RESUMO.-O objetivo do trabalho foi analisar os dados relacionados à transmissão da leishmaniose visceral (LV), no município de Fortaleza, e discutir a respeito da distribuição do vetor, reservatório doméstico e casos humanos ocorridos no The objective was to analyze the data relating to the transmission of visceral leishmaniasis (LV) in Fortaleza municipality, and discuss about the vector distribution, domestic reservoir and human cases from 2009 to 2013.This study is descriptive conducted through a survey of secondary data. The correlation between human and canine cases and sandflies number was taken by Spearman correlation test, with 5% significance level. In the period 2009 to 2013, 941 cases and 55 deaths were confirmed. The mortality rate for the period was 5.84%. There was a higher proportion of cases among men in all the years analyzed. The age group 1-4 years old had the highest percentage of cases, highlighting the year 2010 with 31.5% of the cases. The average distribution of cases by SER demonstrated a predominance in SER I, V and VI. With regard to the vector species found in greater abundance was Lutzomyia (Lutzomyia) longipalpis, followed by Lutzomyia (Lutzomyia) migonei and Lutzomyia (Lutzomyia) lenti. According to the records, 39,626 dogs were serum reagents for LV in the years 2009-2013, these 14,313 were killed. The year 2013 stood out with 17,808 dogs serum reagents, and 1,273 of SER III, 2572 of SER V and 1909 of SER VI. There was no significant correlation between the number of sand flies and the number of canine VL cases (p>0.05). There was a negative correlation between human and canine cases (r = -0.0388) and correlation was observed between human cases and number of sand flies (r= 0.7469). Our findings create prospects for the identification of other factors that may influence the incidence of human and canine cases, such as the participation of other possible reservoirs and vectors in the transmission of the visceral leishmaniasis in Fortaleza.

No abstract

Visceral leishmaniasis (VL) in Brazil is caused by the protozoan Leishmania infantum. This parasite is transmitted by the bite of a female sand fly. The most important sand fly species in VL transmission is Lutzomyia longipalpis. In Fortaleza, the capital of Ceará State, Brazil, the simultaneous occurrence of Lutzomyia migonei and L. longipalpis was detected in localities where VL transmission is observed. The purpose of this study was to determine conclusively if L. migonei can be found naturally infected with L. infantum in key focus in Fortaleza. Using a CDC traps we performed phlebotomine capture during one year. External morphological features and qPCR targeting species-specific gene sequences of Lutzomyia species were used to identify the female phlebotomine sand flies. The molecular identification of the Leishmania species was performed using qPCR targeting species-specific gene sequences of L. infantum and Leishmania braziliensis. The males L. migonei abundance was higher in the rainy season. Humidity and rainfall positively correlated with males L. migonei abundance, while temperature showed a negative correlation. The correlation between the density of L. migonei female with rainfall, relative air humidity, and temperature were not statistically significant. According to the molecular data produced by qPCR amplifications, three positive sand flies were identified as L. longipalpis, and one was identified as L. migonei. The infection rate was 0.35% and 0.18%, respectively. The parasite load was 32,492±2572 L. infantum in L. migonei while the L. longipalpis had parasite loads between 2,444,964.6±116,000 and 6,287,130±124,277. Our findings confirm L. migonei as a potential vector of VL in Fortaleza at a molecular level.

The objective of this study was to perform an epidemiological survey to determine the areas at risk of visceral leishmaniasis through the detection and quantification of natural infection by Leishmania infantum in Lutzomyia longipalpis. The sandflies were captured between February 2009 and January 2010, at 21 sites in four regions of the Fortaleza municipality. Samples were screened for the presence of Leishmania DNA by Real Time PCR (qPCR), amplification of kDNA minicircle sequence. Out of the 123 pools of analyzed sandflies, 45 were positive for L.infantum, and the minimum infection rate was 3.7%. In the north, south, east and west regions, the pool screen assay predicted sand-fly infection prevalence of 3.4%, 4.7%, 4.9% and 8.4%, respectively. The parasite load ranged from 2.45 ± 0.96 to 2,820,246 ± 106,072. No statistical differences were found with respect to the frequency of sand-fly infection between the regions (P=0.3014), seasons (P = 0.3906) or trap locations (P = 0.8486). Statistical differences were found with respect to the frequency of sand-fly infection between the two seasons only in the west region (P=0.0152). The qPCR was able to detect and quantify L. infantum in L. longipalpis, therefore succeeding in identifying the areas of greatest risk of VL transmission.Keywords: Leishmania infantum, Lutzomyia longipalpis, minimum infection rate, qPCR. ResumoO objetivo foi realizar um estudo epidemiológico para determinar as áreas de risco de transmissão de leishmaniose visceral pela detecção e quantificação de infecções naturais por Leishmania infantum em Lutzomyia longipalpis. As coletas foram realizadas entre fevereiro de 2009 e janeiro de 2010 em 21 locais, distribuídos em quatro regiões do município de Fortaleza. As amostras foram testadas quanto à presença de DNA de Leishmania por PCR em tempo real (qPCR). Dos 123 pools de flebotomíneos investigados, 45 foram positivos para L. infantum, e a taxa de infecção mínima foi de 3,7%. Nas regiões Norte, Sul, Leste e Oeste, a prevalência de flebotomíneos infectados foi de 3,4%, 4,7%, 4,9% e 8,4%, respectivamente. A carga de parasitas nos pools variou de 2,45 ± 0,96 a 2.820.246 ± 106.072. Não foram observadas diferenças significativas na frequência de flebotomíneos infectados entre as regiões (P = 0,3014), estação do ano (P = 0,3906) ou localização da armadilha (P = 0,8486). Foram observadas diferenças significativas na frequência de flebotomíneos somente na região oeste durante a estação chuvosa (P = 0,0152). A qPCR foi capaz de detectar e quantificar L. infantum em L. longipalpis, identificando as áreas de maior risco de transmissão de leishmaniose visceral.Palavras-chave: Leishmania infantum, Lutzomyia longipalpis, taxa de infecção mínima, qPCR.

Phlebotomine sand flies are blood-feeding insects of marked medical and veterinary significance. Investigations on the biology of these insects hold great importance for both ecological and epidemiological purposes. The present work describes a new approach for real-time PCR (qPCR) with SYBR Green, named WMG-qPCR, to identify phlebotomine blood meals. The novelty of the assay was to design primers based on the Whole Mitochondrial Genome (WMG) of the potential hosts (human, dog, cat, brown rat and chicken) aiming to amplify through qPCR the regions of mitochondrial DNA (mtDNA) which are less conserved among all species. Initially, the best method for mtDNA extraction to be applied in WMG-qPCR was determined. Afterwards, amplification specificities were accessed by cross-reaction assays with mtDNA samples from all animal species, besides phlebotomine DNA. Finally, the selected primers were also tested for their limit of DNA detection through standard curves constructed by serial dilution of blood DNA obtained for each target animal species. The WMG-qPCR was able to detect as low as 10pL of blood, equivalent to 26, 84, 130, and 320fg DNA of cat, human, dog and rat, respectively. The assay was also capable to amplify as low as 5pL of chicken blood (5pg DNA). In conclusion, WMG-qPCR seems to be a promising tool to identify phlebotomine blood meals, with high species-specificity and sensitivity. Furthermore, as no supplementary techniques are required, this new approach presents minimized costs and simplified technical-training requirements for execution.

Lutzomyia longipalpis is the main vector of Leishmania infantum, the causative agent of visceral leishmaniasis in the Neotropical realm. Its taxonomic status has been widely discussed once it encompasses a complex of species. The knowledge about the genetic structure of insect vector populations helps the elucidation of components and interactions of the disease ecoepidemiology. Thus, the objective of this study was to genotypically analyze populations of the Lu. longipalpis complex from a macrogeographic perspective using Next Generation Sequencing. Polymorphism analysis of three molecular markers was used to access the levels of population genetic structure among nine different populations of sand flies. Illumina Amplicon Sequencing Protocol® was used to identify possible polymorphic sites. The library was sequenced on paired-end Illumina MiSeq platform. Significant macrogeographical population differentiation was observed among Lu. longipalpis populations via PCA and DAPC analyses. Our results revealed that populations of Lu. longipalpis from the nine municipalities were grouped into three clusters. In addition, it was observed that the levels of Lu. longipalpis population structure could be associated with distance isolation. This new sequencing method allowed us to study different molecular markers after a single sequencing run, and to evaluate population and inter-species differences on a macrogeographic scale.