A novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi

We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions.

First record of human trichinosis in Chile associated with consumption of wild boar (Sus scrofa)

The first South American case of human trichinosis, resulting from the consumption of roast wild boar (Sus scrofa) is reported in Chile. The patient presented fever, diarrhea, myalgias, facial edema, sub-conjunctival reddening, photophobia, eosinophilia, and elevated glutamic oxalacetic transaminase. The diagnosis was confirmed by two immunoenzymatic tests (ELISA) using somatic and excretion-secretion antigens.

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H); vector (Triatoma barberi) (RyC-V); and rodent reservoir (Peromyscus peromyscus) (RyC-R). The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes). Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40%) and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization

Clinical forms of Trypanosoma cruzi infected individuals in the chronic phase of Chagas disease in Puebla, Mexico

In Mexico, despite the relatively high seroprevalence of Trypanosoma cruzi infection in humans in some areas, reported morbidity of Chagas disease is not clear. We determined clinical stage in 71 individuals seropositive to T. cruzi in the state of Puebla, Mexico, an area endemic for Chagas disease with a reported seroprevalence of 7.7%. Diagnosis of Chagas disease was made by two standardized serological tests (ELISA, IHA). Individuals were stratified according to clinical studies. All patients were submitted to EKG, barium swallow, and barium enema. Groups were identified as indeterminate form (IF) asymptomatic individuals without evidence of abnormalities (n = 34 cases); those with gastrointestinal alterations (12 patients) including symptoms of abnormal relaxation of the lower esophageal sphincter and absent peristalsis in the esophageal body, grade I megaesophagus, and/or megacolon; patients with clinical manifestations and documented changes of chronic Chagas heart disease who were subdivided as follows: mild (8 patients) - mild electrocardiographic changes of ventricular repolarization, sinus bradychardia); moderate (6 patients) - left bundle branch block, right bundle branch block associated with left anterior fascicular block); severe (8 patients) - signs of cardiomegaly, dilated cardiomyopathy); and the associated form (3 cases) that included presence of both cardiomyopathy and megaesophagus. These data highlight the importance of accurate evaluation of the prevalence and clinical course of Chagas disease in endemic and non-endemic areas of Mexico.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

Isolation and molecular identification of Leishmania ( Viannia ) peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae) in the Peruvian Andes

Leishmania (Viannia) peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru). Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia) sp. was determined by polymerase chain reaction (PCR), and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V.) peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.

Incidence of myiasis in Panama during the eradication of Cochliomyia hominivorax (Coquerel 1858, Diptera: Calliphoridae) (2002-2005)

We present the results of a study on myiasis in Panama during the first years of a Cochliomyia hominivorax eradication program (1998-2005), with the aim of investigating the behavior of the flies that produce myiasis in animals and human beings. The hosts that registered positive for myiasis were cattle (46.4%), dogs (15.3%), humans (14.7%), birds (12%), pigs (6%), horses (4%), and sheep (1%). Six fly species caused myiasis: Dermatobia hominis (58%), Phaenicia spp. (20%), Cochliomyia macellaria (19%), Chrysomya rufifacies (0.4%), and maggots of unidentified species belonging to the Sarcophagidae (3%) and Muscidae (0.3%). With the Dubois index, was no evidence that the absence of C. hominivorax allowed an increase in the cases of facultative myiasis.

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.

Malaria entomological inoculation rates in gold mining areas of Southern Venezuela

A longitudinal study of malaria vectors aiming to describe the intensity of transmission was carried out in five villages of Southern Venezuela between January 1999-April 2000. The man-biting, sporozoite and entomological inoculation rates (EIR) were calculated based on 121 all-night collections of anophelines landing on humans, CDC light traps and ultra violet up-draft traps. A total of 6,027 female mosquitoes representing seven species were collected. The most abundant species were Anopheles marajoara Galvão & Damasceno (56.7%) and Anopheles darlingi Root (33%), which together accounted for 89.7% of the total anophelines collected. The mean biting rate for An. marajoara was 1.27 (SD + 0.81); it was 0.74 (SD + 0.91) for An. darlingand 0.11 (SD + 0.10) for Anopheles neomaculipalpus Curry and the overall biting rate was 2.29 (SD + 1.06). A total of 5,886 mosquitoes collected by all three methods were assayed by ELISA and 28 pools, equivalent to 28 mosquitoes, yielded positive results for Plasmodium spp. CS protein. An. neomaculipalpus had the highest sporozoite rate 0.84% (3/356), followed by An. darlingi 0.82% (16/1,948) and An. marajoara 0.27% (9/3,332). The overall sporozoite rate was 0.48% (28/5,886). The rates of infection by Plasmodium species in mosquitoes were 0.37% (22/5,886) for Plasmodium vivax(Grassi & Feletti) and 0.10% (6/5,886) for Plasmodium falciparum (Welch). The estimated overall EIR for An. darling was 2.21 infective bites/person/year, 1.25 for An. marajoara and 0.34 for An. neomaculipalpus. The overall EIR was four infective bites/person/year. The biting rate, the sporozoite rate and the EIR are too low to be indicators of the efficacy of control campaigns in this area.

