Thiago Vasconcelos dos Santos

Natural Leishmania (Viannia) spp. infections in phlebotomine sand flies (Diptera: Psychodidae) from the Brazilian Amazon region reveal new putative transmission cycles of American cutaneous leishmaniasis

In Amazonian Brazil the etiological agents of American cutaneous leishmaniasis (ACL) belong to at least seven Leishmania species but little is known about the putative phlebotomine sand fly vectors in different biomes. In 2002–2003 a survey of the phlebotomine fauna was undertaken in the “Floresta Nacional do Tapajós”, Belterra municipality, in the lower Amazon region, western Pará State, Brazil, where we recently confirmed the presence of a putative hybrid parasite, L. (V.) guyanensis × L. (V.) shawi shawi. Sand flies were collected from Centers for Disease Control (CDC) light traps, Shannon traps and by aspiration on tree bases. Females were dissected and attempts to isolate any flagellate infections were made by inoculating homogenized midguts into Difco B45 medium. Isolates were characterized by monoclonal antibodies and isoenzyme electrophoresis. A total of 9,704 sand flies, belonging to 68 species or subspecies, were collected. Infections were found in the following sand flies: L. (V.) naiffi with Psychodopygus hirsutus hirsutus (1) and Ps. davisi (2); and L. (V.) shawi shawi with Nyssomyia whitmani (3) and Lutzomyia gomezi (1). These results provide strong evidence of new putative transmission cycles for L. (V.) naiffi and L. (V.) s. shawi.

Natural Leishmania (Viannia) infections of phlebotomines (Diptera: Psychodidae) indicate classical and alternative transmission cycles of American cutaneous leishmaniasis in the Guiana Shield, Brazil

From 1996 to 1999 multi-trapping methods (Center of Diseases Control, CDC) light traps, light-baited Shannon traps, and aspiration on tree bases) were used to study the phlebotomine fauna of the “Serra do Navio” region of the Brazilian State of Amapá, which is part of the Guiana Shield. Fifty-three species were identified among 8,685 captured individuals. The following species, associated with the transmission of American cutaneous leishmaniasis in Amazonian Brazil, were captured: Nyssomyia umbratilis (3,388), Psychodopygus squamiventris maripaensis (995), Ny. anduzei (550), Trichophoromyia ubiquitalis (400), Ny. whitmani (291), Ps. paraensis (116), and Bichromomyia flaviscutellata (50). Flagellate infections were detected in 45 flies. Of the 19 parasites isolated in vitro, 15 were Leishmania (Viannia) guyanensis (13 in Ny. umbratilis, 1 in Ny. whitmani, 1 in Ny. anduzei) and three were L. (V.) naiffi (2 in Ps. s. maripaensis, 1 in Ny. anduzei). The results indicate the participation of three phlebotomine species in the transmission of L. (V.) guyanensis and two species in that of L. (V.) naiffi, and show that the same phlebotomine species is involved in the transmission of different Leishmania (Viannia) species in the Guianan/Amazon region. A review of the literature together with the results of the present study, and other published and unpublished results, indicate that eight phlebotomine species potentially participate in the transmission of Leishmania (Viannia) naiffi in Amazonia.

Preclinical diagnosis of American visceral leishmaniasis during early onset of human Leishmania (L.) infantum chagasi-infection

Abstract American visceral leishmaniasis (AVL) is an infectious disease, often with long-duration evolution, caused by Leishmania (L.) infantum chagasi. However, although the disease is considered the major clinical manifestation of the link between L. (L.) i. chagasi and the human immune response, we have recently identified five clinical–immunological profiles of infection in the Brazilian Amazon: three asymptomatic (Asymptomatic Infection — AI, Sub-clinical Resistant Infection — SRI, and Indeterminate Initial Infection — III), and two symptomatic ones [Symptomatic Infection — SI ( = AVL) and Sub-clinical Oligosymptomatic Infection — SOI]. We confirm here the preclinical diagnosis of AVL through the IgM-antibody response in a case of an early infection (profile III) that evolved to the full disease after 6 weeks.

