Marleny Montilla

Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans.

Genetic variability in the Trypanosoma cruzi I group has recently been revealed in Colombian isolates from humans, reservoirs and vectors. Genomic rearrangements and the polymorphic regions in taxonomic markers, such as the miniexon gene, have led to the development of molecular tools to identify phylogenetic haplotypes in T. cruzi isolates. From genetic polymorphisms found in T. cruzi I isolates, they have been classified into four haplotypes according to their epidemiologic transmission cycles. Haplotype Ia is associated with domestic isolates, from Rhodnius prolixus; haplotype Ib, with the domestic and peridomestic cycle, mainly associated with Triatoma dimidiata; haplotype Ic is a poorly characterized group, which has been associated with the peridomestic cycle; and haplotype Id has been related to the sylvatic cycle. In order to demonstrate that the circulating T. cruzi I isolates in Colombia can be classified in the four proposed haplotypes, specific primers were designed on polymorphic regions of the miniexon genes intergenic sequences. This set of primers allowed the molecular characterization of 33 Colombian isolates, classifying them into three of the four proposed haplotypes (Ia, Ib and Id). Results obtained from maximum parsimony and maximum-likelihood-based phylogenetic analyses correlated with the molecular classification of the isolates and their transmission cycles. This study brings insights into the Chagas disease epidemiology and the parasites transmission dynamics.

Contemporary cryptic sexuality in Trypanosoma cruzi

Clonal propagation is considered to be the predominant mode of reproduction among many parasitic protozoa. However, this assumption may overlook unorthodox, infrequent or cryptic sexuality. Trypanosoma cruzi, which causes Chagas disease, is known to undergo non‐Mendelian genetic exchange in the laboratory. In the field, evidence of extant genetic exchange is limited. In this study, we undertook intensive sampling of T. cruzi Discrete Typing Unit I in endemic eastern Colombia. Using Fluorescence‐activated cell sorting, we generated 269 biological clones from 67 strains. Each clone was genotyped across 24 microsatellite loci. Subsequently, 100 representative clones were typed using 10 mitochondrial sequence targets (3.76 Kbp total). Clonal diversity among humans, reservoir hosts and vectors suggested complex patterns of superinfection and/or coinfection in oral and vector‐borne Chagas disease cases. Clonal diversity between mother and foetus in a congenital case demonstrates that domestic TcI genotypes are infective in utero. Importantly, gross incongruence between nuclear and mitochondrial markers is strong evidence for widespread genetic exchange throughout the data set. Furthermore, a confirmed mosaic maxicircle sequence suggests intermolecular recombination between individuals as a further mechanism of genetic reassortment. Finally, robust dating based on mitochondrial DNA indicates that the emergence of a widespread domestic TcI clade that we now name TcIDOM (formerly TcIa/VENDom) occurred 23 000 ± 12 000 years ago and was followed by population expansion, broadly corresponding with the earliest human migration into the Americas.

Molecular epidemiology of human oral Chagas disease outbreaks in Colombia.

Background Trypanosoma cruzi, the causative agent of Chagas disease, displays significant genetic variability revealed by six Discrete Typing Units (TcI-TcVI). In this pathology, oral transmission represents an emerging epidemiological scenario where different outbreaks associated to food/beverages consumption have been reported in Argentina, Bolivia, Brazil, Ecuador and Venezuela. In Colombia, six human oral outbreaks have been reported corroborating the importance of this transmission route. Molecular epidemiology of oral outbreaks is barely known observing the incrimination of TcI, TcII, TcIV and TcV genotypes. Methodology and Principal Findings High-throughput molecular characterization was conducted performing MLMT (Multilocus Microsatellite Typing) and mtMLST (mitochondrial Multilocus Sequence Typing) strategies on 50 clones from ten isolates. Results allowed observing the occurrence of TcI, TcIV and mixed infection of distinct TcI genotypes. Thus, a majority of specific mitochondrial haplotypes and allelic multilocus genotypes associated to the sylvatic cycle of transmission were detected in the dataset with the foreseen presence of mitochondrial haplotypes and allelic multilocus genotypes associated to the domestic cycle of transmission. Conclusions These findings suggest the incrimination of sylvatic genotypes in the oral outbreaks occurred in Colombia. We observed patterns of super-infection and/or co-infection with a tailored association with the severe forms of myocarditis in the acute phase of the disease. The transmission dynamics of this infection route based on molecular epidemiology evidence was unraveled and the clinical and biological implications are discussed.

Genetic Variability and Phylogenetic Relationships within Trypanosoma cruzi I Isolated in Colombia Based on Miniexon Gene Sequences

Phylogenetic studies of Trypanosoma cruzi have identified the existence of two groups: T. cruzi I and T. cruzi II. There are aspects that still remain unknown about the genetic variability within the T. cruzi I group. Given its epidemiological importance, it is necessary to have a better understanding of T. cruzi transmission cycles. Our purpose was to corroborate the existence of haplotypes within the T. cruzi I group and to describe the genetic variability and phylogenetic relationships, based on single nucleotide polymorphisms (SNPs) found in the miniexon gene intergenic region, for the isolates from different hosts and epidemiological transmission cycles in Colombian regions. 31 T. cruzi isolates were molecularly characterized. Phylogenetic relationships within T. cruzi I isolates showed four haplotype groups (Ia–Id), associated with their transmission cycle. In previous studies, we reported that haplotype Ia is mainly associated with the domestic cycle and domiciliated Rhodnius prolixus. Haplotype Ib is associated with the domestic cycle and peridomestic cycle, haplotype Ic is closely related with the peridomestic cycle, and haplotype Id is strongly associated with the sylvatic cycle. The phylogenetic methodologies applied in this study are tools that bolster the associations among isolates and thus shed light on Chagas disease epidemiology.

