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Aim: Here, we evaluate the ddPCR platform using an evaluated qPCR-based diagnostic assay for the detection of Leishmania infection in Cutaneous Leishmaniasis patients. Methods: A standard curve of cultured Leishmania parasite material and clinical samples of CL patients were tested with ddPCR to determine the analytical and diagnostic performance. Results: The limit of detection of the assay on the ddPCR platform was much higher than the published limit of detection of the same assay on the qPCR platform (100 vs 1 parasites/mL, respectively). Conclusion: While the performance of this assay in ddPCR format was acceptable for research purposes, it is not sufficient for clinical diagnostic purposes. The assay is more suited to the qPCR platform. Keywords: Leishmania, ddPCR, Molecular diagnosis, Cutaneous Leishmaniasis

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. © 2017 León, Muñoz, Hernández, Ayala, Flórez, Teherán, Cubides and Ramírez.

SUMMARYAvian malaria is a vector transmitted disease caused byPlasmodiumand recent studies suggest that variation in its prevalence across avian hosts is correlated with a variety of ecological traits. Here we examine the relationship between prevalence and diversity ofPlasmodiumlineages in southeastern Amazonia and: (1) host ecological traits (nest location, nest type, flocking behaviour and diet); (2) density and diversity of avian hosts; (3) abundance and diversity of mosquitoes; and (4) season. We used molecular methods to detectPlasmodiumin blood samples from 675 individual birds of 120 species. Based on cytochromebsequences, we recovered 89 lineages ofPlasmodiumfrom 136 infected individuals sampled across seven localities.Plasmodiumprevalence was homogeneous over time (dry season and flooding season) and space, but heterogeneous among 51 avian host species. Variation in prevalence among bird species was not explained by avian ecological traits, density of avian hosts, or mosquito abundance. However,Plasmodiumlineage diversity was positively correlated with mosquito abundance. Interestingly, our results suggest that avian host traits are less important determinants ofPlasmodiumprevalence and diversity in southeastern Amazonia than in other regions in which they have been investigated.

Chagas disease is a zoonotic infection widely distributed in tropical and subtropical regions of America, including more than 50% of the Colombian territory. In the last years, an increase of outbreaks of acute Chagas disease has been observed in the east of the country due to environmental changes and mammal movements toward human settlements. Given the importance of dogs (Canis lupus familiaris) as reservoir hosts and sentinels of Trypanosoma cruzi infection across different regions of America, in this study we reported a serological and molecular detection of T. cruzi infection in 242 dogs from an endemic area of Meta department (East of Colombia), with recent emergence of acute Chagas disease outbreaks. The distribution of T. cruzi infection in dogs was not homogeneous, ranging from 0–41.4% and 0–5.1% in different sampling sectors, through serological (ELISA/IFAT) and molecular methods (conventional and real time PCR), respectively. Statistical analysis indicated that dog infection was associated with specific sampling sectors. Our results show a moderate seroprevalence of infection and active circulation of T. cruzi in dogs from this zone, which suggest areas with potential risk of infection to human that must be taken into consideration when Chagas disease control programs need to be implemented. © 2017 Elsevier B.V.

Interest in larval source management (LSM) as an adjunct intervention to control and eliminate malaria transmission has recently increased mainly because long-lasting insecticidal nets (LLINs) and indoor residual spray (IRS) are ineffective against exophagic and exophilic mosquitoes. In Amazonian Peru, the identification of the most productive, positive water bodies would increase the impact of targeted mosquito control on aquatic life stages. The present study explores the use of unmanned aerial vehicles (drones) for identifying Nyssorhynchus darlingi (formerly Anopheles darlingi) breeding sites with high-resolution imagery (~0.02m/pixel) and their multispectral profile in Amazonian Peru. Our results show that high-resolution multispectral imagery can discriminate a profile of water bodies where Ny. darlingi is most likely to breed (overall accuracy 86.73%- 96.98%) with a moderate differentiation of spectral bands. This work provides proof-of-concept of the use of high-resolution images to detect malaria vector breeding sites in Amazonian Peru and such innovative methodology could be crucial for LSM malaria integrated interventions., Author summary The most efficient malaria vector in the Latin American region is Nyssorhynchus darlingi (formerly Anopheles darlingi). In Amazonian Peru, where malaria is endemic, Ny. darlingi feeds both indoors and outdoors (endophagy, exophagy), depending on the local environment, and rests outdoors (exophily). LLINs and IRS, the most common tools employed for vector control, target endophagic and endophilic mosquitoes. Thus, they are only partially effective against Ny. darlingi. Control of the aquatic stages of vector mosquitoes, larval source management (LSM), targets the most productive breeding sites nearest to human habitation. In four riverine communities, we used drones with high-resolution imagery as a key initial step to analyze water bodies within the estimated flight range of Ny. darlingi, ~ 1 km. We found distinctive spectral profiles for water bodies that were positive versus negative for Ny. darlingi. The methodology and analysis reported here provide the basis for testing whether LSM can be combined successfully with LLINs and IRS to contribute to the elimination of transmission in malaria hotspots in the Amazon.