Clara Isabel González

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. Conclusion/Significance This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Evidence of Trypanosoma cruzi II infection in Colombian chagasic patients

Trypanosoma cruzi is genetically classified into at least two major lineages named T. cruzi I (also named Tc I) and T. cruzi II (also named Tc IIb). T. cruzi II is associated with Chagas’ disease in the southern cone of South America, while T. cruzi I is the only one so far identified in chagasic patients of Central America and in the northern part of South America. Herein we identified T. cruzi IIb directly in 9.9% of blood of chronic chagasic patients of Colombia. This finding establishes that in this region, the two T. cruzi lineages are associated with the pathology of Chagas’ disease and have implications in the morbidity and epidemiology of the disease.

Mixed infection of Trypanosoma cruzi I and II in a Colombian cardiomyopathic patient

The Trypanosoma cruzi taxon is composed of 2 major lineages, T cruzi I and T cruzi II. The clinical symptoms of Chagas disease are highly variable, and their geographic distribution is correlated with the distribution of the parasite lineages. In Colombia and northern South America, T cruzi I lineage is associated with chagasic cardiomyopathy. Alternatively, in the countries south cone of South America, there is a predominance of T cruzi II, which is associated with cardiomyopathy and digestive diseases. We report for the first time a mixed infection consisting of both T cruzi I and T cruzi II detected in the esophagus and in the heart, respectively, of a cardiomyopathic patient from an endemic area in Santander, Colombia. This finding has epidemiological relevance related to the association of T cruzi II with the clinical manifestations of Chagas disease and its frequency in Colombia and countries in northern South America.

Direct analysis of genetic variability in Trypanosoma cruzi populations from tissues of Colombian chagasic patients

The clinical symptoms of Chagas disease are highly variable and are correlated with geographical distribution and parasite genetic group. Trypanosoma cruzi group I is associated with chagasic cardiomyopathy in Colombia and other countries in northern South America. However, in southern South America, T cruzi group II predominates and is associated with cardiomyopathy and digestive forms of the disease. The aim of this work was to determine the correlation between the genetic profiles of T cruzi groups circulating in the biological cycle and those present in tissues from patients with Chagas disease. We genotyped T cruzi in 10 heart tissue samples from patients with cardiomyopathy from a highly endemic area of Colombia. The genotyping was performed using nuclear and mitochondrial genes and low-stringency single-specific primer polymerase chain reaction. As expected, the predominant genetic group was T cruzi group I; however, we also detected T cruzi group II. Microsatellite analyses suggested a predominance of monoclonal populations, and sequence alignments showed similarities with Colombian strains. In addition, kinetoplast DNA signatures obtained by low-stringency single-specific primer polymerase chain reaction allowed us to group strains into the 2 genetic groups. Thus, we conclude that both T cruzi genetic groups are producing severe cases of Chagas disease in Colombia. We did not observe any correlation between low-stringency single-specific primer polymerase chain reaction profiles, histopathologic findings, clinical forms, and severity of Chagas disease.

Training model for teaching endoscopic submucosal dissection of gastric tumors

INTRODUCTION The elevated risk of complications and technical complexity of endoscopic submucosal dissection (ESD) has limited its implementation in our medical system. OBJECTIVE To design and evaluate a training program for learning the ESD technique. METHODS Four endoscopists with no experience with ESD underwent a 4-step training program: 1) review of the existing literature, didactic material, and theoretical aspects of ESD; 2) ESD training in an ex-vivo animal model; 3) ESD training in an in-vivo animal model (supervised by ESD expert); and 4) ESD performance in a patient. A standard gastroscope and an ESD knife (IT, Flex or Hook-knife Olympus) were employed. The classical ESD technique was performed: rising of the lesion, circumferential incision, and submucosal dissection. RESULTS Ex-vivo animal model: 6 x swine stomach/esophagus -cost < 100 euro; 6 x ESD: antrum (n = 2), body (n = 3) and fundus/cardia (n = 1)-; size of resected specimen: 4-10 cm; ESD duration: 105-240 minutes; therapeutic success: 100%; complications: perforation (1/6: 16%) sealed with clips. In-vivo animal model: 6 ESD (antrum/body of stomach: 4; esophagus: 2); size: 2-5 cm; duration: 40-165 minutes; success: 100%; complications: 0%. PATIENT ESD of a gastric lesion located in the antrum/body; size: 3 cm; duration 210 minutes; a complete resection was achieved; no complications. CONCLUSIONS The results of the present study support the usefulness of this model for learning ESD in our system.

Role of the IFNG +874T/A polymorphism in Chagas disease in a Colombian population

Abstract Genetic susceptibility to Trypanosoma cruzi infection and the development of cardiomyopathy is complex, heterogeneous, and likely involves several genes. Previous studies have implicated cytokine and chemokine genes in susceptibility to Chagas disease. Here we investigated the association between the interferon-gamma gene (IFNG) +874T/A polymorphism and Chagas disease, focusing on susceptibility and severity. This study included 236 chagasic patients (asymptomatic, n =116; cardiomyopathic, n =120) and 282 healthy controls from a Colombian population where T. cruzi is highly endemic. Individuals were genotyped for functional single nucleotide polymorphism (SNP; rs2430561; A/T) of the IFNG gene by amplification refractory mutational system PCR (ARMS-PCR). Moreover, clinical manifestations of Chagas in patients were analyzed. We found a significant difference in the distribution of the IFNG +874 “A” allele between patients and healthy controls (P =0.003; OR=1.46, 95% CI, 1.13–1.89). The frequency of the IFNG +874 genotype A/A, which is associated with reduced production of interferon-gamma, was increased in the patients relative to controls (38.1% vs. 26.6%). We compared the frequencies of IFNG alleles and genotypes between asymptomatic patients and those with chagasic cardiomyopathy and found no significant difference. Our data suggest that the IFNG +874T/A genetic polymorphism may be involved in susceptibility but not in the progression of Chagas disease in this Colombian population.

Molecular Typing and Virulence Characteristic of Methicillin-Resistant Staphylococcus Aureus Isolates from Pediatric Patients in Bucaramanga, Colombia

Background Staphylococcus aureus is among the most common global nosocomial pathogens. The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide that causes nosocomial and community infections. The goals of this study were to establish the clonal complexes (CC) of the isolates of MRSA obtained from pediatric patients in a university hospital in Colombia and to investigate its molecular characteristics based on the virulence genes and the genes of staphylococcal toxins and adhesins. Methods A total of 53 MRSA isolates from pediatric patients with local or systemic infections were collected. The MRSA isolates were typed based on the SCCmec, MLST, spa and agr genes. The molecular characterization included the detection of Panton-Valentine Leukocidin, superantigenic and exfoliative toxins, and adhesin genes. The correlation between the molecular types identified and the profile of virulence factors was determined for all isolates. Results Four CC were identified, including CC8, CC5, CC80 and CC78. The ST8-MRSA-IVc-agrI was the predominant clone among the isolates, followed by the ST5-MRSA-I-agrII and ST5-MRSA-IVc-agrII clones. Twelve spa types were identified, of which t10796 and t10799 were new repeat sequences. The isolates were carriers of toxin genes, and hlg (100%), sek (92%) and pvl (88%) were the most frequent. Ten toxin gene profiles were observed, and the most frequent were seq-sek-hlg (22.6%), sek-hlg (22.6%), seb-seq-sek-hlg (18.9%) and seb-sek-hlg (15.1%). The adhesion genes were present in most of the MRSA isolates, including the following: clf-A (89%), clf-B (87%), fnb-A (83%) and ica (83%). The majority of the strains carried SCCmec-IVc and were identified as causing nosocomial infection. No significant association between a molecular type and the virulence factors was found. Conclusion Four major MRSA clone complexes were identified among the isolates. ST8-MRSA-IVc-agrI pvl+ (USA300-LV) was the most frequent, confirming the presence of community-associated MRSA in Colombian hospitals.

