Candidate targets for Multilocus Sequence Typing of Trypanosoma cruzi: validation using parasite stocks from the Chaco Region and a set of reference strains.

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 4178 bp 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

Multilocus sequence typing approach for a broader range of species of Leishmania genus: Describing parasite diversity in Argentina

Leishmaniasis is a vector-borne protozoan infection affecting over 350 million people around the world. In Argentina cutaneous leishmaniasis is endemic in nine provinces and visceral leishmaniasis is spreading from autochthonous transmission foci in seven provinces. However, there is limited information about the diversity of the parasite in this country. Implementation of molecular strategies for parasite typing, particularly multilocus sequence typing (MLST), represents an improved approach for genetic variability and population dynamics analyses. We selected six loci as candidates implemented in reference strains and Argentinean isolates. Phylogenetic analysis showed high correlation with taxonomic classification of the parasite. Autochthonous Leishmania (Viannia) braziliensis showed higher genetic diversity than L. (Leishmania) infantum but low support was obtained for intra-L. braziliensis complex variants suggesting the need of new loci that contribute to phylogenetic resolution for an improved MLST or nested-MLST scheme. This study represents the first characterization of genetic variability of Leishmania spp. in Argentina.Fil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Barroso, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Locatelli, Fabricio M.. Kochi University. Kochi Medical School. Departament of Parasitology; JapónFil: Cajal, S. Pamela. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Hoyos, Carlos Lorenzo. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Nevot, Cecilia. Kochi University. Kochi Medical School. Departament of Parasitology; JapónFil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; ArgentinaFil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; ArgentinaFil: Juarez, Marisa. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Estevez, J. Octavio. Veterinaria Del Oeste; ArgentinaFil: Korenaga, Masataka. Kochi University. Kochi Medical School. Departament of Parasitology; JapónFil: Nasser, Julio. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School. Dept.of Parasitology; Japón. Universidad Central del Ecuador y Proyecto Prometeo. Centro de Biomedicina; EcuadorFil: Ruybal, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones En Microbiología y Parasitología Médica; Argentin

Biological behavior of different Trypanosoma cruzi isolates circulating in an endemic area for Chagas disease in the Gran Chaco region of Argentina

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High performance of an enzyme linked immunosorbent assay for American tegumentary leishmaniasis diagnosis with Leishmania (Viannia) braziliensis amastigotes membrane crude antigens.

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

Genetic and clinical characterization of canine leishmaniasis caused by Leishmania (Leishmania) infantum in northeastern Argentina

Leishmaniases comprise zoonotic diseases caused by protozoan flagellates of the Leishmania genus. They are endemic to South America, and the visceral form has been recently reported in Argentina. Dogs can play different roles in the Leishmania transmission cycles, depending mainly on the species of parasite involved. Here we focused on the clinical characterization of canine leishmaniasis (CanL) in Northeast Argentina and on the molecular typing of its etiological agent. The nested polymerase chain reaction and sequence analysis of the Leishmania cytochrome b (cyt b) gene was performed on DNA templates purified from lymph nodes, bone marrow or spleen aspirates obtained from 48 dogs previously diagnosed by the observation of Leishmania amastigotes on smears from these aspirates. Their clinical and epidemiological data were also recorded. Systemic abnormalities were observed in 46 subjects (95.8%), most frequently lymphadenopathy, and emaciation (89.6 and 75%). Furthermore, 87% also presented tegumentary abnormalities, such as alopecia (54.2%) or secondary skin lesions (47.9%), among others. Twenty three dogs were positive for cyt b amplification. The sequence analysis showed the presence of two genotypes, LiA1 and LiA2, assigned to Leishmania (Leishmania) infantum, with 99.9 and 100% homology with the reference strain MHOM/TN/80/IPT1 respectively. LiA1 was identified in 18 cases (78.3%) and LiA2 in five (21.7%). Two cyt b variants of L. (L.) infantum were incriminated as the causative agents of CanL cases from three cities: Posadas, Garupá, and Ituzaingó. All three cities are located in the northeastern area of the country, where these parasites seem to be spreading in urban areas.Fil: Barroso, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Nevot, M. Cecilia. Veterinaria del Oeste; ArgentinaFil: Hoyos, Carlos Lorenzo. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Locatelli, Fabricio M.. Kochi University. Kochi Medical School; JapónFil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Ruybal, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Cardozo, Rubén Marino. Provincia de Salta. Ministerio de Salud Pública. Coordinación de Gestión Epidemiológica; ArgentinaFil: Russo, Pablo D.. Veterinaria del Oeste; ArgentinaFil: Vassiliades, Carola N.. Veterinaria del Oeste; ArgentinaFil: Mora, Maria Celia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Estévez, J. Octavio. Veterinaria del Oeste; ArgentinaFil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School; JapónFil: Korenaga, Masataka. Kochi University. Kochi Medical School; JapónFil: Basombrío, Miguel Ángel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta. Facultad de Ciencias Exactas; Argentin

Parasitemia in peripheral blood of singles and mixed infections.

<p>This variable was measured by microscopic observation, of animals inoculated with single and mixed isolates. (A) Shows difference between TcIII vs TcVI vs TcIII + TcVI, (B) TcVI vs TcV + TcVI and (C) TcIII vs TcIII + TcV. The parasitemia of TcV isolate was sub-patent.</p

Southern blot analyses using the TcIII (LL051-P24) probe.

<p>Each panel shows the electrophoretic pattern of minicircle regions and kDNA transferred to a nylon membrane. Panel A: lane TcIII, TcV, TcVI, TcV* and TcVI* correspond to DNA of parasite culture from LL051-P24 (DTU TcIII), LL055R3cl2 (DTU TcV), CL-Brener (DTU TcVI), LL014–1 (DTU TcV*) and LL040–1 (DTU TcVI*) respectively; and lane 1–4: blood (B) and skeletal muscle (SM) samples of mouse infected with TcIII isolate. The asterisk as superscript of the DTU indicates DNA sample from culture of the same inoculated isolate. Panel B, C and D: blood, skeletal muscle and cardiac muscle, respectively, of animals infected with TcIII + TcV (Lane 5–8) and TcIII + TcVI (Lane 9–11).</p