms which are indistinguishable from kala-azar. The need for a reliable diagnostic method is imperative for both clinical and epidemiological purposes. The direct agglutination test developed by Harith et af. [1] has shown to be both sensitive and specific for the diagnosis of visceral leishmaniasis in Kenya. We therefore decided to evaluate the reliability of this test in the diagnosis of visceral leishmaniasis in Brazil, where the causative agent is Leishmania donovani chagasi. MATERIALS AND METHODS The antigen for the direct agglutination test was prepared at the Royal Tropical Institute as described by Harith et at. [1]. Briefly, L. donovan; donovan; strain I-S were harvested from RPMI-1640 medium (supplemented with 15 % foetal bovine serum), treated at 37°C with 0.4 010 trypsin for 45 min, fixed for 20 h at 4°C with 1 % formalin and stained with Coomassie brilliant blue. The parasite suspension was filtered through a nylon gauze and the concentration was adjusted to 5 X 107 cells/ml in 1 % formalin. Serial dilutions of the sera in 0.9 % saline with 1 % foetal bovine serum were made up in wells of V-bottom microtitre plates; 50 fLI of antigen suspension were then added to each well. The results were read visually against a white background after 18 h. In total, 265 sera were tested, 14 from kala-azar cases: 10 with parasitologicalpositive bone marrow aspirates and 4 with repeatedly positive strong IFAT results. Other serum samples were from patients with Chagas' disease (73), schistosomiasis (5), filariasis (18), malaria (59), mucocutaneous leishmaniasis (2), hanseniasis (4) and from apparently healthy Brazilian donors (90). RESULTS AND DISCUSSION
Table I is a summary of the results obtained in the direct agglutination test. The titres of the kala-azar sera were ~ 51,200. No cross-reactions were observed with chagasic patients' sera (all below 11400), and all other sera were weakly reactive (~1I400). One serum sample from a mucocutaneous leishmaniasis patient (possibly L. brasiliensis infection) showed a titre of 111,600. The titres found in this study with Brazilian Chagas' sera were remarkably lower than those reported with African trypanosomiasis using the same antigen [1]. Titre levels comparable to those reported with East African leishmaniasis [1] were obtained here, which suggests that the surface antigens recognized by human agglutinating antibodies are similar in L. donovani donovani and L. donovani chagasi. In fact, Lemesre et af. [2] have shown that the surface antigens of these two parasites are very similar. The specificity level obtained suggests that the direct agglutination test developed by Harith et af. [1] can be of diagnostic value for American visceral leishmaniasis, even in areas where Chagas's disease co-exists. A cut-off point of 116,400 is to be considered for differential diagnosis with respect to the mucocutaneous form of the disease. A broader evaluation of the test is currently being undertaken in Brazil.
IMMUNODIFFERENTIATION OF LEISHMANIASIS
459
TABLE 1. - Titre distribution in Brazilian kala-azar patients, healthy controls and patients with other infections.
Reciprocal of titre Sera
< 50 50 100 200 400 800 1,600 3,200 6,400 12,800 25,600 ~ 51,200 Total
Kala-azar
14
Chagas'
9 38
Schistosomiasis
2
2
Filariasis
8
7
2
Malaria
4 26
17
10
9
7
73 5 18
12
59
Mucocutaneous leishmaniasis
2
Hanseniasis Controls
14
72
13
2
4
4
90
RESUME
UN TEST D'AGGLUTINATION DIRECTE POUR LE DIAGNOSTIC DE LA LEISHMANIOSE VISCERALE AU BRESIL, DIFFERENTIEL POUR LA MALADIE DE CHAGAS: RESULTATS PRELIMINAIRES On a examine 265 serums provenant du Bresil a l'aide d'un test d'agglutination directe, en employant comme antigene Leishmania donovani donovani souche 1-8 traitee par la trypsine. L'etude montre qu'on peut utiliser ce test pour Ie diagnostic de la leishmaniose viscerale americaine. Les serums des patients atteints de maladie de Chagas n'entrainent d'agglutination qu'aux dilutions ~ 1/400. MOTS-CLES: Leishmaniose, Maladie de Chagas, Agglutination directe; Diagnostic. REFERENCES [1] HARITH, A.E., KOLK, A.H.J., KAGER, P.A., LEEUWENBURG, J., MUiGAI, R.,
KIUGU, S., KIUGU, S. & LAARMAN, J.J., A simple and economical direct agglutination test for serodiagnosis and sera-epidemiological studies of visceral leishmaniasis. Trans. roy. Soc. trap. Med. Hyg., 1986,80,538-587. [2] LEMESRE, J.L., RIZVI, F.S., AFCHAIN, D., SADIGURSKY, M., CAPRON, A. & SANTORO, F., Subspecies-specific surface antigens of pramastigotes of the Leishmania donovani complex. Infect. Immun., 1985, 50, 136-141.
Ann. Inst. PasteurlImmunol. 1987, 138,457-459
ELSEVIER
Paris
1987
PRELIMINARY NOTE
A DIRECT AGGWTINATION TEST DISCRIMINATIVE TOWARD CHAGAS' DISEASE FOR THE DIAGNOSIS OF VISCERAL LEISHMANIASIS IN BRAZIL: PRELIMINARY RESULTS by C.R. Andrade (I), O.A. Silva (2), P.P. Andrade (I), A.H.J. Kolk (3) and A.E. Harith (3) (*) (1) Department of Biophysics and Radiobiology, Federal University of Pernambuco,
Av. Moraes Rego, sin, Recife (Brazil),
(2) Centro de Pesquisas Aggeu Magalhaes (FIOCR UZ), Campus Universitario,
UFPE, A v. Moraes Rego, sin, Recife (Brazil), and
(3) Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam (The Netherlands)
z-o
SUMMARY
Two-hundred and sixty-five human sera from Brazil were tested in a direct agglutination assay using trypsinated Leishmania donovani donovani antigen. The assay proved to be of value for the diagnosis of American visceral leishmaniasis in that it was discriminative toward Chagas' disease. KEY-WORDS:
Diagnosis.
