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TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1994) 88, 658-659

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)

Pierre Marty, Alain Lelievre, Jean-Franqois Quaranta, Amer Rahal, Martine Gari-Toussaint and Yves Le Fichoux Parasitologic-Mycologic, Fact& de Midecine, Giver& de Nice Sophia-Antipolis, Avenue de Valombrose, 06107 Nice Cedex 2, France

Abstract Fifty unselected subjects living in Alpes-Maritimes, France, a high risk area for visceral leishmaniasis due to Leishmania infantum, were examined simultaneously by the leishmanin skin test and the Western blot tech- nique in 1993; 32% and 38%, respectively, gave a positive reaction. The concordance of the 2 methods was 82%. Thus, in this high risk area, a large proportion of inhabitants had been exposed to the parasite. The use of these 2 tests should permit the detection of potential cases of reactivated leishmaniasis in prospective follow-up investigations. -

Introduction The Alpes-Maritimes, in the south of France, is an en-

demic focus of canine leishmaniasis. In some villages, 20% of domestic dogs are seropositive (MARTY et al., 1988). Human cases have increased from 3 diagnosed m the year 1985 to 15 diagnosed in 1992 among a popula- tion of one million (MARTY et al., 1994).

Castagniers is a village in a rural area at an altitude of 350 m, behind the city of Nice. In January 1993,24% of 92 dogs tested were seropositive for Leishmania infantum antigens by enzyme-linked immunosorbent and indirect immunofluorescence assays.

The leishmanin skin test is well known to detect asymptomatic cases (PAMPIGLIONE et al., 1975). More re- cently, Western blot analysis has been proposed for epi- demiological surveys (MARY et al., 1992).

In this study, we examined 50 unselected subjects sim- ultaneously by means of the skin test and the Western blot technique.

Patients and Methods The mean age of the 50 unselected persons tested was

50 years (range 11-88); 56% were female. All the sub- jects were inhabitants of the village of Castagniers, in southern France; the work was done in 1993.

Leishmanin skin test The leishmanin was provided by the World Health Or-

ganization Leishmaniasis Reference Centre in Rome, Italy (Laboratorio di Parassitologia, Istituto Superiore di SanitB). It was a suspension of 5 X lo6 promastigotes of L. infantum (zymodeme MON-1) in 0.5% phenol-saline; 0.1 mL was injected intradermally in the deltoid area using an automatic syringe (Dermojet@). Skin reaction was as- sessed 2 d later by the ball-point pen method (SOKAL, 1975). If the average of 2 perpendicular diameters of the induration was 5 mm or more, the test was considered positive. An erythematous response alone was recorded as negative.

Western blotting The blood specimen for Western blotting was taken on

the same day that the skin test was performed. Sera were separated and tested according to the method described by MARY et al. (1992), with a few modifications.

The antigen used was a strain of L. infantum (zy- modeme MON-l), provided by the Laboratoire de Para- sitologie-Mycologic of the Faculte de Medecine in Mar- seille, France. Promastigotes were solubilized and denatured in Tris buffer (0.5 M, pH 6.8), containing suc- rose, P-mercaptoethanol and sodium dodecyl sulphate, by heating at 100°C for 4 min; 200 pg of antigen proteins were used for each ‘minigel’.

A 14% polyacrylamide ‘minigel’ was used as separating gel, with Tris-glycine buffer (pH 8.3) and Tris-HCl buffer (pH 8.8). A 4% stacking gel (pH 6.8) was poly-

merized on top of the separating gel. Molecular wei ht markers (BioRad@) were used in a separate track. A ter f electrophoresis for about 2 h at 150 V, the antigens were electrotransferred from the gel to a nitrocellulose mem- brane (0.2 pm pore size) using Towbin’s buffer (Tris- glycine, pH 8.3, with 20% methanol). After transfer., the membrane was dried on filter paper and cut into strips 3 mm wide. The strips were saturated with phosphate-buf- fered saline (PBS) containing 1% non-fat dried milk for 15 min on a shaker and incubated for 2 h on a shaker with 50 PL of the serum to be tested, diluted in 500 PL of PBS plus 1% non-fat dried milk. After several washes in PBS, they were incubated with anti-human immuno- globulin G peroxidase conjugate (Sigma@) diluted l/1000 in the same buffer. After several more washes, anti-L. in- fantum antibodies were revealed by addition of the appro- priate substrate (diaminobenzidine+COU+Hz02 in PBS), which generated an insoluble dark grey product.

The test was considered positive when the presence of antibodies directed against the 14 and/or 16 kDa antigens of L. infantum were detected (MARY et al., 1992). Table. Comparison of the leishmanin skin test and the Western blot technique for epidemiological studies of visceral leishmaniasis due to L. infanturn in southern France

Skin test + Total

Western blot” z ‘33 Total 16

6 ::

2 50

‘Western blot was considered positive if antibodies toward 14 and/or 16 kDa antigens were detected.

