Claudiney Biral dos Santos

Evaluation of a novel chromatographic immunoassay based on Dual-Path Platform technology (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is the major source of human visceral leishmaniasis (VL) and is transmitted from dogs to sand flies to humans. To control the spread of this disease, early and accurate detection of infected dogs is critical but challenging. Here we demonstrate the potential of the Dual-Path Platform (DPP(®)) CVL rapid test for detecting K26/K39-reactive antibodies in sera from clinically symptomatic (n=60) and asymptomatic (n=60) Leishmania infantum-infected dogs. For the specificity evaluation, assays were performed using known negative diagnostic serum samples (n=59) and cross-reaction control sera (n=11) from animals born in a VL-free area of Brazil. The diagnostic kit displayed high specificity (96%) but low sensitivity (47%) in identifying parasite-positive dogs without signs of CVL. However, the test sensitivity was significantly higher (98%) in diseased cases, indicating that this convenient test may be useful to identify the most infectious dogs. Efforts should be pursued to obtain a more sensitive DPP-multiplexed test parameter (i.e. based on simultaneous yet separate antibody detection of carefully selected multiple antigens of diagnostic utility) for effective serodiagnosis of early-infected dogs, as this will likely allow more efficient canine removal regimens than those used in practice by public health services.

Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil

Between 1985 and 2000, epidemiological surveys of the American tegumentary leishmaniasis (ATL) were carried out in several rural and urban communities in Espírito Santo, Brazil. A total of 100 stocks of Leishmania (comprising isolates from both human and canine hosts with ATL) were identified by two methods of molecular characterization, using specific monoclonal antibodies and multilocus enzyme electrophoresis. Parasite isolates from 19 municipalities were found to belong to the same zymodeme and serodeme type as of the Leishmania (Viannia) braziliensis reference strain. In contrast, our genotyping studies have shown intra-specific variation among these parasites (comparisons of the variability of the internal transcribed spacers between the small and large subunits of the rRNA genes of the 22 stocks studied revealed at least 11 genotypes). Two main clusters of L. (V.) braziliensis genotypes were observed, representing parasites collected from different endemic regions in the state, where transmission reflects distinct eco-epidemiological features. Infection with this pathogen was associated with the characteristic disease forms, but neither the clinical outcome nor the response to treatment could be related to the genetic polymorphism of the isolates, as defined by using the proposed methodology.

Survey of natural infection by Leishmania in sand fly species collected in southeastern Brazil.

In this study, we sought to identify sand fly vectors of the Leishmania species that circulate in distinct eco-epidemiological disease-endemic rural areas within the Espírito Santo State in southeastern Brazil. PCR amplification of a conserved region of the minicircle kDNA was used to estimate infection rates in field-captured, peridomestic female sand flies. Only 13 of the 1689 female sand fly specimens (0.77%) actually contained Leishmania DNA. Leishmania braziliensis infections were found in Lutzomyia intermedia and Lu. whitmani, and, for the first time, in Lu. fischeri and Lu. ferreirana. Interestingly, the high rate of genetic polymorphism of the L. braziliensis parasites in one of the disease-endemic areas that were studied may reflect specific transmission cycles involving different sand fly vectors.

Criadouros de Aedes (Stegomyia) aegypti (Linnaeus, 1762) em bromélias nativas na Cidade de Vitória, ES

Some insects that are vectors of human diseases have accompanied man in his migrations throughout the world and breed exclusively in the proximity of human dwellings. The mosquito Aedes aegypti has been responsible for epidemics of dengue in Brazil and its presence also constitutes a serious risk for future outbreaks of urban yellow fever. The failure of campaigns to eradicate this species justifies the search for alternative breeding sites, which may be beyond the reach of present control measures. In this study the occurrence of Aedes aegypti breeding sites in native bromeliads on rocky slopes was investigated in five areas of Vitoria, capital of the Brazilian State of Espirito Santo, ES. Water contained in the bromeliads was collected with the aid of a suction apparatus to search for culicid larvae. The degree of infestation of buildings in adjacent urban areas was evaluated simultaneously. Culicid larvae were found in bromeliads in four of the five areas investigated, Aedes aegypti being present in two areas. The presence of breeding sites in bromeliads was not related to indices of infestation of buildings in adjacent areas. Further studies are necessary to define whether breeding sites in bromeliads constitute primary foci of Aedes aegypti, or are a consequence of high infestation levels in urban areas.

