Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20% of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population.

Epidemiological and immunological aspects of human visceral leishmaniasis on Margarita Island, Venezuela

Sixty-five patients were diagnosed with visceral leishmaniasis (VL) on Margarita Island in the decade from 1990 to1999; 86.2% were <= 3 years old. All were leishmanin-negative at diagnosis. Evaluation of 23 cured patients in 1999 revealed that 22/23 had converted to leishmanin-positive; five had persisting antibodies to rK39 antigen, with no clinical evidence of disease. Leishmanin tests were positive in 20.2% of 1,643 healthy individuals from 417 households in endemic areas. Of the positive reactors, 39.8% were identified in 35 (8.4%) of the households, 15 of which had an antecedent case of VL, a serologically positive dog or both. Weak serological activity to rK39 antigen was detected in 3 of 488 human sera from the endemic areas. The presence of micro-foci of intense peri-urban transmission and the apparent absence of other Trypanosomatidae causing human disease offer a unique opportunity for the study of reservoirs, alternative vectors and evaluation of control measures on the Island.

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40%) of 45 SC subjects (mean absorbance of 0.49 ± 0.17). All nine sera from VL patients had such antibody (0.99 ± 0.21), while 11 (65%) of 17 CVL individuals were seropositive (0.46 ± 0.05). Only three (12%) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58%) of 45 SC patients (0.35 ± 0.14), and in all VL patients (0.65 ± 0.29). These antibodies were also detected in 13(76%) of 17 CVL subjects (0.42 ± 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.

Epidemiology of visceral leishmaniasis through spatial analysis, in Belo Horizonte municipality, state of Minas Gerais, Brazil

The geographic information system approach has permitted integration between demographic, socio-economic and environmental data, providing correlation between information from several data banks. In the current work, occurrence of human and canine visceral leishmaniases and insect vectors (Lutzomyia longipalpis) as well as biogeographic information related to 9 areas that comprise the city of Belo Horizonte, Brazil, between April 2001 and March 2002 were correlated and georeferenced. By using this technique it was possible to define concentration loci of canine leishmaniasis in the following regions: East; Northeast; Northwest; West; and Venda Nova. However, as for human leishmaniasis, it was not possible to perform the same analysis. Data analysis has also shown that 84.2% of the human leishmaniasis cases were related with canine leishmaniasis cases. Concerning biogeographic (altitude, area of vegetation influence, hydrographic, and areas of poverty) analysis, only altitude showed to influence emergence of leishmaniasis cases. A number of 4673 canine leishmaniasis cases and 64 human leishmaniasis cases were georeferenced, of which 67.5 and 71.9%, respectively, were living between 780 and 880 m above the sea level. At these same altitudes, a large number of phlebotomine sand flies were collected. Therefore, we suggest control measures for leishmaniasis in the city of Belo Horizonte, giving priority to canine leishmaniasis foci and regions at altitudes between 780 and 880 m.

Abundance of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and urban transmission of visceral leishmaniasis in Campo Grande, state of Mato Grosso do Sul, Brazil

The outspread and urbanization of visceral leishmaniasis (VL) in Campo Grande, state of Mato Grosso do Sul, lead us to undertake the present study over diversity and abundance of sand flies in the urban area to compare with previous search carried out during 1999/2000, before the identification of the disease in the human population.The captures were carried out with automatic light traps, weekly, from February 2004 to February 2005 on three sites including a forested area (Zé Pereira), two peridomicilies (shelters of domestic animals and cultivation areas), and intradomicilie. In the present study 110 collections were obtained during 13 months for 1320 h of collections, resulting in 5004 specimens, 3649 males and 1355 females belonging to the 20 following species: Brumptomyia avellari, Brumptomyia sp., Bichromomyia flaviscutellata, Evandromyia lenti, E. termitophila, E. cortelezzii, E. borrouli, Lutzomyia sp., L. longipalpis, Micropygomyia quinquefer, N. antunesi, N. whitmani, Pintomyia christenseni, Pi. damascenoi, Psathyromyia aragaoi, Ps. campograndensis, Ps. hermanlenti, Ps. shannoni, Pychodopygus claustrei, and Sciopemyia sordellii. L. longipalpis was the most abundant species in the anthropic environment with 92.22% of the captures. This shows an increase of sixty times in the density of L. longipalpis compared to the last sand fly evaluation in 1999/2000. The high density of L. longipalpis in Campo Grande is the main factor of risk in transmission of the disease to human in the urban area. The capture of N. antunesi, typical specie from Amazonian region, in Mato Grosso do Sul is reported for the first time.