Results Leishmanin skin test

32% (16150) of the sera gave a positive result.

Western blotting 38% (19150) of the sera had antibodies against the 14 or

16 kDa antigens; 20% (10/50) had antibodies against both antigens, 8% (4150) against the 14 kDa antigen only, and 10% (5150) against the 16 kDa antigen only.

Concordance of the two techniques Concordance of the 2 methods was assessed by the

ratio No. of sera oositive bv both tests + no. negative bv both tests

Total no. of sera tested

A comparison of the results of the 2 techniques is shown in the Table. The concordance was

13+** = 829/ 50 cl

No subject whose sera reacted with both antigens had a negative skin test.

Discussion The leishmanin skin test is an important tool in epi-

demiological studies of visceral leishmaniasis. It has been used to assess transmission of the parasite, particularly in the foci of Mediterranean visceral leishmaniasis and espe- cially in Italy (PAMPIGLIONE et al., 1975; BETTINI et al., 1983; GRAMICCIA et al., 1990). In the surroundings of Nice, in a highly endemic focus of canine leishmaniasis, 30% of the population gave a positive test (MARTY et al., 1992), similar to the results obtained in the present study. The leishmanin skin test allows one to estimate the extent of contact with the parasite and to detect cryp- tic, asymptomatic infections (PAMPIGLIONE et al., 1975). This test has been proposed to assess cell mediated im- munity .

The Western blot procedure, recently described by MARY et al. (1992), is another valuable tool for epidemio- logical studies. MARY et al. (1992) found antibodies against the 14 and/or 16 kDa antigens in 65% of asymp- tomatic patients living in endemic areas, compared with none of the asymptomatic subjects living in non-endemic areas. This test detects humoral immunity.

The excellent concordance (82%) between the skin test and the Western blot technique suggests that these tests should be able to detect potential cases of reactivated leishmaniasis. Complementary studies are necessary to understand the reasons for this concordance.

In developing countries, the Western blot procedure may be difficult to carry out and the skin test ma

P be ea-

sier in practice. However, the simultaneous use o these 2 tests will be valuable in the study of immunological status of populations in prospective follow-up investiga- tions.

Acknowledgements We thank Dr M. Gramiccia (Laboratorio di Parassitologia,

Istituto Superiore di SanitB, Rome, Italy) for giving us the leish- manin and Dr C. Mary (Laboratoire de Parasitologie-Myco- logie, FacultC de Medecine, Marseille, France) for supplying the antigen used in Western blot analysis.

659

This project was supported by the Groupe d’Action Contre la Leishmaniose. an association of oeoole living in the endemic area in Alpes-harihmes.

. .

References Bettini, S., Gramiccia, M., Gradoni, L., Pozio, E., Mignai, S.

& Maroli, M. (1983). Leishmaniasis in Tuscany (Italy): VII. Human population response in the focus of Monre Argentario (Grosseto) and epidemiological evaluation. Annales de Parasit- ologie Humaine et Cornparke, s&539-547.

Gramiccia, M., Bettini, S., Gradoni, L., Ciarmoli, P., Verelli, M. L., Loddo, S. & Cicalb, C. (1990). Leishmaniasis in Sardinia. 5. Leishmanin reaction in the human population of a focus of low endemicity of canine leishmaniasis. Transactions of the Royal Sociery of Tropical Medicine and Hygiene, 84, 371- 374.

Marty, P., Ozon, C., Jambou, R., Bayada, M., Haas, P. & Le Fichoux, Y. (1988). Nouvelles enquCtes prospectives sur la leishmaniose canine dans le departement des Alpes-Ma- ritimes. Bulletin de la Sock% Franfaise de Parasitologic, 6, 3-6.

Marty, I’., Le Fichoux, Y., Giordana, D. & Brugnetti, A. (1992). Leishmanin reaction in the human population of a highly endemic focus of canine leishmaniasis in Alpes-Ma- ritimes, France. Transactions of the Royal Society of Tropical Medicine and Hygiene, 86,249-250.

Marty, I’., Le Fichoux, Y., Pratlong, F. & Gari-Toussaint, M. (1994). Human visceral leishmaniasis in Alpes-Maritimes (France): epidemiological characteristics for the period 1985- 1992. Transactions of the Royal Society of Tropical Medicine and Hygiene, 88,33-34.

Mary, C., Lamouroux, D., Dunan, S. & Quilici, M. (1992). Western blot analysis of antibodies to Leishmania infanrum antigens: potential of the 14-kD and 16-kD antigens for diag- nosis and epidemiologic purposes. American Journal of Tropi- cal Medicine and Hygiene, 47,76&77 1.

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 azar. Transactions of the Royal Society of Tropical Medicine and Hy- giene, 69,60-68.

Sokal, J. E. (1975). Measurement of delayed skin-test re- sponses. New EnglandJournal ofMedicine, 293,501-502.

Received 21 February 1994; revised 6 April 1994; accepted for publication 6 Apn.11994

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