Characterization and follow-up of Trypanosoma cruzi natural populations refractory to etiological chemotherapy in oral chagas disease patients

We aimed to characterize the genetic constitution of natural T. cruzi populations involved in an Oral Chagas Disease (OCD) outbreak at a rural school of the community of Chichiriviche de la Costa, Venezuela, which affected patients did not respond to the etiological treatment. Peripheral blood samples and/or hemocultures were obtained from twenty-nine OCD patients at time of diagnosis or along nine years of Post-treatment (Tx) follow-up. The IgG serology, T. cruzi discrete typing units (DTU), satellite DNA-qPCR parasitic loads, and minicircle signatures were determined at Pre-Tx and after Tx. The serological titles and parasitic loads changed after treatment, with a significant decrease of IgG titers (Spearman’s r value= -0.961) and median parasite loads from 2.869 [IQR = 2.113 to 3.720] to 0.105 [IQR = -1.147 to 1.761] log10 par eq. /mL at Pre-Tx and Post-Tx, respectively, suggesting infection evolution from acute to chronic phase, without seroconversion or parasitological eradication, which was indicative of treatment failure. All patients were infected with T. cruzi DTU I populations. At Pre-Tx their median Jaccard genetic distances were 0.775 [IQR = 0.708 to 0.882], decreasing in genetic variability towards the end of follow-up (Mann-Whitney U test p= 0.0031). Interestingly, no Post-Tx minicircle signature was identical to its Pre-Tx counterpart population in a same patient, revealing selection of parasite subpopulations between the primary infection and Post-Tx. The parasitic populations isolated from hemocultures showed a lower number of bands in the minicircle signatures with respect to the signatures obtained directly from the patients’ blood samples, demonstrating a process of parasitic selection and reduction of the population variability that initially infected the patients. Decrease of parasitic loads after treatment as well as Pre- and Post-Tx intra-TcI diversity might be a consequence of both, natural evolution of the acute infection to the chronic phase and persistence of refractory populations due to Tx selection.Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Díaz Bello, Zoraida. Universidad Central de Venezuela; VenezuelaFil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela; VenezuelaFil: Noya González, Oscar O.. Universidad Central de Venezuela; VenezuelaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence

Accurate diagnostics tools and surrogate markers of parasitological response to treatment are priority needs for management of Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a Satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (Tc I) and CL-Brener (Tc VI) stocks similar LOD95 values (0.903 (0.745-1.497) and 0.667 (CI 0.113-3.927) copy numbers/μL, respectively), when synthetic DNA was the standard for quantification, thus allowing direct comparison of loads in samples infected with different DTUs. This standard curve was evaluated in 205 samples from 38 acute oral and 19 chronic CD patients from different geographical areas infected with different genotypes, including samples obtained during treatment follow-up, and high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase III clinical trials, to follow-up patients under treatment or at risk of reactivation and in experimental models using different parasite strains.Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Silva Gomess, Natalia Lins. Fundación Oswaldo Cruz; BrasilFil: Apodaca, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela; VenezuelaFil: Díaz Bello, Zoraida. Universidad Central de Venezuela; VenezuelaFil: Quintino Souza, Leticia Rocha. Fundación Oswaldo Cruz; BrasilFil: Tavares Costa, Alexandre Dias. Fundación Oswaldo Cruz; BrasilFil: Britto, Constança. Fundación Oswaldo Cruz; BrasilFil: Moreira, Otacilio Cruz. Fundación Oswaldo Cruz; BrasilFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Geographical Distribution of Trypanosoma cruzi Genotypes in Venezuela

Chagas disease is an endemic zoonosis native to the Americas and is caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The parasite is also highly genetically diverse, with six discrete typing units (DTUs) reported TcI – TcVI. These DTUs broadly correlate with several epidemiogical, ecological and pathological features of Chagas disease. In this manuscript we report the most comprehensive evaluation to date of the genetic diversity of T. cruzi in Venezuela. The dataset includes 778 samples collected and genotyped over the last twelve years from multiple hosts and vectors, including nine wild and domestic mammalian host species, and seven species of triatomine bug, as well as from human sources. Most isolates (732) can be assigned to the TcI clade (94.1%); 24 to the TcIV group (3.1%) and 22 to TcIII (2.8%). Importantly, among the 95 isolates genotyped from human disease cases, 79% belonged to TcI - a DTU common in the Americas, however, 21% belonged to TcIV- a little known genotype previously thought to be rare in humans. Furthermore, were able to assign multiple oral Chagas diseases cases to TcI in the area around the capital, Caracas. We discuss our findings in the context of T. cruzi DTU distributions elsewhere in the Americas, and evaluate the impact they have on the future of Chagas disease control in Venezuela

