Resistance to benzimidazole in South India: Polymorphic variants in the β-tubulin gene of Ascaris lumbricoides and Necator americanus

Objective: Benzimidazole resistance in helminths is linked to single nucleotide polymorphisms (SNPs) at codons 167, 198, and 200. We studied the prevalence of these SNPs in the β-tubulin gene of Ascaris (A.) lumbricoides and Necator (N.) americanus so as to explore the resistance to benzimidazoles in South India. Methods: DNA extracts of stool samples positive for A. lumbricoides and N. americanus were used for identification of SNPs. We utilized 22 samples to examine codon 167 SNPs and 15 samples for codon 198 and 200 SNPs in N. americanus . Three samples were used for SNP analysis of A. lumbricoides . Two conventional PCR assays were standardised for N. americanus (one targeting SNPs at coden 167 and another for 198 and 200), covering three SNPs, while a single PCR was developed for A. lumbricoides . The PCR amplified products were sequenced and compared with published sequences in NCBI for SNP identification. Results: A total of 650 stool samples were collected from pregnant women and screened for the presence of A. lumbricoides and N. americanus , out of which 22 positive samples were used to examine codon 167 SNPs and 15 samples for codon 198 and 200 SNPs in N. americanus , and three samples were used for SNP analysis of A. lumbricoides . Despite thorough analysis, no reported SNPs were identified in A. lumbricoides and N. americanus samples. These findings suggest a potential lack of diversity in the β-tubulin gene within the examined populations. Conclusions: The findings suggest that the parasite species circulating in the South Indian region are susceptible to benzimidazoles. To the best of our knowledge, this is the first attempt in India to evaluate the SNPs in two of the STH parasites using cost-effective conventional PCR and Sanger sequencing. Although biannual deworming has been implemented in India since 2015, no resistance to benzimidazoles has been observed. This method is suitable even in resource-limited settings. However, due to the limited sample size, future research with a larger sample size is essential to confirm these results and provide a more comprehensive understanding of the susceptibility of these parasites to benzimidazoles in the broader population.