Lutzomyia longipalpis in Uruguay: the first report and the potential of visceral leishmaniasis transmission

Phlebotomine captures were performed in February 2010 in Salto (Salto department) and Bella Unión-Cuarein (Artigas department), Uruguay. Bella Unión is located across the Paraná River from Monte Caseros, Argentina, where a focus of canine visceral leishmaniasis (VL) was reported in 2009. No VL cases have ever been recorded in Uruguay and the last reported capture of Phlebotominae was in 1932 (Lutzomyia cortelezzii and Lutzomyia gaminarai). Light traps were placed in peridomestic environments, and Lutzomyia longipalpis, the main vector of visceral leishmaniasis, was found in Salto and Bella Unión. This is a first report of an area of potential VL transmission in Uruguay. Active and coordinated surveillance is required immediately the Uruguay-Argentina-Brazil border area.

Hourly activity of Lutzomyia neivai in the endemic zone of cutaneous leishmaniasis in Tucumán, Argentina: preliminary results

In the present work, the hourly activity of Lutzomyia neivai was studied in the southern part of the province of Tucumán, Argentina, in an area of transmission of cutaneous leishmaniasis during two months of higher activity. In addition, the variables that influenced the abundance of Lu. neivai were evaluated. A total of 1,146 individuals belonging to Lu. neivai (97%) and Lutzomyia migonei (3%) were captured. The hourly activity of Lu. neivai was mainly nocturnal, with a bimodal pattern in both months. In January, the variable that most influenced the abundance of Lu. neivai was the temperature, whereas in April, that variable was humidity. These results may contribute to the design of anti-vectorial control measures at a micro-focal scale.

The distribution pattern of Lutzomyia longipalpis (Diptera: Psychodidae) in the peridomiciles of a sector with canine and human visceral leishmaniasis transmission in the municipality of Dracena, São Paulo, Brazil

The specimen distribution pattern of a species can be used to characterise a population of interest and also provides area-specific guidance for pest management and control. In the municipality of Dracena, in the state of São Paulo, we analysed 5,889 Lutzomyia longipalpis specimens collected from the peridomiciles of 14 houses in a sector where American visceral leishmaniasis (AVL) is transmitted to humans and dogs. The goal was to analyse the dispersion and a theoretical fitting of the species occurrence probability. From January-December 2005, samples were collected once per week using CDC light traps that operated for 12-h periods. Each collection was considered a sub-sample and was evaluated monthly. The standardised Morisita index was used as a measure of dispersion. Adherence tests were performed for the log-series distribution. The number of traps was used to adjust the octave plots. The quantity of Lu. longipalpis in the sector was highly aggregated for each month of the year, adhering to a log-series distribution for 11 of the 12 months analysed. A sex-stratified analysis demonstrated a pattern of aggregated dispersion adjusted for each month of the year. The classes and frequencies of the traps in octaves can be employed as indicators for entomological surveillance and AVL control.

