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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: In Peru, the information regarding sand fly vectors of leishmaniasis and bartonellosis in the Amazon region is limited. In this study, we carried out sand fly collections in Peruvian lowland and highland jungle areas using different trap type configurations and screened them for Leishmania and Bartonella DNA. Methodology/Principal findings: Phlebotomine sand flies were collected in Peruvian Amazon jungle and inter Andean regions using CDC light trap, UV and color LED traps, Mosquito Magnet trap, BG Sentinel trap, and a Shannon trap placed outside the houses. Leishmania spp. screening was performed by kDNA PCR and confirmed by a nested cytochrome B gene (cytB) PCR. Bartonella spp. screening was performed by ITS PCR and confirmed by citrate synthase gene (gltA). The PCR amplicons were sequenced to identify Leishmania and Bartonella species. UV and Blue LED traps collected the highest average number of sand flies per hour in low jungle; UV, Mosquito Magnet and Shannon traps in high jungle; and Mosquito Magnet in inter Andean region. Leishmania guyanensis in Lutzomyia carrerai carrerai and L. naiffi in Lu. hirsuta hirsuta were identified based on cytB sequencing. Bartonella spp. related to Bartonella bacilliformis in Lu. whitmani, Lu. nevesi, Lu. hirsuta hirsuta and Lu. sherlocki, and a Bartonella sp. related to Candidatus B. rondoniensis in Lu. nevesi and Lu. maranonensis were identified based on gltA gene sequencing. Conclusions/Significance: UV, Blue LED, Mosquito Magnet and Shannon traps were more efficient than the BG-Sentinel, Green, and Red LED traps. This is the first report of L. naiffi and of two genotypes of Bartonella spp. related to B. bacilliformis and Candidatus B. rondoniensis infecting sand fly species from the Amazon region in Peru. Author summary: Leishmaniasis and human bartonellosis, two neglected tropical diseases, are endemic in Peruvian Andean regions. However, no human bartonellosis cases has been reported in the Amazon jungle where leishmaniasis has high prevalence. The improvement of sand fly trapping methods and identification of putative Leishmania and Bartonella vectors across Andean and Amazonian regions are important steps towards the design of better control strategies and reducing the risk of transmission in endemic areas. In this paper the authors explored the effectiveness of different trap types, based in LED colors and odor attractants, for sand fly collections in endemic areas of leishmaniasis and bartonellosis. Also, we reported for the first time the detection of Bartonella DNA in sand fly species from the Peruvian Amazon jungle, and the first record in Peru of L. (V.) naiffi infecting Lu. hirsuta hirsuta. The information provided by this manuscript will serve as a baseline for future surveillance and intervention studies about sand fly species as potential vectors of Leishmania and Bartonella in highly endemic areas. [ABSTRACT FROM AUTHOR]

