Practical diagnostic algorithms for Chagas disease: a focus on low resource settings.

INTRODUCTION: Chagas disease, caused by parasite Trypanosoma cruzi, is the most important neglected tropical disease in the Americas. Two drugs are available for treatment, but access to them is challenging, in part due to complex diagnostic algorithms. These are stage-dependent, involve multiple tests, and are ill-adapted to the reality of vast areas where the disease is endemic. Molecular and serologic tools are used to detect acute and chronic infections, with the performance of the latter showing geographic differences. Breakthroughs in the development of new diagnostic tools include the validation of a loop-mediated isothermal amplification assay for acute infections (T. cruzi-LAMP), and the regional validation of several rapid diagnostic tests (RDTs) for chronic infection, which simplify testing in resource-limited settings. The literature search was carried out in the MEDLINE database until 1 August 2023. AREAS COVERED: This review outlines existing algorithms, and proposes new ones focused on point-of-care testing. EXPERT OPINION: Integrating point-of-care testing into existing diagnostic algorithms in certain endemic areas will increase access to timely diagnosis and treatment. However, additional research is needed to validate the use of these techniques across a wider geography, and to better understand the cost-effectiveness of their large-scale implementation.

A Phase 2, Randomized, Multicenter, Placebo-Controlled, Proof-of-Concept Trial of Oral Fexinidazole in Adults With Chronic Indeterminate Chagas Disease.

BACKGROUND: Chagas disease (CD) has significant global health impact, but safe, effective treatments remain elusive. The nitroimidazole fexinidazole is a potential treatment. METHODS: This double-blind, randomized, placebo-controlled, dose-finding, proof-of-concept study was conducted in Bolivia. Adults with serologically confirmed chronic indeterminate CD and positive PCR were randomly assigned to 1 of 6 fexinidazole regimens (1200 or 1800 mg/day for 2, 4, or 8 weeks) or placebo. Target recruitment was 20 patients/arm. The primary endpoint was sustained parasitological clearance by serial negative qPCR from end of treatment (EOT) until 6 months follow-up in the intention-to-treat (ITT) population. Follow-up was extended to 12 months. RESULTS: Enrollment was interrupted after 4/47 patients presented with transient asymptomatic grade 3 and 4 neutropenia. Treatment of ongoing patients was stopped in all patients administered >2 weeks. A total of 40 patients received treatment with fexinidazole from 3 days to 8 weeks. Delayed-onset neutropenia (n = 8) and increased liver enzymes (n = 8) were found in fexinidazole patients vs none in the placebo arm. In the ITT analysis, sustained parasitological clearance from EOT to 12 months follow-up varied between 66.7% (1200 mg-2 week) and 100.0% (1800 mg-2 week). Rapid, sustained clearance of parasitemia was observed in all treated patients with available data, but not in any patients in the placebo group, at 12 months (P = .0056). Further exploratory exposure-response analysis suggested low dosages of fexinidazole may be safe and effective. CONCLUSIONS: Further evaluation is needed to establish fexinidazole's minimum effective dosage and risk-benefit relationship. Results suggest potential for effective treatment regimens <10 days. CLINICAL TRIALS REGISTRATION: NCT02498782.

Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory.

There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels.

Genetic polymorphism of Trypanosoma cruzi bloodstream populations in adult chronic indeterminate Chagas disease patients from the E1224 clinical trial.

