Parasite Burden of Trypanosoma cruzi in Whole Blood and Buffy Coat Determined by Real-Time PCR in Individuals with Chronic Chagas Disease.

The objective of this study was to compare, by qPCR, the circulating blood parasite load of Trypanosoma cruzi in the buffy coat, and in whole blood mixed with boiled and unboiled guanidine hydrochloride-EDTA buffer, of individuals with chronic ChD. The concentration and purity of DNA were evaluated in a Nanodrop Denovix DS-11FX Series Spectrophotometer (DeNovix Inc., Wilmington, NC, USA). The parasite load was determined with the Taqman® qPCR system using a Stratagene Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA, USA) with Cruzi 1 and Cruzi 2 satellite primers. Student's t-test with Bonferroni correction, Chi-squared (χ2) tests and Spearman's correlation coefficient were applied. The concentration and purity of DNA were higher in the buffy coat. Parasite DNA was detected and quantifiable in the three types of samples in seven patients, without statistically significant differences in the parasite load obtained. Higher correlations were found between the total DNA concentrations and the parasite loads obtained in the samples of the buffy coat.

Discrete Typing Units of Trypanosoma cruzi Identified by Real-Time PCR in Peripheral Blood and Dejections of Triatoma infestans Used in Xenodiagnosis Descriptive Study.

Chagas disease (ChD) is a vector zoonosis native to the American continent caused by the protozoan parasite Trypanosoma cruzi; the biological vectors are multiple species of hematophagous insects of the family Triatominae. A relevant aspect in the host-parasite relationship is the identification of the various genotypes of T. cruzi called discrete typing units (DTU) that circulate in mammals and vectors. In Chile, it has been described that the DTUs TcI, TcII, TcV, and TcVI circulate in infected humans, vectors, and wild animals. Identifying DTUs has acquired clinical importance, since it has been suggested that different genotypes could cause distinct pathologies, circulate in different geographical areas, and present different sensitivities to trypanocidal drugs. In this study, circulating T. cruzi DTUs in peripheral blood and Triatoma infestans dejections used in xenodiagnosis (XD) were amplified by qPCR in 14 Chilean patients with chronic ChD from highly endemic areas. More positive samples were detected by XD compared to peripheral blood samples, and 64.28% of the cases were simple infections and 35.72% mixed, with a statistically significant difference in the frequency of TcV DTU. This study would suggest that T. infestans from Chile is more competent to amplify one DTU over others, probably due to a process of co-evolution.

Clinical, electrocardiographic and echocardiographic evolution of chronic Chagas disease treated with nifurtimox on prolonged follow-up in Chile: observational study.

OBJECTIVES: This study aimed to describe the electrocardiographic and echocardiographic status of chronic Chagas disease (cChD) patients treated with nifurtimox. METHODS: An observational study was performed in 146 cChD patients followed over a mean of 7.9 years. RESULTS: Of the 146 patients, 41 (28.1%) with normal electrocardiogram (ECG) at baseline maintained this condition, 34 (23.3%) with altered ECG at baseline normalised the alterations, and 46 (31.5%) with ECG abnormalities at baseline maintained this condition [23 (15.8%) with small alterations]. Finally, 25 cases (17.1%) in indeterminate phase altered the ECG. Differences before and after follow-up (P < 0.001) were found. The percentage of beneficial treatment was different than expected by chance (Z = 4.8; P < 0.001) and the annual percentage of cases that developed ECG alterations was lower than that of a historical cohort of untreated patients (P < 0.001). An echocardiogram was performed in 68 patients with baseline ECG alterations. The ejection fraction (EF) was normal in 57 (83.8%) and abnormal in 11 (16.2%). In 38 patients with ECG abnormalities that did not progress after treatment, EF and segmental motility (SM) were normal in 31 (81.6%) and 26 (68.4%), respectively. In 17 patients with ECG abnormalities, EF and SM were normal in 15 (88.2%) and 14 (82.4%) cases, respectively. CONCLUSION: Less progression to cardiomyopathy compared with a historical untreated cohort as well as the EF/SM results in patients with abnormal ECG that did not progress and in indeterminate cChD that altered the ECG suggests a beneficial effect of nifurtimox.

[Effects of climate change on reproductive number of Chagas disease]. / Estimando el efecto del cambio climático sobre el riesgo de la enfermedad de Chagas en Chile por medio del número reproductivo.

