Application of the mammalian glyceraldehyde-3-phosphate dehydrogenase gene for sample quality control in multiplex PCR for diagnosis of leishmaniasis

Leishmaniasis is a neglected disease endemic in five continents. It is a severe disease that may lead to death, and its early detection is important to avoid severe damage to affected individuals. Molecular methods to detect Leishmania are considered alternatives to overcome the limitations presented by conventional methods. The aim of this study was to develop multiplex PCR systems able to detect small amounts of target DNA of Leishmania infantum and Leishmania braziliensis, and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (G3PD) in mammals, enabling quality evaluation of the sample simultaneously with detection of the specific target. The systems created for G3PD recognition were combined with detection systems for L. infantum and L. braziliensis to compose multiplex PCR systems for visceral (mVL) and cutaneous (mACL) leishmaniasis diagnosis. The multiplex PCR systems developed were assessed in blood samples from five different species of mammal reservoirs involved in the disease cycle in Brazil, and 96 and 52 human samples from patients with suspected visceral leishmaniasis (VL) and cutaneous leishmaniasis (ACL), respectively. Three G3PD detection systems were created (G3PD1, G3PD2 and G3PD3) with different product sizes, G3PD2 was chosen for the formation of multiplex PCR systems. The two multiplex PCR systems (mVL and mACL) were reproducible in all species evaluated. Results of test samples (sensitivity, specificity and efficiency) suggest its use in routine diagnosis, research activities in medicine and veterinary medicine. Additionally, the systems designed to detect the G3PD gene are capable of combining with other targets used for molecular diagnosis of infectious diseases. Concerning leishmaniasis, the multiplex PCR systems can be used in epidemiological studies for the detection of new and classic reservoirs, which may contribute to the reliability of results and development of actions to control the disease.(AU)

Lutzomyia naftalekatzi, a new species of phlebotomine sand fly (Diptera: Psychodidae) from Zona da Mata region, Pernambuco, Brazil.

Lutzomyia naftalekatzi, a new phlebotomine sand fly belonging to the L. aragaoi species group, is described from specimens collected in Zona da Mata region of Pernambuco, Brazil.

Domesticity of Lutzomyia whitmani (Diptera: psychodidae) populations: field experiments indicate behavioural differences.

The sandfly Lutzomyia whitmani (Antunes & Coutinho) is an important vector for cutaneous leishmaniasis throughout much of Brazil, and has recently been shown to consist of more than one mitochondrial lineage. It has frequently been asserted that the degree of adaptation of L. whitmani to human environments varies across its range. As a standardized test of indoor feeding for three geographically distant populations of L. whitmani, catches inside experimental chicken sheds of varying degrees of wall closure (0%, 33%, 67% and 98%) were compared. Each increment in shed closure reduced catches of females (relative to the most open shed) by a similar degree for each population: geometric mean catches dropped by 11-40% with 33% closure, by 41-62% with 67% closure, and by 69-100% with 98% closure. Geometric mean catches of males from the two more northerly populations also decreased with increasing shed closure, by 18% and 22% for 33% closure, 58% and 69% for 67% closure, 91% and 93% for 98% closure. Males from the most southerly population showed significantly different behaviour, with 33% closure causing a 54% increase in geometric mean catch, 67% closure causing a 6% increase, and 98% closure causing a 32% reduction. For this southerly population, sex ratios became more male biased with increasing density in more closed sheds, suggesting aggregation driven by intra-specific communication. Lutzomyia intermedia (Lutz & Neiva) was relatively more likely than L. whitmani to approach baits in the three more closed sheds, rather than the most open shed, offering a behavioural explanation for observed differences in indoor biting rates between the species.

Morphometric and phenetic studies of five geographical populations of Lutzomyia whitmani (Diptera: Psychodidae) in Brazil.

A morphometric survey examined adult specimens of Lutzomyia whitmani (Antunes & Coutinho) captured at 5 municipalities in southeastern and northeastern Brazil to compare the populations. The localities were Ilhéus (Bahia), Martinho Campos (Minas Gerais), Corte de Pedra (Bahia), Baturité (Ceará), and Amaraji (Pernambuco): all are known foci of American cutaneous leishmaniasis. Fifteen males and 15 females from each population were analyzed morphometrically for 42 and 37 characters, respectively. Statistical data alone were insufficient to discriminate among the 5 populations. Further analysis generated phenograms that indicated there were 2 spatial clusters: the 1st was composed of specimens from Ilhéus (Bahia) and Baturité (Ceará) and the 2nd of specimens from Martinho Campos (Minas Gerais), Corte de Pedra (Bahia), and Amaraji (Pernambuco). Although insufficient to define the taxonomic status of the populations studied, the results delineated the existence of biogeographical structuring within L. whitmani. Complementary studies on the susceptibility to Leishmania braziliensis infection in the 5 populations are in progress to clarify the relationship between the 2 biogeographical clusters and American cutaneous leishmaniasis transmission in those Brazilian regions.

Experimental comparison of anthropophily between geographically dispersed populations of Lutzomyia whitmani (Diptera: Psychodidae).

