Background. Children have a lower response rate to antimonial drugs and higher elimination rate of antimony (Sb) than adults. Oral miltefosine has not been evaluated for pediatric cutaneous leishmaniasis. Methods. A randomized, noninferiority clinical trial with masked evaluation was conducted at 3 locations in Colombia where Leishmania panamensis and Leishmania guyanensis predominated. One hundred sixteen children aged 2–12 years with parasitologically confirmed cutaneous leishmaniasis were randomized to directly observed treatment with meglumine antimoniate (20 mg Sb/kg/d for 20 days; intramuscular) (n = 58) or miltefosine (1.8–2.5 mg/kg/d for 28 days; by mouth) (n = 58). Primary outcome was treatment failure at or before week 26 after initiation of treatment. Miltefosine was noninferior if the proportion of treatment failures was ≤15% higher than achieved with meglumine antimoniate (1-sided test, α = .05). Results. Ninety-five percent of children (111/116) completed follow-up evaluation. By intention-to-treat analysis, failure rate was 17.2% (98% confidence interval [CI], 5.7%–28.7%) for miltefosine and 31% (98% CI, 16.9%–45.2%) for meglumine antimoniate. The difference between treatment groups was 13.8%, (98% CI, −4.5% to 32%) (P = .04). Adverse events were mild for both treatments. Conclusions. Miltefosine is noninferior to meglumine antimoniate for treatment of pediatric cutaneous leishmaniasis caused by Leishmania (Viannia) species. Advantages of oral administration and low toxicity favor use of miltefosine in children. Clinical Trial Registration. NCT00487253.

Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis. The endosymbiosis of viruses in microbes is a well-described and prevalent environmental partnership, where viruses offer their cellular host incentives of fitness in exchange for the use of their metabolic machinery. We have recently exposed this as an important factor in certain metastatic leishmaniases of South America, where the nucleic acid of a virus residing within some Leishmania parasites acts as a potent innate immunogen causing a destructive inflammatory response, which worsens disease. Leishmania RNA Virus (LRV) exists within many species of Leishmania as a stable infection; these LRV positive strains have been found throughout South America in cutaneous leishmaniases that are often complicated by the occurrence of infectious metastasis with an underlying hyperinflammatory response. In this report, we describe the use of an anti-dsRNA monoclonal antibody (J2), which specifically recognizes dsRNA in a quantitative and sequence-independent fashion. Refined versions of these methods could be transferred to the field as diagnostic tools for detecting the presence of LRV (or other dsRNA viruses), and potentially assessing the LRV-related risk of complicated cutaneous leishmaniasis.

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 ofLeishmaniaDNA 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 forLeishmaniaDNA, 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.

Pentavalent 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. 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 (Sb^V); 163, (80%) were evaluated for both drugs. Additionally, susceptibility to Sb^V 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 Sb^V. Resistance to HePC and Sb^V occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to Sb^V. Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to Sb^V 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. Large 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. Treatment of dermal leishmaniasis is unsuccessful in an important proportion of cases and evidence of loss of susceptibility to antimonial drugs and miltefosine has been demonstrated in some cases of treatment failure with these medications. Despite the variability in the clinical outcome of treatment, little is known about the susceptibility of the different species and diverse populations of these species circulating in areas of high transmission. Based on 402 strains isolated from patients, the susceptibility of the Leishmania species most frequently causing dermal leishmaniasis in Colombia was determined to first and second line medications commonly used in Latin America. The results showed that susceptibility to each drug varied among the species and populations of each species, geographically between regions east and west of the Andes mountain range, during different time periods over 30 years, and within different epidemiological circumstances. The findings provide a comprehensive picture of drug susceptibility of dermal leishmaniasis in Colombia and baseline information for monitoring the emergence of drug resistance.

Toll-like receptors (TLRs) play a central role in macrophage activation and control of parasitic infections. Their contribution to the outcome ofLeishmaniainfection is just beginning to be deciphered. We examined the interaction ofLeishmania panamensiswith TLRs in the activation of host macrophages.L. panamensisinfection resulted in upregulation of TLR1, TLR2, TLR3, and TLR4 expression and induced tumor necrosis factor alpha (TNF-α) secretion by human primary macrophages at comparable levels and kinetics to those of specific TLR ligands. The TLR dependence of the host cell response was substantiated by the absence of TNF-α production in MyD88/TRIF^−/−murine bone marrow-derived macrophages and mouse macrophage cell lines in response to promastigotes and amastigotes. Systematic screening of TLR-deficient macrophages revealed that TNF-α production was completely abrogated in TLR4^−/−macrophages, consistent with the increased intracellular parasite survival at early time points of infection. TNF-α secretion was significantly reduced in macrophages lacking endosomal TLRs but was unaltered by a lack of TLR2 or MD-2. Together, these findings support the participation of TLR4 and endosomal TLRs in the activation of host macrophages byL. panamensisand in the early control of infection.