Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

Horizontal stratification of the sand fly fauna (Diptera: Psychodidae) in a transitional vegetation between caatinga and tropical rain forest, state of Bahia, Brazil

A study about the horizontal stratification of the sand fly fauna in two distinct ecosystems, caatinga area, endemic for visceral leishmaniasis, and the tropical rain forest area, endemic for cutaneous leishmaniasis, was performed in the state of Bahia, Brazil. Lutzomyia longipalpis was predominant in the caatinga, and following it came the species L. capixaba and L. oswaldoi. In the tropical rain forest other species were found, such as L. intermedia, L. migonei, L. whitmani, L. yuilli, L.fischeri, L. damascenoi, L. evandroi, L. monticola, and L. lenti. It was found that the geographical limits of the vector species of visceral and cutaneous leishmaniasis are clearly defined by the biological and phytogeographic characteristics.

Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs

The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9%; in lymph node cultures it was 25%. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test) in symptomatic (28 out of 33 animals) than in asymptomatic animals (19 out of 31 animals). These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection.

Tegumentary leishmaniasis outbreak in Bella Vista City, Corrientes, Argentina during 2003

Bella Vista City, Corrientes, Argentina, reported an epidemic outbreak of tegumentary leishmaniasis during 2003. The mean age of the 31 cases was 25.0 ± 13.7 years old, with a sex ratio male:female 1.8, and without mucosal involvement. They clustered in two contiguous neighbourhoods, 96% in the periurban border and 4% in the peripheral outskirts. The transmission peak was estimated to have occurred during April 2003. Four species (3608 sand flies) were captured in nine sites: Lutzomyia neivai (90.1%), Lu. pessoai (8.9%), Lu. migonei (0.8 %), and Brumptomyia avellari (0.2 %). The outskirts/rural capture ratio of Lu. neivai was up to 3, and the outskirts/periurban up to 200. Therefore, the 'urban' transmission in this southernmost known focus is still an ecotone-border associated risk. The changes in human distribution or activities, patches of the secondary vegetation, periurban streams, rainfall of the previous year, and river period floods could all contribute to 'urban' outbreaks in the region. Tegumentary leishmaniasis risk should be assessed for any project that involves changes in land use throughout an endemic area.

Clinical versus ultrasound examination in the evaluation of hepatosplenic schistosomiasis mansoni in endemic areas

The best way to appraise the size of abdominal organs remains undefined. Herein we compare the size of liver and spleen in hepatosplenic schistosomiasis using clinical and ultrasound (US) examination, and the size of the organs measured by US with their visualization below the costal margin ("palpable by US"). For this study, 411 individuals from an endemic area for schistosomiasis mansoni in Brazil have been selected. We found that palpable spleens and left liver lobes are larger than non palpable ones. Also, 23% of normal spleens measured by US were palpable on clinical examination, and 22% of spleens increased in size on US were non palpable. A total of 21% of normal spleens were "palpable by US". We also found 54% of normal sized right liver lobes palpable on clinical examination, whilst 54% of the increased livers, measured by US, were non palpable. About 76% of normal right liver lobes were "palpable by US". We conclude that the association of clinical, ultrasound and magnetic resonance imaging (MRI) examinations, in the near future, should give the investigators the necessary tools to perform a more accurate clinical diagnosis of hepatosplenic schistosomiasis mansoni.

RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

First encounter of subclinical human Leishmania (Viannia) infection in State of Rio Grande do Sul, Brazil

The objective of the present study was to evaluate the specificity of the Montenegro skin test (MST) in an area in Brazil, state of Grande do Sul State (RS), which was considered to be non-endemic for leishmaniasis. Sixty subjects presented a positive MST and were reevaluated by clinical examination, serology and polymerase chain reaction (PCR) of peripheral blood for the detection of subclinical Leishmania infection. None of the subjects presented clinical signs or symptoms of current leishmaniasis or a history of the disease.Leishmania (Viannia) DNA was detected in blood by PCR and hybridization in one subject. The PCR skin test-positive individual remained asymptomatic throughout the study. Clinical examination showed no scars suggestive of past cutaneous leishmaniasis. Human subclinical infection with Leishmania (Viannia) in RS was confirmed by PCR. This is the first report of subclinical infection with this parasite in the human population of this area.

