690

TRANSACTIONS OF THE ROYAL SOCIETY OF TR~PKAL MEUICINE AND HYGIENE (1995) 89,690-691

Detection by Western blot of four antigens characterizing acute clinical leishmaniasis due to Leishmania infanturn

Pierre Marty, Alain Leli&re, Jean-Ftangois Quaranta, Isabelle Suffla, Maria Eulalio, Martine Gari-Toussaint, Yves Le Fichoux and Joanna Kubar Groupe de Recherche en Immunopathologie de la Leishmaniose, Fact& de Mkdecim, Universitk de Nice-Sophia Antipolis, Avenue de Valombrose, 06107 Nice Cedex 2, France

Abstract Western blot analysis of sera from 32 patients with acute clinical leishmaniasis due to Leishmania infanturn showed the simultaneous presence of antibodies against 4 antigens with molecular masses of 18, 2 1, 23, 3 1 kDa. The simultaneous presence of these 4 antigens was specific to the clinical disease and it was not de- tected in 47 sera from asymptomatic individuals living in the leishmaniasis endemic area of Alpes-Maritimes (southern France) or in 37 sera from patients with other protozoan infections.

Keywords: leishmaniasis, Leishmania infanturn, characteristic antigens, France

Introduction Results Western blot analysis has been used in the study of

sera from patients infected with Leishmania infantum in southern France by MARY et al. (1992) and MARTY et al. (1994). In this paper we report a comparison of the West- ern blot patterns of sera from patients with clinical cuta- neous or visceral leishmaniasis due to L. infantum with sera from asymptomatic individuals living in an endemic area (Alpes-Maritimes) in France who never had clinical leishmaniasis but some of whom had a positive leish- manin skin test, and from other groups of subjects.

Materials and Methods Blood samples were obtained from 4 groups of sub-

jects: (i) 47 asymptomatic subjects living in an area of Alpes-Maritimes, France, endemic for L. infantum; 22 had a positive leishmanin skin test (6 females and 16 males, mean age 54 years, range 20-85) and 25 had a neg- ative test (15 females, 10 males, mean age 40 years, range 14-74); (ii) 32 patients (11 females and 21 males, mean age 30 years, range l-86), 29 with visceral leishmaniasis and 3 with cutaneous leishmaniasis, living in the same area (diagnosis was by detection of amastigotes in bone marrow or skin; cases of human immunodeficiency virusleishmania co-infections were excluded); (iii) sera from 37 patients with other protozoan infections-recent malaria (7), recent toxoplasmosis (7), hepatic amoebiasis (5), African trypanosomiasis (11 patients from the Congo Republic), and Chagas disease (7 patients from a region in Bolivia free of leishmaniasis); and (iv) sera from 21 asymptomatic African subjects living in an area free of leishmaniasis and trypanosomiasis in Bafang, Cameroon.

The sera of patients with symptomatic leishmaniasis, cutaneous or visceral, reacted with many antigens, giving multiple bands (Figure., B). Some bands were not res- ent in all sera or were dlffcult to differentiate. We t K ere- fore selected 4 bands which were well differentiated and cross reacted less with other groups of sera and which were all present in all the 32 sera from clinical leishma- niasis cases. These bands have molecular masses of 18, 21, 23 and 31 kDa. None of the sera from asymptomatic subjects reacted with all 4 of those antigens, although some sera from the latter group had antibodies against 1 or, rarely, 2 of the antigens, but never against 3 or 4 (Figure, A).

Some sera from patients infected with other protozoa had 1, or sometimes 2, bands corresponding to those antigens, but never more than 2. Only m sera from cases of Chagas disease was the presence of 2 bands rather fre- quent.

The band most often detected by asymptomatic sub- jects living in endemic areas was that of 18 kDa*, with which 22 sera reacted (Table).