BACKGROUND: The role that the genetic diversity of natural Trypanosoma cruzi populations plays in response to trypanocidal treatment of chronic Chagas disease (CD) patients remains to be understood. We analysed the genetic polymorphisms of parasite bloodstream populations infecting chronic CD patients enrolled in the E1224 clinical trial. METHODS: A total of 506 baseline and post-treatment follow-up samples from 188 patients were analysed. T. cruzi satellite DNA (satDNA) was amplified and sequenced using cruzi1/cruzi2 primers, and samples with TcI/III, TcII, TcIV or hybrid satDNA sequences were identified. Minicircle signatures were obtained after kinetoplast DNA amplification using 121/122 primers and restriction enzyme digestion. Genetic distances between baseline and post-treatment minicircle signatures were estimated using the Jaccard coefficient. RESULTS: At baseline, 74.3% TcII, 17.9% hybrid and 7.8% TcI/III satDNA sequences were found, whereas at the end of follow-up the distribution was 55.2% TcII, 35.2% hybrid and 9.5% TcI/III. The placebo arm was the treatment group with the highest variation of satDNA sequences between baseline and post-treatment follow-up. Genetic distances between baseline and post-treatment minicircle signatures were similar among all treatment arms. No association between minicircle signature variability and satDNA type distribution was found. CONCLUSIONS: Genetic variability of T. cruzi bloodstream populations during post-treatment follow-up did not differ from that observed during chronic infection in the absence of treatment, suggesting that there were no selection events of E1224-resistant parasite populations. This is the first report documenting the genetic polymorphism of natural T. cruzi populations in chronic patients in the context of clinical trials with trypanocidal drugs.

Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory

There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels.

Prospective multicenter evaluation of real time PCR Kit prototype for early diagnosis of congenital Chagas disease.

BACKGROUND: Current algorithm for Congenital Chagas Disease (cCD) diagnosis is unsatisfactory due to low sensitivity of the parasitological methods. Moreover, loss to follow-up precludes final serodiagnosis after nine months of life in many cases. A duplex TaqMan qPCR kit for Trypanosoma cruzi DNA amplification was prospectively evaluated in umbilical cord (UCB) and peripheral venous blood (PVB) of infants born to CD mothers at endemic and non-endemic sites of Argentina. METHODS: We enrolled and followed-up 370 infants; qPCR was compared to gold-standard cCD diagnosis following studies of diagnostic accuracy guidelines. FINDINGS: Fourteen infants (3·78%) had cCD. The qPCR sensitivity and specificity were higher in PVB (72·73%, 99·15% respectively) than in UCB (66·67%, 96·3%). Positive and negative predictive values were 80 and 98·73% and 50 and 98·11% for PVB and UCB, respectively. The Areas under the Curve (AUC) of ROC analysis for qPCR and micromethod (MM) were 0·81 and 0·67 in UCB and 0·86 and 0·68 in PVB, respectively. Parasitic loads ranged from 37·5 to 23,709 parasite equivalents/mL. Discrete typing Unit Tc V was identified in five cCD patients and in six other cCD cases no distinction among Tc II, Tc V or Tc VI was achieved. INTERPRETATION: This first prospective field study demonstrated that qPCR was more sensitive than MM for early cCD detection and more accurate in PVB than in UCB. Its use, as an auxiliary diagnostic tool to MM will provide more accurate records on cCD incidence. FUNDING: FITS SALUD 001-CHAGAS (FONARSEC, MINCyT, Argentina) to the Public-Private Consortium (INGEBI-CONICET, INP-ANLIS MALBRAN and Wiener Laboratories); ERANET-LAC-HD 328 to AGS and PICT 2015-0074 (FONCYT, MinCyT) to AGS and FA.

Congenital Transmission of Trypanosoma cruzi: A Review About the Interactions Between the Parasite, the Placenta, the Maternal and the Fetal/Neonatal Immune Responses.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, is considered a neglected tropical disease by the World Health Organization. Congenital transmission of CD is an increasingly relevant public health problem. It progressively becomes the main transmission route over others and can occur in both endemic and non-endemic countries. Though most congenitally infected newborns are asymptomatic at birth, they display higher frequencies of prematurity, low birth weight, and lower Apgar scores compared to uninfected ones, and some suffer from severe symptoms. If not diagnosed and treated, infected newborns are at risk of developing disabling and life-threatening chronic pathologies later in life. The success or failure of congenital transmission depends on interactions between the parasite, the placenta, the mother, and the fetus. We review and discuss here the current knowledge about these parameters, including parasite virulence factors such as exovesicles, placental tropism, potential placental defense mechanisms, the placental transcriptome of infected women, gene polymorphism, and the maternal and fetal/neonatal immune responses, that might modulate the risk of T. cruzi congenital transmission.