Cytokines and visceral leishmaniasis: a comparison of plasma cytokine profiles between the clinical forms of visceral leishmaniasis

It is not well established whether cytokine production differs in response to different clinical forms of visceral leishmaniasis (VL). In this work, we performed a cross-sectional study to investigate the plasma levels of cytokines [interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-10 and IL-12] involved in the pathogenesis of VL in 80 subjects from VL endemic areas, including subjects with active VL, subjects with asymptomatic infection, subjects with cured VL and uninfected controls. The patients were recruited by sampling from a referral hospital and by random selection from a population-based cohort study. The results showed significant differences in the plasma concentration of all cytokines between the groups (p < 0.05). Patients with the active disease had higher plasma levels of IL-10, IL-4, INF-γ and TNF-α relative to the other groups and they produced more IL-12 than asymptomatic and cured subjects. Only the IL-2 concentration was higher in the asymptomatic and cured subjects relative to the patients with active disease (p < 0.05). Our results suggest that these cytokines can be used as markers in epidemiological studies conducted in endemic areas to distinguish between different clinical forms of VL. However, their usefulness should be confirmed in investigations conducted in other endemic areas.

Inflammation in disseminated lesions: an analysis of CD4+, CD20+, CD68+, CD31+ and vW+ cells in non-ulcerated lesions of disseminated leishmaniasis

Disseminated leishmaniasis (DL) differs from other clinical forms of the disease due to the presence of many non-ulcerated lesions (papules and nodules) in non-contiguous areas of the body. We describe the histopathology of DL non-ulcerated lesions and the presence of CD4-, CD20-, CD68-, CD31- and von Willebrand factor (vW)-positive cells in the inflamed area. We analysed eighteen biopsies from non-ulcerated lesions and quantified the inflamed areas and the expression of CD4, CD20, CD68, CD31 and vW using Image-Pro software (Media Cybernetics). Diffuse lymphoplasmacytic perivascular infiltrates were found in dermal skin. Inflammation was observed in 3-73% of the total biopsy area and showed a significant linear correlation with the number of vW+ vessels. The most common cells were CD68+ macrophages, CD20+ B-cells and CD4+ T-cells. A significant linear correlation between CD4+ and CD20+ cells and the size of the inflamed area was also found. Our findings show chronic inflammation in all DL non-ulcerated lesions predominantly formed by macrophages, plasmacytes and T and B-cells. As the inflamed area expanded, the number of granulomas and extent of the vascular framework increased. Thus, we demonstrate that vessels may have an important role in the clinical evolution of DL lesions.

Visceral leishmaniasis and hemophagocytic syndrome

A 11 months old female infant from Portugal, free of family history, consults for apathy, weight loss, tachycardia, tachypnea, petechiae, pallor without icterus and hepatoslenomegaly. Seven months earlier, while being in Portugal, she presented a persistent bluish pimple on her buttock. Laboratory results showed anemia (35 g/l), leucopenia (3.3 G/l), thrombocytopenia (13 G/l), impaired coagulation (INR 1.4, PTT 41 sec.), hyponatremia (124 mmol/l), elevated CRP (139 mg/l), high ferritin (34.775 μg/l) and high triglycerides (5.22 mmol/l). After correction of vital parameters, a bone marrow aspiration and biopsy (BMB) revealed both the etiological diagnosis, namely a visceral leishmaniasis (VL) as well as one of its potential complications, the hemophagocytic syndrome (HS). Transfusions of whole blood, platelets and fresh frozen plasma were immediately started. Dexamethasone (10 mg/m2) and amphotericin B (3 mg/kg/day) have also been administrated. Visceral leishmaniasis is caused by a protozoan (Leishmania donovani) transmitted by the female sandfly. It is endemic in the Mediterranean basin (including France, Italy, Spain and Portugal), South America, sub-Saharan Africa as well as in India and Bangladesh. The parasite infects macrophages and, after several weeks of incubation, the disease occurs by affection of bloodlines (anemia, leucopenia, thrombocytopenia), hepatosplenomegaly, cachexia, gastrointestinal damage. The complications of the disease may lead to death. Liposomal amphotericin B is the currently recommended treatment. HS is caused by the proliferation and activation of macrophages in the marrow in response to a cytokine storm. It may be of primary cause. When it is secondary, it may be related to infections such as leishmaniasis. Patients present with fever and laboratory diagnostic criteria include cytopenia, hypertriglyceridemia, high ferritin and hemophagocytosis in the BMB. The treatment consists among other in the administration of high doses corticosteroids and, in secondary cases, in the treatment of the underlying cause. In conclusion, the clinical and biological features of VL may mimic haematological disorders as leukemia, but an enlargement of the liver and especially of the spleen should remind in this parasitic infection and its potential fatal complication, the HS.