BACKGROUND: Reproductive number (R0)-maps estimate risk zones of vector-borne diseases and geographical distribution changes under climate change. AIM: To map R0 aiming to estimate the epidemiological risk of Chagas disease in Chile, its distribution and possible changes due to the global climate change. MATERIAL AND METHODS: We used a relationship between R0 and entomological parameters of vectors as function of environmental variables, to map the risk of Chagas disease in Chile, under current and projected future environmental conditions. RESULTS: We obtained a geographical R0 estimation of Chagas disease in Chile. The highest R0averages correspond to the Central-Northern regions of Chile. T. cruzi transmission area could increase in the future due to climate changes. Independent of the future condition, both for optimistic and pessimistic climate change scenarios, the area of potential risk for Chagas disease transmission would increase. The estimated R0 values suggest that, if a control of T. infestans is not maintained, Chagas disease endemic status will persist or increase, independently of the climate change scenarios. CONCLUSIONS: Mapping R0 values is an effective method to assess the risk of Chagas disease. The eventual increase in the transmission area of the disease is worrisome.

Chronic Chagas disease: Quantification of Trypanosoma cruzi in peripheral blood and dejections of Triatoma infestans fed by xenodiagnosis in patients with and without cardiopathy.

It is not currently known which individuals with chronic Chagas disease (ChD) will develop cardiopathy in a determined period and which will be maintained asymptomatic with normal routine laboratory tests all their lives. The parasite burden is a factor that could explain this different evolution. The objective of this study was to quantify Trypanosoma cruzi burden by real-time PCR in blood (qPCR-B) and dejections of triatomines fed by xenodiagnosis (qPCR-XD) in 90 individuals with chronic ChD untreated, classified according to XD results and the presence or absence of cardiopathy. All individuals came from hyperendemic areas of Chile and participated in the study under Informed Consent. The standard qPCR curves for qPCR-B and qPCR-XD were elaborated with a mixture of known concentrations of T. cruzi strains, performing DNA serial dilutions (1/10) with a dynamic range between 105 and 10-1 parasite equivalents/mL. The TaqManⓇ detection system was applied in a Stratagene Mx3000P thermocycler (Agilent Technologies, USA) with cruzi 1 and cruzi 2 satellite primers. 22.2% and 15.6% of cases with cardiopathy or without cardiopathy were XD positive. There was no significant difference between the groups. The positivity of qPCR-B and qPCR-XD in the positive XD group was 82.35% and 100%, respectively, while in the negative XD group was 55.26% and 42.10%, respectively. A superior qPCR value in chronic ChD patients with and without cardiopathy was determined for qPCR in cases with positive XD and positive qPCR-XD. The receiver operating characteristic (ROC) curve analyses show better accuracy for detecting parasite burden (area under the curve, AUC) for qPCR-XD in comparison to qPCR-B. That is to say, major performance in DNA samples obtained of positive XD (gold standard for viable T. cruzi) detected and quantified by qPCR-XD. A high percentage of cases with XD and qPCR-XD positive (80-100%) have result concordant with qPCR-B. In absence of XD, future challenges are especially related to the low parasitic load of chronic ChD patients treated with trypanocidal drugs and post-therapy parasitological evaluations by qPCR-B. Finally, no statistically significant differences were found between presence or absence of cardiopathy and XD, qPCR-B or qPCR-XD.

Detection of Trypanosoma cruzi by PCR in adults with chronic Chagas disease treated with nifurtimox.

Chagas disease, a vector-borne parasitosis caused by Trypanosoma cruzi, is endemic to Latin America and has spread to other countries due to immigration of infected persons. It is estimated that 160,000 people are infected in Chile, most of them in the chronic phase and without etiological treatment. The infection is confirmed by conventional serological methods while molecular methods have become in valuable tools to evaluate parasitemia in treated and non-treated chronic Chagas disease patients. The objective of this study was to determine, by conventional Polymerase Chain Reaction, the presence of T. cruzi kinetoplastid DNA in peripheral blood samples from 114 adult individuals with confirmed chronic Chagas disease, before and 6.6 years (average) after treatment with nifurtimox. The samples were received and preserved in guanidine-EDTA until DNA purification. Conventional PCR assays were performed in triplicate with T. cruzi kinetoplastid DNA primers 121 and 122. The amplified products were fractionated by electrophoresis in 2% agarose gels. A 330 bp product represented a positive assay. 84.2% (96 cases) and 6.1% (7 cases) of the samples taken before and after the treatment, respectively, were positive. The McNemar test showed a statistically significant difference between the groups of samples (p<0.001). Since serological negativization (the current cure criterion) delay many years after therapy and positive parasitological results represent a treatment failure, the conversion of pre-therapy positive conventional PCR is a qualitative and complementary tool that could be included in protocols of prolonged follow-up.

Estimando el efecto del cambio climático sobre el riesgo de la enfermedad de Chagas en Chile por medio del número reproductivo / Effects of climate change on reproductive number of Chagas disease

Background: Reproductive number (R0)-maps estimate risk zones of vector-borne diseases and geographical distribution changes under climate change. Aim: To map R0 aiming to estimate the epidemiological risk of Chagas disease in Chile, its distribution and possible changes due to the global climate change. Material and Methods: We used a relationship between R0 and entomological parameters of vectors as function of environmental variables, to map the risk of Chagas disease in Chile, under current and projected future environmental conditions. Results: We obtained a geographical R0 estimation of Chagas disease in Chile. The highest R0averages correspond to the Central-Northern regions of Chile. T. cruzi transmission area could increase in the future due to climate changes. Independent of the future condition, both for optimistic and pessimistic climate change scenarios, the area of potential risk for Chagas disease transmission would increase. The estimated R0 values suggest that, if a control of T. infestans is not maintained, Chagas disease endemic status will persist or increase, independently of the climate change scenarios. Conclusions: Mapping R0 values is an effective method to assess the risk of Chagas disease. The eventual increase in the transmission area of the disease is worrisome.

Quantification of Immunoglobulin G against Trypanosoma cruzi in Individuals with Chronic Chagas Disease Treated with Nifurtimox and Evaluated in Prolonged Follow-Up.

In the indeterminate chronic period of Chagas disease (ChD) the treatment has not been conclusive, because the serological negativization requires many years. This study aims to evaluate the efficacy of nifurtimox (NF) in the treatment of chronic ChD in prolonged follow-up by serological techniques of indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) IgG comparing 2 groups of patients, treated and non treated. Mann-Whitney test was performed for ELISA and IFA, with significant difference between the groups (P < 0.05). IgG levels were lower in individuals treated compared with untreated patients, indicating chemotherapeutic efficacy in prolonged follow-up.

Course of serological tests in treated subjects with chronic Trypanosoma cruzi infection: A systematic review and meta-analysis of individual participant data.

OBJECTIVE: To determine the course of serological tests in subjects with chronic Trypanosoma cruzi infection treated with anti-trypanosomal drugs. METHODS: A systematic review and meta-analysis was conducted using individual participant data. Survival analysis and the Cox proportional hazards regression model with random effects to adjust for covariates were applied. The protocol was registered in the PROSPERO database (http://www.crd.york.ac.uk/PROSPERO; CRD42012002162). RESULTS: A total of 27 studies (1296 subjects) conducted in eight countries were included. The risk of bias was low for all domains in 17 studies (63.0%). Nine hundred and thirteen subjects were assessed (149 seroreversion events, 83.7% censored data) for enzyme-linked immunosorbent assay (ELISA), 670 subjects (134 events, 80.0% censored) for indirect immunofluorescence assay (IIF), and 548 subjects (99 events, 82.0% censored) for indirect hemagglutination assay (IHA). A higher probability of seroreversion was observed within a shorter time span in subjects aged 1-19 years compared to adults. The chance of seroreversion also varied according to the country where the infection might have been acquired. For instance, the pooled adjusted hazard ratio between children/adolescents and adults for the IIF test was 1.54 (95% confidence interval 0.64-3.71) for certain countries of South America (Argentina, Bolivia, Chile, and Paraguay) and 9.37 (95% confidence interval 3.44-25.50) for Brazil. CONCLUSIONS: The disappearance of anti-T. cruzi antibodies was demonstrated along the course of follow-up. An interaction between age at treatment and country setting was found.

Discrete typing units of Trypanosoma cruzi detected by real-time PCR in Chilean patients with chronic Chagas cardiomyopathy.

