Iso-enzymatic variability of Leishmania infantum in Spain

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Annals of Tropical Medicine & Parasitology, Vol. 97, Supplement No. 1, S57–S64 (2003)

Iso-enzymatic variability of Leishmania infantum in Spain C. CHICHARRO, M. I. JIME´NEZ and J. ALVAR WHO Collaborating Centre for Leishmaniasis, Servicio de Parasitologõ´a, Centro Nacional de Microbiologõ´a, Instituto de Salud Carlos III, Carretera Majadahonda–Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain Received and accepted 30 May 2003

In many areas of the Mediterranean basin, leishmaniasis can now be found in HIV-positive individuals. Such cases of Leishmania/HIV co-infection are relatively common in southern Europe, Spain being the country that has reported the greatest number. Since 1984, 359 Spanish isolates of Leishmania infantum have been characterized at the Instituto de Salud Carlos III in Madrid. Most (94.6%) of the isolates came from HIV-positive patients. The results of iso-enzymatic analysis indicated a high level of variability among the isolates, the visceralization in HIVpositive individuals of variants considered to be dermotropic in the immunocompetent, and the appearance of new zymodemes among the HIV-positive human population.

Leishmania infantum, which may cause either cutaneous leishmaniasis (CL) or visceral leishmaniasis (VL) in humans living around the Mediterranean, has been shown to be an important opportunistic parasite in HIVpositive patients (Alvar, 1994; Dereure et al., 1995). As with other Leishmania species causing human disease, the variability of L. infantum has been studied by iso-enzyme analysis. Such analysis, and the subsequent identiŽ cation of distinct zymodemes, constitutes an extremely useful taxonomic tool that has already contributed much to the epidemiology of leishmaniasis (Le Blancq, 1986). In immunocompetent patients, certain zymodemes of L. infantum (MON-27, MON-28, MON-72, MON-77 and MON-187) only cause VL, whereas others cause only CL (MON-11, MON-24, MON-29, MON-33, MON-78 and MON-111) or may cause either VL or CL (MON-1, MON-34, MON80 and MON-190). The MON-1 zymodeme normally predominates, causing about 90% Reprint requests to: J. Alvar. E-mail: [email protected]; fax: +34 91 5097034. © 2003 The Liverpool School of Tropical Medicine DOI: 10.1179/000349803225002534

of the VL cases and 20% of the CL cases attributed to L. infantum infection in HIVnegatives. Some other zymodemes, such as MON-27, MON-72, MON-111, MON187 and MON-189, are relatively rare in immunocompetent patients and have not yet been found in any HIV-positive individuals (Alvar et al., 1997). Curiously, the L. infantum zymodemes commonly found in those co-infected with HIV may be found rarely, if at all, in the immunocompetent, and parasites of the same zymodeme may show diVerent tropism according to the HIV status of their host. MON-1 is only responsible for 50% of VL cases among HIV-positives, for example, and MON-24, MON-28, MON-29, MON-33, MON-34, MON-77 and MON-78 are more frequently isolated from HIVpositives with VL than from HIV-negatives with this form of leishmaniasis. Several zymodemes (MON-136, MON-183, MON185, MON-188, MON-198, MON-199 and MON-201) have been found only in HIV-positive individuals. Co-infected patients frequently have both viscerotropic and dermotropic zymodemes of L. infantum in their

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bone marrow ( Jime´nez et al., 1991; Marty et al., 1991; Gramiccia et al., 1992; Campino et al., 1994). The main aim of the present report was to summarize the results of investigations of the L. infantum strains, isolated in Spain from both HIV-positive and HIV-negative patients, carried out in the Instituto de Salud Carlos III, in Madrid, since 1984. MATERIALS AND METHODS Leishmania Stocks The 358 isolates investigated were collected from VL and CL cases in various areas of Spain between 1984 and 2001. Most of the isolates had been recovered from NNN cultures of bone-marrow or blood (Table 1). Two isolates had been made from each of 127 of the patients with VL/HIV, either one before and one after a post-treatment relapse (43 cases) or two at the same time (the concurrent bone-marrow and blood cultures for each of 84 cases both being positive). To give suYcient material for the iso-enzyme analysis, each isolate was mass cultured in RPMI 1640 medium supplemented with 10% foetal calf serum. Iso-enzyme Characterization Electrophoresis was carried out in starch gel (Rioux et al., 1990), using a panel of 13 enzymes (covering 15 enzymatic loci):

