Linking In Vitro and In Vivo Survival of Clinical Leishmania donovani Strains

BACKGROUND: Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these 'hostile' environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain. CONCLUSIONS/SIGNIFICANCE: The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesi

American Tegumentary Leishmaniasis: Is Antimonial Treatment Outcome Related to Parasite Drug Susceptibility?

BackgroundAntimonials are the first drug of choice for the treatment of American tegumentary leishmaniasis (ATL); however, their efficacy is not predictable, and this may be linked to parasite drug resistance. We aimed to characterize the in vitro antimony susceptibility of clinical isolates of Peruvian patients with ATL who were treated with sodium stibogluconate and to correlate this in vitro phenotype with different treatment outcomes MethodsThirty-seven clinical isolates were obtained from patients with known disease and treatment histories. These isolates were typed, and the susceptibility of intracellular amastigotes to pentavalent (SbV) and trivalent (SbIII) antimonials was determined ResultsWe observed 29 SbV-resistant isolates among 4 species of subgenus Viannia most of which exhibited primary resistance; isolates resistant only to SbIII; and 3 combinations of in vitro phenotypes: (1) parasites sensitive to both drugs, (2) parasites resistant to both drugs, and (3) parasites resistant to SbV only (the majority of isolates fell into this category). There was no correlation between in vitro susceptibility to both antimonials and the clinical outcome of therapy ConclusionAntimony insensitivity might occur in a stepwise fashion (first to SbV and then to SbIII). Our data question the definition of true parasite resistance to antimonials. Further studies of treatment efficacy should apply standardized protocols and definitions and should also consider host factor

Influence of Leishmania (Viannia) Species on the Response to Antimonial Treatment in Patients with American Tegumentary Leishmaniasis

Background. Pentavalent antimonials (SbV) are the first-line chemotherapy for American tegumentary leishmaniasis (ATL). There are, however, reports of the occurrence of treatment failure with these drugs. Few studies in Latin America have compared the response to SbV treatment in ATL caused by different Leishmania species. Methods. Clinical parameters and response to SbV chemotherapy were studied in 103 patients with cutaneous leishmaniasis (CL) in Peru. Leishmania isolates were collected before treatment and typed by multilocus polymerasechain-reaction restriction fragment—length polymorphism analysis. Results. The 103 isolates were identified as L. (Viannia) peruviana (47.6%), L. (V.) guyanensis (23.3%), L. (V.) braziliensis (22.3%), L. (V.) lainsoni (4.9%), L. (Leishmania) mexicana (1%), and a putative hybrid, L. (V.) braziliensis/L. (V.) peruviana (1%). L. (V.) guyanensis was most abundant in central Peru. Of patients infected with the 3 former species, 21 (21.9%) did not respond to SbV chemotherapy. The proportions of treatment failure (after 12 months of follow-up) were 30.4%, 24.5%, and 8.3% in patients infected with L. (V.) braziliensis, L. (V.) peruviana, and L. (V.) guyanensis, respectively. Infection with L. (V.) guyanensis was associated with significantly less treatment failure than L. (V.) braziliensis, as determined by multiple logistic regression analysis (odds ratio, 0.07 [95% confidence interval, 0.007-0.8]; P = .03). Conclusions. Leishmania species can influence SbV treatment outcome in patients with CL. Therefore, parasite identification is of utmost clinical importance, because it should lead to a species-oriented treatmen

Clinical and Parasite Species Risk Factors for Pentavalent Antimonial Treatment Failure in Cutaneous Leishmaniasis in Peru

Background. Treatment for cutaneous leishmaniasis (CL) with standard pentavalent antimonial therapy is hampered by cumbersome administration, toxicity, and potential failure. Knowledge of factors influencing treatment outcome is essential for successful management. Methods. A case-control study of incident cases was performed with patients experiencing their first CL episode. The standard treatment for CL for these patients was 20 mg/kg/day of sodium stibogluconate for 20 days. Clinical and epidemiological data were recorded, and parasite isolates were species typed. Patients were followed up for 6 months to assess treatment outcome. Clinical cure was defined as complete wound closure and re-epithelization without inflammation or infiltration; new lesions, wound reopening, or signs of activity were classified as treatment failure. Descriptive, bivariate, and logistic regression analyses were performed. Results. One hundred twenty-seven patients were recruited; 63 (49.6%) were infected with Leishmania (Viannia) peruviana, 29 (22.8%) were infected with Leishmania (Viannia) braziliensis, 27 (21.3%) were infected with Leishmania (Viannia) guyanensis, and 8 (6.3%) were infected with other species. Only patients infected with the 3 most common species were selected for risk-factor analysis (n=119). Final failure rate at 6 months was 24.4% (95% confidence interval [CI], 16.5%-32.1%), with 96% of failures occurring within the first 3 months of follow-up assessment. Risk factors for treatment failure identified in the final multivariate model were age (per year, odds ratio [OR], 0.95; 95% CI, 0.92-0.99; P=.017), stay of <72 months in area of disease acquisition (OR, 30.45; 95% CI, 2.38-389.25; P=.009), duration of disease <5 weeks (OR, 4.39; 95% CI, 1.12-17.23; P=.034), additional lesion (per lesion, OR, 2.06; 95% CI, 1.3-3.28; P=.002), infection with L. (V.) peruviana (OR, 9.85; 95% CI, 1.01-95.65; P=.049), and infection with L. (V.) braziliensis (OR, 22.36; 95% CI, 1.89-263.96; P=.014). Conclusions. The identification of parasite species and clinical risk factors for antimonial treatment failure should lead to an improved management of CL in patients in Per

