Alejandro Llanos-Cuentas

Successful Treatment of Drug-Resistant Cutaneous Leishmaniasis in Humans by Use of Imiquimod, an Immunomodulator

Treatment failures for leishmaniasis with pentavalent antimonials, including meglumine antimonate, are increasingly common in many endemic areas. Imiquimod (Aldara; 3M Pharmaceuticals) is a novel immune response-activating compound, approved by the United States Food and Drug Administration, that is currently used to treat cervical warts and has been shown to activate macrophage killing of Leishmania species. Therefore, an open-label, prospective study was conducted of combined imiquimod plus meglumine antimonate therapy in 12 patients with cutaneous leishmaniasis who had previously not responded to meglumine antimonate therapy. All of the patients responded well to this combination therapy, and 90% were found to be cured at the 6-month follow-up period.

Tafenoquine plus chloroquine for the treatment and relapse prevention of Plasmodium vivax malaria (DETECTIVE): a multicentre, double-blind, randomised, phase 2b dose-selection study

BACKGROUND Clinical effectiveness of previous regimens to treat Plasmodium vivax infection have been hampered by compliance. We aimed to assess the dose-response, safety, and tolerability of single-dose tafenoquine plus 3-day chloroquine for P vivax malaria radical cure. METHODS In this double-blind, randomised, dose-ranging phase 2b study, men and women (aged ≥16 years) with microscopically confirmed P vivax monoinfection (parasite density >100 to <100,000 per μL blood) were enrolled from community health centres and hospitals across seven sites in Brazil, Peru, India, and Thailand. Patients with glucose-6-phosphate dehydrogenase enzyme activity of less than 70% were excluded. Eligible patients received chloroquine (days 1-3) and were randomly assigned (1:1:1:1:1:1) by a computer-generated randomisation schedule to receive single-dose tafenoquine 50 mg, 100 mg, 300 mg, or 600 mg, primaquine 15 mg for 14 days, or chloroquine alone. Randomisation was stratified by baseline parasite count (≤7500 and >7500 per μL blood). The primary efficacy endpoint was relapse-free efficacy at 6 months from initial dose (ie, clearance of initial infection without subsequent microscopically confirmed infection), analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01376167. FINDINGS Between Sept 19, 2011, and March 25, 2013, 329 patients were randomly assigned to a treatment group (chloroquine plus tafenoquine 50 mg [n=55], 100 mg [n=57], 300 mg [n=57], 600 mg [n=56]; or to chloroquine plus primaquine [n=50]; or chloroquine alone [n=54]). Relapse-free efficacy at 6 months was 57·7% (95% CI 43-70) with tafenoquine 50 mg, 54·1% (40-66) with tafenoquine 100 mg, 89·2% (77-95) with tafenoquine 300 mg, 91·9% (80-97) with tafenoquine 600 mg, 77·3% (63-87) with primaquine, and 37·5% (23-52) with chloroquine alone. Tafenoquine 300 mg and 600 mg had better efficacy than chloroquine alone (treatment differences 51·7% [95% CI 35-69], p<0·0001, with tafenoquine 300 mg and 54·5% [38-71], p<0·0001, with tafenoquine 600 mg), as did primaquine (treatment difference 39·9% [21-59], p=0·0004). Adverse events were similar between treatments. 29 serious adverse events occurred in 26 (8%) of 329 patients; QT prolongation was the most common serious adverse event (11 [3%] of 329), occurring in five (2%) of 225 patients receiving tafenoquine, four (8%) of 50 patients receiving primaquine, and two (4%) of 54 patients receiving chloroquine alone, with no evidence of an additional effect on QT of chloroquine plus tafenoquine coadministration. INTERPRETATION Single-dose tafenoquine 300 mg coadministered with chloroquine for P vivax malaria relapse prevention was more efficacious than chloroquine alone, with a similar safety profile. As a result, it has been selected for further clinical assessment in phase 3. FUNDING GlaxoSmithKline, Medicines for Malaria Venture.