TABLE 1. The organs or tissues cultured in the isolation of the Leishmania infantum investigated Organ or tissue Bone marrow Blood Skin Spleen Liver Lymph node Ascitic  uid Tongue Unknown Any

No. and (%) of isolates 245 69 24 3 2 2 1 1 11 358

(68.4) (19.3) (6.7) (0.8) (0.6) (0.6) (0.3) (0.3) (3.1)

malate dehydrogenase (MDH; EC 1.1.1.37), malic enzyme (ME; EC 1.1.1.40), glucose6-phosphate dehydrogenase (G6PD; EC 1.1.1.49), 6-phosphogluconate dehydrogenase (6PGD; EC 1.1.1.44), isocitrate dehydrogenase (ICD; EC 1.1.1.42), nucleoside purine phosphorylase (NP1 and NP2; EC 2.4.2.1), phosphoglucomutase (PGM; EC 2.7.5.1), diaphorase (DIA; EC 1.6.2.2), glutamate– oxalacetate transaminase (GOT1 and GOT2; EC 2.6.1.1), mannose phosphate isomerase (MPI; EC 5.3.1.8), glucose-phosphate isomerase (GPI; EC 5.3.19), fumarate hydratase (FH; EC 4.2.1.2) and glutamate dehydrogenase (GLUD; EC 1.4.1.3). Four reference strains of L. infantum, representing the MON-1 (MHOM/FR/78/LEM-75), MON-29 (MHOM/ES/81/LEM-307), MON24 (MHOM/DZ/82/LIPA-59) and MON-183 zymodemes (MHOM/FR/91/LEM-2298), were run in parallel with the fresh isolates from Spain. RESULTS Characterized Strains Almost all (339) of the isolates investigated came from 212 HIV-positive patients, the other 19 (5.4%) been collected from 19 immunocompetent subjects. The predominance of HIV-negative donors re ects the incidence of VL in Spain, most cases of the disease now being associated with HIV infection. Since 1999, the annual number of L. infantum isolates characterized in Madrid has been falling, largely, it appears, as the result of the introduction of highly active antiretroviral therapy (HAART) in 1997 (Fig. 1). Enzymatic Characterization Leishmania infantum from immunocompetent patients Of the 19 isolates from HIV-negative patients investigated, 15 (79%) came from cases of VL and four (21%) from cases of CL. The 19 isolates belonged to seven zymodemes

ISO-ENZYMES OF SPANISH Leishmania infantum

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FIG. 1. The temporal changes in the numbers of Leishmania infantum isolates characterized at the Instituto de Salud Carlos III, in Madrid, between 1984 and 2001.

(Table 2) including one, MON-285, that had never being detected before in humans, dogs or sand ies. Leishmania infantum from HIV-infected patients When the 339 L. infantum isolates from 212 HIV-positive patients were analysed they were found to belong to 20 diVerent zymodemes (Figure 2 and Table 3). The TABLE 2. The zymodemes of the Leishmania infantum isolates from immunocompetent patients with visceral leishmaniasis ( VL ) or cutaneous leishmaniasis (CL ) Zymodeme

No. of isolates

MON-1 MON-24 MON-34 MON-105 MON-253 MON-285 GR13* Any

8 4 2 1 2 1 1 19

Clinical feature VL and CL VL and CL CL VL VL VL VL

*According to the nomenclature adopted by Martõ´nSa´nchez et al. (1995).