Detection of anti-Trypanosoma cruzi antibodies by chimeric antigens in chronic Chagas disease-individuals from endemic South American countries.

BACKGROUND: Laboratory diagnosis of chronic Chagas disease is a troubling factor due to lack of reference tests. The WHO suggests the use of two distinct commercial serological tests in parallel. The performance of commercial immunoassays might fluctuate depending on the antigenic matrices and the local strains of T. cruzi in different geographical settings. The use of antigenic matrices based on chimeric proteins can solve these limitations. Here, we evaluated the diagnostic performance of two chimeric T. cruzi antigens (IBMP-8.1 and -8.4) to diagnose chronic Chagas disease in individuals from endemic South American countries. METHODOLOGY/PRINCIPAL FINDINGS: IBMP-8.1 and IBMP-8.4 chimeric antigens were expressed as soluble proteins in E. coli and purified using chromatography methods. Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house ELISA with sera from 122 non-infected and 215 T. cruzi-infected individuals from Argentina, Bolivia, and Paraguay. Cut-off values were based on ROC curves and performance parameters were determined using a dichotomous approach. Area under the curve values were > 99.7% for both IBMP-8.1 and IBMP-8.4 antigens. IgG levels in T. cruzi-positive and negative samples were higher for IBMP-8.4 than IBMP-8.1. Both IBMP-8.1 and -8.4 were 100% specific, while IBMP-8.4 were 100% sensitive compared to IBMP-8.1 (95.3%). Admitting RI values of 1.0 ± 0.10 as the inconclusive interval, 6.2% of the samples tested using IBMP-8.1 and 2.1% using IBMP-8.4 fell inside the grey zone. Based on accuracy and diagnostic odds ratio values, IBMP-8.4 presented the best performance. Differences in sensitivity and IgG levels among the samples from Argentina, Bolivia, and Paraguay were not significant. CONCLUSIONS/SIGNIFICANCE: Our findings showed a notable performance of IBMP-8.1 and -8.4 chimeric antigens in diagnosing chronic Chagas disease in individuals from endemic South American countries, confirming our hypothesis that these antigens could be used in geographical areas where distinct T. cruzi DTUs occur.

Association study between CCR2-CCR5 genes polymorphisms and chronic Chagas heart disease in Wichi and in admixed populations from Argentina.

Several studies have proposed different genetic markers of susceptibility to develop chronic Chagas cardiomyopathy (CCC). Many genes may be involved, each one making a small contribution. For this reason, an appropriate approach for this problematic is to study a large number of single nucleotide polymorphisms (SNPs) in individuals sharing a genetic background. Our aim was to analyze two CCR2 and seven CCR5 SNPs and their association to CCC in Argentina. A case-control study was carried out in 480 T. cruzi seropositive adults from Argentinean Gran Chaco endemic region (Wichi and Creole) and patients from Buenos Aires health centres. They were classified according to the Consensus on Chagas-Mazza Disease as non-demonstrated (non-DC group) or demonstrated (DC group) cardiomyopathy, i.e. asymptomatic or with CCC patients, respectively. Since, after allelic analysis, 2 out of 9 studied SNPs did not fit Hardy-Weinberg equilibrium in the unaffected non-DC group from Wichi patients, we analyzed them as a separate population. Only rs1800024T and rs41469351T in CCR5 gene showed significant differences within non-Wichi population (Creole + patients from Buenos Aires centres), being the former associated to protection, and the latter to risk of CCC. No evidence of association was observed between any of the analyzed CCR2-CCR5 gene polymorphisms and the development of CCC; however, the HHE haplotype was associated with protection in Wichi population. Our findings support the hypothesis that CCR2-CCR5 genes and their haplotypes are associated with CCC; however, depending on the population studied, different associations can be found. Therefore, the evolutionary context, in which the genes or haplotypes are associated with diseases, acquires special relevance.