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.

Identification of the natural breeding sites of sandflies (Diptera: Psychodidae: Phlebotominae), potential vectors of leishmaniasis, in the province of Chaco, Argentina

The aim of this work was to identify the natural breeding sites of sandflies in the province of Chaco, Argentina, for the first time. Preliminary studies were conducted in two different phytogeographic regions: dry Chaco (Parque Provincial Pampa del Indio), in January 2010, and humid Chaco (Resistencia, Margarita Belén and Colonia Benítez), from May-September 2010. A total of 127 samples were collected (Pampa del Indio: 15, Resistencia: 37, Margarita Belén: 36, Colonia Benítez: 39). A female of Migonemyia migonei was found in Pampa del Indio at the base of a bromeliad in the summer (January) and a pupal exuvium of a phlebotomine fly was found in Resistencia, in a place where dogs rested, in the winter (July). These findings highlighted these two sites as potential breeding sites. Because the existence of potential natural breeding sites for sandflies has been demonstrated in both forest and periurban areas, expanding the search efforts and characterising these sites will enable the development of specific study designs to gain insight into the spatial distribution of the risks posed by these vectors. The resulting information will serve as a basis for proposing and evaluating vector control measures.

Lutzomyia longipalpis abundance in the city of Posadas, northeastern Argentina: variations at different spatial scales

The distribution of Lutzomyia longipalpis is heterogeneous with a pattern of high abundance areas (HAAs) embedded in a matrix of low abundance areas (LAAs). The objective of this study was to describe the variability in the abundance of Lu. longipalpis at two different spatial levels and to analyse the relationship between the abundance and multiple environmental variables. Of the environmental variables analysed in each household, the condition that best explained the differences in vector abundance between HAA-LAA was the variable "land_grass", with greater average values in the peridomestic environments within the LAA, and the variables "#sp tree", "#pots" and "dist_water" that were higher in the HAA. Of the environmental variables analysed in the patches, the variable "unpaved_streets" was higher in the LAAs and the variable "prop_inf_dogs" was higher in the HAAs. An understanding of the main environmental variables that influence the vector distribution could contribute to the development of strategies for the prevention and control of visceral leishmaniasis (VL). This is the first work in which environmental variables are analysed at the micro-scale in urban areas at the southern edge of the current range of Lu. longipalpis. Our results represent a significant contribution to the understanding of the abundance of the vector in the peridomestic habitats of the region.

Vector control intervention towards interruption of transmission of Chagas disease by Rhodnius prolixus, main vector in Guatemala

In Guatemala, the Ministry of Health (MoH) began a vector control project with Japanese cooperation in 2000 to reduce the risk of Chagas disease infection. Rhodnius prolixus is one of the principal vectors and is targeted for elimination. The control method consisted of extensive residual insecticide spraying campaigns, followed by community-based surveillance with selective respraying. Interventions in nine endemic departments identified 317 villages with R. prolixus of 4,417 villages surveyed. Two cycles of residual insecticide spraying covered over 98% of the houses in the identified villages. Fourteen villages reinfestated were all resprayed. Between 2000-2003 and 2008, the number of infested villages decreased from 317 to two and the house infestation rate reduced from 0.86% to 0.0036%. Seroprevalence rates in 2004-2005, when compared with an earlier study in 1998, showed a significant decline from 5.3% to 1.3% among schoolchildren in endemic areas. The total operational cost was US$ 921,815, where the cost ratio between preparatory, attack and surveillance phases was approximately 2:12:1. In 2008, Guatemala was certified for interruption of Chagas disease transmission by R. prolixus. What facilitated the process was existing knowledge in vector control and notable commitment by the MoH, as well as political, managerial and technical support by external stakeholders.

Seasonal variation and natural infection of Lutzomyia antunesi (Diptera: Psychodidae: Phlebotominae), an endemic species in the Orinoquia region of Colombia

Lutzomyia antunesi has been commonly reported in outbreaks of cutaneous leishmaniasis (CL) in the Orinoquia region of Colombia. The bionomics of this species were studied in the municipality of Villavicencio (Meta, Colombia). Sandflies were captured over the course of one week per month for one year in intradomiciliary, peridomiciliary and extradomiciliary housing areas. The captures were performed from 06:00 pm-06:00 am using CDC light traps and the females were processed for polymerase chain reaction (PCR) to detect Leishmania spp. A total of 22,097 specimens and 19 species were captured of which Lu. antunesi (89%) and Lutzomyia walkeri (5%) were the most abundant. Other species recognised as anthropophilic (Lutzomyia panamensis, Lutzomyia gomezi, Lutzomyia flaviscutellata and Lutzomyia fairtigi) were present in very low abundance (< 2%). Natural infection with Leishmania spp was detected using PCR in Lu. antunesi, Lu. panamensis and Lu. flavicutellata, showing infection rates of 1%, 4.8% and 7.5%, respectively. The present paper provides information on various ecological aspects of Lu. antunesi. An analysis of seasonality shows that this species increases in abundance in the hottest months (December, January and February), directly correlating with the maximum temperature and inversely correlating with precipitation. The natural infection rate is associated with the peaks of highest abundance.