SHORT COMMUNICATION - A mucin like gene different from the previously reported members of the mucin like gene families is transcribed in Trypanosoma cruzi but not in Trypanosoma rangeli

Trypanosoma cruzi expresses mucin like glycoproteins encoded by a complex multigene family. In this work, we report the transcription in T. cruzi but not in T. rangeli of a mucin type gene automatically annotated by the T. cruzi genome project. The gene showed no nucleotide similarities with the previously reported T. cruzi mucin like genes, although the computational analysis of the deduced protein showed that it has the characteristic features of mucins: a signal peptide sequence, O-glycosylation sites, and glycosylphosphatidylinositol (GPI) anchor sequence. The presence in this gene of N- terminal and C- terminal coding sequences common to other annotated mucin like genes suggests the existence of a new mucin like gene family

Genetic diversity of natural populations of Trypanosoma cruzi in clinical samples from patients with oral Chagas disease in Venezuela: Follow-up after treatment with trypanocidal drugs

Oral transmission of Chagas disease (OChD) is an increasingly important aspect in the epidemiology. Venezuela has reported the two largest outbreaks described so far, affecting a total of 192 people mostly children. The long-term impact after treatment on the dynamics of infection in natural populations of T. cruzi is still unclear. In this sense, we proposed a genetic characterization of T. cruzi populations present in peripheral blood in order to differentiate responder or non-responder patients and predict the response to treatment (Tmt). We performed quantification of the parasitic load by qPCR, genetic typing of T. cruzi populations by multiplex qPCR of nuclear genome markers and RFLP-PCR for the hypervariable region of T. cruzi kDNA to demonstrate changes in Minicircle signatures (Ms) of the parasite populations present in 41 clinical samples from 15 patients. To reflect the genetic diversity found, Jaccard distances (Jd) values were compared. This clinical monitoring confirmed the presence of T. cruzi DNA in 26 post-treatment samples up to 9 years after Tmt. These results reveal 100 % therapeutic failure for both outbreaks of OChD, classifying these patients as non-responders to Tmt. All samples showed homogeneity at the DTU level, being typified as TcI. The Ms showed a high degree of polymorphism, with 73 % of total post-Tmt samples with Jd values close to 1. Analyzing the dynamics of each patient`s population separately, in all post-Tmt samples the change in Ms variability respect to pre-Tmt sample was not statistically significant. This variability does not reflect a natural or induced clonal selection process driven by the etiological Tmt; on the contrary, it could be associated with the clonal histotropism process evidenced in natural T. cruzi infections. In conclusion, these strategies of molecular characterization of parasite DNA were useful to detect Tmt failure and find out the lack of parasite population selection with Tmt in these OChD settings.Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Díaz Bello, Zoraida. Instituto de Medicina Tropical "Dr. Felix Pifano"; VenezuelaFil: Alarcón de Noya, Belkisyolé. Instituto de Medicina Tropical "Dr. Felix Pifano"; VenezuelaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaReunión Anual de Sociedades de Biociencia 2019; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; IX Reunión Anual de la Asociación Argentina de Nanomedicinas; VI Reunión Científica Regional de la Asociación Argentina de Ciencia y Tecnología de Animales de LaboratorioMar del PlataArgentinaSociedad Argentina de Investigación ClínicaAsociación Argentina de Farmacología ExperimentaSociedad Argentina de BiologíaSociedad Argentina de ProtozoologíaAsociación Argentina de NanomedicinasAsociación Argentina de Ciencia y Tecnología de Animales de Laboratori