Nitric oxide synthase and oxidative-nitrosative stress play a key role in placental infection by Trypanosoma cruzi.

PROBLEM: The innate immune response of the placenta may participate in the congenital transmission of Chagas disease through releasing reactive oxygen and nitrogen intermediates. METHOD OF STUDY: Placental explants were cultured with 1 × 106 and 1 × 105 trypomastigotes of Tulahuen and Lucky strains and controls without parasites, and with the addition of nitric oxide synthase inhibitor Nω-Nitro-l-arginine methyl ester (l-NAME) and N-acetyl cysteine (NAC) as the reactive oxygen species (ROS) scavenger. Detachment of the syncytiotrophoblast (STB) was examined by histological analysis, and the nitric oxide synthase, endothelial (eNOS), and nitrotyrosine expressions were analyzed by immunohistochemistry, as well as the human chorionic gonadotrophin (hCG) levels in the culture supernatant through ELISA assays. Parasite load with qPCR using Taqman primers was quantified. RESULTS: The higher number of T. cruzi (106 ) increased placental infection, eNOS expression, nitrosative stress, and STB detachment, with the placental barrier being injured by oxidative stress. CONCLUSION: The higher number of parasites caused deleterious consequences to the placental barrier, and the inhibitors (l-NAME and NAC) prevented the damage caused by trypomastigotes in placental villi but not that of the infection. Moreover, trophoblast eNOS played a key role in placental infection with the highest inoculum of Lucky, demonstrating the importance of the enzyme and nitrosative-oxidative stress in Chagas congenital transmission.

Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region.

The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease.

Molecular characterization of trypanosomatid infections in wild howler monkeys (Alouatta caraya) in northeastern Argentina.

The transmission of Trypanosoma cruzi by vectors is confined to the Americas, and the infection circulates in at least two broadly defined transmission cycles occurring in domestic and sylvatic habitats. This study sought to detect and characterize infection by T. cruzi and other trypanosomes using PCR strategies in blood samples from free-ranging howler monkeys, Alouatta caraya, in the northeastern Argentina. Blood samples were collected at four sites with variable levels of habitat modification by human activity. PCR was conducted using primers for kinetoplast DNA, satellite DNA and ribosomal DNA of the trypanosomatid parasites. Ribosomal and satellite DNA fragments were sequenced to identify the trypanosomatid species and to characterize the discrete typing units (DTUs) of T. cruzi. Overall, 46% (50/109) of the howlers were positive according to the kDNA-PCR assay, but only 7 of the howlers were positive according to the SatDNA-PCR protocol. We sequenced the amplicons of the satellite DNA obtained from five specimens, and the sequences were 99% and 100% similar to T. cruzi. A sequence typical of DTU T. cruzi I was found in one howler monkey from the "remote" site, while sequences compatible with DTUs II, V, and VI were found in howlers from the "remote", "rural" and "village" sites. We detected 96% positive samples for RibDNA-PCR, 9 of which were sequenced and displayed 99% identity with Trypanosoma minasense, while none showed identity with T. cruzi. The results demonstrated the presence of T. cruzi and a species closely related to T. minasense in blood samples from free-ranging A. caraya, belonging to different T. cruzi DTUs circulating in these howler monkey populations. The results obtained in this study could help evaluate the role of A. caraya as a reservoir of T. cruzi in regions where Chagas disease is hyper-endemic and where the human-wildlife interface is increasing.

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.

Pharmacokinetic and pharmacodynamic responses in adult patients with Chagas disease treated with a new formulation of benznidazole.