Transforming growth factor beta 1 (TGFβ1) gene polymorphisms and Chagas disease susceptibility in Peruvian and Colombian patients

Susceptibility to Chagas disease infections and its clinical manifestations may be influenced by host genetic factors. Among cytokines, the multifunctional transforming growth factor beta 1 (TGFbeta1), plays a major role in the establishment and pathogenesis of Trypanosoma cruzi infection, the etiological agent of Chagas disease. Several single-nucleotide polymorphisms (SNP) in the TGFbeta1 gene that may affect cytokine production have been described. We investigated, by PCR methods, five SNP in the TGFbeta1 gene of known or suggested functional significance (-988 C/A; -800 G/A; -509 C/T; 10 T/C and 263 C/T) in 347 seropositive (asymptomatic, n=175; cardiomyopathic, n=172) and 279 seronegative unrelated individuals from a Peruvian and a Colombian population where T. cruzi is highly endemic. We found a significant difference in the distribution of the TGFbeta1 10T and 10C alleles between patients and healthy controls in both cohorts, analyzed independently and combined. The frequency of the high TGFbeta1 producer genotype 10 C/C was increased in the patients groups of both populations. Our data suggests that TGFbeta1 genetic polymorphisms at codon 10 may be involved in a differential susceptibility to T. cruzi infection in these South American samples.

Association of the macrophage migration inhibitory factor −173G/C polymorphism with Chagas disease

Our aim was to evaluate the association of functional polymorphism of macrophage migration inhibitory factor (MIF) gene with Chagas disease. Our study includes two independent cohorts: 240 chagasic patients and 199 controls from Colombia; and 74 chagasic patients and 85 controls from Peru. The single nucleotide polymorphism (SNP) -173 G/C of MIF gene was determined using a polymerase chain reaction (PCR) system with pre-developed TaqMan assay. We observed a statistically significant difference in the distribution of -173*C allele of MIF gene between patients and controls in the Colombian cohort (OR = 1.6, 95% CI = 1.12-2.18, p = 0.006). Similar association was found in the Peruvian cohort (OR = 2.4, 95% CI = 1.31-4.38, p = 0.003). A meta-analysis of the Colombian and Peruvian cohorts demonstrated that the -173 C allele confers a risk effect in chagasic patients (pooled OR = 1.75, 95% CI = 1.30-2.33, p = 0.0002). In addition, a gene dose of the MIF -173 C allele was observed (pooled OR = 4.01, 95% CI = 1.25-12.85, p = 0.004). Our results suggest that the MIF -173G/C polymorphism confers susceptibility to Chagas disease in the populations under study.

Interleukin 4, interleukin 4 receptor-α and interleukin 10 gene polymorphisms in Chagas disease

In this study, we investigated the association between single‐nucleotide polymorphisms (SNPs) of the interleukin‐4 (IL4), interleukin‐4 receptor‐α (IL4RA) and interleukin‐10 (IL10) genes with the development of chagasic heart disease. This study included 260 patients from Colombia who were serologically positive for Trypanosoma cruzi antigens (cardiomyopathic, n = 130; asymptomatic, n = 130). Genotypes were determined by polymerase chain reaction (PCR)–restriction fragment length polymorphism or sequence‐specific primer methods. We found statistically significant differences in the distribution of the IL4RA +148 AA (P = 0·025, OR = 1·89, CI = 1·04–3·43) genotype when comparing asymptomatic and symptomatic patients. No statistically significant differences in the genotype and allele frequency of IL4 and IL10 gene polymorphisms between symptomatic and asymptomatic patients were observed. Our experimental evidence suggests that the IL4RA +148 AA genotype has a weak association with the development of chagasic cardiomyopathy in the population under study.