Enfermedad de Chagas aguda en Colombia, una entidad poco sospechada. Informe de 10 casos presentados en el periodo 2002 a 2005

Introduction. In Colombia, reported cases of acute Chagas disease are sporadic. Objective. Ten cases were described that had been reported to the Parasitology Laboratory of the Colombian National Health Institute between December 2002 and November 2005. Materials and methods. Information from clinical records, epidemiological report forms, laboratory and blood tests was collated. In addition the following data were compiled: demographic variables, clinical findings, results of laboratory tests and other exams (such as peripheral blood smears), IFAT for IgG antibodies, isolation in culture medium, inoculation in mice, polymerase chain reaction tests and isoenzyme eletrophoresis. Results. All the cases presented in known endemic areas for Chagas disease transmission in Colombia. Three cases were from Putumayo Province, two each from the provinces of Arauca, Casanare, Norte de Santander and one from Santander Province. The probable mode of transmission was vector-borne. Seven cases presented in adults aged 18 to 50, three in children aged 6 months to 2 years. Seven were male and three were female. The most frequent symptom was fever in nine cases. Signs of portal of entry were rare; only one patient presented a possible Romana´s sign. Three patients presented myocarditis, two acute cardiac failure and one cardiac tamponade. Parasitemia was evident in nine cases; five had positive IgG serological tests; five cases were confirmed through parasite isolation; isoenzyme electrophoresis showed Trypanosoma cruzi group I. Conclusions. Clinical variability prevailed. In none of the cases was a clinical diagnosis suspected. The diagnosis was made and confirmed through laboratory tests alone. The results highlight the importance of including this disease in the differential diagnosis of febrile syndrome in endemic regions due to its good response to etiological treatment and thereby preventing its progression to the chronic phase.

Characterization of a candidate Trypanosoma rangeli small nucleolar RNA gene and its application in a PCR-based parasite detection

In this study, we report the isolation and characterization of a candidate Trypanosoma rangeli small nucleolar RNA (snoRNA) gene, and the development of a PCR assay for detection of the parasite based on its nucleotide sequence. This gene, isolated from a T. rangeli genomic sub-library, was named snoRNA-cl1 and is encoded by a multi-copy gene of 801bp in length. Computer sequence analysis of snoRNA-cl1 showed the presence of two sequence motifs, box C and box D, as well as of two long stretches that perfectly complement the universal core region of the mature rRNA 28S, suggesting that cl1 encodes for a Box C/D snoRNA from the parasite. Hybridization analysis using cl1 as probe, showed a weak hybridization signal with Trypanosoma cruzi DNA, demonstrating the existence of differences in this locus between these two species. Two oligonucleotide primers from this gene, which specifically amplified a 620-bp fragment in KP1 (+) and KP1 (-) strains of T. rangeli, were used in a PCR assay. The amplification allowed the detection of 1pg of DNA in the presence of heterologous DNA and no amplification was observed with different T. cruzi strains (groups I and II). In addition, the PCR assay reported here is able to detect T. rangeli in the presence of T. cruzi DNA, and is useful for detection of the parasite in samples from infected vectors.

First Report of Human Trypanosoma cruzi Infection Attributed to TcBat Genotype

Chagas disease is an endemic disease of the American continent caused by Trypanosoma cruzi and divided into six discrete typing units (TcI – TcVI). Nearly 10 million people harbour the infection representing a serious issue in public health. Epidemiological surveillance allowed us to detect a bat‐related T. cruzi genotype (henceforth named TcBat) in a 5‐year‐old female living in a forest area in northwestern Colombia. Molecular tools determined a mixed infection of T. cruzi I and TcBat genotypes. This represents the first report of TcBat infection in humans; the epidemiological consequences of this finding are discussed herein.

Natural and emergent Trypanosoma cruzi I genotypes revealed by mitochondrial (Cytb) and nuclear (SSU rDNA) genetic markers

Chagas disease is a tropical and systemic disease caused by the parasite Trypanosoma cruzi. This parasite has been divided into six Discrete Typing Units (DTUs) due to its high genetic diversity. T. cruzi I (TcI) is the most prevalent DTU in Colombia and recently associated to cardiomyopathies. The aim of this study was to unravel the genetic variability among a set of 70 cell-single TcI clones from different geographical regions and hosts using the sequences of Cytb and SSU rDNA. The results showed two genotypes associated to transmission cycles of Chagas disease in Colombia and supports the previous descriptions using SL-IR. Phylogenetic networks were developed detecting recombination events within TcI. We also tested the phylogenetic relationships beneath TcI clones and TcIII/TcIV sequences observing the high relatedness of TcI clones from sylvatic cycle with TcIII/TcIV. We corroborate the high genetic diversity displayed by TcI, the plausible recombination within this DTU supporting the previous model of genetic exchange proposed in T. cruzi populations. We conclude inquiring the need to pursue new studies to elucidate the genetic structure of TcI across Chagas disease endemic countries.

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100% of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71% with TrF/R2 and in 6% with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100% with both PCRs and T. rangeli in 14% with TrF/R2 and 10% with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

Isoenzymatic Characterization of Colombian Strains of Trypanosoma cruzi

Laboratorio de Parasitología Molecular, Departamento de Microbiología, Facultad de Ciencias, Universidad Javeriana, Carrera 7 No. 43-82, Ed. Felix Restrepo, Of.111, Santafé de Bogotá, Colombia *Laboratorio de Parasitología, Instituto Nacional de Salud, Av. El Dorado con Carrera 50, Santafé de Bogotá, Colombia **Instituto de Genética, Facultad de Medicina, Universidad Javeriana, Carrera 7 No. 40-62, Santafé de Bogotá, Colombia