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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. Author summary: Human cutaneous leishmaniasis affects low-income populations living in places far from health services. The early sampling and detection of the parasite are necessary for timely treatment, however there are no uniform sampling criteria, thus the sensitivity of molecular tests may vary due to various factors such as the type of sample, the time of the disease and the parasite load in the lesion. In this study, we compared the performance of three sampling methods for molecular identification of the genus and Leishmania (Viannia) species in Peru. Several analytical methods, including composite reference standards and latent class models, suggested that lancet scraping might be the best approach for parasite genus detection by kDNA-PCR and for parasite species determination by FRET probes-based Nested Real-Time PCR. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax is the most predominant malaria species in Latin America, constituting 71.5% of malaria cases in 2021. With several countries aiming for malaria elimination, it is crucial to prioritize effectiveness of national control programs by optimizing the utilization of available resources and strategically implementing necessary changes. To support this, there is a need for innovative approaches such as genomic surveillance tools that can investigate changes in transmission intensity, imported cases and sources of reintroduction, and can detect molecular markers associated with drug resistance. Methodology/Principal findings: Here, we apply a modified highly-multiplexed deep sequencing assay: Pv AmpliSeq v2 Peru. The tool targets a newly developed 41-SNP Peru barcode for parasite population analysis within Peru, the 33-SNP vivaxGEN-geo panel for country-level classification, and 11 putative drug resistance genes. It was applied to 230 samples from the Peruvian Amazon (2007–2020), generating baseline surveillance data. We observed a heterogenous P. vivax population with high diversity and gene flow in peri-urban areas of Maynas province (Loreto region) with a temporal drift using all SNPs detected by the assay (nSNP = 2909). In comparison, in an indigenous isolated area, the parasite population was genetically differentiated (FST = 0.07–0.09) with moderate diversity and high relatedness between isolates in the community. In a remote border community, a clonal P. vivax cluster was identified, with distinct haplotypes in drug resistant genes and ama1, more similar to Brazilian isolates, likely representing an introduction of P. vivax from Brazil at that time. To test its applicability for Latin America, we evaluated the SNP Peru barcode in P. vivax genomes from the region and demonstrated the capacity to capture local population clustering at within-country level. Conclusions/Significance: Together this data shows that P. vivax transmission is heterogeneous in different settings within the Peruvian Amazon. Genetic analysis is a key component for regional malaria control, offering valuable insights that should be incorporated into routine surveillance. Author summary: Latin America is aiming towards malaria elimination. Genomic surveillance is crucial for a country's malaria strategy, aiding in understanding and stopping the spread of the disease. While widely used for another malaria species (Plasmodium falciparum), limited tools exist for tracking P. vivax, a significant player in malaria-endemic areas outside of Africa, and the primary cause of malaria in Latin America. In this study, we used a new tool, Pv AmpliSeq v2 Peru assay, to examine the genetic makeup of malaria parasites in the Peruvian Amazon. This tool helps us see how the parasites from different areas are connected and tracks markers that could indicate resistance to drugs. We found that the parasites from remote areas in the Amazon were genetically different from parasites in areas surrounding the main city of Iquitos, and parasites in a remote border community were genetically more similar to Brazilian parasites. We also show that the Pv AmpliSeq v2 Peru assay can be used to study parasites from other countries in Latin America, highlighting the broader application in the region. Considering that parasites are not constrained by borders and can easily spread between neighboring countries, a regional approach can be crucial for malaria elimination. [ABSTRACT FROM AUTHOR]