Pharmacological treatment of Chagas disease with benznidazole (BNZ) is effective in children in all stages, but it is controversial in chronically infected adults. We report the pharmacokinetics and pharmacodynamics in six adult patients with Chagas disease treated with the new BNZ formulation (ABARAX®) in doses between 2.5-5.5 mg/Kg/day. All but one patient had plasmatic BNZ concentrations within the expected range. All patients finalised treatment with nondetectable Trypanosoma cruzi quantitative polymerase chain reaction, which remained nondetectable at the six month follow-up. Our data suggests parasitological responses with the new BNZ and supports the hypothesis that treatment protocols with lower BNZ doses may be effective.

Pharmacokinetic and pharmacodynamic responses in adult patients with Chagas disease treated with a new formulation of benznidazole

Pharmacological treatment of Chagas disease with benznidazole (BNZ) is effective in children in all stages, but it is controversial in chronically infected adults. We report the pharmacokinetics and pharmacodynamics in six adult patients with Chagas disease treated with the new BNZ formulation (ABARAX®) in doses between 2.5-5.5 mg/Kg/day. All but one patient had plasmatic BNZ concentrations within the expected range. All patients finalised treatment with nondetectable Trypanosoma cruziquantitative polymerase chain reaction, which remained nondetectable at the six month follow-up. Our data suggests parasitological responses with the new BNZ and supports the hypothesis that treatment protocols with lower BNZ doses may be effective.

A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi.

Chagas disease is a major unsolved health issue in Latin America and an emerging threat worldwide. New drugs are urgently needed for chemotherapy as those available (benznidazole and nifurtimox) have variable efficacy and elevated toxicity. Efforts are actually oriented to improve tools and technologies (e.g. transgenic parasites, flow cytometry or image-based systems) for the screening of large numbers of candidate compounds for their activity against Trypanosoma cruzi (T. cruzi). Methods that test drug efficacy and selectivity in the same assay are suitable to accelerate the process of drug discovery. Here, we developed a GFP expressing T. cruzi from a moderate virulence stock and confirmed that the transgenic parasite retained the biological characteristics of the parental strain. With this tool, we established a flow cytometer-based method to simultaneously test drug activity against intracellular amastigotes and toxicity to the host cell. This one-step procedure allows determining the selectivity index of the tested compound in a sensitive and accurate manner even with low infection rates. This method can provide additional information on the interactions between drug, parasites and host cell and could be adapted to other trypanosomatids and protozoa with intracellular multiplication.

Geographic variation of Trypanosoma cruzi discrete typing units from Triatoma infestans at different spatial scales.

We assessed the diversity and distribution of Trypanosoma cruzi discrete typing units (DTU) in Triatoma infestans populations and its association with local vector-borne transmission levels at various geographic scales. At a local scale, we found high predominance (92.4%) of TcVI over TcV in 68 microscope-positive T. infestans collected in rural communities in Santiago del Estero province in northern Argentina. TcV was more often found in communities with higher house infestation prevalence compatible with active vector-borne transmission. Humans and dogs were the main bloodmeal sources of the TcV- and TcVI-infected bugs. At a broader scale, the greatest variation in DTU diversity was found within the Argentine Chaco (227 microscope-positive bugs), mainly related to differences in equitability between TcVI and TcV among study areas. At a country-wide level, a meta-analysis of published data revealed clear geographic variations in the distribution of DTUs across countries. A correspondence analysis showed that DTU distributions in domestic T. infestans were more similar within Argentina (dominated by TcVI) and within Bolivia (where TcI and TcV had similar relative frequencies), whereas large heterogeneity was found within Chile. DTU diversity was lower in the western Argentine Chaco region and Paraguay (D=0.14-0.22) than in the eastern Argentine Chaco, Bolivia and Chile (D=0.20-0.68). Simultaneous DTU identifications of T. cruzi-infected hosts and triatomines across areas differing in epidemiological status are needed to shed new light on the structure and dynamics of parasite transmission cycles.