Table. Reactivity of leishmaniasis patients, asymptomatic subjects living in areas endemic for leishmaniasis, patients with other protozoan infections, and control subjects towards the 18.21.23 and 31 kDa antigens of Leishmania infantum

Subiects

The technique used has been described by MARTY et al. (1994). The promastigotes used were derived from cultures of a strain of L. infanturn, zymodeme MON-1 (MHOM/FR/gl/LPN 5).

“;ggg?$g$4g

Positiye 22: 20 pwt,, 2 tl 2 ;

Clinical leishmaniasis iz Sf 3:: 3s 320 Ot$nrfecnons

Toxoplasmosis Hepatic amoebiasis African tfypanosomiasis 11 C&gas disease

Control subjectsa

KDa

97,4 +

68,2 e

21,5 t

B KDa KDa

-23 - 21

(Hw) s-r+ (uw) PATIENTS

Figure. Western blot patterns of (A) asymptomatic skin test-positive sub- jects living in an endemic area for leishmaniasis and (B) clinical leishma- niasis patients. The lanes labelled (MW) are molecular mass markers.

NO. Antigens

(molecular mass in kDa) tested 18 21 23 31

aAsymptomatic residents of Bafang, Cameroon-an area free of trypanosomiasis and leishmaniasis.

Almost all the sera of asymptomatic subjects which re- acted with the 18 kDa antigen were derived from sub- jects with a positive skin test. This is consistent with pre- vious work showing a good concordance between skin test positivity and the presence of the 18 and/or 14 kDa bands (MARTY et al., 1994), possibly related to asympto- matic infection or contact with the Darasite (PAMPI- GLIONE et al., 1975).

The 18 kDa antigen was present in all the 7 Chagas disease sera tested, but in only one of the African trypan-

‘The 18 kDa antigen is probably the same as that reported by MARY et al. (1992) as having a molecular mass of 16 kDa.

691

osomiasis sera. Another band, 14 kDa, was present in all the Chagas disease sera and in most clmical leishmaniasis sera (but not all of them, so it was not taken into account in this study). This 14 kDa band has also been reported by MARY et al. (1992).

Conclusion We consider that the simultaneous presence of the 18,

21, 23 and 31 kDa bands is sufficient to define clinical letshmaniasis in this endemic area. In current practice, when a patient presents few specific symptoms of leish- maniasis, serological tests are done before trying to find the parasite in bone marrow or skin. This Western blot technique, easy to perform, may prove useful in diag- nosis and may enable better selection of patients for bio- PSY*

The nature of these antigenic fractions has yet to be defined.

Acknowledgements We thank Dr J.L. Lemesre (ORSTOM, Montpellier, France)

for the sera from patients with Chagas disease and Dr B. Bou-

1 Announcements1

teille (Faculte de M&lecine, Limoges, France) for the sera from patients with African trypanosomiasis.

References Many, P., Lelievre, A., Quaranta, J.-F., Bahal, A., Gari-Tous-

saint, M. & Le Fichoux, Y. (1994). Use of the leishmanin skin test and Western blot analysis for epidemiological studies in visceral leishmaniasis areas: experience in a highly endemic focus in Alpes-Maritimes (France). Transactions of the Royal Socie

Mary, 8 of Tropical Medicine and Hygiene, 88,658-659.

Lamouroux, D., Dunan, S. & Quihci, M. (1992). Weste; blot analysis of antibodies to Leishmania infanturn antigens: potential of the 14 kD and the 16 kD antigens for diagnosis and epidemiologic purposes. American 3ournal of Tropical Medicine and Hygiene, 47, X4-771.

Pampiglione, S., Manson-Bahr, P. E. C., La Placa, M., Bor- gatti, M. A. & Musumeci, S. (1975). Studies in Mediter- ranean leishmaniasis 3. The leishmanin skin test in kala-axar. Transactions of the Royal Society of Tropical Medicine and Hy- giene, 69,60-G.

Received 15 December 1994; revised 12 April 1995; accepted for publication 25 May 1995

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