The Effect of Removing Potentially Infectious Dogs on the Numbers of Canine Leishmania infantum Infections in an Endemic Area with High Transmission Rates

To assess the effect of the rapid removal of potentially infectious dogs on the prevalence and incidence of canine infections, a prospective study was undertaken in an area endemic for Leishmania infantum. We used serological testing based on the rapid DPP rK28 fusion protein chromatographic immunoassay for this dog screening-and-culling intervention trial. The outcome was evaluated by measuring seropositivity and sero-conversion/-reversion rates for canine infection. Our estimates indicated that concomitant detection and elimination of seropositive dogs with active disease may affect the numbers of canine infections and disease burden temporarily, although it is insufficient as a measure to interrupt the zoonotic L. infantum transmission. However, most of the asymptomatic, seropositive dogs continuously exhibit low levels of antibodies and/or reverted, remaining seronegative thereafter. In the process of waiting for an effective vaccine, one option for canine reservoir control may be to identify these possibly genetically resistant animals and promote their expansion in the population.

Peridomiciliary colonies of Triatoma vitticeps (Stal, 1859) (Hemiptera, Reduviidae, Triatominae) infected with Trypanosoma cruzi in rural areas of the state of Espírito Santo, Brazil

In Brazil, the colonization of human dwellings by triatomines occurs in areas with native vegetation of the caatinga or cerrado types. In areas of Atlantic forest such as in the Brazilian state of Espírito Santo, there are no species adapted to live in human habitations. The few autochthonous cases of Chagas disease encountered in Espírito Santo have been attributed to adult specimens of Triatoma vitticeps that invade houses from forest remnants. In recent years, the entomology unit of the Espírito Santo State Health Secretariat has recorded nymphs infected with flagellates similar to Trypanosoma cruzi in rural localities. Entomological surveys were carried out in the residences and outbuildings in which the insects were found, and serological examinations for Chagas disease performed on the inhabitants. Four colonies were found, all associated with nests of opossums (Didelphis aurita), 111 specimens of T. vitticeps, and 159 eggs being collected. All the triatomines presented flagellates in their frass. Mice inoculated with the faeces presented trypomastigotes in the circulating blood and groups of amastigotes in the cardiac muscle fibres. Serological tests performed on the inhabitants were negative for T. cruzi. Even with the intense devastation of the forest in Espírito Santo, there are no indications of change in the sylvatic habits of T. vitticeps. Colonies of this insect associated with opossum nests would indicate an expansion of the sylvatic environment into the peridomicile.

Phlebotomine sand flies (Diptera, Psychodidae) in an American tegumentary leishmaniasis transmission area in northern Espírito Santo State, Brazil.

American tegumentary leishmaniasis is endemic to the Espírito Santo State, Brazil, where it is widely distributed. The composition of the phlebotomine sand fly fauna in an American tegumentary leishmaniasis focus was determined by monthly sampling, using Shannon light traps in an Atlantic Forest reserve and adjacent habitat that had been modified by human activity. Seasonal fluctuations in numbers of the most abundant species were also monitored from June 2004 to May 2006. Of the 6,176 specimens collected, 47.4% were captured in the forest and 52.6% in the disturbed habitat. Although Lutzomyia davisi (60.8%) predominated in specimens from the forest, those captured near human dwellings consisted almost entirely of Lu. choti (72%) and Lu. intermedia (24.3%). All three species occurred throughout the year. Based on our findings, Lu. intermedia probably acts as the principal domestic Leishmania vector in the study area.

Effects of the installation of the Rosal hydroelectric power station, Itabapoana River, States of Espírito Santo and Rio de Janeiro, on anophelinae, planorbidae and phlebotominae

The construction of dams usually changes the composition of the fauna within their areas of influence. In the area where the Rosal hydroelectric power station has been installed, in the States of Espírito Santo and Rio de Janeiro, changes to the abundance of anophelinae, planorbidae and phlebotominae were evaluated before (1998-2000) and after (2000-2005) the damming. Nine sample collection points were defined, each represented by a home and its outbuildings, shelters for domestic animals and water collection points within a radius of 150m. One hundred and three adult anophelinae were collected before the damming and 313 afterwards; 200 immature anophelinae before and 708 afterwards; 868 planorbidae before and 486 afterwards; and 2,979 phlebotominae before and 912 afterwards. The vector record, consisting of anophelinae, planorbidae and phlebotominae, shows the potential of this area for transmission of malaria, schistosomiasis and American cutaneous leishmaniasis. However, the environmental changes did not increase the risk of the occurrence of these diseases.

