Patricio Diosque

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.

Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I Reveals Phylogeographic Structure and Specific Genotypes Linked to Human Infection

Trypanosoma cruzi is the most important parasitic infection in Latin America and is also genetically highly diverse, with at least six discrete typing units (DTUs) reported: Tc I, IIa, IIb, IIc, IId, and IIe. However, the current six-genotype classification is likely to be a poor reflection of the total genetic diversity present in this undeniably ancient parasite. To determine whether epidemiologically important information is “hidden” at the sub-DTU level, we developed a 48-marker panel of polymorphic microsatellite loci to investigate population structure among 135 samples from across the geographic distribution of TcI. This DTU is the major cause of resurgent human disease in northern South America but also occurs in silvatic triatomine vectors and mammalian reservoir hosts throughout the continent. Based on a total dataset of 12,329 alleles, we demonstrate that silvatic TcI populations are extraordinarily genetically diverse, show spatial structuring on a continental scale, and have undergone recent biogeographic expansion into the southern United States of America. Conversely, the majority of human strains sampled are restricted to two distinct groups characterised by a considerable reduction in genetic diversity with respect to isolates from silvatic sources. In Venezuela, most human isolates showed little identity with known local silvatic strains, despite frequent invasion of the domestic setting by infected adult vectors. Multilocus linkage indices indicate predominantly clonal parasite propagation among all populations. However, excess homozygosity among silvatic strains and raised heterozygosity among domestic populations suggest that some level of genetic recombination cannot be ruled out. The epidemiological significance of these findings is discussed.

Trypanosoma cruzi: adaptation to its vectors and its hosts

American trypanosomiasis is a parasitic zoonosis that occurs throughout Latin America. The etiological agent, Trypanosoma cruzi, is able to infect almost all tissues of its mammalian hosts and spreads in the environment in multifarious transmission cycles that may or not be connected. This biological plasticity, which is probably the result of the considerable heterogeneity of the taxon, exemplifies a successful adaptation of a parasite resulting in distinct outcomes of infection and a complex epidemiological pattern. In the 1990s, most endemic countries strengthened national control programs to interrupt the transmission of this parasite to humans. However, many obstacles remain to the effective control of the disease. Current knowledge of the different components involved in elaborate system that is American trypanosomiasis (the protozoan parasite T. cruzi, vectors Triatominae and the many reservoirs of infection), as well as the interactions existing within the system, is still incomplete. The Triatominae probably evolve from predatory reduvids in response to the availability of vertebrate food source. However, the basic mechanisms of adaptation of some of them to artificial ecotopes remain poorly understood. Nevertheless, these adaptations seem to be associated with a behavioral plasticity, a reduction in the genetic repertoire and increasing developmental instability.

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.

Multilocus Sequence Typing (MLST) for Lineage Assignment and High Resolution Diversity Studies in Trypanosoma cruzi

Background Multilocus sequence typing (MLST) is a powerful and highly discriminatory method for analysing pathogen population structure and epidemiology. Trypanosoma cruzi, the protozoan agent of American trypanosomiasis (Chagas disease), has remarkable genetic and ecological diversity. A standardised MLST protocol that is suitable for assignment of T. cruzi isolates to genetic lineage and for higher resolution diversity studies has not been developed. Methodology/Principal Findings We have sequenced and diplotyped nine single copy housekeeping genes and assessed their value as part of a systematic MLST scheme for T. cruzi. A minimum panel of four MLST targets (Met-III, RB19, TcGPXII, and DHFR-TS) was shown to provide unambiguous assignment of isolates to the six known T. cruzi lineages (Discrete Typing Units, DTUs TcI-TcVI). In addition, we recommend six MLST targets (Met-II, Met-III, RB19, TcMPX, DHFR-TS, and TR) for more in depth diversity studies on the basis that diploid sequence typing (DST) with this expanded panel distinguished 38 out of 39 reference isolates. Phylogenetic analysis implies a subdivision between North and South American TcIV isolates. Single Nucleotide Polymorphism (SNP) data revealed high levels of heterozygosity among DTUs TcI, TcIII, TcIV and, for three targets, putative corresponding homozygous and heterozygous loci within DTUs TcI and TcIII. Furthermore, individual gene trees gave incongruent topologies at inter- and intra-DTU levels, inconsistent with a model of strict clonality. Conclusions/Significance We demonstrate the value of systematic MLST diplotyping for describing inter-DTU relationships and for higher resolution diversity studies of T. cruzi, including presence of recombination events. The high levels of heterozygosity will facilitate future population genetics analysis based on MLST haplotypes.

