SUMMARY Variation in clinical accuracy of molecular diagnostic methods for cutaneous leishmaniasis (CL) is commonly observed depending on the sample source, the method of DNA recovery and the molecular test. Few attempts have been made to compare these variables. Two swab and aspirate samples from lesions of patients with suspected CL (n = 105) were evaluated alongside standard diagnosis by microscopic detection of amastigotes or culture of parasites from lesion material. Three DNA extraction methods were compared: Qiagen on swab and aspirate specimens, Isohelix on swabs and Boil/Spin of lesion aspirates. Recovery of Leishmania DNA was evaluated for each sample type by real-time polymerase chain reaction detection of parasitic 18S rDNA, and the diagnostic accuracy of the molecular method determined. Swab sampling combined with Qiagen DNA extraction was the most efficient recovery method for Leishmania DNA, and was the most sensitive (98%; 95% CI: 91–100%) and specific (84%; 95% CI: 64–95%) approach. Aspirated material was less sensitive at 80% (95% CI: 70–88%) and 61% (95% CI: 50–72%) when coupled to Qiagen or Boil-Spin DNA extraction, respectively. Swab sampling of lesions was painless, simple to perform and coupled with standardized DNA extraction enhances the feasibility of molecular diagnosis of CL.

IntroductionReports of therapeutic failure to meglumine antimoniate (MA) and miltefosine in cutaneous leishmaniasis (CL) varies between species, populations and geographic regions. This study aimed to determine the clinical, drug-related factors, and Leishmania species associated with treatment failure in children and adults with cutaneous leishmaniasis.MethodsA cohort study was performed with children (2–12 years old) and adults (18–65 years old) with CL, who have participated in clinical studies at CIDEIM Cali, Tumaco and Chaparral. Incidence of therapeutic failure was estimated by treatment and age groups. Descriptive, bivariate, and multiple logistic regression analyses were performed for the complete cohort and pediatric patients.ResultsTwo hundred and thirty patients were included (miltefosine: 112; MA: 118), of which 60.4% were children and 83.9% were infected with L.V. panamensis. Overall incidence of therapeutic failure was 15.65% (95%CI: 10.92–20.38), and was lower for miltefosine than for MA (8.92%, 95%CI: 3.59–14.26 versus 22.03%, 95%CI:14.48–29.58, p = 0.006). Treatment failure was associated with age ≤8 years (OR: 3.29; 95%CI: 1.37–7.89), disease duration ≤1 month (OR: 3.29; 95%CI: 1.37–7.89), regional lymphadenopathy (OR: 2.72; 95%CI: 1.10–6.70), treatment with MA (OR: 3.98; 95%CI: 1.66–9.50), and adherence <90% (OR: 3.59; 95%CI: 1.06–12.11). In children, higher Z-score of height/age was a protective factor (OR: 0.58; 95%CI: 0.36–0.93), while treatment with MA was a risk factor (OR: 40.82; 95%CI: 2.45–677.85), demonstrating significant interaction with age (p = 0.03).ConclusionsClinical and drug-related factors determine therapeutic failure in CL. High risk of failure in children treated with MA indicates the need to reconsider this drug as first line treatment in this population.Trial registrationClinical trial registration: NCT00487253Clinical trial registration: NCT01462500Clinical trial registration: NCT01464242

An open-label pharmacokinetics (PK) clinical trial was conducted to comparatively assess the PK and explore the pharmacodynamics (PD) of miltefosine in children and adults with cutaneous leishmaniasis (CL) in Colombia. Sixty patients, 30 children aged 2 to 12 years and 30 adults aged 18 to 60 years, were enrolled. Participants received miltefosine (Impavido) at a nominal dose of 2.5 mg/kg/day for 28 days. Miltefosine concentrations were measured in plasma and peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry of samples obtained during treatment and up to 6 months following completion of treatment, when therapeutic outcome was determined. Fifty-two patients were cured, 5 pediatric patients failed treatment, and 3 participants were lost to follow-up. Leishmania (Viannia) panamensis predominated among the strains isolated (42/46; 91%). Noncompartmental analysis demonstrated that plasma and intracellular miltefosine concentrations were, overall, lower in children than in adults. Exposure to miltefosine, estimated by area under the concentration-time curve and maximum concentration, was significantly lower in children in both the central and intracellular compartments (P < 0.01). Leishmania persistence was detected in 43% of study participants at the end of treatment and in 27% at 90 days after initiation of treatment. Clinical response was not dependent on parasite elimination. In vitro miltefosine susceptibility was similar for Leishmania strains from adults and children. Our results document PK differences for miltefosine in children and adults with cutaneous leishmaniasis that affect drug exposure and could influence the outcome of treatment, and they provide bases for optimizing therapeutic regimens for CL in pediatric populations. (This study has been registered at ClinicalTrials.gov under identifier NCT01462500.)

BackgroundThe contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking.MethodsWe investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed.Principal FindingsPersistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus.ConclusionsOur results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.

BackgroundThe determinants of parasite persistence or elimination after treatment and clinical resolution of cutaneous leishmaniasis (CL) are unknown. We investigated clinical and parasitological parameters associated with the presence and viability of Leishmania after treatment and resolution of CL caused by L. Viannia.MethodsSeventy patients who were treated with meglumine antimoniate (n = 38) or miltefosine (n = 32) and cured, were included in this study. Leishmania persistence and viability were determined by detection of kDNA and 7SLRNA transcripts, respectively, before, at the end of treatment (EoT), and 13 weeks after initiation of treatment in lesions and swabs of nasal and tonsillar mucosa.ResultsSixty percent of patients (42/70) had evidence of Leishmania persistence at EoT and 30% (9/30) 13 weeks after treatment initiation. A previous episode of CL was found to be a protective factor for detectable Leishmania persistence (OR: 0.16, 95%CI: 0.03–0.92). kDNA genotyping could not discern differences between parasite populations that persisted and those isolated at diagnosis.ConclusionsLeishmania persist in skin and mucosal tissues in a high proportion of patients who achieved therapeutic cure of CL. This finding prompts assessment of the contribution of persistent infection in transmission and endemicity of CL, and in disease reactivation and protective immunity.