Chagas disease is a major public health problem in Latin America and has spread to other countries due to immigration of infected persons. 10-30% of patients with chronic Chagas disease will develop cardiomyopathy. Chagas cardiomyopathy is the worst form of the disease, due to its high morbidity and mortality. Because of its prognostic value and adequate medical monitoring, it is very important to identify infected people who could develop Chagas cardiomyopathy. The aim of this study was to determine if discrete typing units (DTUs) of Trypanosoma cruzi are related to the presence of heart disease in patients with chronic Chagas disease. A total of 86 untreated patients, 41 with cardiomyopathy and 45 without heart involvement were submitted to clinical study. Electrocardiograms and echocardiograms were performed on the group of cardiopaths, in which all important known causes of cardiomyopathy were discarded. Sinus bradycardia and prolonged QTc interval were the most frequent electrocardiographic alterations and patients were classified in group I (46%) and group II (54%) of New York Hearth Association. In all cases real-time PCR genotyping assays were performed. In the group with cardiomyopathy, the most frequent DTU was TcI (56.1%), followed by TcII (19.5%). Mixed infections TcI + TcII were observed in 7.3% of the patients. In the group without cardiac pathologies, TcI and TcII were found at similar rates (28.9 and 31.1%, respectively) and mixed infections TcI + TcII in 17.8% of the cases. TcIII and TcIV were not detected in any sample. Taken together, our data indicate that chronic Chagas cardiomyopathy in Chile can be caused by strains belonging to TcI and TcII.

[What do the numbers tell us about the temporal evolution of Chagas' disease?] / ¿Qué dicen los números de la evolución temporal de la enfermedad de Chagas?

Chagas disease remains highly prevalent in Chile, especially between the regions of Arica and Parinacota, and Coquimbo. Since 1999 it is considered that in Chile the vector transmission was interrupted. Under this premise, the epidemiological dynamics should be changing. We analyzed the evolution of the prevalence of Chagas' disease analyzing 64,995 xenodiagnosis performed in the laboratory of Parasitology of the Faculty of Medicine of the University of Chile between 1949 and 2014. The evolution of the mortalities and incidences from the databases of the Ministry of Health in the periods in which it was analyzed. The rates of domiciliary infestation and the number of vector insects sent to the Public Health Institute and its trypano-triatomine indices were also analyzed. The prevalence of Chagas' disease in inhabitants of risk areas remained stable in this period as well as mortality. The incidence rate shows a progressive increase with a tendency towards stabilization. A significant decrease in sampling effort was found, declining by two orders of magnitude, especially since 2000. The progressive increase in morbidity had no clear relation to the interruption of the vector chain nor to the greater diagnostic effort occurred in 2009, since it was evident from before. While home infestation declines, reports of intrusion of solitary individuals and wild foci of T. infestans have increased. Trypano-triatomine indices were maintained with high values in all vector species. This study shows a worrying situation, for while on the one hand the interruption of the vector transmission and improvement in the research systems is emphasized, the concern for this disease seems to be decreasing with less diagnostic efforts and lower education at the higher level, and by the other hand the numbers show that the problem if it is not increasing, at least maintains its careless historical magnitude.

¿Qué dicen los números de la evolución temporal de la enfermedad de Chagas? / What do the numbers tell us about the temporal evolution of Chagas’ disease?

Chagas disease remains highly prevalent in Chile, especially between the regions of Arica and Parinacota, and Coquimbo. Since 1999 it is considered that in Chile the vector transmission was interrupted. Under this premise, the epidemiological dynamics should be changing. We analyzed the evolution of the prevalence of Chagas’ disease analyzing 64,995 xenodiagnosis performed in the laboratory of Parasitology of the Faculty of Medicine of the University of Chile between 1949 and 2014. The evolution of the mortalities and incidences from the databases of the Ministry of Health in the periods in which it was analyzed. The rates of domiciliary infestation and the number of vector insects sent to the Public Health Institute and its trypano-triatomine indices were also analyzed. The prevalence of Chagas’ disease in inhabitants of risk areas remained stable in this period as well as mortality. The incidence rate shows a progressive increase with a tendency towards stabilization. A significant decrease in sampling effort was found, declining by two orders of magnitude, especially since 2000. The progressive increase in morbidity had no clear relation to the interruption of the vector chain nor to the greater diagnostic effort occurred in 2009, since it was evident from before. While home infestation declines, reports of intrusion of solitary individuals and wild foci of T. infestans have increased. Trypano-triatomine indices were maintained with high values in all vector species. This study shows a worrying situation, for while on the one hand the interruption of the vector transmission and improvement in the research systems is emphasized, the concern for this disease seems to be decreasing with less diagnostic efforts and lower education at the higher level, and by the other hand the numbers show that the problem if it is not increasing, at least maintains its careless historical magnitude.

Resumen Introducción: La enfermedad de Chagas sigue siendo altamente prevalente en Chile, especialmente entre las regiones de Arica y Parinacota y de Coquimbo. Desde 1999 se considera que en Chile se encuentra interrumpida la transmisión vectorial. Bajo esta premisa, la dinámica epidemiológica se debiera estar modificando. Objetivo: Analizar la evolución temporal de la enfermedad de Chagas en Chile Material y Métodos: Analizamos la evolución de la prevalencia de la enfermedad de Chagas a través del análisis de resultados de 64.995 xenodiagnós-ticos realizados en el laboratorio de Parasitología de la Facultad de Medicina de la Universidad de Chile, entre 1949 y 2014. Se estudió la evolución de las mortalidades e incidencias disponibles en las bases de datos del Ministerio de Salud en los períodos en que fue posible. Se analizaron las tasas de infestación domiciliaria y el número de insectos vectores enviados al Instituto de Salud Pública y sus índices tripano-tratominos. Resultados: La prevalencia de la enfermedad de Chagas en habitantes de zonas de riesgo se mantuvo estable en este período, al igual que la mortalidad. La tasa de incidencia muestra un incremento progresivo con tendencia a la estabilización. Se encontró un significativo decrecimiento del esfuerzo de muestreo, decayendo dos órdenes de magnitud, especialmente desde 2000. El aumento progresivo de la morbilidad no tiene clara relación con el corte de la cadena vectorial ni con el mayor esfuerzo diagnóstico ocurrido en 2009, ya que era evidente desde antes. Mientras que la infestación domiciliaria disminuye, han aumentado los reportes de intromisión de individuos solitarios y los focos silvestres de T. infestans. Los índices tripano triatominos se mantienen con valores altos en todas las especies vectores. Discusión: Este estudio muestra una situación preocupante, ya que mientras por una parte se destaca el corte de la transmisión vectorial y mejora en los sistemas de pesquisa, la preocupación por esta enfermedad parece ir decreciendo con menores esfuerzos diagnósticos y menor enseñanza a nivel superior, y por otra parte los números muestran que el problema si es que no está aumentando, al menos mantiene su descuidada magnitud histórica.

Real-time PCR strategy for the identification of Trypanosoma cruzi discrete typing units directly in chronically infected human blood.

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a major public health problem in Latin America. This parasite has a complex population structure comprised by six or seven major evolutionary lineages (discrete typing units or DTUs) TcI-TcVI and TcBat, some of which have apparently resulted from ancient hybridization events. Because of the existence of significant biological differences between these lineages, strain characterization methods have been essential to study T. cruzi in its different vectors and hosts. However, available methods can be laborious and costly, limited in resolution or sensitivity. In this study, a new genotyping strategy by real-time PCR to identify each of the six DTUs in clinical blood samples have been developed and evaluated. Two nuclear (SL-IR and 18S rDNA) and two mitochondrial genes (COII and ND1) were selected to develop original primers. The method was evaluated with eight genomic DNA of T. cruzi populations belonging to the six DTUs, one genomic DNA of Trypanosoma rangeli, and 53 blood samples from individuals with chronic Chagas disease. The assays had an analytical sensitivity of 1-25fg of DNA per reaction tube depending on the DTU analyzed. The selectivity of trials with 20fg/µL of genomic DNA identified each DTU, excluding non-targets DTUs in every test. The method was able to characterize 67.9% of the chronically infected clinical samples with high detection of TcII followed by TcI. With the proposed original genotyping methodology, each DTU was established with high sensitivity after a single real-time PCR assay. This novel protocol reduces carryover contamination, enables detection of each DTU independently and in the future, the quantification of each DTU in clinical blood samples.

Quantification by real-time PCR of Trypanosoma cruzi DNA in samples of Triatoma infestans used in xenodiagnosis of chronic Chagas disease patients.