Urban distribution of Phlebotominae in a cutaneous leishmaniasis focus, Argentina

Urbanization and vector domestication are currently proposed as factors that contributed to the recent increase of American cutaneous leishmaniasis (ACL). Is likely also urban transmission? Oran is the main city in the Argentinean hyper-endemic area of ACL, and human cases in urban residences are usually reported. In order to assess the spatial distribution of risk, phlebotomine traps were located in different environments of Oran. A total of 7,787 sand flies were captured: Lutzomyia neivai (98.1%), Lutzomyia migonei (1.2%), Lutzomyia cortelezzii (0.7%), and one Lutzomyia shannoni. During the season of transmission (April-May) a single sand fly was obtained in one out of five urban sites, while a trap in a peri-urban pigsty captured up to 2,985 Lu. neivai/night. Captures performed in the other season of vector activity (September-October) revealed that small-scale changes in the pigsty environment resulted in noticeable changes in the abundance of Lu. neivai. In addition, in a new neighbourhood, on the fringe of the city, 1,073 Lu. neivai/site were captured in the forested edge but one in the yard of the houses. Therefore, in this urban ACL focus the human-vector effective contact risk is still associated with peri-urban vegetation and ecotone modifications despite the urban residence of the cases.

Genus-specific kinetoplast-DNA PCR and parasite culture for the diagnosis of localised cutaneous leishmaniasis: applications for clinical trials under field conditions in Brazil

The positivities of two methods for the diagnosis of localised cutaneous leishmaniasis (CL) were estimated in 280 patients enrolled in a clinical trial. The trial was conducted in an endemic area of Leishmania (Viannia) braziliensis and trial participants were patients with skin ulcers and positive leishmanin skin tests. Patients underwent aspirative skin punctures of the ulcerated lesions and lymph nodes for in vitro cultures, which were processed under field conditions at the local health centre. Skin lesion biopsies were tested at a reference laboratory using kinetoplastid DNA (kDNA)-PCR to detect DNA. The median time required to obtain a positive culture from the skin samples was seven days and the contamination rate of the samples was 1.8%. The positivities of the cultures from skin lesions, kDNA-PCR and the combination of the two methods were 78.2% (95% CI: 73-82.6%), 89.3% (95% CI: 85.1-92.4%) and 97.1% (95% CI: 94.5-98.5%). We conclude that parasite culture is a feasible method for the detection of Leishmania in field conditions and that the combination of culture and PCR has a potential role for the diagnosis of CL in candidates for clinical trials.

Effect of untreated bed nets on blood-fed Phlebotomus argentipes in kala-azar endemic foci in Nepal and India

Observational studies in the Indian subcontinent have shown that untreated nets may be protective against visceral leishmaniasis (VL). In this study, we evaluated the effect of untreated nets on the blood feeding rates of Phlebotomus argentipes as well as the human blood index (HBI) in VL endemic villages in India and Nepal. The study had a "before and after intervention" design in 58 households in six clusters. The use of untreated nets reduced the blood feeding rate by 85% (95% CI 76.5-91.1%) and the HBI by 42.2% (95% CI 11.1-62.5%). These results provide circumstantial evidence that untreated nets may provide some degree of personal protection against sand fly bites.

Delimitation of kala-azar risk areas in the district of Vaishali in Bihar (India) using a geo-environmental approach

Remote sensing and geographical information technologies were used to discriminate areas of high and low risk for contracting kala-azar or visceral leishmaniasis. Satellite data were digitally processed to generate maps of land cover and spectral indices, such as the normalised difference vegetation index and wetness index. To map estimated vector abundance and indoor climate data, local polynomial interpolations were used based on the weightage values. Attribute layers were prepared based on illiteracy and the unemployed proportion of the population and associated with village boundaries. Pearson's correlation coefficient was used to estimate the relationship between environmental variables and disease incidence across the study area. The cell values for each input raster in the analysis were assigned values from the evaluation scale. Simple weighting/ratings based on the degree of favourable conditions for kala-azar transmission were used for all the variables, leading to geo-environmental risk model. Variables such as, land use/land cover, vegetation conditions, surface dampness, the indoor climate, illiteracy rates and the size of the unemployed population were considered for inclusion in the geo-environmental kala-azar risk model. The risk model was stratified into areas of "risk"and "non-risk"for the disease, based on calculation of risk indices. The described approach constitutes a promising tool for microlevel kala-azar surveillance and aids in directing control efforts.

Appraisal of Phlebotomus argentipes habitat suitability using a remotely sensed index in the kala-azar endemic focus of Bihar, India

Visceral leishmaniasis, or kala-azar, is recognised as a serious emerging public health problem in India. In this study, environmental parameters, such as land surface temperature (LST) and renormalised difference vegetation indices (RDVI), were used to delineate the association between environmental variables and Phlebotomus argentipes abundance in a representative endemic region of Bihar, India. The adult P. argentipes were collected between September 2009-February 2010 using the hand-held aspirator technique. The distribution of P. argentipes was analysed with the LST and RDVI of the peak and lean seasons. The association between environmental covariates and P. argentipes density was analysed a multivariate linear regression model. The sandfly density at its maximum in September, whereas the minimum density was recorded in January. The regression model indicated that the season, minimum LST, mean LST and mean RDVI were the best environmental covariates for the P. argentipes distribution. The final model indicated that nearly 74% of the variance of sandfly density could be explained by these environmental covariates. This approach might be useful for mapping and predicting the distribution of P. argentipes, which may help the health agencies that are involved in the kala-azar control programme focus on high-risk areas.

