Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused by Leishmania braziliensis in Peru and Bolivia.

Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis.

Multilocus genotyping reveals a polyphyletic pattern among naturally antimony-resistant Leishmania braziliensis isolates from Peru.

In order to understand the epidemiological dynamics of antimonial (Sb(V)) resistance in zoonotic tegumentary leishmaniasis and its link with treatment outcome, we analyzed the population structure of 24 Peruvian Leishmania braziliensis clinical isolates with known in vitro antimony susceptibility and clinical phenotype by multilocus microsatellite typing (14 microsatellite loci). The genetic variability in the Peruvian isolates was high and the multilocus genotypes were strongly differentiated from each other. No correlation was found between the genotypes and in vitro drug susceptibility or clinical treatment outcome. The finding of a polyphyletic pattern among the Sb(V)-resistant L. braziliensis might be explained by (i) independent events of drug resistance emergence, (ii) sexual recombination and/or (iii) other phenomena mimicking recombination signals. Interestingly, the polyphyletic pattern observed here is very similar to the one we observed in the anthroponotic Leishmania donovani (Laurent et al., 2007), hereby questioning the role of transmission and/or chemotherapeutic drug pressure in the observed population structure.

Immunological determinants of clinical outcome in Peruvian patients with tegumentary leishmaniasis treated with pentavalent antimonials.

The mechanisms linking the immune response to cutaneous and mucosal leishmaniasis (CL and ML, respectively) lesions and the response to treatment are incompletely understood. Our aims were to prospectively assess, by quantitative reverse transcription-PCR, the levels of mRNA for gamma interferon, tumor necrosis factor alpha, interleukin-10 (IL-10), IL-4, and IL-13, as well as the presence of T cells (CD2) and macrophages (CD68), in CL and ML lesions and to follow their changes in response to treatment with pentavalent antimonials. The leishmanin skin test (LST) was performed on all CL and ML patients before treatment. The patient population included individuals living in areas of Peru where the disease is endemic, i.e., 129 with CL and 43 with ML. Compared to CL patients, the LST induration size was larger, the levels of all cytokine mRNAs but IL-10 were higher, T-cell mRNA was similar, and macrophage mRNA was lower in ML patients. The proportion of CL patients with an LST induration size of >8 mm was higher among responders to treatment. In CL, the pretreatment levels of cytokine mRNAs did not discriminate between responders and nonresponders; however, treatment was more often accompanied by a reduction in the levels of T-cell and cytokine mRNAs in responders than in nonresponders. Furthermore, the production of cytokines per T cell and macrophage decreased with treatment but IL-10 production remained high in nonresponders. Overall, these findings point to complex relationships among New World Leishmania parasites, skin and mucosal immune responses, and treatment outcome. The persistence of high levels of IL-10 in CL is characteristically associated with a poor response to treatment.

PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar.

OBJECTIVE: To compare a PCR assay and direct agglutination test (DAT) for the detection of potential markers of Leishmania infection in 231 healthy subjects living in a kala-azar endemic focus of Nepal. METHODS: The sample was composed of 184 (80%) persons without any known history of KA and not living in the same house as known kala-azar cases (HNK), 24 (10%) Healthy Household Contacts (HHC) and 23 (10%) past kala-azar cases which had been successfully treated (HPK). RESULTS: PCR and DAT positivity scores were, respectively: HNK, 17.6% and 5.6%; HHC, 12.5% and 20.8%; HPK, 26.1% and 95.7%. The ratio PCR-positives/DAT-positives was significantly higher in HNK (ratio = 3.1) than in HHC (ratio = 0.6, P = 0.036) and in HPK (ratio = 0.2, P = 0.012). The ratio PCR-positives/DAT-positives did not significantly differ between HHC (ratio = 0.6) and HPK (ratio = 0.2, P = 0.473). The positive agreement index between PCR and DAT in HNK was 5%; in HHC, 0%; in HPK, 43%. CONCLUSIONS: Our study highlights the specific character of PCR and DAT for the exploration of Leishmania asymptomatic infections. PCR is probably more informative for very recent infections among HNK, while DAT provides more information among HHC and HPK, a feature likely related to the power of serology to track less recent infections.

Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection.

