Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

Genetic diversity and population structure of Leishmania (Viannia) braziliensis in the Peruvian jungle.

BACKGROUND: Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is highly prevalent in the Peruvian jungle, where it affects military forces deployed to fight against drug trafficking and civilian people that migrate from the highland to the lowland jungle for economic activities such as mining, agriculture, construction, and chestnut harvest. We explored the genetic diversity and population structure of 124 L. (V.) braziliensis isolates collected from the highland (Junín, Cusco, and Ayacucho) and lowland Peruvian jungle (Loreto, Ucayali, and Madre de Dios). All samples were genotyped using Multilocus Microsatellite Typing (MLMT) of ten highly polymorphic markers. PRINCIPAL FINDINGS: High polymorphism and genetic diversity were found in Peruvian isolates of L. (V.) braziliensis. Most markers are not in Hardy-Weinberg equilibrium; this deviation is most likely caused by local inbreeding, as shown by the positive FIS values. Linkage Disequilibrium in subpopulations was not strong, suggesting the reproduction was not strictly clonal. Likewise, for the first time, two genetic clusters of this parasite were determined, distributed in both areas of the Peruvian jungle, which suggested a possible recent colonization event of the highland jungle from the lowland jungle. CONCLUSIONS: L. (V.) braziliensis exhibits considerable genetic diversity with two different clusters in the Peruvian jungle. Migration analysis suggested a colonization event between geographical areas of distribution. Although no human migration was observed at the time of sampling, earlier displacement of humans, reservoirs, or vectors could have been responsible for the parasite spread in both regions.