Leishmaniasis, Chagas' disease, Direct agglutination;
INTRODUCTION
The immunological diagnosis of visceral leishmaniasis in Brazil is difficult since, in currently employed assays (IFAT and ELISA), cross-reactions with Chagas' disease, endemic in South America, are frequent. The clinical diagnosis is also rendered difficult when patients harbouring Schistosoma mansoni and suffering from prolonged septicaemic salmonellosis (a common finding in
Submitted November 4, 1986, accepted February 14, 1987. (*) Address for correspondence.
458
C.R. ANDRADE AND COLL.
endemic regions) have clinical signs and symptoms which are indistinguishable from kala-azar. The need for a reliable diagnostic method is imperative for both clinical and epidemiological purposes. The direct agglutination test developed by Harith et af. [1] has shown to be both sensitive and specific for the diagnosis of visceral leishmaniasis in Kenya. We therefore decided to evaluate the reliability of this test in the diagnosis of visceral leishmaniasis in Brazil, where the causative agent is Leishmania donovani chagasi. MATERIALS AND METHODS The antigen for the direct agglutination test was prepared at the Royal Tropical Institute as described by Harith et at. [1]. Briefly, L. donovan; donovan; strain I-S were harvested from RPMI-1640 medium (supplemented with 15 % foetal bovine serum), treated at 37°C with 0.4 010 trypsin for 45 min, fixed for 20 h at 4°C with 1 % formalin and stained with Coomassie brilliant blue. The parasite suspension was filtered through a nylon gauze and the concentration was adjusted to 5 X 107 cells/ml in 1 % formalin. Serial dilutions of the sera in 0.9 % saline with 1 % foetal bovine serum were made up in wells of V-bottom microtitre plates; 50 fLI of antigen suspension were then added to each well. The results were read visually against a white background after 18 h. In total, 265 sera were tested, 14 from kala-azar cases: 10 with parasitologicalpositive bone marrow aspirates and 4 with repeatedly positive strong IFAT results. Other serum samples were from patients with Chagas' disease (73), schistosomiasis (5), filariasis (18), malaria (59), mucocutaneous leishmaniasis (2), hanseniasis (4) and from apparently healthy Brazilian donors (90). RESULTS AND DISCUSSION
Table I is a summary of the results obtained in the direct agglutination test. The titres of the kala-azar sera were ~ 51,200. No cross-reactions were observed with chagasic patients' sera (all below 11400), and all other sera were weakly reactive (~1I400). One serum sample from a mucocutaneous leishmaniasis patient (possibly L. brasiliensis infection) showed a titre of 111,600. The titres found in this study with Brazilian Chagas' sera were remarkably lower than those reported with African trypanosomiasis using the same antigen [1]. Titre levels comparable to those reported with East African leishmaniasis [1] were obtained here, which suggests that the surface antigens recognized by human agglutinating antibodies are similar in L. donovani donovani and L. donovani chagasi. In fact, Lemesre et af. [2] have shown that the surface antigens of these two parasites are very similar. The specificity level obtained suggests that the direct agglutination test developed by Harith et af. [1] can be of diagnostic value for American visceral leishmaniasis, even in areas where Chagas's disease co-exists. A cut-off point of 116,400 is to be considered for differential diagnosis with respect to the mucocutaneous form of the disease. A broader evaluation of the test is currently being undertaken in Brazil.
IMMUNODIFFERENTIATION OF LEISHMANIASIS
459
TABLE 1. - Titre distribution in Brazilian kala-azar patients, healthy controls and patients with other infections.
Reciprocal of titre Sera
< 50 50 100 200 400 800 1,600 3,200 6,400 12,800 25,600 ~ 51,200 Total
Kala-azar
14
Chagas'
9 38
Schistosomiasis
2
2
Filariasis
8
7
2
Malaria
4 26
17
10
9
7
73 5 18
12
59
Mucocutaneous leishmaniasis
2
Hanseniasis Controls
14
72
13
2
4
4
90
RESUME
UN TEST D'AGGLUTINATION DIRECTE POUR LE DIAGNOSTIC DE LA LEISHMANIOSE VISCERALE AU BRESIL, DIFFERENTIEL POUR LA MALADIE DE CHAGAS: RESULTATS PRELIMINAIRES On a examine 265 serums provenant du Bresil a l'aide d'un test d'agglutination directe, en employant comme antigene Leishmania donovani donovani souche 1-8 traitee par la trypsine. L'etude montre qu'on peut utiliser ce test pour Ie diagnostic de la leishmaniose viscerale americaine. Les serums des patients atteints de maladie de Chagas n'entrainent d'agglutination qu'aux dilutions ~ 1/400. MOTS-CLES: Leishmaniose, Maladie de Chagas, Agglutination directe; Diagnostic. REFERENCES [1] HARITH, A.E., KOLK, A.H.J., KAGER, P.A., LEEUWENBURG, J., MUiGAI, R.,
KIUGU, S., KIUGU, S. & LAARMAN, J.J., A simple and economical direct agglutination test for serodiagnosis and sera-epidemiological studies of visceral leishmaniasis. Trans. roy. Soc. trap. Med. Hyg., 1986,80,538-587. [2] LEMESRE, J.L., RIZVI, F.S., AFCHAIN, D., SADIGURSKY, M., CAPRON, A. & SANTORO, F., Subspecies-specific surface antigens of pramastigotes of the Leishmania donovani complex. Infect. Immun., 1985, 50, 136-141.