Lutzomyia whitmani, a major vector of cutaneous leishmaniasis in Brazil, occupies diverse habitats from the Amazon forest canopy to suburban animal pens. Three mitochondrial lineages of Lu. whitmani ('Amazonian', 'North-South' and 'North-east') have parapatric distributions coinciding with different ecological zones. We assessed the host preferences of populations representing the three lineages in standardized field experiments, and found that Lu. whitmani in all sites were significantly more attracted to humans than to dogs or chickens. Females from a southerly population of the North-South lineage showed the greatest degree of anthropophily. Lu. whitmani from Amazonia were also strongly attracted to human baits, contradicting previously published accounts. Intraspecific comparisons in non-Amazonian sites suggest that Lu. whitmani is less anthropophilic than Lu. intermedia but more so than Lu. longipalpis. No significant difference was detected in anthropophily between Lu. whitmani in the Amazon and either Lu. dendrophyla or Lu. gomezi. Anthropophilic behaviour was demonstrated in the same site for Lu. complexa, Lu. flaviscutellata and Lu. brachyphalla, but not for Lu. infraspinosa.

Epidemiological surveys confirm an increasing burden of cutaneous leishmaniasis in north-east Brazil.

Health service records for north-east Brazil suggest a consistent rise in numbers of cases of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis over the past decade. In a study site in Pernambuco, prospective, cross-sectional and retrospective epidemiological surveys of infection (a positive Montenegro skin test response) and/or clinical symptoms confirmed a high current force of infection (0.092/year), and an approximately 10-fold increase in transmission during the last 10 years. Cross-sectional analysis indicated that the incidence rate among children (aged < or = 15 years) was lower than that among adult immigrants exposed for similar time periods, but there was no apparent difference in transmission rate according to gender. Coupled with the known behaviour of the local sandfly vector, Lutzomyia whitmani, this suggests that most people are infected outside their houses, rather than either indoors or while visiting remnant rainforest. The estimated proportion of infections which lead to cutaneous lesions (0.81-0.87) is relatively high for L. braziliensis areas. However, an unusually low proportion of clinical infections (0.0042) apparently leads to metastasis.

[American cutaneous leishmaniasis in a military training center located in Zona da Mata, Pernambuco, Brazil]. / Leishmaniose tegumentar americana em centro de treinamento militar localizado na zona da mata de pernambuco, Brasil.

An outbreak of American cutaneous leishmaniasis has been occurred in military training unit localized in 'Zona da Mata' of Pernambuco State, Brazil, where were registered 26 human cases. An epidemiological survey was carried out by entomological investigation and Montenegro skin test (MST). Lutzomyia choti presents predominance (89.9%) in sandflies identified. Out of 545 men who realized training activities were 24.12% positive to MST.

Leishmaniose tegumentar americana em centro de treinamento militar localizado na zona da mata de pernambuco, Brasil / American cutaneous leishmaniasis in a military training center located in Zona da Mata, Pernambuco, Brazil

An outbreak of American cutaneous leishmaniasis has been occurred in military training unit localized in 'Zona da Mata' of Pernambuco State, Brazil, where were registered 26 human cases. An epidemiological survey was carried out by entomological investigation and Montenegro skin test (MST). Lutzomyia choti presents predominance (89.9%) in sandflies identified. Out of 545 men who realized training activities were 24.12% positive to MST.

Um surto de leishmaniose tegumentar americana ocorreu em 1996 em unidade de treinamento militar situada na Zona da Mata de Pernambuco, com o registro de 26 casos humanos. Um inquérito epidemiológico foi realizado através da realização de levantamento entomológico e da aplicação do Teste de Montenegro. Lutzomyia choti apresentou predominância de 89,9% dos flebótomos identificados. De 545 homens que participaram de treinamentos no período, 24,1% (incluindo os casos clínicos) foram positivo para o Teste de Montenegro.

American cutaneous leishmaniasis in Pernambuco, Brazil: eco-epidemiological aspects in 'Zona da Mata' region.

American cutaneous leishmaniasis (ACL) has been increasing in Pernambuco, thus becoming an important problem for Public Health. The incindence is predominant in the region called 'Zona da Mata', in the east of this state. This region corresponds geographically to the primitive area of the Atlantic forest. In order to characterize the eco-epidemiology expression of ACL in this region, two localities situated in the municipalities of Amaraji e Cortes have been selected by the criterion of higher incindence of human cases. Five stocks of patients were characterized and identified on the basis of enzyme profiles as a new variant of Leishmania (V.) braziliensis. A survey of wild and domestic animals was carried out by means of a parasitological and serological diagnosis. Through the analysis of the spleen and liver imprints, were detected amastigotes compatible with Leishmania in five Nectomys s. squamipes, five Bolomys 1. pixuna, two Rattus r. alexandrinus and one Rattus r. frugivorus. For two years we carried out monthly sandflies captures using CDC light traps as well as manual captures. Lutzomyia whitmani was predominant, which accounted for 97.4% of the total. These data indicate a strong evidence on the vector and the potential reservoirs of L. braziliensis in this region.