A new species of phlebotomine, Trichophoromyia adelsonsouzai (Diptera: Psychodidae) of Brazilian Amazonia

The phlebotomine sandfly Trichophoromyia adelsonsouzai sp. nov. is described and illustrated based on the male and female morphological characteristics of specimens collected at Km 27 of the Trans-Amazonian Highway, municipality of Vitória do Xingu, state of Pará, Brazilian Amazonia. This is an area subject to the direct influence of Belo Monte hydroelectric system. With the description of this new species the number of Trichophoromyia sandflies recorded in Brazil is increased to 20.

Further evidence associating IgG1, but not IgG2, with susceptibility to canine visceral leishmaniasis caused by Leishmania (L.) infantum chagasi-infection

We present here a cross-sectional study analyzing the IgG1 and IgG2 immune responses to natural canine Leishmania (L.) infantum chagasi-infection and their relationships with delayed-type hypersensitivity (DTH) in 50 mongrel dogs with previous positive serodiagnoses (IFAT-IgG) (56% with subclinical status [= apparently healthy] and 44% clinically sick), living in endemic areas for visceral leishmaniasis in the Brazilian Amazon. IgG1 and IgG2 responses were measured using commercial polyclonal antibodies in ELISA, while DTH was elicited by intradermal skin test using cultured promastigotes L. (L.) i. chagasi-antigen. Data analyses used Chi-square and Pearsons r coefficient (95% confidence interval). Regarding DTH and the clinical statuses of dogs, it was noted that 100% of the animals showing positive DTH (n = 8) were from the subclinical group, while 100% showing negative DTH were from the clinically sick group; higher IgG2 than IgG1 responses were observed in both clinical groups. However, when this comparison was made between the subclinical and sick groups, higher IgG1 responses were noted in the dogs from the sick rather than the subclinical group, while no differences were noted between the IgG2 responses in the dogs from both clinical groups. Additionally, we found lower IgG1 responses in dogs from the subclinical group showing positive DTH than in the dogs from the subclinical or sick groups with negative DTH; no differences were found between the IgG2 responses of these two clinical groups. These findings suggest that the IgG1, but not the IgG2, response is associated with susceptibility to canine visceral leishmaniasis (CVL).

Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil

Leishmaniasis are worldwide diseases that occur in 98 countries including Brazil, transmitted by the bite of female phlebotomines during blood feeding. In Brazil it is known that some species of sand flies as Lutzomyia longipalpis sensun latum (vector of Leishmania infantum chagasi), Lutzomyia flaviscutellata (vector of Leishmania (Leishmania) amazonensis) and Lutzomyia antunesi [suspected vector of Leishmania (Viannia) lindenbergi] are incriminated of transmitting the parasite Leishmania for the vertebrate host. The phlebotomine-parasite is mediated by the attachment of the promastigote lipophosphoglycan (LPG) to the midgut epithelium. However, another mechanism that is LPG-independent and mediated by N-acetyl-galactosamine (GalNAc) seems to occur in some species of phlebotomines that are classified as permissive. The aim of this study was to characterize the carbohydrate residues that, probably, play a role in parasite attachment to the midgut of phlebotomine from colony and field populations from the Brazilian Amazonian region. We observed the presence of GalNAc, mannose, galactose and GlcNAc in all phlebotomine species. A binding assay between L. (L.) amazonensis and L. i.chagasi to the midguts of different species of phlebotomines was performed. The attachment of both Leishmania and vector species suggests the presence of GalNAc on the midgut surfaces. Thus, these results suggested that GalNAc is a possible binding sites of Leishmania in sand flies from the Brazilian Amazonian region.