The Peruvian-Brazilian border is a highly endemic tegumentary leishmaniasis region in South America. The interoceanic highway is a commercial route that connects Peru and Brazil through Madre de Dios and has raised concerns about its impact on previously undisturbed areas. In order to assess leishmaniasis transmission risk along this highway, we conducted a surveillance study of the sand fly populations in this area. Sand flies were collected between 2009 and 2010 along transects at 200 m, 600 m and 1000 m from six study sites located along the highway (Iberia, La Novia, Alto Libertad, El Carmen, Florida Baja, Mazuko and Mavila) and an undisturbed area (Malinowski). Collected specimens were identified based on morphology and non-engorged females of each species were pooled and screened by kinetoplast PCR to detect natural Leishmania infections. A total of 9,023 specimens were collected belonging to 54 different Lutzomyia species including the first report of Lu. gantieri in Peru. Four species accounted for 50% of all specimens (Lutzomyia carrerai carrerai, Lu. davisi, Lu. shawi and Lu. richardwardi). El Carmen, Alto Libertad, Florida Baja and Malinowski presented higher Shannon diversity indexes (H = 2.36, 2.30, 2.17 and 2.13, respectively) than the most human disturbed sites of Mazuko and La Novia (H = 1.53 and 1.06, respectively). PCR detected 10 positive pools belonging to Lu. carrerai carrerai, Lu. yuilli yuilli, Lu. hirsuta hirsuta, Lu. (Trichophoromyia) spp., and Lu. (Lutzomyia) spp. Positive pools from 1,000 m transects had higher infectivity rates than those from 600 m and 200 m transects (9/169 = 5.3% vs 0/79 = 0% and 1/127 = 0.8%, p = 0.018). El Carmen, accounted for eight out of ten positives whereas one positive was collected in Florida Baja and Mazuko each. Our study has shown differences in sand fly diversity, abundance and species composition across and within sites. Multiple clustered Lutzomyia pools with natural Leishmania infection suggest a complex, diverse and spotty role in leishmaniasis transmission in Madre de Dios, with increased risk farther from the highway. Author summary: Leishmaniasis is a neglected disease that is endemic in tropical and subtropical regions affecting up to 2 million people every year. Currently, there is concern about the effects of human-made environmental changes on the transmission of leishmaniasis due to increased exposure to the sand fly vector. In this paper, the authors explored changes in the distribution of sand flies and natural leishmania infection across communities located along the interoceanic highway that connects Peru and Brazil. The study found differences in sand fly species composition and abundance between and within sites as well as a lower sand fly diversity in communities with high human disturbance. Ten pools belonging to Lutzomyia shawi, Lu. carrerai carrerai, Lu. yuilli yuilli, Lu. hirsuta hirsuta, Lu. (Helcocyrtomyia) spp. and Lu. (Lutzomyia) spp. were found positive for Leishmania spp. The majority of these positive pools were collected from a single community and far from the highway. The information provided by this manuscript will serve as a baseline to assess the effects of human activities in the region and guide future surveillance and intervention strategies. [ABSTRACT FROM AUTHOR]

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mannose phosphate isomerase (mpi) gene was applied to 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru for identification of the infecting parasite at the species level, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Although most results (121/134) including 4 hybrids of Leishmania (Viannia) braziliensis and L. (V.) peruviana corresponded to those obtained in the previous study, PCR-RFLP analyses revealed the distribution of putative hybrid strains between L. (V.) peruviana and L. (V.) lainsoni in two samples, which has never been reported. Moreover, parasite strains showing discordance between kinetoplast and nuclear genes (kDNA and nDNA), so-called mito-nuclear discordance, were identified in 11 samples. Of these, six strains had the kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) guyanensis, and three strains had the kDNAs of L. (V.) shawi and nDNAs of L. (V.) braziliensis. The rest were identified as mito-nuclear discordance strains having kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) lainsoni, and kDNAs of L. (V.) lainsoni and nDNAs of L. (V.) braziliensis. The results demonstrate that Leishmania strains in Peru are genetically more complex than previously considered. Author summary: Protozoan parasites of the genus Leishmania are able to undergo genetic exchange during their growth. The previous description of hybrids in Peru and the recent discovery of unexpected genetically complex strains having characteristics of both hybrid and mito-nuclear discordance in its neighbouring country (Ecuador) with a similar eco-epidemiological situation led us to consider that the genetic structure of Leishmania strains in Peru is more complicated than previously thought. In an effort to revise the data on Leishmania strain dispersion in Peru and to search for evidence of genetic recombination, the present study was conducted. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis targeting the mannose phosphate isomerase (mpi) gene sequence was performed to identify the infecting parasite at the species level in 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Most results (121/134) including 4 hybrids between L. (V.) braziliensis and L. (V.) peruviana showed agreement between PCR-RFLP of the mpi gene and cyt b gene sequence analysis; however, 13 of 134 samples revealed the distribution of strains with hybrids and mito-nuclear discordance. The results demonstrate that genetically complex Leishmania strains are present in Peru. These findings indicate that Leishmania strain dispersion in Peru is genetically more complex than previously considered. Further prospective studies including larger samples and the isolation of parasite strains are required to update the available data. [ABSTRACT FROM AUTHOR]

To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru. [ABSTRACT FROM AUTHOR]