Congenital Chagas disease involves Trypanosoma cruzi sub-lineage IId in the northwestern province of Salta, Argentina

Trypanosoma cruzi is genetically classified into six discrete phylogenetic lineages on the basis of different genetic markers. Identifying lineages circulating among humans in different areas is essential to understand the molecular epidemiology of Chagas disease. In the present study, 18 T. cruzi isolates from congenitally infected newborns in the northwestern province of Salta-Argentina were studied by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD). All isolates were typed by MLEE and RAPD as belonging to T. cruzi IId. Analysis of minor variants of TcIId using probes hybridizing with hypervariable domains of kDNA minicircles, detected three variants with a similar distribution among the isolates. Our findings confirm the presence of T. cruzi IId among congenitally infected newborns in northwestern Argentina and support the assumption that human infection by T. cruzi in the Southern Cone countries of Latin America is due principally to T. cruzi II.

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.

Candidate targets for Multilocus Sequence Typing of Trypanosoma cruzi: Validation using parasite stocks from the Chaco Region and a set of reference strains

A Multilocus Sequence Typing (MLST) scheme was designed and applied to a set of 20 Trypanosoma cruzi stocks belonging to three main discrete typing units (T. cruzi I, V and VI) from a geographically restricted Chagas disease endemic area in Argentina, 12 reference strains comprising two from each of the six main discrete typing units of the parasite (T. cruzi I-VI), and one T. cruzi marinkellei strain. DNA fragments (≅400-bp) from 10 housekeeping genes were sequenced. A total of 4178 bp were analyzed for each stock. In all, 154 polymorphic sites were identified. Ninety-five sites were heterozygous in at least one analyzed stock. Seventeen diploid sequence types were identified from 32 studied T. cruzi stocks (including the reference strains). All stocks were correctly assigned to their corresponding discrete typing units. We propose this MLST scheme as provisional, with scope for improvement by studying new gene targets on a more diverse sample of stocks, in order to define an optimized MLST scheme for T. cruzi. This approach is an excellent candidate to become the gold standard for T. cruzi genetic typing. We suggest that MLST will have a strong impact on molecular epidemiological studies of Chagas disease and the phylogenetics of its causative agent.

MLSTest: novel software for multi-locus sequence data analysis in eukaryotic organisms.

Multi-locus sequence typing (MLST) is a frequently used genotyping method whose goal is the unambiguous assignment of microorganisms to genetic clusters. MLST typically involves analysis of DNA sequence results generated from several house-keeping gene loci. MLST remains the gold standard for molecular typing of many bacterial pathogens. Eukaryotic pathogens have also been the subject of MLST, however, few tools are available to deal with diploid sequence data. Here we present novel software for MLST data analysis tailored towards diploid Eukaryotes: MLSTest. This software meets various methods used in MLST and introduces some novel methodologies for the evaluation of the data set. In addition to construction of allelic profiles and basic clustering analysis, the MLSTest looks for network structures that suggest genetic exchange in BURST graphs. Additionally, it uses several simple methods for tree construction with the advantage of managing heterozygous or three-state sites. Additionally, the software analyses whether concatenation of fragments from different genes is suitable for the data set using different tests (bionj-incongruence length difference test, Templeton test). It evaluates how the incongruence is distributed across the tree using a variation of the localized incongruence length difference test based on a modified neighbour joining algorithm. We tested the last method in simulated datasets. We showed that is conservative (adequate type I error rate) and moderately to highly powerful as well as useful to localize incongruences in two bacterial and two eukaryotic MLST datasets. MLSTest was also designed for developing MLST schemes. It thus has tools to optimize locus combinations and to reduce the number of targets required for typing. MLSTest also analyses whether the discriminatory power of the typing scheme is increased by including more loci. We evaluated the software over simulated and real datasets from bacterial and eukaryotic microorganisms. The software is freely available at http://www.ipe.unsa.edu.ar/software.

Interest and limitations of Spliced Leader Intergenic Region sequences for analyzing Trypanosoma cruzi I phylogenetic diversity in the Argentinean Chaco.

Internal and geographical clustering within Trypanosoma cruzi I (TcI) has been recently revealed by using Multilocus Microsatellite Typing and sequencing of the Spliced-Leader Intergenic Region (SL-IR). In the present work, 14 isolates and 11 laboratory-cloned stocks obtained from a geographically restricted area in Chaco Province, Argentina, were analyzed by PCR and sequencing of SL-IR. We were able to differentiate 8 different genotypes that clustered into 4 groups. One of these groups was classified within the formerly described haplotype A and another one within the recently described SL-IR group E. Both were phylogenetically well-supported. In contrast, none of the stocks from the Chaco province were grouped within the cluster previously named haplotype D despite the fact that they shared a similar microsatellite motif in the SL-IR. No evidence of recombination or gene conversion within these stocks was found. On the other hand, multiple ambiguous alignments in the microsatellite region of SL-IR, affecting the tree topology and relationships among groups were detected. Finally, since there are multiple copies of the SL-IR, and they are arranged in tandem, we discuss how molecular processes affecting this kind of sequences could mislead phylogenetic inference.