Faustino Torrico

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 IIc: phylogenetic and phylogeographic insights from sequence and microsatellite analysis and potential Impact on emergent chagas disease

Trypanosoma cruzi, the etiological agent of Chagas disease, is highly genetically diverse. Numerous lines of evidence point to the existence of six stable genetic lineages or DTUs: TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe. Molecular dating suggests that T. cruzi is likely to have been an endemic infection of neotropical mammalian fauna for many millions of years. Here we have applied a panel of 49 polymorphic microsatellite markers developed from the online T. cruzi genome to document genetic diversity among 53 isolates belonging to TcIIc, a lineage so far recorded almost exclusively in silvatic transmission cycles but increasingly a potential source of human infection. These data are complemented by parallel analysis of sequence variation in a fragment of the glucose-6-phosphate isomerase gene. New isolates confirm that TcIIc is associated with terrestrial transmission cycles and armadillo reservoir hosts, and demonstrate that TcIIc is far more widespread than previously thought, with a distribution at least from Western Venezuela to the Argentine Chaco. We show that TcIIc is truly a discrete T. cruzi lineage, that it could have an ancient origin and that diversity occurs within the terrestrial niche independently of the host species. We also show that spatial structure among TcIIc isolates from its principal host, the armadillo Dasypus novemcinctus, is greater than that among TcI from Didelphis spp. opossums and link this observation to differences in ecology of their respective niches. Homozygosity in TcIIc populations and some linkage indices indicate the possibility of recombination but cannot yet be effectively discriminated from a high genome-wide frequency of gene conversion. Finally, we suggest that the derived TcIIc population genetic data have a vital role in determining the origin of the epidemiologically important hybrid lineages TcIId and TcIIe.

Congenital Chagas Disease: Recommendations for Diagnosis, Treatment and Control of Newborns, Siblings and Pregnant Women

In May 2010, the sixty-third World Health Assembly adopted resolution WHA63.20 on the control and elimination of Chagas disease, highlighting the need “to promote the development of public health measures in disease-endemic and disease non-endemic countries, with special focus on endemic areas, for the early diagnosis of congenital transmission and management of cases” [1]. This article summarizes the recommendations of the Technical Group IVa on “Prevention and Control of Congenital Transmission and Case Management of Congenital Infections” of the World Health Organizations Programme on Control of Chagas disease (infection with Trypanosoma cruzi). The present recommendations derive from those obtained in the meetings listed in Box 1. Box 1. Meetings from Which the Present Recommendations Derive Meeting ULB (Belgium)/UMSS (Bolivia)), Cochabamba, Bolivia, November 6–8, 2002: “Congenital Infection with Trypanosoma cruzi: From Mechanisms of Transmission to Strategies for Diagnosis And Control”, Carlier Y and Torico F, Revista da Sociedade Brasileira de Medicina Tropical 2003, 6: 767–771. Meeting PAHO/CLAP/ULB (Belgium)/IRD (France), Montevideo, Uruguay, June 24–25, 2004: “Congenital Chagas Disease: Its Epidemiology and Management”, http://www.paho.org/English/AD/DPC/CD/dch-chagas-congenita-2004.htm Meeting PAHO/CLAP/ULB (Belgium), Montevideo, Uruguay, May 17–18, 2007: “Information, Education and Communication in Congenital Chagas Disease”, http://www.paho.org/English/AD/DPC/CD/dch-congenita-iec-07.doc Meeting WHO, Geneva, Switzerland, July 4–6, 2007: “Revisiting Chagas Disease: From a Latin American Health Perspective to a Global Health Perspective” Meeting of the WHO TG IVa (congenital and paediatric Chagas disease), New Orleans, Louisiana, United States, December 11, 2008, satellite meeting to the ASTMH 57th annual meeting Meeting of the 6th European Congress of Tropical Medicine and International Health, Verona, Italy, September 6–10, 2009: “Chagas Disease in Europe” Meeting of WHO-HQ and the WHO regional office for Europe, Geneva, Switzerland, December 17–18, 2009: “Consultation on Chagas Disease in Europe”

Towards a Paradigm Shift in the Treatment of Chronic Chagas Disease

ABSTRACT Treatment for Chagas disease with currently available medications is recommended universally only for acute cases (all ages) and for children up to 14 years old. The World Health Organization, however, also recommends specific antiparasite treatment for all chronic-phase Trypanosoma cruzi-infected individuals, even though in current medical practice this remains controversial, and most physicians only prescribe palliative treatment for adult Chagas patients with dilated cardiomyopathy. The present opinion, prepared by members of the NHEPACHA network (Nuevas Herramientas para el Diagnóstico y la Evaluación del Paciente con Enfermedad de Chagas/New Tools for the Diagnosis and Evaluation of Chagas Disease Patients), reviews the paradigm shift based on clinical and immunological evidence and argues in favor of antiparasitic treatment for all chronic patients. We review the tools needed to monitor therapeutic efficacy and the potential criteria for evaluation of treatment efficacy beyond parasitological cure. Etiological treatment should now be mandatory for all adult chronic Chagas disease patients.

Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.

Congenital infection with Trypanosoma cruzi: from mechanisms of transmission to strategies for diagnosis and control

The transmission rate of congenital T. cruzi infection (number of congenital cases/number of chagasic mothers) in the Southern Cone countries varies widely, from 1% in Brazil to 4 to 12% Argentina, Bolivia, Chile and Paraguay. These differences were discussed at length, and attributed to the different methodologies used for detection of congenital cases, and/or possible special characteristics from the infecting parasites, differences in the immunological, genetical or nutritional status of the mother, or specific epidemiological situations remaining to be studied.

Congenital transmission of Chagas disease: a clinical approach.

Chagas disease is caused by the protozoan Trypanosoma cruzi and is an endemic zoonosis in the American continent. Thanks to the successful implementation of national programs for reducing the vectorial infestation and the strict control of blood-borne transmission of Chagas disease, the relative importance of congenital transmission has recently increased. Nowadays, in areas without vectorial transmission, congenital transmission has become the main way by which the disease has spread. This article reviews current knowledge on Chagas disease, focusing on the congenital transmission route. The public health implications of the increasing number of T. cruzi-infected immigrants and congenital transmission in nonendemic areas is also analyzed.

Maternal Trypanosoma cruzi Infection Upregulates Capacity of Uninfected Neonate Cells To Produce Pro- and Anti-Inflammatory Cytokines

ABSTRACT The possibility of maternal in utero modulation of the innate and/or adaptive immune responses of uninfected newborns fromTrypanosoma cruzi-infected mothers was investigated by studying the capacity of their whole blood cells to produce cytokines in response to T. cruzi lysate or lipopolysaccharide-plus-phytohemagglutinin (LPS-PHA) stimulation. Cells of such newborns occasionally released gamma interferon (IFN-γ) and no interleukin-2 (IL-2) and IL-4 upon specific stimulation, while their mothers responded by the production of IFN-γ, IL-2, and IL-4. Infection in mothers was also associated with a hyperactivation of maternal cells and also, strikingly, of cells of their uninfected neonates, since their release of proinflammatory (IL-1β, IL-6, and tumor necrosis factor alpha [TNF-α]) as well as of anti-inflammatory (IL-10 and soluble TNF receptor) cytokines or factors was upregulated in the presence of LPS-PHA and/or parasite lysate. These results show that T. cruzi infection in mothers induces profound perturbations in the cytokine response of their uninfected neonates. Such maternal influence on neonatal innate immunity might contribute to limit the occurrence and severity of congenital infection.

Maternal and congenital syphilis in Bolivia, 1996: prevalence and risk factors

OBJECTIVES The present study was carried out in seven maternity hospitals to determine the prevalence of maternal syphilis at the time of delivery and the associated risk factors, to conduct a pilot project of rapid syphilis testing in hospital laboratories, to assure the quality of syphilis testing, and to determine the rate of congenital syphilis in infants born to women with syphilis at the time of delivery--all of which would provide baseline data for a national prevention programme in Bolivia. METHODS All women delivering either live-born or stillborn infants in the seven participating hospitals in and around La Paz, El Alto, and Cochabamba between June and November 1996 were eligible for enrolment in the study. FINDINGS A total of 61 out of 1428 mothers (4.3%) of live-born infants and 11 out of 43 mothers (26%) of stillborn infants were found to have syphilis at delivery. Multivariate analysis showed that women with live-born infants who had less than secondary-level education, who did not watch television during the week before delivery (this was used as an indicator of socioeconomic status), who had a previous history of syphilis, or who had more than one partner during the pregnancy were at increased risk of syphilis. While 76% of the study population had received prenatal care, only 17% had syphilis testing carried out during the pregnancy; 91% of serum samples that were reactive to rapid plasma reagin (RPR) tests were also reactive to fluorescent treponemal antibody-absorption (FTA-ABS) testing. There was 96% agreement between the results from local hospital laboratories and national reference laboratories in their testing of RPR reactivity of serum samples. Congenital syphilis infection was confirmed by laboratory tests in 15% of 66 infants born to women with positive RPR and FTA-ABS testing. CONCLUSION These results indicate that a congenital syphilis prevention programme in Bolivia could substantially reduce adverse infant outcomes due to this disease.