Antimonial Resistance in Leishmania donovani Is Associated with Increased In Vivo Parasite Burden

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis (VL). Antimonials (SSG) have long been the first-line treatment against VL, but have now been replaced by miltefosine (MIL) in the Indian subcontinent due to the emergence of SSG-resistance. Our previous study hypothesised that SSG-resistant L. donovani might have increased in vivo survival skills which could affect the efficacy of other treatments such as MIL. The present study attempts to validate these hypotheses. Fourteen strains derived from Nepalese clinical isolates with documented SSG-susceptibility were infected in BALB/c mice to study their survival capacity in drug free conditions (non-treated mice) and in MIL-treated mice. SSG-resistant parasites caused a significant higher in vivo parasite load compared to SSG-sensitive parasites. However, this did not seem to affect the strains' response to MIL-treatment since parasites from both phenotypes responded equally well to in vivo MIL exposure. We conclude that there is a positive association between SSG-resistance and in vivo survival skills in our sample of L. donovani strains which could suggest a higher virulence of SSG-R strains compared to SSG-S strains. These greater in vivo survival skills of SSG-R parasites do not seem to directly affect their susceptibility to MIL. However, it cannot be excluded that repeated MIL exposure will elicit different adaptations in these SSG-R parasites with superior survival skills compared to the SSG-S parasites. Our results therefore highlight the need to closely monitor drug efficacy in the field, especially in the context of the Kala-azar elimination programme ongoing in the Indian subcontinent

Comparative Gene Expression Analysis throughout the Life Cycle of Leishmania braziliensis: Diversity of Expression Profiles among Clinical Isolates

Leishmania is a group of parasites (Protozoa, Trypanosomatidae) responsible for a wide spectrum of clinical forms. Among the factors explaining this phenotypic polymorphism, parasite features are important contributors. One approach to identify them consists in characterizing the gene expression profiles throughout the life cycle. In a recent study, the transcriptome of 3 Leishmania species was compared and this revealed species-specific differences, albeit in a low number. A key issue, however, is to ensure that the observed differences are indeed species-specific and not specific of the strains selected for representing the species. In order to illustrate the relevance of this concern, we analyzed here the gene expression profiles of 5 clinical isolates of L. braziliensis at seven time points of the life cycle. Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics: one Leishmania strain is not necessarily representative of a given species

The sensitivity of clinical isolates of Leishmania from Peru and Nepal to miltefosine.

Clinical isolates of Leishmania, from visceral leishmaniasis (VL) cases in Nepal and from cutaneous leishmaniasis (CL) cases in Peru, were cultured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to type species and strain. Promastigotes from 38 isolates, within eight passages from isolation, were used to infect mouse peritoneal macrophage cultures in vitro, and the amastigote sensitivity to miltefosine was determined. The concentration required to kill 50% of intracellular amastigotes from Nepalese VL isolates, all typed as Leishmania (L.) donovani (N = 24) from both Sbv responders and nonresponders, ranged from 8.7 to 0.04 microg/mL. In contrast, the concentration required to kill 50% intracellular amastigotes from isolates from Peru, typed as L.(V.) braziliensis (N = 8), was > 30 to 8.4 microg/mL, L.(V.) guyanensis (N = 2) > 30 to 1.9 microg/mL, L.(L.) mexicana (N = 1) > 30 microg/mL, and L. (V.) lainsoni (N = 4) was 3.4 to 1.9 microg/mL. This demonstrates a notable difference in the intrinsic sensitivity of Leishmania species to miltefosine in vitro. If this model can be correlated to therapeutic outcome, it may have implications for the interpretation of clinical trials

Diagnostic accuracy of a new Leishmania PCR for clinical visceral leishmaniasis in Nepal and its role in diagnosis of disease

To develop a new PCR for Leishmania detection and to estimate its diagnostic accuracy in a visceral leishmaniasis (VL) endemic area.status: publishe

Gene Expression Analysis of the Mechanism of Natural Sb(V) Resistance in Leishmania donovani Isolates from Nepal

Control of visceral leishmaniasis (VL) is being challenged by the emergence of natural resistance against the first line of treatment, pentavalent antimonials [Sb(V)]. An insight into the mechanism of natural Sb(V) resistance is required for the development of efficient strategies to monitor the emergence and spreading of Sb(V) resistance in countries where VL is endemic. In this work, we have focused on the mechanism of natural Sb(V) resistance emerging in Nepal, a site where anthroponotic VL is endemic. Based on the current knowledge of Sb(V) metabolism and of the in vitro trivalent antimonial [Sb(III)] models of resistance to Leishmania spp., we selected nine genes for a comparative transcriptomic study on natural Sb(V)-resistant and -sensitive Leishmania donovani isolates. Differential gene expression patterns were observed for the genes coding for 2-thiol biosynthetic enzymes, gamma-glutamylcysteine synthetase (GCS) and ornithine decarboxylase (ODC), and for the Sb(III) transport protein aquaglyceroporin 1 (AQP1). The results indicate that the mechanism for natural Sb(V) resistance partially differs from the mechanism reported for in vitro Sb(III) resistance. More specifically, we hypothesize that natural Sb(V) resistance results from (i) a changed thiol metabolism, possibly resulting in inhibition of Sb(V) activation in amastigotes, and (ii) decreased uptake of the active drug Sb(III) by amastigotes