BACKGROUND: Trypanosoma cruzi multiplies and differentiates in the digestive tract of triatomine insects. Xenodiagnosis (XD) is a parasitological tool in which the insect vectors acts as a biological culture medium to amplify and detect T. cruzi infection in mammals. The sensitivity of XD has been overcome by the application of PCR in fecal samples (FS) of XD (PCR-XD). In this study, T. cruzi amplified in Triatoma infestans fed by XD on individuals with chronic Chagas disease (CChD) is quantified by real-time PCR (qPCR-XD). FINDINGS: Under informed consent, 100 individuals were evaluated. In 21 of them XD, PCR-XD and qPCR-XD were positive. For the contrary, 79 were negative XD. In 58 (73.4 %) and 66 cases (83.5 %) of them, PCR-XD (Fisher's exact test P = 0.005) and qPCR-XD (Fisher's exact test: P = 0.037) respectively, were positive. In cases with positive XD, qPCR-XD allowed to establish that in 9/21 cases (42.9 %) the parasite burden fluctuated between 100 and 1,000 par. eq./ml. Otherwise, in 32/79 (40.5 %) cases with negative XD, a parasite burden between 1 and 10 par. eq./ml was determined. All samples showed amplification of exogenous internal control (X12, Ct average: 31.8), so problems in the DNA extraction (excess or loss of genetic material), unspecific amplification and/or inhibition in qPCR-XD reactions were ruled out. Additionally, in all the patients qPCR in blood (qPCR-B) was performed. In the cases with positive XD, the concordance between the positivity of qPCR-XD and qPCR-B was 100 %, nevertheless, the parasite burden in blood was lower and different than XD (Chi-square test: χ (2) = 91.82, df = 5, P = 0.0001). In the cases with negative XD the ranges of qPCR-XD and qPCR-B were similar (Chi-square test: χ (2) = 6.71, df = 5, P = 0.1520). CONCLUSIONS: This study allowed the detection and quantification of T. cruzi by qPCR-XD in FS of Tr. infestans fed on patients with CChD. The highest parasite burden was observed in positive XD cases. qPCR-XD could be used in different studies related with the complex T. cruzi-vector-host interactions.

The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles.

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite's genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite.

Chronic Chagas cardiopathy in Chile. Importance of Trypanosoma cruzi burden and clinical evaluation.

Currently there are no biological markers to indicate which individuals with chronic indeterminate period of Chagas disease develop heart disease and who will remain all his life in this phase. The aim of this survey was to determine if Trypanosoma cruzi burden is related to the presence of heart disease in patients with chronic Chagas disease. 200 patients who had not been treated, 100 with cardiopathy and 100 without, groups A and B respectively, were submitted to clinical study and electrocardiogram, Echo-Doppler was performed for group A in which all important known causes of cardiopathy were discarded. In both groups xenodiagnosis, conventional PCR and quantitative PCR were undertaken. The 100 cardiopaths had 133 electrocardiographic alterations most of them in grade II of the New York Heart Association classification. 98 cardiopaths were classified in grade I by Echo-Doppler and only 2 cases were in grade III due to low ejection fraction. The difference in average parasitemia in patients of group A and B was not significant and no statistically differences were observed between average parasitemia of cardiopaths grade II versus grade I of NYHA. This results allow to characterize same clinical, electrocardiographical and parasitological features in chagasic cardiopaths of Chile.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Trypanosoma cruzi burden, genotypes, and clinical evaluation of Chilean patients with chronic Chagas cardiopathy.

There are currently no biomarkers to assess which patients with chronic indeterminate Chagas disease will develop heart disease and which will spend their entire life in this state. We hypothetize that the parasite burden and Trypanosoma cruzi genotypes are related to the presence of heart disease in patients with Chagas disease. This study is aimed to investigate the parasite burden and T. cruzi genotypes in chagasic cardiopaths versus chagasic individuals without cardiac involvement according to the New York Heart Association. Patients with chronic Chagas disease, 50 with and 50 without cardiopathy (controls), groups A and B, respectively, were submitted to anamnesis, physical examination, and electrocardiogram. Echo-Doppler was performed for group A; all important known causes of cardiopathy were discarded. Xenodiagnosis, conventional PCR, and quantitative PCR were performed on patients of both groups. T. cruzi genotyping was done for 25 patients of group A and 20 of group B. The 50 cardiopaths had 80 electrocardiographic alterations, most of them in grade II of the New York Heart Association classification; 49 were classified in grade I by Echo-Doppler, and only one patient was in grade III. The difference in average parasitemia in patients of groups A and B was not significant. The most frequent T. cruzi DTU found was TcV. The parasite burden and genotype of the groups with and without cardiopathy were similar. Graphical abstract Imagen 1 Chronic chagas cardiopathy chest X-ray heart enlargement Figure 2 Chronic Chagas cardiopathy microaneurism of left ventricle. Cineangiography.