Molecular biological identification of monoxenous trypanosomatids and Leishmania from antropophilic sand flies (Diptera: Psychodidae) in Southeast Brazil

The application of new molecular techniques to the genomic study of leishmanial parasites will have a substantial impact on our understanding of the dynamics of specific transmission cycles of clinically important Leishmania species (such as Leishmania braziliensis and Leishmania infantum), particularly in the various regions of Brazil where they are endemic. In this study, we used a polymerase chain reaction (PCR) technique targeting the conserved region of minicircle kDNA, aiming to detect Leishmania DNA in a sample of 430 female sand flies identified as Lutzomyia intermedia (171), Lutzomyia whitmani (115), Lutzomyia fischeri (76), Lutzomyia monticola (31), Lutzomyia migonei (15), Lutzomyia firmatoi (8), Lutzomyia pessoai (8) and Lutzomyia salesi (6). Sand flies were collected in peridomiciliary dependencies within an endemic area of L. braziliensis cutaneous leishmaniasis in Southeast Brazil (Afonso Cláudio, Espírito Santo). Of the sand fly species examined, 18 of 76 L. fischeri were found to be naturally infected. After sequence analysis, Leishmania and monoxenous lower trypanosomatid parasites were determined to be associated with the insect infections. The results indicate that a positive PCR is not sufficient in itself to indicate Leishmania infection and that the false detection of leishmanial infection within suspected vectors of human leishmaniasis would overestimate the true transmission rate of this parasite. More than 350 different phlebotomine sand fly species (Diptera: Psychodidae) from the Americas are known, but less than 40 of these have been implicated as proven or suspected vectors of human leishmaniasis (Grimaldi and Tesh 1993). Hence, monitoring natural Leishmania infections in sand fly populations is an important epidemiological parameter for controlling transmission, the estimation of which depends on the reliable identification of infected sand flies. False detection of leishmanial infection within suspected vectors of human leishmaniasis, however, will overestimate the true epidemiologic risk of parasite transmission. Several studies on natural infections of sand fly vectors with Leishmania and other trypanosomatids have been reported (Wallace and Hertig 1968; Arias et al. 1985; Naiff et al. 1989). Because direct methods to detect parasites in sand flies are limited, polymerase chain reaction (PCR)based assays have been used for detecting Leishmaniainfected sand fly vectors in many regions where the disease is endemic (Paiva et al. 2006; Kato et al. 2007; Savani et al. 2009). The reported performances of these tests, however, indicate that some probes appear limited in distinguishing between Leishmania and other trypanosomatids (Degrave et al. 1994). Further application of a combination of molecular approaches (such as RFLP, DNA hybridisation and sequencing) should help identify parasitic species in these microorganisms. L. de Souza Rocha :G. Grimaldi Jr. : E. Cupolillo (*) Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz—FIOCRUZ, Pavilhão Leônidas Deane sala 509, Av. Brasil 4365, Manguinhos, Rio de Janeiro, Rio de Janeiro, Brazil 21045-900 e-mail: ecupoli@ioc.fiocruz.br

Richness and Diversity of Sand Flies (Diptera, Psychodidae) in an Atlantic Rainforest Reserve in Southeastern Brazil

ABSTRACT: Our objective was to study and evaluate the richness and diversity of Phlebotominae fauna in the Duas Bocas Biological Reserve (DBBR) in the state of Espírito Santo, in southeastern Brazil. Sand fly collections were carried out during four consecutive nights each month between August 2007 and July 2008 at DBBR by using CDC automatic light traps and an illuminated Shannon trap. Specific richness (S) and Shannon diversity index (H) was calculated for each trap. We collected 18,868 sand flies belonging to 29 species and 13 genera. Nyssomyia yuilli yuilli was the most abundant species followed by Psychodopygus ayrozai, Ps. hirsutus, Psathyromyia pascalei, and Ps. matosi. We recorded Brumptomyia cardosoi, Br. troglodytes, and Ps. geniculatus for the first time in the state of Espírito Santo. We discuss the differences in diversity and richness of the sand flies in both traps and in relation to other Brazilian localities and biomes. We also discuss the possibility of wild transmission of Leishmania in the DBBR and the influence of the sand fly species in leishmaniasis transmission to the adjacent areas of the reserve.