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BackgroundLeishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between Leishmania isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk.ResultsA SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between Leishmania subgenera. A panel of reference strains representing subgenera Leishmania and Viannia was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95°C ± 0.01 and 77.36°C ± 0.02 for Leishmania and Viannia, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together L. (V.) braziliensis and L. (V.) shawii with a bootstrap value of 100%; while for L. (L.) infantum and L. (L.) amazonensis, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus Viannia amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus Leishmania (average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected Lutzomyia spp. captured in two Brazilian localities.ConclusionsFor all tested samples, a characteristic amplicon melting profile was evidenced for each Leishmania subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil.

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BackgroundBrazilian populations of Lutzomyia longipalpis may constitute a complex of cryptic species, and this report investigates the distribution and number of potential sibling species. One of the main differences observed among Brazilian populations is the type of acoustic signal produced by males during copulation. These copulation song differences seem to be evolving faster than neutral molecular markers and have been suggested to contribute to insemination failure observed in crosses between these sibling species. In previous studies, two main types of copulation songs were found, burst-type and pulse-type. The latter type can, in turn, be further subdivided into five different patterns.MethodsWe recorded male song from 13 new populations of the L. longipalpis complex from Brazil and compared the songs with 12 already available.ResultsOut of these 25 populations, 16 produce burst-type and 9 produce pulse-type songs. We performed a principal component analysis in these two main groups separately and an additional discriminant analysis in the pulse-type group. The pulse-type populations showed a clear separation between the five known patterns with a high correspondence of individuals to their correct group, confirming the differentiation between them. The distinctiveness of the burst-type subgroups was much lower than that observed among the pulse-type groups and no clear population structure was observed. This suggests that the burst-type populations represent a single species.ConclusionOverall, our results are consistent with the existence in Brazil of at least six species of the L. longipalpis complex, one with a wide distribution comprising all the populations with burst-type songs, and five more closely related allopatric siblings with different pulse-type song patterns and more restricted distribution ranges.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0900-8) contains supplementary material, which is available to authorized users.

The eco-epidemiological scenario of spotted fever (SF), a tick-borne disease that affects humans and other animals in several countries around the world, was analyzed in Rio de Janeiro (RJ) State, Brazil. During the last 34 years, 990 SF cases were reported in RJ (the Brazilian state with the highest population density), including 116 cases confirmed by serology (RIFI) or PCR, among 42.39% of the municipalities with reported cases of SF. The epidemiologic dynamics of SF in RJ State are very heterogeneous in time and space, with outbreaks, high mortality rates and periods of epidemiological silence (no SF cases reported). Furthermore, it exhibited a changing epidemiological profile from being rural to becoming an urban disease. This study identified arthropods infected with Rickettsia felis, R. bellii and R. rickettsii, and found that the abundance of ectoparasites was associated with specific hosts. The R. rickettsii-vector-host relationship was most evident in species-specific parasitism. This suggests that the association between dogs, cattle, horses, capybaras and their main ectoparasites, Rhipicephalus sanguineus and Ctenocephalides felis, Rhipicephalus microplus, Dermacentor nitens, and Amblyomma dubitatum, respectively, has a key role in the dynamics of R. rickettsii transmission in enzootic cycles and the maintenance of carrier ectoparasites, thus facilitating the existence of endemic areas with the ability to produce epidemic outbreaks of SF in RJ. This study found confirmed human infections for only the R. rickettsii carrier Amblyomma sculptum, which reinforces the importance of this species as a vector of the pathogen in Brazil. This study can be adapted to different eco-epidemiological scenarios of spotted fever throughout the Americas.