The phlebotomine fauna (Diptera: Psychodidae) of Guaraí, state of Tocantins, with an emphasis on the putative vectors of American cutaneous leishmaniasis in rural settlement and periurban areas

Phlebotomine sandflies were captured in rural settlement and periurban areas of the municipality of Guaraí in the state of Tocantins (TO), an endemic area of American cutaneous leishmaniasis (ACL). Forty-three phlebotomine species were identified, nine of which have already been recognised as ACL vectors. Eleven species were recorded for the first time in TO. Nyssomyia whitmani was the most abundant species, followed by Evandromyia bourrouli, Nyssomyia antunesi and Psychodopygus complexus. The Shannon-Wiener diversity index and the evenness index were higher in the rural settlement area than in the periurban area. The evaluation of different ecotopes within the rural area showed the highest frequencies of Ev. bourrouli and Ny. antunesi in chicken coops, whereas Ny. whitmani predominated in this ecotope in the periurban area. In the rural settlement area, Ev. bourrouli was the most frequently captured species in automatic light traps and Ps. complexus was the most prevalent in Shannon trap captures. The rural settlement environment exhibited greater phlebotomine biodiversity than the periurban area. Ps. complexus and Psychodopygus ayrozai naturally infected with Leishmania (Viannia) braziliensis were identified. The data identified Ny. whitmani as a potential ACL vector in the periurban area, whereas Ps. complexus was more prevalent in the rural environment associated with settlements.

Spatial and temporal changes in Lutzomyia longipalpis abundance, a Leishmania infantum vector in an urban area in northeastern Argentina

This study aimed to analyse changes in the spatial distribution of Lutzomyia longipalpis in Posadas, an urban area located in northeastern Argentina. Data were obtained during the summer of 2007 and 2009 through two entomological surveys of peridomiciles distributed around the city. The abundance distribution pattern for 2009 was computed and compared with the previous pattern obtained in 2007, when the first human visceral leishmaniasis cases were reported in the city. Vector abundance was also examined in relation to micro and macrohabitat characteristics. In 2007 and 2009, Lu. longipalpis was distributed among 41.5% and 31% of the households in the study area, respectively. In both years, the abundance rates at most of the trapping sites were below 30 Lu. longipalpis per trap per night; however, for areas exhibiting 30-60 Lu. longipalpis and more than 60 Lu. longipalpis, the areas increased in both size and number from 2007-2009. Lu. longipalpis was more abundant in areas with a higher tree and bush cover (a macrohabitat characteristic) and in peridomiciles with accumulated unused material (a microhabitat characteristic). These results will help to prioritise and focus control efforts by defining which peridomiciles display a potentially high abundance of Lu. longipalpis.

Malaria in Brazil: what happens outside the Amazonian endemic region

Brazil, a country of continental proportions, presents three profiles of malaria transmission. The first and most important numerically, occurs inside the Amazon. The Amazon accounts for approximately 60% of the nation’s territory and approximately 13% of the Brazilian population. This region hosts 99.5% of the nation’s malaria cases, which are predominantly caused by Plasmodium vivax (i.e., 82% of cases in 2013). The second involves imported malaria, which corresponds to malaria cases acquired outside the region where the individuals live or the diagnosis was made. These cases are imported from endemic regions of Brazil (i.e., the Amazon) or from other countries in South and Central America, Africa and Asia. Imported malaria comprised 89% of the cases found outside the area of active transmission in Brazil in 2013. These cases highlight an important question with respect to both therapeutic and epidemiological issues because patients, especially those with falciparum malaria, arriving in a region where the health professionals may not have experience with the clinical manifestations of malaria and its diagnosis could suffer dramatic consequences associated with a potential delay in treatment. Additionally, because the Anopheles vectors exist in most of the country, even a single case of malaria, if not diagnosed and treated immediately, may result in introduced cases, causing outbreaks and even introducing or reintroducing the disease to a non-endemic, receptive region. Cases introduced outside the Amazon usually occur in areas in which malaria was formerly endemic and are transmitted by competent vectors belonging to the subgenus Nyssorhynchus (i.e., Anopheles darlingi, Anopheles aquasalis and species of the Albitarsis complex). The third type of transmission accounts for only 0.05% of all cases and is caused by autochthonous malaria in the Atlantic Forest, located primarily along the southeastern Atlantic Coast. They are caused by parasites that seem to be (or to be very close to) P. vivax and, in a less extent, by Plasmodium malariae and it is transmitted by the bromeliad mosquito Anopheles (Kerteszia) cruzii. This paper deals mainly with the two profiles of malaria found outside the Amazon: the imported and ensuing introduced cases and the autochthonous cases. We also provide an update regarding the situation in Brazil and the Brazilian endemic Amazon.