Deep-amplicon sequencing of the complete beta-tubulin gene in Trichuris trichiura before and after albendazole treatment

8 páginas, 2 figuras, 1 tabla.

Concerns about the emergence of benzimidazole resistance in soil-transmitted helminths (STH) infections, particularly against Trichuris trichiura, have arisen. Previous studies of veterinary nematodes have linked benzimidazole resistance to single-nucleotide polymorphisms (SNPs) at three specific codons in the beta-tubulin gene, but similar associations in STH have not been consistently observed. In this work, we screened the complete beta-tubulin gene previously linked to benzimidazole resistance in T. trichiura by deep-amplicon sequencing to identify genetic variants and associate levels of diversity with drug response to albendazole. We used 99 DNA samples extracted from T. trichiura pooled eggs, previously semi-purified from human stool samples collected in Manhiça district, Mozambique. We obtained a set of 39 amplicons of the complete gene by subjecting the pooled eggs to long-read PCR and subsequently sequencing them. Of those amplicons, 22 and 17 were obtained from stool samples collected before, and 21 days after albendazole treatment, respectively. We observed genetic variation across the whole gene sequence, in both exons and introns; however, none were associated with the previously proposed resistance-associated SNPs, and none were predicted to significantly affect protein function. No significant differences in genetic diversity were observed between pre- and post-treatment samples. Using publicly available genome-wide data, we also analysed a second beta-tubulin isotype in the T. trichiura genome. We focused on detecting the canonical SNPs and assessing for signatures of genetic selection around this second isotype gene. This analysis did not reveal evidence supporting this second isotype's role in anthelmintic resistance. Despite the limitations of our study, such as a small sample size, particularly paired pre- and post-treatment samples (n = 6), or a restricted geographical area, we found no evidence linking either of the two beta-tubulin genes to benzimidazole resistance in T. trichiura, suggesting that genetic markers of drug resistance likely exist outside the beta-tubulin genes.

The authors are grateful to this study's participants, community leaders, and local government authorities for their unconditional support and collaboration. Special thanks to all CISM's staff, particularly the Demography and Social Sciences department, for their indispensable assistance and to those involved in data collection and laboratory testing. This research was conducted with the support of the Stopping Transmission Of intestinal Parasites (STOP) consortium funded by the EDCTP2 programme (grant number RIA2017NCT-1845 STOP), Horizon 2020European Union Funding for Research and Innovation, and Mundo Sano Foundation. The ISGlobal authors acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the ‘Centro de Excelencia Severo Ochoa 2019–2023’ Program (CEX2018-000806-S), CIBER-Consorcio Centro de Investigación Biomédica en Red-(CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea and support from the Generalitat de Catalunya through the CERCA Program. CISM receives core funding from the “Agencia Española de Cooperación Internacional para el Desarollo” (AECID). MCP discloses support from: 'Pla de Doctorats Industrials' (DIN2022) of Departament de Recerca i Universitats de la Generalitat de Catalunya; ‘Ayudas a proyectos de colaboración público-privada’ (CPP2022-009773) funded by the MCIN/AEI/10.13039/501100011033 and the European Union ‘NextGenerationEU’/PRTR; and ‘Ayudas para la formación de doctores en empresas « Doctorados Industriales»’ (DIN2022-012602) funded by MCIN/AEI/10.13039/501100011033. JM also acknowledges support from the Hospital Clinic of Barcelona and the Spanish Ministry of Science and Innovation through the programs PID2020-116770RJ-I00 and CPP2021-008658. SRD is supported by a UKRI Future Leaders Fellowship (MR/T020733/1), Wellcome via core funding of the Wellcome Sanger Institute [220540/Z/20/A], and a grant (101103089). For the purpose of Open Access, the authors have applied a CC BY public copyright Licence to any Author Accepted Manuscript version arising from this submission.

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