TABLE 3. The zymodemes of the Leishmania infantum isolates from HIV-positive patients, all of whom had visceral leishmaniasis Zymodeme MON-1 MON-24 MON-27 MON-28 MON-29 MON-33 MON-34 MON-80 MON-108 MON-183 MON-185 MON-190 MON-198 MON-199 MON-228 MON-253 MON-282 MON-283 MON-284 GR13* Any

No. and (%) of isolates 152 63 3 6 15 8 36 2 2 19 1 1 1 1 2 12 2 2 1 9 339

(44.8) (18.6) (0.9) (1.8) (4.4) (2.4) (10.6) (0.6) (0.6) (5.6) (0.3) (0.3) (0.3) (0.3) (0.6) (3.5) (0.6) (0.6) (0.3) (2.7)

*According to the nomenclature adopted by Martõ´nSa´nchez et al. (1995).

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FIG. 2. The numbers of HIV-positive cases found infected with the various zymodemes of Leishmania infantum in Spain.

most common zymodeme encountered was MON-1, responsible for 44.8% of the HIVinfected cases. Although the 20 zymodemes detected included variants that are dermatropic in immunocompetent individuals, all 212 cases of Leishmania/HIV co-infection investigated had VL. The post-treatment isolate collected from each of 43 HIVpositive cases who had leishmanial relapses was found to be of the same zymodeme as the corresponding pre-treatment sample: MON-1 (20 cases), MON-24 (10), MON-34 (Ž ve), MON-183 (two), MON-253 (two), MON-29 (one), MON-108 (one), MON228 (one) or MON-283 (one). new zymodemes of Leishmania infantum Six of the L. infantum zymodemes detected, since 1984, during the present study had never been observed before, in humans, dogs or sand ies (Table 4). One of these,

TABLE 4. The electrophoretic mobilities (ME ) of some iso-enzyme markers of the ‘new’ zymodemes, relative to those of Leishmania infantum zymodeme MON-1 Iso-enzyme* Zymodeme

MDH

G6PD

NP1

GPI

FH

MON-198 MON-199 MON-282 MON-283 MON-284 MON-285

104 104 100 104 104 104

105 102 100 100 100 105

140 140 100 130 100 100

115 105 100 105 105 115

100 100 105 100 100 100

*All six zymodemes had M of 100 for the other 10 isoE enzymatic loci studied.

MON-285, came from a HIV-negative case in 1996 — an 8-year-old Andalucian girl. The other Ž ve — MON-198, MON-199, MON-282, MON-283 and MON-284 — were from HIV-positive cases. Although the six new zymodemes each had iso-enzymatic

ISO-ENZYMES OF SPANISH Leishmania infantum

proŽ les similar to those of the zymodemes responsible for CL in immunocompetent patients (MDH ; G6PD ; NP1 ; > 100 104 105 FH ), all were isolated from bone> 100 marrow aspirates of individuals with VL. Geographical Distribution The geographical distribution of L. infantum is not homogeneous in Spain, although transmission of the parasite probably occurs in all regions except the north–west and the Canary Islands (Figure 3 and Table 5); the single cases of leishmaniasis detected in Galicia (MON-24) and the Canary Islands (MON-1) were probably imported, each occurring in individuals who had travelled to other endemic areas of the country. Although more zymodemes (15) have been detected in the Madrid region than elsewhere, this is probably a re ection of

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the in ux of citizens from other areas of the country and/or the large sample size. The level of iso-enzymatic variability is second highest for Andalucia, with 12 zymodemes detected among 52 isolates (Table 5). Of the six ‘new’ zymodemes, three (MON-198, MON-199 and MON-282) were isolated from patients who lived in Madrid, and the other three (MON-283, MON-284 and MON-285) from individuals who resided in Andalucia.

DISCUSSION In the Mediterranean basin, up to 70% of adult cases of VL are co-infected with HIV (Desjeux, 1998). It is therefore perhaps not surprising to Ž nd that the present L. infantum isolates came from 212 HIV-positives with

FIG. 3. Geographical distribution of the Leishmania infantum zymodemes identiŽ ed in Spain, as the result of the isolate characterizations at the Instituto de Salud Carlos III, in Madrid, between 1984 and 2001. Two of the MON-1 cases (*) are thought to have been imported from another region.

