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Visceral and cutaneous leishmaniasis is caused by the protozoan parasite Leishmania. In the Mediterranean region, L. infantum is responsible for visceral and cutaneous leishmaniasis and it uses hematophagous phlebotomine sand flies as vectors and dogs as reservoir hosts.
Traditionally, pentavalent antimonial drugs are used for treatment. However, relapses are common in patients infected with human immunodeficiency virus (HIV), but rare in patients not known to be immunosuppressed if given incomplete treatment.1 In the majority of relapses up to now, the same Leishmania strain was isolated before and after treatment months or years after the first episode2. However, in two HIV-positive patients from Catalonia, Spain with repeated clinical episodes, a second Leishmania strain was isolated 20 and 29 months after the first treatment.3
In relapses, a second drug is usually administered because of the possibility that the parasite has developed resistance to the first drug.4 In such cases, we do not know whether the elimination of clinical symptoms is due to the new drug, which eliminated the now resistant parasite, or to the fact that the original infection was caused by more than one strain of Leishmania, each with different drug sensitivity. In the case of an HIV-negative patient, we report for the first time that a mixed infection by two L. infantum strains was responsible for therapeutic failure.
A Leishmania isolate (MHOM/GR/2001/GH6), was obtained from the bone marrow of a 64-year-old man with visceral leishmaniasis and identified as L. infantum zymodeme MON-98 by starch gel electrophoresis using 15 enzymatic systems according the method of Rioux and others.5 The patient was treated with meglumine antimoniate (20 mg/kg of body weight for 30 days, 2 courses with a 15-day interval). Because symptoms persisted 20 days after the first isolation of the parasite, a second isolation was attempted. Strain (MHOM/GR/2001/GH8) was isolated from the bone marrow and identified as L. infantum zymodeme MON-1. After 75 days, and no improvement in clinical signs, liposomal amphotericin B was administered (total dose = 1.5 grams) and this produced a cure. The patient was informed of all actions taken for the isolation of the parasite and he consented. Four years later, the patient remains free of leishmaniasis.
Both clinical and experimental evidence point to the conclusion that visceral leishmaniasis in this patient was due to a mixed infection. The two Leishmania strains isolated from the patient 20 days apart exhibited different behavior in vitro. First, MON-98 produced a positive culture in both NNN and RPMI 1640 culture media three days after inoculation of a bone marrow sample, while MON-1 required 26 days in the same culture media to grow. In the first isolation, MON-98 was the only strain detected by isoenzyme analysis since MON-1 did not have time to grow to detectable numbers after two weeks of culture. Second and most importantly, the two strains showed different sensitivity to meglumine antimoniate in vitro. Using stationaryphase promastigotes from the established Leishmania cultures and host cells of the human cell line THP-16 at a host cell to parasite ratio of 1:6, we estimated that MON-98 required a 50% effective dose (the concentration of meglumine antimoniate at which 50% of the Leishmania parasites survived [ED50]4) of 13.0 µg/mL, while MON-1 required 279.2 µg/mL. These results were consistent with the clinical findings: MON-98 was eliminated in 20 days by meglumine antimoniate, while MON-1 did not respond even after 60 days of treatment with this drug. For this reason, MON-98 was not detected in the second isolation with isoenzyme analysis, while MON-1 was not detected in the first isolation since it was overgrown by MON-98.
It is unlikely that reinfection took place in this patient because the patient remained in the hospital (Red Cross Hospital, Athens, Greece) with persisting symptoms of leishmaniasis throughout the treatment period and did not recover until liposomal amphotericin B was administered.
While zymodeme MON-1 is found predominantly in the Mediterranean Basin and is the only zymodeme reported in Greece in humans, dogs, and sand flies, zymodeme MON-98 is rare and this is the first time it has been reported from Greece. The zymodeme MON-98 has been found in Egypt (isolated from a sand fly,7 dogs,8 and a human with visceral leishmaniasis9) and in Portugal (isolated from a dog10).
In the case of visceral leishmaniasis, it is important to consider that relapses in dogs given treatment are very common. Stray dogs usually live in packs in areas where sand fly colonies develop. This is the case in the outskirts of Athens, an endemic area for leishmaniasis,11 where our patient lives. Such dogs are exposed to a large numbers of sand fly bites, which increases the possibility of infection by different strains of the parasite. Moreover, mixed infections of the same macrophage by different species of Leishmania are possible, as shown by experimental results,12 and this fact may lead to the generation of genetic diversity within natural Leishmania populations.13,14 The fact that different L. infantum isolates with the same zymodeme profile have different ED504 values could be a result of such genetic exchange. This matter becomes of great concern when we consider that a multidrug resistance gene is present in Leishmania.15 Therefore, mixed infections by different Leishmania strains could be a frequent phenomenon. They may explain differences in the clinical course of leishmaniasis in many patients and may be the reason for treatment failures.
Received November 12, 2003. Accepted for publication March 16, 2004.
Acknowledgment: We thank Ippokratis Messaritakis for technical assistance.
Financial support: This work is part of a project supported by the European Commission Research Directorates (Contract no. QLK-CT-2001-01810).
Authors’ addresses: Maria Antoniou and Y. Tselentis, Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, PO Box 1393, Heraklion, Crete 71409, Greece, Telephone: 30-2810-394-746, Fax: 30-2810-394-740, E-mails: antoniou{at}med.uoc.gr and tselendi{at}med.uoc.gr C. Doulgerakis: Department of Nephrology, Red Cross Hospital, Athens, Greece, Telephone: 30-22610 20051, Fax: 30-22610-24753. F. Pratlong and J. P. Dedet, Laboratoire de Parasitologie and Centre National de Référence des Leishmania, Centre Hospitalier Universitaire de Montpellier, 163 Rue Auguste Broussonet, 34090 Montpellier, France. Telephone: 33-4-67-63-27-51, Fax: 33-4-67-63-00-49, E-mail: parasito{at}univ-montp1.fr.
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