Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs

12 páginas, 3 tablas, 3 figuras.There is an urgent need for an extensive evaluation of benzimidazole efcacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequenc‑ ing, and standardized it for large-scale benzimidazole efcacy screening use.This study was funded by the ‘Stopping Transmission Of intestinal Parasites (STOP)’ project (EDCTP2 programme supported by the European Union; RIA2017NCT-1845-STOP). JG was personally supported at the beginning of the work by the Ramón Areces Foundation and is now funded by the Spanish ‘Juan de la Cierva’ Programme, Ministry of Economy and Competitiveness (FJC-2018-38305). MCP is supported by the Junta de Castilla y León and Fondo Social Europeo, and MMV by the Spanish ‘Ramón y Cajal’ Programme, Ministry of Economy and Competitiveness (RYC-2015-18368).Peer reviewe

Single-nucleotide polymorphisms in the beta-tubulin gene and its relationship with treatment response to albendazole in human soil-transmitted helminths in southern Mozambique

9 páginas, 2 tablas, 3 figuras.Soil-transmitted helminth (STH) cornerstone control strategy is mass drug administration (MDA) with benzimidazoles. However, MDA might contribute to selection pressure for anthelmintic resistance, as occurred in livestock. The aim of this study is to evaluate the treatment response to albendazole and the relationship with the presence of putative benzimidazole resistance single-nucleotide polymorphisms (SNPs) in the b-tubulin gene of STH in Southern Mozambique. After screening 819 participants, we conducted a cohort study with 184 participants infected with STH in Manhic¸ a district, Southern Mozambique. A pretreatment and a posttreatment stool samples were collected and the STH infection was identified by duplicate Kato-Katz and quantitative polymerase chain reaction (qPCR). Cure rate and egg reduction rates were calculated. Putative benzimidazole resistance SNPs (F167Y, F200T, and E198A) in Trichuris trichiura and Necator americanus were assessed by pyrosequencing. Cure rates by duplicate Kato-Katz and by qPCR were 95.8% and 93.6% for Ascaris lumbricoides, 28% and 7.8% for T. trichiura, and 88.9% and 56.7% for N. americanus. Egg reduction rate by duplicate Kato-Katz was 85.4% for A. lumbricoides, 34.9% for T. trichiura, and 40.5% for N. americanus. Putative benzimidazole resistance SNPs in the b-tubulin gene were detected in T. trichiura (23%) and N. americanus (21%) infected participants at pretreatment. No statistical difference was observed between pretreatment and posttreatment frequencies for none of the SNPs. Although treatment response to albendazole was low, particularly in T. trichiura, the putative benzimidazole resistance SNPs were not higher after treatment in the population studied. New insights are needed for a better understanding and monitoring of human anthelmintic resistance.This work was supported by the British Society for Antimicrobial Chemotherapy (Bsac-2018-0018) and Mundo Sano Foundation (www.mundosano.org). We acknowledge support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018- 000806-S), and support from the “Generalitat de Catalunya” through the CERCA Program. CISM is supported by the Government of Mozambique and the Spanish Agency for International Development (AECID). M. C. P. was funded by Junta de Castilla y Leon and Fondo Social Europeo (LE-135-19). J. G. was personally supported at the beginning of the work by the Ramon Areces Foundation and is now funded by the Spanish “Juan de la Cierva” Programme, Ministry of Economy and Competitiveness (FJC-2018-38305). M. M. V. by the “Ramon y Cajal ” Programme (RYC-2015-18368), Spanish Ministry of Economy, Industry and Competitiveness (MINECO). This project was in part funded by the EDCTP2 program supported by the European Union (Grant no. RIA2017NCT-1845-STOP; www.stoptheworm.org) Horizon 2020 European Union Funding for Research and Innovation.Peer reviewe

Molecular analysis of Strongyloides stercoralis beta-tubulin gene and the possible effects on albendazole efficacy