LA TRANSMISIÓN ORAL EN LA ENFERMEDAD DE CHAGAS

La Enfermedad de Chagas se transmite al hombre por varios mecanismos participando en algunos, el vector de maneradirecta ó indirecta. En otras ocasiones, la transmisión de hombre a hombre ocurre a través de transfusiones, trasplantesde órganos y transplacentaria, y menos frecuente por la manipulación de tejidos, líquidos de animales infectados ó accidentesde laboratorio. La transmisión oral por contaminación de alimentos con el contenido intestinal de triatominos infectadoscon Trypanosoma cruzi ha sido un mecanismo demostrado experimentalmente en animales. Esta particular vía, probablementela más común entre los animales silvestres, asociado a la constitución bioquímica de los aislados, ha sidoresponsable de numerosos brotes en Brasil. En Venezuela se han descrito cuatro episodios desde 2007 con 228 casos y 6fallecimientos. Las medidas de vigilancia epidemiológica y control sanitario deben basarse en el estudio del comportamiento de los vectores, identificación de factores de riesgo y la concientización del personal de salud y autoridades sanitarias de que es una modalidad de transmisión de T cruzi por alimentos, definitivamente demostrada en VenezuelaABSTRACT: Chagas Disease is transmitted to humans through various mechanisms in which the vector directly or indirectly can participate.In other circumstances, infection from man to man occurs through blood transfusions, organ transplants and transplacentalthrough food contamination with the intestinal content of triatomines infected with Trypanosoma cruzi has been demonstratedexperimentally in animals. This particular way, probably the most common among wild animals, will depend on the biochemicalconstitution of the isolates and it has been responsible for numerous outbreaks in Brazil. In Venezuela, four episodeshave been reported since 2007 with 228 cases and 6 deaths. The measures of surveillance and disease control by the health authoritiesshould by based on the study of the behaivor of the vectors, identification of the main risk factors for the human population and awereness of the health staff and health authorities, that this way of transmission is definitely establishedin Venezuela

Diagnóstico de la Toxoplasmosis en la mujer embarazada y en el recién nacido

La infección aguda por Toxoplasma gondii durante el embarazo puede tener un resultado trágico para el recién nacido a pesar de que se puede prevenir. La infección puede ser adquirida por la ingestión de carne infectada o alimentos contaminados. La transmisión al feto se produce casi exclusivamente en las mujeres que adquieren la infección primaria durante la gestación y puede dar lugar a la pérdida de visión y audición, retraso mental y psicomotor, convulsiones o la muerte. Sistemática en la educación y el diagnóstico serológico de las embarazadas son las estrategias más confiables para la prevención, diagnóstico y tratamiento precoz de la infección en los bebes.AbstractAcute infection by Toxoplasma gondii during pregnancy can have a tragic outcome for the baby even though it is preventable. The infection can be acquired by eating infected meat or contaminated food. The transmission to the fetus occurs almost exclusively in women who acquire primary infection during pregnancy and can lead to loss of vision and hearing, mental and psychomotor retardation, seizures or death. Systematic education and serological diagnosis of pregnant women are the most reliable strategies for prevention, early diagnosis and treatment of infection in infants

Genetic Characterization of Trypanosoma cruzi I Populations from an Oral Chagas Disease Outbreak in Venezuela: Natural Resistance to Nitroheterocyclic Drugs

The oral transmission of Chagas disease (oCD) in Venezuela announced its appearance in 2007. Different from other populations affected by oCD and despite close supervision during treatment with nitroheterocyclic drugs, the result was treatment failure. We studied genetic features of natural bloodstream parasite populations and populations after treatment of nine patients of this outbreak. In total, we studied six hemoculture isolates, eight Pre-Tx blood samples, and 17 samples collected at two or three Post-Tx time-points between 2007 and 2015. Parasitic loads were determined by quantitative polymerase chain reaction (qPCR), and discrete typing units (DTU), minicircle signatures, and Tcntr-1 gene sequences were searched from blood samples and hemocultures. Half-maximal inhibitory concentration (IC50) values were measured from the hemocultures. All patients were infected by TcI. Significant decrease in parasitic loads was observed between Pre-Tx and Post-Tx samples, suggesting the evolution from acute to chronic phase of Chagas disease. 60% of intra-DTU-I variability was observed between Pre-Tx and Post-Tx minicircle signatures in the general population, and 43 single-nucleotide polymorphisms (SNPs) were detected in a total of 12 Tcntr-1 gene sequences, indicative of a polyclonal source of infection. SNPs in three post-Tx samples produced stop codons giving rise to putative truncated proteins or displaced open reading frames, which would render resistance genes. IC50 values varied from 5.301 ± 1.973 to 104.731 ± 4.556 μM, demonstrating a wide range of susceptibility. The poor drug response in the Pre-Tx parasite populations may be associated with the presence of naturally resistant parasite clones. Therefore, any information that can be obtained on drug susceptibility from in vitro assays, in vivo assays, or molecular characterization of natural populations of Trypanosoma cruzi becomes essential when therapeutic guidelines are designed in a given geographical area.Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ramirez, José Luis. Fundación Instituto de Estudios Avanzados ; VenezuelaFil: Díaz Bello, Zoraida. Universidad Central de Venezuela Facultad de Medicina; VenezuelaFil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela Facultad de Medicina; VenezuelaFil: Noya, Oscar. Universidad Central de Venezuela Facultad de Medicina; Venezuela. Ministerio del Poder Popular Para la Salud; VenezuelaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin