Absence of β-tubulin SNPs Associated with Drug Resistance in Ascaris lumbricoides Infections of School-Age Children in Bungoma County, Kenya

Abstract

Aims: This study compared the prevalence of Ascaris lumbricoides infections pre- and post-deworming in school-age children in Bungoma County, western Kenya, to detect β-tubulin gene single nucleotide polymorphisms (SNPs) associated with drug resistance in these infections. Study Design: A longitudinal study design was adopted. Place and Duration of Study: The research was conducted in Bungoma County, western Kenya. Stool samples were collected one month before (October 2021) and two weeks after the mass drug administration (MDA) deworming exercise (January 2022). Methodology: 414 school-aged children were recruited for the study. Two hundred and two stool samples were collected pre-MDA while 212 were obtained post-MDA. Prevalence rates and infection intensities were determined using the Kato-Katz technique at both time points. Genomic DNA was extracted from selected pre- and all post-deworming positive A. lumbricoides eggs. Standard PCR generated a 564bp amplicon that surrounded the target codons 167, 198, and 200 of the A. lumbricoides β-tubulin gene which have been associated with the drug-resistant phenotype. Sixteen amplicons were sequenced using Sanger sequencing to detect the presence of SNPs at these loci. Results: The overall pre-MDA and post-MDA STH prevalence rates were 33% and 6%, respectively, whereas the prevalence of A. lumbricoides infections alone was 31% and 4%, respectively. A. lumbricoides was the dominant infecting helminth species. However, a few samples (<5)  had hookworms and T. trichiura. Sixteen sequences were generated covering codons 167, 198, and 200 of the β-tubulin gene of A. lumbricoides. Sequencing of the DNA samples revealed that no β-tubulin SNPs were present in the A. lumbricoides infections. Conclusion: The persistence of some A. lumbricoides infections following MDA using mebendazole suggests the drug-resistant phenotype, but the possibility of less than 100% MDA coverage in schools could be an alternative explanation. However, it is noteworthy that even after over ten years of use of benzimidazole drugs in the Kenya school-based deworming program, there is no evidence of drug resistance SNPs. This study therefore supports the continued use of the current anti-helmintic  drugs in this setting. We recommend continued use of molecular tools for regular surveillance of drug-resistant parasites as this is essential to ensure the effectiveness of the deworming program.