Trabajo presentado al: 12th European Congress on Tropical Medicine and International Health (ECTMIH). Norway. 28 september-1 octubrer. Virtual meeting.Peer reviewe

Single-Nucleotide Polymorphisms in the Beta-Tubulin Gene and Its Relationship with Treatment Response to Albendazole in Human Soil-Transmitted Helminths in Southern Mozambique

Soil-transmitted helminth (STH) cornerstone control strategy is mass drug administration (MDA) with benzimidazoles. However, MDA might contribute to selection pressure for anthelmintic resistance, as occurred in livestock. The aim of this study is to evaluate the treatment response to albendazole and the relationship with the presence of putative benzimidazole resistance single-nucleotide polymorphisms (SNPs) in the 3-tubulin gene of STH in Southern Mozambique. After screening 819 participants, we conducted a cohort study with 184 participants infected with STH in Manhiga district, Southern Mozambique. A pretreatment and a posttreatment stool samples were collected and the STH infection was identified by duplicate Kato-Katz and quantitative polymerase chain reaction (qPCR). Cure rate and egg reduction rates were calculated. Putative benzimidazole resistance SNPs (F167Y, F200T, and E198A) in Trichuris trichiura and Necator americanus were assessed by pyrosequencing. Cure rates by duplicate Kato-Katz and by qPCR were 95.8% and 93.6% for Ascaris lumbricoides, 28% and 7.8% for T. trichiura, and 88.9% and 56.7% for N. americanus. Egg reduction rate by duplicate Kato-Katz was 85.4% for A. lumbricoides, 34.9% for T. trichiura, and 40.5% for N. americanus. Putative benzimidazole resistance SNPs in the 3-tubulin gene were detected in T. trichiura (23%) and N. americanus (21%) infected participants at pretreatment. No statistical difference was observed between pretreatment and posttreatment frequencies for none of the SNPs. Although treatment response to albendazole was low, particularly in T. trichiura, the putative benzimidazole resistance SNPs were not higher after treatment in the population studied. New insights are needed for a better understanding and monitoring of human anthelmintic resistance

Evaluation of antibody serology to determine current helminth and Plasmodium falciparum infections in a co-endemic area in Southern Mozambique

BackgroundSoil-transmitted helminths (STH), Schistosoma spp. and Plasmodium falciparum are parasites of major public health importance and co-endemic in many sub-Saharan African countries. Management of these infections requires detection and treatment of infected people and evaluation of large-scale measures implemented. Diagnostic tools are available but their low sensitivity, especially for low intensity helminth infections, leaves room for improvement. Antibody serology could be a useful approach thanks to its potential to detect both current infection and past exposure.MethodologyWe evaluated total IgE responses and specific-IgG levels to 9 antigens from STH, 2 from Schistosoma spp., and 16 from P. falciparum, as potential markers of current infection in a population of children and adults from Southern Mozambique (N = 715). Antibody responses were measured by quantitative suspension array Luminex technology and their performance was evaluated by ROC curve analysis using microscopic and molecular detection of infections as reference.Principal findingsIgG against the combination of EXP1, AMA1 and MSP2 (P. falciparum) in children and NIE (Strongyloides stercoralis) in adults and children had the highest accuracies (AUC = 0.942 and AUC = 0.872, respectively) as markers of current infection. IgG against the combination of MEA and Sm25 (Schistosoma spp.) were also reliable markers of current infection (AUC = 0.779). In addition, IgG seropositivity against 20 out of the 27 antigens in the panel differentiated the seropositive endemic population from the non-endemic population, suggesting a possible role as markers of exposure although sensitivity could not be assessed.ConclusionsWe provided evidence for the utility of antibody serology to detect current infection with parasites causing tropical diseases in endemic populations. In addition, most of the markers have potential good specificity as markers of exposure. We also showed the feasibility of measuring antibody serology with a platform that allows the integration of control and elimination programs for different pathogens