Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction

Background: In resource-limited countries, stool microscopy is the diagnostic test of choice for intestinal parasites (soil-transmitted helminths and/or intestinal protozoa). However, sensitivity and specificity is low. Improved diagnosis of intestinal parasites is especially important for accurate measurements of prevalence and intensity of infections in endemic areas. Methods: The study was carried out in Orán, Argentina. A total of 99 stool samples from a local surveillance campaign were analyzed by concentration microscopy and McMaster egg counting technique compared to the analysis by multi-parallel quantitative real-time polymerase chain reaction (qPCR). This study compared the performance of qPCR assay and stool microscopy for 8 common intestinal parasites that infect humans including the helminths Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, and the protozoa Giardia lamblia, Cryptosporidium parvum/hominis, and Entamoeba histolytica, and investigated the prevalence of polyparasitism in an endemic area. Results: qPCR showed higher detection rates for all parasites as compared to stool microscopy except T. trichiura. Species-specific primers and probes were able to distinguish between A. duodenale (19.1 %) and N. americanus (36.4 %) infections. There were 48.6 % of subjects co-infected with both hookworms, and a significant increase in hookworm DNA for A. duodenale versus N. americanus (119.6 fg/μL: 0.63 fg/μL, P∈<∈0.001) respectively. qPCR outperformed microscopy by the largest margin in G. lamblia infections (63.6 % versus 8.1 %, P∈<∈0.05). Polyparasitism was detected more often by qPCR compared to microscopy (64.7 % versus 24.2 %, P∈<∈0.05). Conclusions: Multi-parallel qPCR is a quantitative molecular diagnostic method for common intestinal parasites in an endemic area that has improved diagnostic accuracy compared to stool microscopy. This first time use of multi-parallel qPCR in Argentina has demonstrated the high prevalence of intestinal parasites in a peri-urban area. These results will contribute to more accurate epidemiological survey, refined treatment strategies on a public scale, and better health outcomes in endemic settings.Fil: Cimino, Rubén Oscar. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jeun, Rebecca. Baylor College Of Medicine; Estados UnidosFil: Juarez, Marisa. Universidad Nacional de Salta; ArgentinaFil: Cajal, Pamela S.. Universidad Nacional de Salta; ArgentinaFil: Vargas Flores, Paola Andrea. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Echazú, Adriana. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bryan, Patricia E.. Baylor College Of Medicine; Estados UnidosFil: Nasser, Julio Rubén. Universidad Nacional de Salta; ArgentinaFil: Krolewiecki, Alejandro Javier. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mejia, Rojelio. Baylor College Of Medicine; Estados Unidos. Universidad Nacional de Salta; Argentin

Impact of intestinal parasites on microbiota and cobalamin gene sequences: A pilot study

Background: Approximately 30% of children worldwide are infected with gastrointestinal parasites. Depending on the species, parasites can disrupt intestinal bacterial microbiota affecting essential vitamin biosynthesis. Methods: Stool samples were collected from 37 asymptomatic children from a previous cross-sectional Argentinian study. A multi-parallel real-time quantitative PCR was implemented for Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, Cryptosporidium spp., Entamoeba histolytica and Giardia duodenalis. In addition, whole-genome sequencing analysis was conducted for bacterial microbiota on all samples and analyzed using Livermore Metagenomic Analysis Toolkit and DIAMOND software. Separate analyses were carried out for uninfected, Giardia-only, Giardia + helminth co-infections, and helminth-only groups. Results: For Giardia-only infected children compared to uninfected children, DNA sequencing data showed a decrease in microbiota biodiversity that correlated with increasing Giardia burden and was statistically significant using Shannon's alpha diversity (Giardia-only > 1 fg/μl 2.346; non-infected group 3.253, P = 0.0317). An increase in diversity was observed for helminth-only infections with a decrease in diversity for Giardia + helminth co-infections (P = 0.00178). In Giardia-only infections, microbiome taxonomy changed from Firmicutes towards increasing proportions of Prevotella, with the degree of change related to the intensity of infection compared to uninfected (P = 0.0317). The abundance of Prevotella bacteria was decreased in the helminths-only group but increased for Giardia + helminth co-infections (P = 0.0262). Metagenomic analysis determined cobalamin synthesis was decreased in the Giardia > 1 fg/μl group compared to both the Giardia < 1 fg/μl and the uninfected group (P = 0.0369). Giardia + helminth group also had a decrease in cobalamin CbiM genes from helminth-only infections (P = 0.000754). Conclusion: The study results may provide evidence for an effect of parasitic infections enabling the permissive growth of anaerobic bacteria such as Prevotella, suggesting an altered capacity of vitamin B12 (cobalamin) biosynthesis and potential impact on growth and development in children.Fil: Mejia, Rojelio. Baylor College of Medicine; Estados Unidos. Universidad Nacional de Salta; ArgentinaFil: Damania, Ashish. Baylor College of Medicine; Estados UnidosFil: Jeun, Rebecca. Baylor College of Medicine; Estados UnidosFil: Bryan, Patricia E.. Baylor College of Medicine; Estados UnidosFil: Vargas, Paola. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Juarez, Marisa del Valle. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Cajal, Silvana Pamela. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Nasser, Julio Rubén. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Krolewiecki, Alejandro Javier. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; ArgentinaFil: Lefoulon, Emilie. New England Biolabs; Estados UnidosFil: Long, Courtney. New England Biolabs; Estados UnidosFil: Drake, Evan. New England Biolabs; Estados UnidosFil: Cimino, Rubén Oscar. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; ArgentinaFil: Slatko, Barton. New England Biolabs; Estados Unido