Mouse cachexia induced by trehalose dimycolate from Nocardia asteroides.

Trehalose dimycolate (TDM) isolated from Nocardia asteroides induced in mice a severely wasted condition known as cachexia. Intraperitoneal injection of mice with five 10 micrograms doses of TDM in mineral oil at intervals of 2 d killed 90% of the animals within 26 d. Death followed a precipitous weight loss and an inflammatory process in the peritoneal cavity. When mice were injected intraperitoneally with a single 10 micrograms dose of TDM, 48 h later, they had begun to lose weight and exhibited extreme hypertriglyceridaemia and hypoglycaemia. Tumour necrosis factor (or cachectin) was detected in the plasma from animals injected with TDM. This cytokine released by mononuclear phagocytes may be involved in the induction of cachexia by TDM.

Studies on the pathogenesis of actinomycotic mycetoma in animals injected with fractions isolated from Nocardia brasiliensis.

In the present study the participation was evaluated of isolated fractions of Nocardia brasiliensis in the genesis of the inflammatory response observed in actinomycotic mycetoma. Subcutaneous injection in mice of a suspension containing a polysaccharide fraction F1 obtained by treating cell walls with sodium hydroxide induced an inflammatory response at the inoculation site which was characterized by a large influx of polymorphonuclear (PMN) and mononuclear (MN) leucocytes between the 2nd and 4th days. On the 8th day, a typical granulomatous reaction was observed involving large numbers of epithelioid cells. Intravenous injection of the lipid extract adsorbed to charcoal particles into mice induced an inflammatory reaction around the particles embolized in the pulmonary microcirculation which was similar to that described above. The kinetics of the inflammatory cell migration was studied by total and differential counts of leukocytes that migrated to the peritoneal cavity of rats inoculated intraperitoneally with the F1 and lipid fractions. Both fractions initially induced intense PMN migration, which was later reduced, with a simultaneous increase in mononuclear cells. The present results demonstrate that a polysaccharide fraction (F1) and the lipid fraction reproduce the fundamental lesion of actinomycotic mycetoma.

The role of fractions from Paracoccidioides brasiliensis in the genesis of inflammatory response.

The influx of inflammatory cells towards the peritoneal cavity in rats inoculated intraperitoneally with subcellular preparations of the fungus Paracoccidioides brasiliensis was studied. In addition to the dead fungus, also fractions F1 of the cell wall, which mainly consisted of polysaccharides and the lipid extract, induced intense cell migration 4 hr after inoculation, with a greatly increased number of polymorphonuclear leucocytes (PMN). Study of the kinetics of cell influx showed that both fraction F1 and the lipid extract initially induced intense PMN migration between the 4th and 24th hr after inoculation of these agents, followed by migration of mononuclear cells (MN) around the 48th hr. We also observed that migration of these cells increased gradually after inoculation of growing doses of fraction F1. The present data suggest that polysaccharides and lipids isolated from P. brasiliensis may participate in the initial phase of the inflammatory response in paracoccidioidomycosis.

Mouse toxicity induced by lipids and cell walls isolated from actinomycetes.

The possibility was examined that the toxicity induced in mice by Actinomadura madurae, 'Streptomyces pelletieri' and Nocardia brasiliensis was due to lipid and cell-wall constituents. Mice were inoculated intraperitoneally with heat-killed bacteria, lipid extracts and cell-wall preparations emulsified in mineral oil: toxicity was evaluated by recording weight loss and deaths. Killed cells and cell-wall preparations of all three actinomycetes produced a pronounced loss of body weight, tissue necrosis, splenomegaly, a granulomatous inflammation and sometimes death. Mice inoculated with lipid extracts from A. madurae and 'S. pelletieri' neither died nor showed toxic effects, but mice injected with lipids isolated from N. brasiliensis did suffer toxic effects. They showed more marked wasting symptoms than observed after inoculation of heat-killed bacteria or of the cell-wall preparation.

Cord factor is associated with the maintenance of the chronic inflammatory reaction caused by mycobacteria.

The distribution of an aqueous suspension of cord factor (CF) from Mycobacterium bovis BCG in several mouse organs was examined after intravenous injection, and the correlation between evolution of the inflammatory granulomatous reaction and the presence of CF in these organs was determined. CF was preferentially deposited in the lungs and liver, and the kinetics of the pulmonary and hepatic inflammatory reaction, evaluated by determining the indices for these organs, showed a gradual increase on day 2 after injection, reached a peak around the fifth day, and declined thereafter. Histological analysis showed that on day 5 both the lungs and the liver were diffusely damaged by a mononuclear inflammatory infiltrate arranged in a granulomatous manner and consisting predominantly of histiocytes. CF elimination was more marked in the liver than in the lungs: 2 d after injection 76% of the material deposited in the liver had been eliminated. Little or no CF was detected in the liver and lungs by day 16, when the inflammatory reaction was also substantially decreased. A second CF dose administered 8 d after the first exacerbated the inflammatory process in both the lungs and the liver, indicating that the intensity of this process depends on CF concentration in the lesion site.