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TABLE 5. Geographical distribution of the Leishmania infantum zymodemes detected in Spain Geographical area

Zymodeme

No. of isolates

Andalucia

MON-1 MON-24 MON-29 MON-34 MON-183 MON-185 MON-228 MON-283 MON-284 MON-285 GR-13* MON-1 MON-24 MON-27 MON-29 MON-34 MON-80 MON-1 MON-1 MON-1 MON-24 MON-27 MON-29 MON-34 MON-1 MON-24 MON-105 MON-253 MON-1 MON-28 GR-13* MON-24 MON-1 MON-24 MON-28 MON-29 MON-33 MON-34 MON-80 MON-105 MON-108 MON-183 MON-198 MON-199 MON-253 MON-282 GR13* MON-1 MON-28 MON-29 MON-33 MON-183 MON-190 MON-253 GR-13* MON-1 MON-24 MON-28 MON-29

8 5 1 14 13 1 2 2 1 1 4 49 14 2 2 8 1 1 2 6 4 1 1 1 28 2 1 10 1 1 1 2 55 38 1 3 6 15 1 1 2 5 1 1 3 2 2 6 3 5 2 1 1 1 3 4 2 1 3

Balearic Islands

Canary Islands Castille-Leo´n Castille-La Mancha

Catalonia

Extremadura Galicia Madrid

Valencia

Unknown

*According to the nomenclature adopted by Martõ´nSa´nchez et al. (1995).

VL but only 19 HIV-negatives (15 with VL and four with CL). A tendency to relapse after treatment is one of the features of Leishmania/HIV co-infection and the 43 cases of leishmanial relapse investigated here were all HIV-positive. That, in each of these 43 cases, the zymodeme of the parasites causing the relapse infection appeared the same as that of the parasites in the primary infection indicates that the relapses are caused by recrudescence, not re-infection. New biochemical and molecular tools may help to distinguish between recrudescences and re-infection with more accuracy (Morales et al., 2001, 2002). The results of the iso-enzymatic characterization of L. infantum strains isolated from patients living in Spain ( present study) indicate that the parasites detected in HIV-positives have a greater variability than those detected in HIV-negatives, that zymodemes that are dermotropic in the immunocompetent may cause VL in the immunodeŽ cient, and that zymodemes causing symptomatic leishmaniasis in the HIV-positive do not occur, or occur very rarely, in the immunocompetent. Similar observations have been made before in Spain ( Jime´nez et al., 1995) and in Italy (Gramiccia et al., 1995) and, to a lesser extent, in France, Portugal, Greece and Algeria (Pratlong et al., 1995). There are several reasons why the L. infantum parasites in HIV-positive cases appear particularly diverse. ‘New’ zymodemes in HIV-positive VL cases could be dermotropic and/or non-virulent (and therefore undetected) in the immunocompetent. The tropism of L. infantum variants, for skin or viscera, and their virulence could be more in uenced by the immunological status of the host than by the biochemical characteristics of the variant (Alvar, 1994; Gradoni and Gramiccia, 1994; Jime´nez et al., 1995). In immunocompetent individuals, the low-virulence variants could be eliminated, simplifying the spectrum of zymodemes found in these subjects.

ISO-ENZYMES OF SPANISH Leishmania infantum

The repeated discovery in HIV-positive individuals of zymodemes that have not been seen in immunocompetent humans, dogs or sand ies may be evidence of anthroponotic (human–human) transmission cycles in which those with Leishmania/HIV co-infection serve as reservoir hosts (Alvar et al., 1996). Transmission of L. infantum from the co-infected may be via sand ies or, since intravenousdrug users (IVDU) are the main risk group for HIV infection, via contaminated syringes. Those with Leishmania/HIV have particularly high numbers of amastigotes in their blood (Lo´pez-Ve´lez et al., 1995) and many needles used by IVDU in Spain contain Leishmania DNA (Cruz et al., 2002). In conclusion, although the epidemiological pattern of L. infantum infection may be zoonotic in immunocompetent patients, it may well become anthroponotic among those made immunodeŽ cient by HIV infection.

acknowledgements. This study was partially supported by the Control of Tropical Diseases Section of the World Health Organization (L3/181/42) and Laboratorios Dr. Esteve S. A. ( protocol 97/154). The authors thank the staV of the Laboratoire d’Ecologie Me´dicale, Montpellier, France, for conŽ rming the identity of the six new zymodemes.

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