Biological markers for evaluating therapeutic efficacy in Chagas disease, a systematic review.

The most neglected aspects of Chagas disease (CD) have been patient care and treatment. Despite recent progress in the development of potentially improved drugs, there is no consensus among different research groups on the lack of therapeutic response markers to evaluate efficacy of newly proposed drugs early after treatment. A systematic review of current evidence regarding molecules which are potential biomarkers for therapeutic response has been conducted using quality assessment and target responses as primary criteria. The review provides a panorama of the cumulative evidence and specific needs for development of a battery of complementary biomarkers which together fulfill ideal or acceptable criteria to evaluate early responses to treatment for chronic CD. There are several marker candidates which together may fulfill acceptable criteria to indicate the efficacy of a trypanocidal treatment. Data from ongoing studies are considered essential to improve assessment of existing markers and to identify those for early follow-up of treated patients.

Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation.

BACKGROUND: One hundred years after the discovery of Chagas disease, it remains a major neglected tropical disease. Chronic Chagas heart disease (cChHD) is the most severe manifestation. Heart transplantation is the proper treatment for end-stage heart failure, although reactivation of disease may result after receipt of immunosuppressive therapy. T. cruzi strains cluster into 6 discrete typing units (DTUs; I-VI) associated with different geographical distribution, transmission cycles and varying disease symptoms. In the southern cone of South America, T. cruzi II, V, and VI populations appear to be associated with Chagas disease and T. cruzi I with sylvatic cycles. METHODS: Molecular characterization of DTUs, T. cruzi I genotypes (on the basis of spliced-leader gene polymorphisms), and minicircle signatures was conducted using cardiac explant specimens and blood samples obtained from a cohort of 16 Argentinean patients with cChHD who underwent heart transplantation and from lesion samples obtained from 6 of these patients who presented with clinical reactivation of Chagas disease. RESULTS: Parasite persistence was associated with myocarditis progression, revealing T. cruzi I (genotype Id) in 3 explant samples and T. cruzi II, V, or VI in 5 explant samples. Post-heart transplantation follow-up examination of bloodstream DTUs identified T. cruzi I in 5 patients (genotypes Ia or Id) and T. cruzi II, V, or VI in 7 patients. T. cruzi I, V, and VI were detected in skin chagoma specimens, and T. cruzi V and VI were detected in samples obtained from patients with myocarditis reactivations. Multiple DTUs or genotypes at diverse body sites and polymorphic minicircle signatures at different cardiac regions revealed parasite histotropism. T. cruzi I infections clustered in northern Argentina (latitude, 23 degrees S-27 degrees S), whereas T. cruzi II, V, or VI DTUs were more ubiquitous. CONCLUSIONS: Multiple DTUs coexist in patients with Chagas disease. The frequent finding of T. cruzi I associated with cardiac damage was astounding, revealing its pathogenic role in cChHD at the southern cone.

Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients.

BACKGROUND: This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence. METHODOLOGY/PRINCIPAL FINDINGS: The Q-PCR has a detection limit of 0.1 and 0.01 parasites/mL, with a dynamic range of 10(6) and 10(7) for Silvio X10 cl1 (T. cruzi I) and Cl Brener stocks (T. cruzi IIe), respectively, an efficiency of 99%, and a coefficient of determination (R(2)) of 0.998. In order to express accurately the parasitic loads: (1) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; (2) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and (3) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol (Lg-PCR). The Q-PCR strategy was applied (1) to estimate basal parasite loads in 43 pediatric Chagas disease patients, (2) to follow-up 38 of them receiving treatment with benznidazole, and (3) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression. CONCLUSION/SIGNIFICANCE: All together, the high analytical sensitivity of the Q-PCR strategy, the low levels of intra- and inter-assay variations, as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients.