Natural Resistance of Leishmania infantum to Miltefosine Contributes to the Low Efficacy in the Treatment of Visceral Leishmaniasis in Brazil.

In India, visceral leishmaniasis (VL) caused by Leishmania donovani has been successfully treated with miltefosine with a cure rate of > 90%. To assess the efficacy and safety of oral miltefosine against Brazilian VL, which is caused by Leishmania infantum, a phase II, open-label, dose-escalation study of oral miltefosine was conducted in children (aged 2-12 years) and adolescent-adults (aged 13-60 years). Definitive cure was assessed at a 6-month follow-up visit. The cure rate was only 42% (6 of 14 patients) with a recommended treatment of 28 days and 68% (19 of 28 patients) with an extended treatment of 42 days. The in vitro miltefosine susceptibility profile of intracellular amastigote stages of the pretreatment isolates, from cured and relapsed patients, showed a positive correlation with the clinical outcome. The IC50 mean (SEM) of eventual cures was 5.1 (0.4) µM, whereas that of eventual failures was 12.8 (1.9) µM (P = 0.0002). An IC50 above 8.0 µM predicts failure with 82% sensitivity and 100% specificity. The finding of L. infantum amastigotes resistant to miltefosine in isolates from patients who eventually failed treatment strongly suggests natural resistance to this drug, as miltefosine had never been used in Brazil before this trial was carried out.

A Leishmania infantum genetic marker associated with miltefosine treatment failure for visceral leishmaniasis.

BACKGROUND: Miltefosine has been used successfully to treat visceral leishmaniasis (VL) in India, but it was unsuccessful for VL in a clinical trial in Brazil. METHODS: To identify molecular markers that predict VL treatment failure whole genome sequencing of 26 L. infantum isolates, from cured and relapsed patients allowed a GWAS analysis of SNPs, gene and chromosome copy number variations. FINDINGS: A strong association was identified (p = 0·0005) between the presence of a genetically stable L. infantumMiltefosine Sensitivity Locus (MSL), and a positive response to miltefosine treatment. The risk of treatment failure increased 9·4-fold (95% CI 2·11-53·54) when an isolate did not have the MSL. The complete absence of the MSL predicted miltefosine failure with 0·92 (95% CI 0·65-0·996) sensitivity and 0·78 (95% CI 0·52-0·92) specificity. A genotyping survey of L. infantum (n = 157) showed that the frequency of MSL varies in a cline from 95% in North East Brazil to <5% in the South East. The MSL was found in the genomes of all L. infantum and L. donovani sequenced isolates from the Old World (n = 671), where miltefosine can have a cure rate higher than 93%. INTERPRETATION: Knowledge on the presence or absence of the MSL in L. infantum will allow stratification of patients prior to treatment, helping to establish better therapeutic strategies for VL treatment. FUND: CNPq, FAPES, GCRF MRC and Wellcome Trust.

Use of VHH antibodies for the development of antigen detection test for visceral leishmaniasis.

We have recently developed a sensitive and specific urine-based antigen detection ELISA for the diagnosis of visceral leishmaniasis (VL). This assay used rabbit IgG and chicken IgY polyclonal antibodies specific for the Leishmania infantum proteins iron superoxide dismutase 1 (Li-isd1), tryparedoxin1 (Li-txn1) and nuclear transport factor 2 (Li-ntf2). However, polyclonal antibodies have limitations for upscaling and continuous supply. To circumvent these hurdles, we began to develop immortalized monoclonal antibodies. We opted for recombinant camelid VHHs because the technology for their production is well established and they do not have Fc, hence providing less ELISA background noise. We report here an assay development using VHHs specific for Li-isd1 and Li-ntf2. This new assay was specific and had analytical sensitivity of 15-45 pg/mL of urine. The clinical sensitivity was comparable to that obtained with the ELISA assembled with conventional rabbit and chicken antibodies to detect these two antigens. Therefore, similar to our former studies with conventional antibodies, the future inclusion of VHH specific for Li-txn1 and/or other antigens should further increase the sensitivity of the assay. These results confirm that immortalized VHHs can replace conventional antibodies for the development of an accurate and reproducible antigen detection diagnostic test for VL.

Proteomic analysis of the soluble proteomes of miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates obtained from Brazilian patients with different treatment outcomes.