Geometric morphometry of the head in sand flies (Diptera: Psychodidae: Phlebotominae), an alternative approach to taxonomy studies

Almost 30% of female American sand flies are morphologically indistinguishable from one another. These females belong to species-complexes or groups with closely related species, making species-level identification difficult or even impossible. This fact has implications for public health since several of these groups include species which are vectors of human pathogens. Thus, new tools are needed to minimize species-level identification problems. In this context, our research sought to use geometric morphometry in the study of the head of females of closely related species of the genus Psychodopygus, Chagasi series, which includes the following species: P. chagasi, P. complexus, P. squamiventris maripaensis, P. squamiventris squamiventris, and P. wellcomei. We have used ten landmarks distributed on the head of these species. All species were clearly distinguished using the centroid size and shape together. These results highlight the head analysis as an effective tool for future geometric morphometry studies in sand flies.

Toll-like receptors 2, 4, and 9 expressions over the entire clinical and immunopathological spectrum of American cutaneous leishmaniasis due to Leishmania (V.) braziliensis and Leishmania (L.) amazonensis

Leishmania (V.) braziliensis and Leishmania(L.) amazonensis are the most pathogenic agents of American Cutaneous Leishmaniasis in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including: localized cutaneous leishmaniasis (LCLDTH+/++), borderline disseminated cutaneous leishmaniasis (BDCLDTH±), anergic diffuse cutaneous leishmaniasis (ADCLDTH-), and mucosal leishmaniasis (MLDTH++++). It has recently been demonstrated, however, that while L. (V.) braziliensis shows a clear potential to advance the infection from central LCL (a moderate T-cell hypersensitivity form) towards ML (the highest T-cell hypersensitivity pole), L. (L.) amazonensis drives the infection in the opposite direction to ADCL (the lowest T-cell hypersensitivity pole). This study evaluated by immunohistochemistry the expression of Toll-like receptors (TLRs) 2, 4, and 9 and their relationships with CD4 and CD8 T-cells, and TNF-α, IL-10, and TGF-β cytokines in that disease spectrum. Biopsies of skin and mucosal lesions from 43 patients were examined: 6 cases of ADCL, 5 of BDCL, and 11 of LCL caused byL. (L.) amazonensis; as well as 10 cases of LCL, 4 of BDCL, and 6 of ML caused byL. (V.) braziliensis. CD4+ T-cells demonstrated their highest expression in ML and, in contrast, their lowest in ADCL. CD8+ T-cells also showed their lowest expression in ADCL as compared to the other forms of the disease. TNF-α+showed increased expression from ADCL to ML, while IL-10+and TGF-β+ showed increased expression in the opposite direction, from ML to ADCL. With regards to TLR2, 4, and 9 expressions, strong interactions of TLR2 and 4 with clinical forms associated with L. (V.) braziliensis were observed, while TLR9, in contrast, showed a strong interaction with clinical forms linked to L. (L.) amazonensis. These findings strongly suggest the ability of L. (V.) braziliensis and L. (L.) amazonensis to interact with those TLRs to promote a dichotomous T-cell immune response in ACL.

Catalogue of the type material of Phlebotominae (Diptera, Psychodidae) deposited in the Instituto Evandro Chagas, Brazil

Abstract The available type material of Phlebotominae (Diptera, Psychodidae) deposited in the “Coleção de Flebotomíneos” of the Instituto Evandro Chagas (ColFleb IEC) is now presented in an annotated catalogue comprising a total of 121 type specimens belonging to 12 species as follow: Nyssomyia richardwardi (2 female paratypes), Nyssomyia shawi (9 male and 25 female paratypes), Nyssomyia umbratilis (female holotype and 1 female paratype), Nyssomyia yuilli yuilli (1 male and 1 female paratypes), Pintomyia gruta (1 male and 2 female paratypes), Psychodopygus lainsoni (2 male syntypes), Psychodopygus leonidasdeanei (male holotype, female “allotype” and 45 female paratypes), Psychodopygus llanosmartinsi (2 female paratypes), Psychodopygus wellcomei (1 male and 4 female “syntypes”), Trichophoromyia readyi (male holotype, female “allotype” and 1 male paratype), Trichophoromyia adelsonsouzai (male holotype, 13 male 5 female paratypes), and Trichophoromyia brachipyga (1 male paratype).