Safety and effectiveness of amphotericin B deoxycholate for the treatment of visceral leishmaniasis in Uganda

Between September 2003 and April 2004, the supply of antimonial drugs to Amudat Hospital, in north-eastern Uganda, was interrupted and all cases of visceral leishmaniasis presenting at the hospital could only be treated with amphotericin B deoxycholate (AmB). This allowed the safety and effectiveness of the AmB to be evaluated, in comparison with an historical cohort of patients treated, at the same hospital, with meglumine antimoniate (Sb-V). Demographic and clinical data were collected before and after treatment. Adverse effects were recorded passively in all the subjects, and actively, using a standardized questionnaire, in a sub-group of the patients given AmB. The in-hospital case-fatality 'rates' were 4.8% [95% confidence interval (CI) =2.4%-8.8%] among the 210 patients treated with AmB and 3.7% (CI=1.4%-7.9%) among the 161 patients treated with Sb-V (P>0.20). Adverse effects requiring treatment interruption were rare in both cohorts. Treatment failures (i.e. non-responses or relapses) were observed in 2.9% (CI= 1.2%-6.4%) of the patients treated with AmB and 1.2% (CI=0.1%-4.4%) of the patients treated with Sb-V (P>0.20). For the treatment of visceral leishmaniasis in Uganda, AmB therefore had a similar effectiveness and safety profile to that of meglumine antimoniate.

Spatial distribution of canine Visceral Leishmaniasis in Ilha Solteira, São Paulo, Brazil

Visceral Leishmaniasis (VL) is caused by protozoan of genus Leishmania and transmitted by sand flies of genus Lutzomyia, which has been adapted to the peridomicile environment where dogs are their mainly food source, increasing the risk for human cases. In this study, techniques of geoprocessing and spatial statistics were utilized as a contribution to understanding the epidemiological dynamics of VL in the urban area of Ilha Solteira, SP.

Serological diagnosis of visceral leishmaniasis by an enzyme immunoassay using protein A in naturally infected dogs

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Leishmania chagasi using total antigen from lysed promastigotes. Fifty symptomatic mixed breed dogs from a region of high incidence of visceral leishmaniasis in Brazil were examined. The results showed that in the positive animals, diagnosed by cytological examination, the ELISA using protein A assay system (mean optical density ± SD / 2.078 ± 0.631) detected more antibodies than the anti-IgG assay (mean optical density ± SD / 1.008 ± 0.437), while in the negative animals, the results by both systems were similar. These results suggest that the ELISA assay using protein A peroxidase conjugated could be useful to detect early infected animals in endemic areas, and thus help to control the spread of the infection.

Outbreak of autochthonous canine visceral leishmaniasis in Santa Catarina, Brazil

The present study reports the first outbreak of autochthonous canine visceral leishmaniasis in Florianópolis, Santa Catarina, southern Brazil. Following the report of two cases of CVL, the Control Center of Zoonotic Diseases conducted a serological survey by ELISA and IFAT assays in seven districts of the Santa Catarina Island. Eleven seropositive dogs of autochthonous transmission were used in the present study. Infection by Leishmania sp. was confirmed by parasitological examination of bone marrow, liver, spleen and lymph nodes, culture in Schneider's medium and PCR. Leishmania sp. isolates were characterized by PCR-RFLP and hybridization with specific probes, allowing for the identification of Leishmania infantum. Autochthonous transmission of this disease in an area with high tourist traffic presents a major public health concern and signifies the emergence of an important zoonosis in southern Brazil. Therefore, the implementation of surveillance and control measures is imperative to prevent the spread of the disease among the canine population as well as transmission to the human population.