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Background. Endemic areas of tegumentary leishmaniasis (TL) in Salta, Argentina, present some overlap zones with the geographical distribution of Chagas disease, with mixed infection cases being often detected. Objectives. The purpose of this study was to determine the magnitude of Leishmania sp. infection and potential associated risk factors, the serologic prevalence of T. cruzi, and the presence of T. cruzi-Leishmania sp. mixed infection in a region of the northwest of Argentina. Methods. Cross-sectional studies were conducted to detect TL prevalence and T. cruzi seroprevalence. A case-control study was conducted to examine leishmaniasis risk factors. Results. Prevalence of TL was 0.17%, seroprevalence of T. cruzi infection was 9.73%, and mixed infection proportion—within the leishmaniasic patients group—was 16.67%. The risk factors associated with TL transmission were sex, age, exposure to bites at work, staying outdoors more than 10 hours/day, bathing in the river, and living with people who had lesions or were infected during the study. Discussion. The endemic pattern of TL seems to involve exposure of patients to vectors in wild as well as peridomestic environment. Cases of T. cruzi infection are apparently due to migration. Therefore, a careful epidemiological surveillance is necessary due to the contraindication of antimonial administration to chagasic patients. [ABSTRACT FROM AUTHOR]

Background: The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients. Methodology/Principal Findings: We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%–99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%–99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%–74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%–84.4%) for the kDNA OligoC-Test. Conclusions/Significance: Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT. Author Summary: Accurate diagnosis of Chagas disease is challenging due to the latent character of the infection and the low parasite load in the blood. Molecular tests such as the polymerase chain reaction (PCR) detect the parasite's DNA and are generally very sensitive and specific. In this study we evaluated two prototypes of a standardized PCR diagnostic kit: the satellite DNA (satDNA) OligoC-TesT and the kinetoplast (kDNA) OligoC-TesT. Sensitivities and specificities of both tests were estimated in a multi-cohort phase II evaluation study with 187 chronic Chagas patients and 88 healthy endemic controls from Argentina, Chile and Spain, and 26 non-endemic controls from D.R. Congo and Sudan with potentially cross-reacting diseases. Specificities in the control persons were high (>97%) and the sensitivity of the kDNA OligoC-TesT (79.1%) was significantly higher than of the satDNA OligoC-Test (67.9%). In a next phase, the kDNA OligoC-TesT should be evaluated in specific niches where standard serological tools have their limitations, e.g. follow-up after treatment and diagnosing newborns and HIV co-infected patients. [ABSTRACT FROM AUTHOR]

Trypanosomes are a group of parasitic flagellates with medical and veterinary importance. Despite many species having been described in this genus, little is known about many of them. Here, we report a genetic and morphological characterization of trypanosomatids isolated from wild mammals from the Argentine Chaco region. Parasites were morphologically and ultrastructurally characterized by light microscopy and transmission electron microscopy. Additionally, 18s rRNA and gGAPDH genes were sequenced and analyzed using maximum likelihood and Bayesian inference. Morphological characterization showed clear characteristics associated with the Trypanosoma genus. The genetic characterization demonstrates that the studied isolates have identical sequences and a pairwise identity of 99% with Trypanosoma lainsoni, which belongs to the clade of lizards and snakes/rodents and marsupials. To date, this species had only been found in the Amazon region. Our finding represents the second report of T. lainsoni and the first record for the Chaco region. Furthermore, we ultrastructurally described for the first time the species. Finally, the host range of T. lainsoni was expanded (Leopardus geoffroyi, Carenivora, Felidae; and Calomys sp., Rodentia, Cricetidae), showing a wide host range for this species. [ABSTRACT FROM AUTHOR]

Abstract: The biological behavior of the different Trypanosoma cruzi strains is still unclear and the importance of exploring the relevance of these differences in natural isolates is of great significance. Herein we describe the biological behavior of four T. cruzi isolates circulating sympatrically in a restricted geographic area in Argentina endemic for Chagas Disease. These isolates were characterized as belonging to the Discrete Typing Units (DTUs) TcI, TcIII, TcV and TcVI as shown by Multilocus Enzyme Electrophoresis and Multilocus Sequence Typing. In order to study the natural behavior of the different isolates and to preserve their natural properties, we developed a vector transmission model that allows their maintenance in the laboratory. The model consisted of serial passages of these parasites between insect vectors and mice. Vector-derived parasite forms were then inoculated in C57BL/6J mice and number of parasite in peripheral blood, serological response and histological damage in acute and chronic phases of the infection were measured. Parasites from DTUs TcI, TcIII and TcVI were detected by direct fresh blood examination, while TcV parasites could only be detected by Polimerase Chain Reaction. No significant difference in the anti-T. cruzi antibody response was found during the chronic phase of infection, except for mice infected with TcV parasites where no antibodies could be detected. Histological sections showed that TcI isolate produced more damage in skeletal muscle while TcVI induced more inflammation in the heart. This work shows differential biological behavior among different parasite isolates obtained from the same cycle of transmission, permitting the opportunity to formulate future hypotheses of clinical and epidemiological importance. [Copyright &y& Elsevier]

Abstract: A Multilocus Sequence Typing (MLST) scheme was designed and applied to a set of 20 Trypanosoma cruzi stocks belonging to three main discrete typing units (T. cruzi I, V and VI) from a geographically restricted Chagas disease endemic area in Argentina, 12 reference strains comprising two from each of the six main discrete typing units of the parasite (T. cruzi I–VI), and one T. cruzi marinkellei strain. DNA fragments (≅400-bp) from 10 housekeeping genes were sequenced. A total of 4178bp were analyzed for each stock. In all, 154 polymorphic sites were identified. Ninety-five sites were heterozygous in at least one analyzed stock. Seventeen diploid sequence types were identified from 32 studied T. cruzi stocks (including the reference strains). All stocks were correctly assigned to their corresponding discrete typing units. We propose this MLST scheme as provisional, with scope for improvement by studying new gene targets on a more diverse sample of stocks, in order to define an optimized MLST scheme for T. cruzi. This approach is an excellent candidate to become the gold standard for T. cruzi genetic typing. We suggest that MLST will have a strong impact on molecular epidemiological studies of Chagas disease and the phylogenetics of its causative agent. [Copyright &y& Elsevier]

Abstract: Internal and geographical clustering within Trypanosoma cruzi I (TcI) has been recently revealed by using Multilocus Microsatellite Typing and sequencing of the Spliced-Leader Intergenic Region (SL-IR). In the present work, 14 isolates and 11 laboratory-cloned stocks obtained from a geographically restricted area in Chaco Province, Argentina, were analyzed by PCR and sequencing of SL-IR. We were able to differentiate 8 different genotypes that clustered into 4 groups. One of these groups was classified within the formerly described haplotype A and another one within the recently described SL-IR group E. Both were phylogenetically well-supported. In contrast, none of the stocks from the Chaco province were grouped within the cluster previously named haplotype D despite the fact that they shared a similar microsatellite motif in the SL-IR. No evidence of recombination or gene conversion within these stocks was found. On the other hand, multiple ambiguous alignments in the microsatellite region of SL-IR, affecting the tree topology and relationships among groups were detected. Finally, since there are multiple copies of the SL-IR, and they are arranged in tandem, we discuss how molecular processes affecting this kind of sequences could mislead phylogenetic inference. [Copyright &y& Elsevier]

Abstract: Trypanosoma cruzi is genetically classified into six discrete phylogenetic lineages on the basis of different genetic markers. Identifying lineages circulating among humans in different areas is essential to understand the molecular epidemiology of Chagas disease. In the present study, 18 T. cruzi isolates from congenitally infected newborns in the northwestern province of Salta-Argentina were studied by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD). All isolates were typed by MLEE and RAPD as belonging to T. cruzi IId. Analysis of minor variants of TcIId using probes hybridizing with hypervariable domains of kDNA minicircles, detected three variants with a similar distribution among the isolates. Our findings confirm the presence of T. cruzi IId among congenitally infected newborns in northwestern Argentina and support the assumption that human infection by T. cruzi in the Southern Cone countries of Latin America is due principally to T. cruzi II. [Copyright &y& Elsevier]

The diagnosis of American tegumentary leishmaniasis (ATL) still requires the design of more effective tools. Leishmania (Viannia) braziliensis is the causal agent of the 90% of Argentinean ATL cases. Considering the current knowledge, an ELISA based crude antigen (CA) for the diagnosis was designed. Ninety-nine subjects diagnosed as ATL, 27 as no-ATL, and 84 donors from non-ATL-endemic areas were included in this study. The current ATL diagnosis was based four techniques, dermal smear microscopic examination (parasitological test), PCR, Leishmanin skin test, and clinical records. We obtained CA extracts from promastigotes and amastigotes from macrophage cultures of different zymodemes of endemic Leishmania species circulating in the study area. Crude antigens from the 'local' main zymodeme of L. (V.) braziliensis showed the highest reactivity against anti-Leishmania antibodies compared to the other included species. The CA of amastigotes of this zymodeme was 3.4 fold more reactive than promastigotes one. Moreover, amastigote-membrane CA (MCA) were 3.6 fold more reactive than the soluble antigens. The MCA-ELISA reached a sensitivity and specificity of 98% (CI = 94.7%-100%) and 63.6% (53.9-73.1), respectively. When anti-Trypanosoma cruzi reactive sera were excluded, the specificity reached 98.4% (94.4-100), while the sensitivity was similar, with a positive predictive value (PV) of 98.6% (94.6-100) and negative PV of 96.3% (91.6-100). The performance of the MCA-ELISA results strongly contribute to the final diagnostic decision, since a non-reactive serological result almost discards the suspected ATL, because of its high negative PV. The developed MCA-ELISA showed a high diagnostic performance, which makes it a good candidate for ATL diagnosis, for seroprevalence studies, or for monitoring treatments efficacy., Competing Interests: The authors have declared that no competing interests exist.

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales, the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study, we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area. Finally, considering our findings we discuss the scenario for the genetic structure of TcI., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We evaluated the recombinant antigen SAPA (Shed Acute Phase Antigen) for the detection of Trypanosoma cruzi antibodies in sera from naturally infected dogs. The technique used was ELISA and the antigens were a homogenate of parasite T. cruzi (ELISA-H) and the recombinant SAPA (ELISA-SAPA). We analyzed 93 sera from dogs by ELISA-H and ELISA-SAPA, which were grouped as follows: G1: 11 negative control sera from the city of Salta, G2: 11 positive control sera from dogs naturally infected with T. cruzi and G3: 71 samples of dogs belonging to a Chagas disease-endemic area. The sensitivity and specificity of ELISA-SAPA were 100 %. The kappa index between ELISA-H and ELISA-SAPA was 0,85. These results confirm the use of SAPA antigen in the diagnosis of infection with T. cruzi in dogs.

We used kernel density and scan statistics to examine the spatial distribution of cases of pediatric and adult American cutaneous leishmaniasis in an urban disease-endemic area in Salta Province, Argentina. Spatial analysis was used for the whole population and stratified by women > 14 years of age (n = 159), men > 14 years of age (n = 667), and children < 15 years of age (n = 213). Although kernel density for adults encompassed nearly the entire city, distribution in children was most prevalent in the peripheral areas of the city. Scan statistic analysis for adult males, adult females, and children found 11, 2, and 8 clusters, respectively. Clusters for children had the highest odds ratios (P < 0.05) and were located in proximity of plantations and secondary vegetation. The data from this study provide further evidence of the potential urban transmission of American cutaneous leishmaniasis in northern Argentina.

We studied the seroprevalence of antibodies against Trypanosoma cruzi in the human population along with domiciliary infestation by triatomine bugs in an area endemic for Chagas disease in the Chaco Province of Argentina. In addition, we carried out parasitologic surveys in patients, dogs, wild mammals, and vectors. The mean seroprevalence in humans was 27.81% (109 of 392) and 24.14% (63 of 261) in 1-15-year-old children. The minimum domiciliary infestation rate was 13.33%, with certain areas reaching 53.85%. The prevalence was 15.09% (16 of 106) in dogs and 35.71% (10 of 28) in opossums. Infection with T. cruzi was detected in 30.10% (59 of 196) of the Triatoma infestans tested. Compared with nationwide studies, our data suggest that 1) there are zones requiring immediate sanitary action, and 2) nationwide estimates are based on very heterogeneous epidemiologic situations. This heterogeneity emphasizes the importance of in-depth studies of restricted areas to provide additional information for a better understanding of the present status of Chagas disease in Argentina.

A set of 65 Trypanosoma cruzi stocks from dogs, opossums, insect vectors and humans was isolated in a geographically restricted endemic area for Chagas' disease in Argentina and was analysed by multilocus enzyme electrophoresis for 15 loci. The results show that at least five multilocus genotypes (clonets) circulate in the study area, one belonging to T. cruzi IIe, one to T. cruzi IId and three clonets belonging to T. cruzi I; and they confirm the presence of these lineages in the country. The three clonets attributed to T. cruzi I were identical to each other for all loci except for Sod-2, where three different patterns were identified. These patterns suggest the presence of two homozygous genotypes and one heterozygous genotype. Our results also suggest association of clonet IIe with dogs, clonet IId with humans and the three T. cruzi I clonets with Didelphis albiventris. On the other hand, there was no significant association between Triatoma infestans and any particular clonet circulating in the area. These findings are consistent with the hypothesis of natural selection, from mixed populations of T. cruzi in vectors, toward more restricted populations in mammals. The epidemiological implications of the possible selection of different clonets by different mammal hosts and the significance of two homozygous genotypes and one heterozygous genotype for the Sod-2 locus are discussed.