Resistance to antimonial drugs has been documented inL ong-term use of antimonial drugs as monotherapy for leishmaniasis and nonadherence to treatment have contributed to the increase in treatment failures (3,5). In addition to intrinsic differences in the susceptibility of different Leishmania populations (18, 23), acquired resistance has also been reported (12,15). Consequent variations in drug efficacy impact clinical management and leishmaniasis control programs, yet little is known about the prevalence of tolerant and/or resistant populations (11) and the impact of treatment policies on drug susceptibility.Evaluation of drug susceptibility of clinical strains of Leishmania has been constrained by the unavailability of an efficient method that yields consistent results. Intracellular amastigotes are the target of treatment, are strikingly more susceptible to antimony than promastigotes (2, 11, 21), and importantly, are killed in vitro at clinically achievable drug concentrations. However, drug susceptibility evaluation of clinical strains using intracellular amastigotes is a slow, low-throughput biological assay that is influenced by numerous poorly understood variables. Individual clinical strains are heterogeneous; even strains having a low 50% effective dose (ED 50 ) often contain subpopulations that are effectively resistant (15). Hence, although cloned parasites transfected with reporter genes have proven useful for drug screening (17), the protracted, intensive in vitro antibiotic selection required to recover and maintain the transfected population may alter the original composition of clinical strains and, consequently, the susceptibility phenotype.Conventional susceptibility assays are based on the determination of the ED 50 using murine peritoneal macrophages or macrophage cell lines under diverse conditions of infection with different numbers and intervals of drug concentrations, periods of drug exposure, and readout parameters. Since these factors influence the outcome of the evaluation (20), results of drug susceptibility testing have been neither consistent nor generalizable.We report a novel approach to drug susceptibility assessment of clinical strains of Leishmania based on quantification of parasite burden after exposure to single discriminatory concentrations of two representative antileishmanial drugs, meglumine antimoniate (Sb V ) and miltefosine (HePC). MATERIALS AND METHODSExperimental strategy. Susceptibility of clinical strains of Leishmania (Viannia) species to Sb V and HePC was determined in parallel by conventional dose-response assay and reduction of intracellular parasite burden at a single drug concentration. Discriminatory drug concentrations for susceptibility were defined as follows: the antimony concentration of 32 g Sb V /ml was based on the estimated maximum concentration (C max ) in plasma (6), and the miltefosine concentration of 16 M was chosen because it clearly and consistently distinguished the susceptible wild-type (WT) control strain and an experimentally selected...

BackgroundPentavalent antimonials have been the first line treatment for dermal leishmaniasis in Colombia for over 30 years. Miltefosine is administered as second line treatment since 2005. The susceptibility of circulating populations of Leishmania to these drugs is unknown despite clinical evidence supporting the emergence of resistance.Methodology/Principal Findings In vitro susceptibility was determined for intracellular amastigotes of 245 clinical strains of the most prevalent Leishmania Viannia species in Colombia to miltefosine (HePC) and/or meglumine antimoniate (SbV); 163, (80%) were evaluated for both drugs. Additionally, susceptibility to SbV was examined in two cohorts of 85 L. V. panamensis strains isolated between 1980–1989 and 2000–2009 in the municipality of Tumaco. Susceptibility to each drug differed among strains of the same species and between species. Whereas 68% of L. V. braziliensis strains presented in vitro resistance to HePC, 69% were sensitive to SbV. Resistance to HePC and SbV occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to SbV. Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to SbV were discerned among L. V. panamensis strains isolated during 1980–1990 in Tumaco where resistant strains belonged to zymodeme 2.3, and sensitive strains to zymodeme 2.2.Conclusions/SignificanceLarge scale evaluation of clinical strains of Leishmania Viannia species demonstrated species, population, geographic, and epidemiologic differences in susceptibility to meglumine antimoniate and miltefosine, and provided baseline information for monitoring susceptibility to these drugs. Sensitive and resistant clinical strains within each species, and zymodeme as a proxy marker of antimony susceptibility for L. V. panamensis, will be useful in deciphering factors involved in susceptibility and the distribution of sensitive and resistant populations.

BackgroundPrevious findings indicate that susceptibility to Leishmania (Viannia) panamensis infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome.Methodology/Principal FindingsTo understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to L. (V.) panamensis. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of L. (V.) panamensis. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other Leishmania species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with L. (Leishmania) chagasi indicate differences in the regulation of genes involved in signaling, motility and the immune response.ConclusionsResults show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to L. (Viannia) panamensis is not quiescent, in contrast to published reports examining later response times (48–96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by L. (Viannia) panamensis illustrate the dynamics of these interactions and the distinct biologic responses to different Leishmania species from the outset of infection within their primary host cell.

We consider PCR-miniexon as a diagnostic method of first choice for mucocutaneous leishmaniasis due to its excellent diagnostic performance and its ability to discriminate between Leishmania and Viannia subgenera as well as between species belonging to Leishmania subgenus.

Introduction: Multilocus enzyme electrophoresis (MLEE) is the reference standard for the characterization of Leishmania species. The test is restricted to specialized laboratories due to its technical complexity, cost, and time required to obtain results. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify Leishmania species. Objective: To establish the concordance between the two tests as identifying methods for circulating species in Colombia. Materials and methods: A total of 96 isolates from patients with cutaneous or mucosal leishmaniasis were selected and identified by MLEE and PCR-RFLP with miniexon and hsp70 as the molecular targets, which were used sequentially. Restriction enzymes HaeIII and BccI were similarly applied. Cohen's kappa coefficient and the 95% confidence interval (CI) were calculated. Results: The kappa coefficient and the 95% CI between MLEE and PCR-RFLP displayed "very good" concordance with a coefficient of 0.98 (CI95%: 0.98 to 1.00). The identified species were Leishmania Viannia braziliensis, Leishmania Viannia panamensis, Leishmania Viannia guyanensis and Leishmania Leishmania amazonensis. A total of 80 of the 96 isolates were sequenced and the results obtained by PCR-RFLP were confirmed. Conclusion: Due to the concordance obtained between tests results with the amplification of the genes miniexon and hsp70, PCR-RFLP is proposed as an alternative for identifying circulating Leishmania species in Colombia.

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