The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in experimental resistant lines; however, studies using clinical isolates with different miltefosine susceptibilities are still needed. In our study, we used a proteomic 2D-DIGE/MS approach to study different protein abundances in miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates from visceral leishmaniasis patients with different miltefosine treatment outcomes. The high-resolution proteome obtained from these isolates showed 823 matched spots and 46 spots exhibited different abundances between the isolates. Out of these differentially expressed spots, 26 (56.5%) showed greater and 20 (43.5%) showed lower expression of the resistant isolate compared to the sensitive isolate. MALDI/TOF-TOF mass spectrometry allowed the identification of 32 spots with unique protein identification correspondent to 22 non-redundant proteins. Most of the proteins up-regulated in the proteome miltefosine-resistant isolates were associated with redox homeostasis, stress response, protection to apoptosis, and drug translocation. These differentially expressed proteins are likely involved in miltefosine natural resistance and suggest that the miltefosine-resistance mechanism in Leishmania is multifactorial. BIOLOGICAL SIGNIFICANCE: Visceral leishmaniasis (VL) is a serious disease with a challenging treatment plan requiring the prolonged and painful applications of poorly tolerated toxic drugs. Therefore, the identification of miltefosine, an effective and safe oral drug, was considered a significant advancement in leishmaniasis therapy. However, different sensitivities to miltefosine in Leishmania have been observed in clinically relevant species, and the biological mechanism by which clinical isolates of Leishmania acquire drug resistance is poorly understood. Our work aims to elucidate the mechanism of natural resistance to miltefosine in Leishmania by studying the isolates from VL patients who displayed different miltefosine treatment outcomes.

Prevalência de adultos infectados por Leishmania leishmania chagasi entre doadores de sangue do Hemocentro Regional de Montes Claros, Minas Gerais, Brasil / Prevalence of adults infected with Leishmania (Leishmania) chagasi among blood donors of the Hemominas Foundation in Montes Claros, Minas Gerais, Brazil

O objetivo deste trabalho foi estudar a prevalência de adultos infectados por L. L. chagasi entre doadores de sangue do Hemocentro Regional de Montes Claros/MG .Realizou-se estudo epidemiológico, transversal e quantitativo, no período de 16/09/08 a 13/11/08. Participaram da pesquisa 421 doadores aptos na triagem clínica, sendo realizada imunofluorescência indireta para L.L.chagasi. Aqueles que apresentaram resultados positivos foram submetidos ao teste rápido antígeno-específico para Leishmania donovani. A análise das variáveis gênero, faixa etária, procedência, número de doações, resultados sorológicos para leishmaniose e chagas, foi realizada pelos testes estatísticos qui-quadrado (x2), x2 com tendência linear e teste Fisher. Foi considerado o nível de significância de 5 por cento (p<0,05). O perfil da amostra foi semelhante ao perfil geral dos doadores. Os participantes, em sua maioria procedentes da zona urbana (92,7 por cento), residentes em Montes Claros (67,9 por cento), homens (61,3 por cento), com faixa etária de 18 a 29 anos. Em relação aos resultados sorológicos, 23 (5,5 por cento) apresentaram positividade para imunofluorescência indireta e nenhum destes foi positivo no teste rápido. Ao comparar os resultados da imunofluorescência para leishmaniose e a sorologia Elisa chagas, dois foram positivos para ambos os testes, sendo demonstrada correlação estatística significante (p=0,003). Porém, 21 foram positivos para leishmaniose e negativos para chagas. Os resultados permitiram conhecer a prevalência da infecção por L. l. chagasi em indivíduos assintomáticos, adultos, doadores de sangue do Hemocentro Regional de Montes Claros/MG e apontam para a necessidade de maiores estudos quanto ao possível risco de transmissão transfusional da doença.

The objective of this work was to study the prevalence of adults infected by Leishmania (Leishmania) chagasi among blood donors of the Hemominas Foundation in Montes Claros, Brazil. A cross-sectional, quantitative epidemiological study was performed of 421 blood donors from September 16 2008 to November 13 2008. The L. l. chagasi indirect immunofluorescence test (RIFI) was utilized. Donors that presented with positive results in RIFI were retested using the fast immunochromatographic test (Trald). The gender, age, place of origin, number of donations, leishmania and chagas disease serum results were studied with statistical correlations being analyzed utilizing the chi-square test (x2), x2 with linear tendency and the Fisher test; a level of significance of 5 percent (p <0.05) was considered acceptable. The profile of the study sample was similar to the overall donor profile. The participants were mostly donors from urban areas (92.7 percent), living in Montes Claros (67.9 percent), men (61.3 percent) and with ages between 18 and 29 years old. In relation to the serum results, 23 (5.5 percent) were positive according to the RIFI however none of them were positive by the Trald. On comparing the results of RIFI and the chagas disease serum test (Elisa), two individuals were positive for both tests, thereby giving a statistically significant correlation (p=0.003). However the other 21 were positive only by RIFI and negative for chagas disease. The results show the prevalence of infection by L. l. chagasi in asymptomatic, adult blood donors in the Hemominas Foundation in Montes Claros, and highlight the need for further studies on the possible risk of disease transmission via blood transfusion.