We used the cysteine proteinase B (cpb) gene family of the trypanosomatid genus Leishmania as a target to develop rapid, specific, and easy-to-use polymerase chain reaction (PCR) tests to discriminate Leishmania infantum, Leishmania donovani, Leishmania tropica, Leishmania aethiopica, and Leishmania major. Identification of all 5 Old World species and validation of intraspecies variability are features lacking in other species-specific PCRs. Amplicon analysis was done on agarose gels and was further simplified by using an oligochromatography dipstick to detect L. infantum and L. donovani products. Because the analytical sensitivity is lower than that of certain other species- and genus-specific PCRs, our assays are especially valuable for use on cultured isolates or directly on cryostabilates. As such, they can be implemented by research and health centers having access to culturing, DNA isolation, and PCR.

[PCR-RFLP and RAPD for typing neotropical Leishmania]. / PCR-RFLP y RAPD para la tipificación de Leishmania neotropical.

INTRODUCTION: The analysis of the PCR-restriction fragment length polymorphism and random amplified polymorphic DNA have been useful tools for Leishmania identification. OBJECTIVES: Molecular procedures were demonstrated for identification and typing of reference strains of New World Leishmania and their applicability was validated for clinical samples. MATERIALS AND METHODS: DNA was extracted from 16 reference strains of Latin American Leishmania as well as from clinical samples of leishmaniasis patients. A sequence coding for cysteine proteinase B was amplified by PCR and subjected to restriction fragment length polymorphism analysis. The enzyme used was Taq1. For eight of the reference strains, the random amplified polymorphic desoxyribonucleic acid technique (RAPD) was applied. Band patterns for Leishmania species differentiation were established each each method. The sample size of the clinical sample was of 5. RESULTS: PCR products of the cysteine proteinase B gene were obtained for L. braziliensis, L. peruviana, L. panamensis and L. guyanensis. For the other species, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, no amplification occurred. The patterns of restriction fragments revealed band patterns in common for L. peruviana, L. guyanensis and L. panamensis, whereas L. braziliensis had a distinctive pattern. When human samples were examined, amplification occurred for all cases, and the profiles corresponded to the common profile of L. peruviana, L. guyanensis and L. panamensis. The RAPD technique demonstrated reproducible and distinctive patterns for each of the 8 reference strains, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, making possible to differentiate all them. The advantages and limitations of each procedure are discussed. CONCLUSIONS: The combination of RFP and RAPD methodologies provide useful tools to identify medical important species of Leishmania by recognizing DNA sequences characteristic of each species.

Epidemiological dynamics of antimonial resistance in Leishmania donovani: genotyping reveals a polyclonal population structure among naturally-resistant clinical isolates from Nepal.

Pentavalent antimonials (SbV) are the first line drug against leishmaniasis worldwide, but drug resistance is increasingly reported, particularly in the Indian sub-continent, where it represents a major threat for the control of anthroponotic visceral leishmaniasis (VL). In order to understand the epidemiological dynamics of antimonial resistance in anthroponotic VL, we analysed here the population structure of 24 Leishmania donovani stocks isolated from anthroponotic VL-patients from Eastern Nepal: 13 SbV-naturally resistant and 11 SbV-sensitive, as demonstrated by in vitro drug susceptibility assays. The parasites were genotyped by PCR-RFLP analysis of kDNA minicircles and by microsatellite analysis and the encountered polymorphism revealed a polyclonal structure among resistant isolates. Furthermore, analysis of paired samples obtained from the same patients before treatment and after failure revealed primary as well as acquired resistance. The hypothesis of independent events of drug resistance emergence is proposed and confronted to alternative explanations. Our results show the dynamics of drug resistance epidemiology and highlight the importance of surveillance networks.

Fluorogenic assay for molecular typing of the Leishmania donovani complex: taxonomic and clinical applications.

We describe a new fluorogenic assay for the identification of species and intraspecies groups within the Leishmania donovani complex. The assay combined (1) 2 polymerase chain reactions targeting the 2 cysteine proteinase b isogenes and (2) a fluorescence-resonance energy transfer/melting curve analysis of the polymorphisms within a 31-nt region. All strains within the L. donovani complex were distinguished from L. tropica, L. major, and L. aethiopica, and 5 distinct groups were identified within the L. donovani complex. Discrepancies were observed with the present taxonomy on the basis of isoenzyme analysis and concerned East African strains, which suggests the need for a systematic reevaluation of the taxonomy. The capacity to type parasites directly from clinical samples was demonstrated with blood and bone marrow samples. This rapid and high-throughput alternative for molecular diagnosis and epidemiological studies of visceral leishmaniasis could be adapted for use with other Leishmania species.