Efficacy and Toxicity of Sodium Stibogluconate for Mucosal Leishmaniasis

OBJECTIVE To determine the efficacy and toxicity of the World Health Organizations (WHO) recommended treatment for mucosal leishmaniasis: antimony, 20 mg/kg body weight per day for 28 days. DESIGN Open trial with 12-month follow-up. SETTING Inpatient unit of a regional referral hospital in a developing country. PATIENTS Twenty-nine consecutive eligible patients with culture-confirmed infection of the mucosa with Leishmania species who were otherwise healthy. Eight patients (28%) had mild to moderate disease (confined to the nasal mucosa). Twenty-one patients (72%) had severe disease (including the oropharynx as well as the nasal mucosa). INTERVENTION Antimony, 20 mg/kg body weight intravenously every day for 28 days. Patients received antimony in the form of sodium stibogluconate. MEASUREMENTS AND MAIN RESULTS Initial results of therapy were as follows: 63 of 72 lesions (88%) healed or markedly improved; all lesions were culture-negative for parasites; and 18 of 29 patients (62%) showed complete clinical and parasitologic cure of all lesions. By the 12-month follow-up examinations, however, 37 lesions had recurred, 8 new lesions had appeared, and only 8 patients (30%) showed clinical cure of all lesions. Of the 8 patients with mild to moderate disease, 6 were cured compared with only 2 of the 21 patients with severe disease. Side effects of this treatment regimen included T-wave inversion on electrocardiogram (4 patients), abnormal liver function tests (10 patients), and musculoskeletal pain (24 patients). No side effects occurred during week 1 of therapy. CONCLUSIONS The only recommended treatment for mucosal leishmaniasis is ineffective in patients with severe disease. The acceptable toxicity of the regimen suggests that longer courses of therapy with antimony, or that trials with other antileishmanial agents alone or combined with antimony be evaluated as initial therapy for this disease.

Extreme inbreeding in Leishmania braziliensis

Leishmania species of the subgenus Viannia and especially Leishmania braziliensis are responsible for a large proportion of New World leishmaniasis cases. The reproductive mode of Leishmania species has often been assumed to be predominantly clonal, but remains unsettled. We have investigated the genetic polymorphism at 12 microsatellite loci on 124 human strains of Leishmania braziliensis from 2 countries, Peru and Bolivia. There is substantial genetic diversity, with an average of 12.4 ± 4.4 alleles per locus. There is linkage disequilibrium at a genome-wide scale, as well as a substantial heterozygote deficit (more than 50% the expected value from Hardy−Weinberg equilibrium), which indicates high levels of inbreeding. These observations are inconsistent with a strictly clonal model of reproduction, which implies excess heterozygosity. Moreover, there is large genetic heterogeneity between populations within countries (Wahlund effect), which evinces a strong population structure at a microgeographic scale. Our findings are compatible with the existence of population foci at a microgeographic scale, where clonality alternates with sexuality of an endogamic nature, with possible occasional recombination events between individuals of different genotypes. These findings provide key clues on the ecology and transmission patterns of Leishmania parasites.

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 Peru.

Randomized, double-blind clinical trial of topical imiquimod 5% with parenteral meglumine antimoniate in the treatment of cutaneous leishmaniasis in Peru.

BACKGROUND Current treatments for cutaneous leishmaniasis are limited by their toxicity, high cost, and discomfort and the emergence of drug resistance. New approaches, including combination therapies, are urgently needed. We performed a double-blind, randomized trial of therapy with parenteral antimony plus topical imiquimod, an innate immune-response modulator, versus therapy with antimony alone, in subjects with cutaneous leishmaniasis for whom an initial course of antimony therapy had failed. METHODS Forty subjects with clinical resistance to antimony were recruited in Lima, Peru, between February 2001 and December 2002. All subjects received meglumine antimoniate (20 mg/kg/day im or iv) and were randomized to receive either topical imiquimod 5% cream (Aldara; 3M Pharmaceuticals) or vehicle control every other day for 20 days. Lesions and adverse events were evaluated during treatment and at 1, 2, 3, 6, and 12 months after the treatment period. RESULTS The mean number of lesions was 1.2 per person; 71% of the lesions were facial and 76% were ulcerative. There were no major differences between the groups, and all but 2 subjects completed therapy. Mild adverse events were reported by 73% of the subjects, but only erythema occurred more commonly in the imiquimod group (P < or = .02). Lesions resolved more rapidly in the imiquimod group: 50% of the imiquimod group achieved cure at 1 month after the treatment period versus 15% of the vehicle cream group (P < or = .02); 61% of the imiquimod group at 2 months versus 25% of the vehicle cream group (P < or = .03); and 72% of the imiquimod group at 3 months versus 35% of the vehicle cream group (P < or = .02). Residual scarring in the imiquimod group was less prominent than in the vehicle cream group. CONCLUSIONS Combined antimony plus imiquimod treatment was well tolerated, accelerated healing of lesions, and improved scar quality. This therapy may have particular advantages for subjects with facial lesions.

Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes

Plasmodium vivax causes 25–40% of malaria cases worldwide, yet research on this human malaria parasite has been neglected. Nevertheless, the recent publication of the P. vivax reference genome now allows genomics and systems biology approaches to be applied to this pathogen. We show here that whole-genome analysis of the parasite can be achieved directly from ex vivo-isolated parasites, without the need for in vitro propagation. A single isolate of P. vivax obtained from a febrile patient with clinical malaria from Peru was subjected to whole-genome sequencing (30× coverage). This analysis revealed over 18,261 single-nucleotide polymorphisms (SNPs), 6,257 of which were further validated using a tiling microarray. Within core chromosomal genes we find that one SNP per every 985 bases of coding sequence distinguishes this recent Peruvian isolate, designated IQ07, from the reference Salvador I strain obtained in 1972. This full-genome sequence of an uncultured P. vivax isolate shows that the same regions with low numbers of aligned sequencing reads are also highly variable by genomic microarray analysis. Finally, we show that the genes containing the largest ratio of nonsynonymous-to-synonymous SNPs include two AP2 transcription factors and the P. vivax multidrug resistance-associated protein (PvMRP1), an ABC transporter shown to be associated with quinoline and antifolate tolerance in Plasmodium falciparum. This analysis provides a data set for comparative analysis with important potential for identifying markers for global parasite diversity and drug resistance mapping studies.

Putative Leishmania hybrids in the Eastern Andean valley of Huanuco, Peru

During an outbreak of tegumentary leishmaniasis that developed in the 1990s in the Eastern Andean valley of Huanuco, Peru, the coexistence of Andean (uta) and sylvatic leishmaniases was suspected for ecological and geographical reasons, and sympatric sampling was carried out. Seven human isolates of Leishmania were characterized by multilocus enzyme electrophoresis, random amplification of polymorphic DNA and molecular karyotyping. The three methods identified 3 isolates as L. braziliensis, and 4 isolates as putative hybrids with characters of L. braziliensis and L. peruviana. Data from Huanuco are compared to previous results from other areas endemic for uta. Biological and epidemiological implications are discussed.

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

BACKGROUND Antimonials 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. METHODS Thirty-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. RESULTS We 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. CONCLUSION Antimony 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 factors.

Role of Imiquimod and Parenteral Meglumine Antimoniate in the Initial Treatment of Cutaneous Leishmaniasis

BACKGROUND Cutaneous leishmaniasis is a serious public health problem in the developing world. The main therapeutic agent--pentavalent antimony, developed >50 years ago--is expensive, often accompanied by severe adverse effects, and complicated by the emergence of drug resistance. Better therapies are urgently needed. In the present pilot study, we compared the use of imiquimod, an immunomodulatory molecule, to the use of meglumine antimoniate alone and in combination for the initial treatment of cutaneous leishmaniasis. MATERIALS AND METHODS Patients with newly diagnosed cutaneous leishmaniasis were enrolled from a single referral center in Lima, Peru, from August 2005 through October 2005. Patients were randomly assigned to 1 of 3 treatment groups and received either imiquimod 7.5% cream administered topically every other day for 20 days, intravenous meglumine antimoniate administered at a dosage of 20 mg/kg per day every day for 20 days, or combination therapy with both intravenous meglumine antimoniate and imiquimod 7.5% cream. Patients were evaluated weekly and at 1 and 3 months after treatment. Patients who had healed lesions at 3 months were considered to be clinically cured. RESULTS Although several patients showed initial resolution of symptoms with imiquimod treatment alone, all of these patients experienced relapse after treatment discontinuation. Four (57%) of 7 patients treated with meglumine antimoniate alone and 7 (100%) of 7 patients treated with combination therapy were cured. Combination therapy was not only more effective than the other 2 treatments (P<.05) but also led to faster healing and better cosmetic results. CONCLUSION Combination therapy with imiquimod and meglumine antimoniate is a promising regimen for the initial treatment of cutaneous leishmaniasis that warrants additional larger studies.