BackgroundA 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/FindingsAn 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/SignificanceThis study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Plasmodium falciparum entered into the Peruvian Amazon in 1994, sparking an epidemic between 1995 and 1998. Since 2000, there has been sustained low P. falciparum transmission. The Malaria Immunology and Genetics in the Amazon project has longitudinally followed members of the community of Zungarococha (N = 1,945, 4 villages) with active household and health center-based visits each year since 2003. We examined parasite population structure and traced the parasite genetic diversity temporally and spatially. We genotyped infections over 5 years (2003–2007) using 14 microsatellite (MS) markers scattered across ten different chromosomes. Despite low transmission, there was considerable genetic diversity, which we compared with other geographic regions. We detected 182 different haplotypes from 302 parasites in 217 infections. Structure v2.2 identified five clusters (subpopulations) of phylogenetically related clones. To consider genetic diversity on a more detailed level, we defined haplotype families (hapfams) by grouping haplotypes with three or less loci differences. We identified 34 different hapfams identified. The Fst statistic and heterozygosity analysis showed the five clusters were maintained in each village throughout this time. A minimum spanning network (MSN), stratified by the year of detection, showed that haplotypes within hapfams had allele differences and haplotypes within a cluster definition were more separated in the later years (2006–2007). We modeled hapfam detection and loss, accounting for sample size and stochastic fluctuations in frequencies overtime. Principle component analysis of genetic variation revealed patterns of genetic structure with time rather than village. The population structure, genetic diversity, appearance/disappearance of the different haplotypes from 2003 to 2007 provides a genome-wide “real-time” perspective of P. falciparum parasites in a low transmission region.

We assessed whether mutations in the Plasmodium falciparum multidrug-resistance gene 1 (pfmdr1) (C1034S, D1042N, and Y1246D) would predict treatment outcome during a 28-day in vivo treatment trial in the Peruvian Amazon. Mefloquine (MQ) was compared with mefloquine-artesunate (MQ-AS) in a randomized, multi-clinic protocol for the first time in the Americas. Of 115 patients enrolled in the in vivo arm, 97 patients were eligible for molecular analysis. All 97 patients remained parasite-free during 28 days of follow-up (MQ, n = 46; MQ-AS, n = 51), indicating 100% clinical efficacy of the MQ and MQ-AS treatment regimens. The reported MQ-sensitive alleles (C1034, D1042, and Y1246) were present in 48.5% (n = 47) of the cases, whereas 49 isolates (50.5%) contained the D1246 mutation reported to confer MQ resistance in vitro. However, neither this mutation nor a double mutation (S1034, D1246; n = 16) was predictive of MQ treatment outcome.

Bióloga, magíster en Epidemiología; b nutricionista, magíster en Nutrición; c médico epidemiólogo y salubrista; d médico especialista en Gestión en Salud, d médico magíster en Epidemiología.

RESUMENEn el presente artículo se reporta la experiencia obtenida por el Instituto Nacional de Salud de Perú, en la utilidad del desarrollo de diálogos deliberativos para la formulación de recomendaciones, como uno de los componentes del proceso de adaptación de Guías de Práctica Clínica basadas en evidencia, utilizando el sistema GRADE (por sus siglas en ingles: Grading of Recommendations, Assessment, Development and Evaluations). Describe los temas de salud pública que fueron deliberados y la participación de los actores implicados con un enfoque multidisciplinario. Presenta los aspectos que fueron tomados en cuenta de forma previa y durante los diálogos. Finalmente, propone los desafíos que serán abordados en las siguientes experiencias a fin de optimizar estos procesos y colaborar en la interacción entre las evidencias científicas y el valor agregado que otorgan los actores implicados a través de su experiencias, perspectivas y conocimientos para el desarrollo de recomendaciones en salud. Palabras clave: Práctica clínica basada en la evidencia, Política de salud, Gestión del conocimiento, Perú (fuente: DeCS BIREME) USE OF DELIBERATIVE DIALOGUES FOR THE FORMULATION OF RECOMMENDATIONS FOR CLINICAL PRACTICE GUIDELINES ABSTRACTThis study reports the experience obtained by the National Institute of Health of Peru in the use of deliberative dialogues for the formulation of recommendations as one of the stages of the process of adaptation of evidence-based clinical practice guidelines using the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) system. Using a multidisciplinary approach, this study describes: 1. the public health problems addressed in the dialogues; 2. the role of the involved parties; 3. the parameters that were considered before and during the discussions; 4. the challenges for optimizing these dialogues and stimulating the interaction between scientific evidence and the added value provided by the involved parties through their experiences, perspectives, and knowledge for the development of recommendations in health.