BackgroundAmerican cutaneous leishmaniasis (ACL) is a complicated disease producing about 67.000 new cases per year. The severity of the disease depends on the parasite species; however in the vast majority of cases species confirmation is not feasible. WHO suggestion for ACL produced by Leishmania braziliensis, as first line treatment, are pentavalent antimonial derivatives (Glucantime or Sodium Stibogluconate) under systemic administration. According to different authors, pentavalent antimonial derivatives as treatment for ACL show a healing rate of about 75% and reasons for treatment failure are not well known.MethodsIn order to characterise the clinical and parasitological features of patients with ACL that did not respond to Glucantime, a cross-sectional observational study was carried out in a cohort of 43 patients recruited in three of the Colombian Army National reference centers for complicated ACL. Clinical and paraclinical examination, and epidemiological and geographic information were recorded for each patient. Parasitological, histopathological and PCR infection confirmation were performed. Glucantime IC50 and in vitro infectivity for the isolated parasites were estimated.ResultsPredominant infecting Leishmania species corresponds to L. braziliensis (95.4%) and 35% of the parasites isolated showed a significant decrease in in vitro Glucanatime susceptibility associated with previous administration of the medicament. Lesion size and in vitro infectivity of the parasite are negatively correlated with decline in Glucantime susceptibility (Spearman: r = (-)0,548 and r = (-)0,726; respectively).ConclusionA negative correlation between lesion size and parasite resistance is documented. L. braziliensis was found as the main parasite species associated to lesion of patients that underwent treatment failure or relapse. The indication of a second round of treatment in therapeutic failure of ACL, produced by L. braziliensis, with pentavalent antimonial derivatives is discussable.

The discrimination of Leishmania species from patient samples has epidemiological and clinical relevance. In this study, different gene target PCR-restriction fragment length polymorphism (RFLP) protocols were evaluated for their robustness as Leishmania species discriminators in 61 patients with cutaneous leishmaniasis. We modified the hsp70-PCR-RFLP protocol and found it to be the most reliable protocol for species identification.H uman infections by Leishmania spp. produce a pleiomorphic syndrome in which symptomatology depends on the parasite species and the immunological stage of the host. The symptoms range from completely asymptomatic to cutaneous, mucocutaneous, and visceral (1). Several authors have reported differences in the treatment outcomes linked to the parasite species (2-5). Furthermore, mucocutaneous leishmaniasis (MCL) is a belated complication associated with specific parasite species (3, 6) most commonly occurring in infections caused by the Leishmania (Viannia) subgenus. American cutaneous leishmaniasis (ACL) cases are usually the result of infections produced by this subgenus, and species identification is useful for treatment and prognosis. Molecular techniques may become a routine way to confirm suspected cases of ACL (7-10); the present study describes the best PCR-restriction fragment length polymorphism (RFLP) gene target for determining the species of Leishmania present in clinical samples from ACL lesions in a set of Colombian patients.The study was approved by the boards of ethical conduct of the Hospital Militar Central-Bogota-Colombia (HOMIC) and Centro Dermatologico Federico Lleras Acosta Bogota-Colombia (CDFLL) in accordance with national (resolution 008430 of the Colombian Health Ministry) and international (Declaration of Helsinki and amendments, World Medical Association, South Korea, 2008) guidelines. DNA was extracted from skin biopsy specimens from the internal border of the lesions from 42 adult patients with a clinical diagnosis of ACL. The diagnosis was confirmed microscopically in 35 patients and by PCR detection of the parasite in 7 patients. All patients voluntarily participated in the study and signed an informed consent.The CDFLL biobank provided 19 Giemsa-stained slide smears from cutaneous lesions. In 17 of them, the presence of Leishmania sp. amastigotes was microscopically confirmed, and in 2 smears, the detection of the parasite was established by PCR. DNA was recovered from the Giemsa-stained smears.All PCRs performed included DNA from 2 negative-control patients from CDFLL (with confirmed diagnoses of sporotrichosis and ecthyma gangrenosum) and from three healthy volunteers. The entire group of patients had been infected within the Colombian borders.We selected genes and sequences previously reported to be useful markers for species identification by PCR-RFLP of Leishmania species for further evaluation. We analyzed zinc-metalloprotease (gp63) (11), spliced leader (SL) (12), cysteine protease B1 (cpb) (13), and heat shock protein 70 (hsp70) (14, 15) for the...