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

Abstract

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.

Keywords

β-tubulin gene / Benzimidazole / Anti-helminthic resistance

Conflict of interest statement

The authors claim there is no conflict of interest.

Funding

The study was supported by the intramural grant provided by Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India. Grant number: JIP/ Res/ Intramural/ phs2/ 2020-21, dated 23/12/2020.

Data availability statement

The data supporting the findings of this study are available from the corresponding author upon request.

Authors’ contributions

Ulaganeethi R, Rajkumari N, Chinnakali P, Saya GK were involved in concept and design of the study. Ulaganeethi R had done the data acquisition, analysis and interpretation of data and drafted the first version of the paper. Jasmine P helped in data acquisition and interpretation of results. Rajkumari N, Chinnakali P, Dorairajan G and Saya GK reviewed the paper critically and all the authors gave their consent to the version submitted for publication.

References

[1] Chopra P, Shekhar S, Dagar VK, Pandey S. Prevalence and risk factors of soil-transmitted helminthic infections in the pediatric population in India: A systematic review and meta-analysis. J Lab Physicians 2023; 15(1): 4-19.

[2] Rahimi BA, Rafiqi N, Tareen Z, Kakar KA, Wafa MH, Stanikzai MH, et al. Prevalence of soil-transmitted helminths and associated risk factors among primary school children in Kandahar, Afghanistan: A cross-sectional analytical study. PLoS Negl Trop Dis 2023; 17(9): e0011614.

[3] Montresor A, Mupfasoni D, Mikhailov A, Mwinzi P, Lucianez A, Jamsheed M, et al. The global progress of soil-transmitted helminthiases control in 2020 and World Health Organization targets for 2030. PLoS Negl Trop Dis 2020; 14(8): e0008505.

[4] Chai JY, Jung BK, Hong SJ. Albendazole and mebendazole as anti-parasitic and anti-cancer agents: An update. Korean J Parasitol 2021; 59(3): 189-225.

[5] Werkman M, Wright JE, Truscott JE, Oswald WE, Halliday KE, Papaiakovou M, et al. The impact of community-wide, mass drug administration on aggregation of soil-transmitted helminth infection in human host populations. Parasit Vectors 2020; 13(1): 290.

[6] Diawara A, Drake LJ, Suswillo RR, Kihara J, Bundy DAP, Scott ME, et al. Assays to detect beta-tubulin codon 200 polymorphism in Trichuris trichiura and Ascaris lumbricoides. PLoS Negl Trop Dis 2009; 3(3): e397.

[7] Diawara A, Halpenny CM, Churcher TS, Mwandawiro C, Kihara J, Kaplan RM, et al. Association between response to albendazole treatment and β-tubulin genotype frequencies in soil-transmitted helminths. PLoS Negl Trop Dis 2013; 7(5): e2247.

[8] Rashwan N, Bourguinat C, Keller K, Gunawardena NK, de Silva N, Prichard R. Isothermal diagnostic assays for monitoring single nucleotide polymorphisms in Necator americanus associated with benzimidazole drug resistance. PLoS Negl Trop Dis 2016; 10(12): e0005113.

[9] Zuccherato LW, Furtado LF, Medeiros C da S, Pinheiro C da S, Rabelo ÉM. PCR-RFLP screening of polymorphisms associated with benzimidazole resistance in Necator americanus and Ascaris lumbricoides from different geographical regions in Brazil. PLoS Negl Trop Dis 2018; 12(9): e0006766.

[10] Grau-Pujol B, Gandasegui J, Escola V, Marti-Soler H, Cambra-Pellejà M, Demontis M, et al. Single-nucleotide polymorphisms in the beta-tubulin gene and its relationship with treatment response to albendazole in human soil-transmitted helminths in Southern Mozambique. Am J Trop Med Hyg 2022; 107(3): 649-657.

[11] Orr AR, Quagraine JE, Suwondo P, George S, Harrison LM, Dornas FP, et al. Genetic markers of benzimidazole resistance among human hookworms (Necator americanus) in Kintampo North Municipality, Ghana. Am J Trop Med Hyg 2019; 100(2): 351-356.

[12] Ulaganeethi R, Shettikothanuru Ramachandrappa VK, Rajkumari N, Dorairajan G, Saya GK. Performance of microscopy compared to conventional PCR in identification of soil-transmitted helminth infections among antenatal women in a low-prevalence setting. Indian J Med Microbiol 2023; 46: 100427.

[13] Sarai RS. Ho MF, McCarthy JS. Necator americanus beta-tubulin isotype I gene, partial cds - Nucleotide - NCBI. [Online] Available from: https://www.ncbi.nlm.nih.gov/nuccore/EU182348.1?report=fasta. [Accessed on 17 April 2024].

[14] Albonico M. W and B. Necator americanus beta tubulin mRNA, partial cds - Nucleotide - NCBI. [Online] Available from: https://www.ncbi.nlm.nih.gov/nuccore/AF453524.1?report=fasta, [Accessed on 17 April 2024].

[15] Diawara A. Drake LJ, Suswillo RR, Kihara J, Bundy DA, Scott ME, et al. Ascaris lumbricoides beta tubulin gene, partial cds - Nucleotide - NCBI. [Online] Available from: https://www.ncbi.nlm.nih.gov/nuccore/FJ501301.1. [Accessed on 17 April 2024].

[16] Shao Z, Zhao H, Zhao H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 2009; 37(2): e16.

[17] Alzohairy, Ahmed Mansour. BioEdit: An important software for molecular biology. GERF Bull Biosci 2011; 2(1): 60-61.

[18] Roose S, Avramenko RW, Pollo SMJ, Wasmuth JD, Ame S, Ayana M, et al. Characterization of the β-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance. PLoS Negl Trop Dis 2021; 15(9): e0009777.

[19] Furtado LFV, Medeiros C da S, Zuccherato LW, Alves WP, de Oliveira VNGM, da Silva VJ, et al. First identification of the benzimidazole resistance-associated F200Y SNP in the beta-tubulin gene in Ascaris lumbricoides. PLoS One 2019; 14(10): e0224108.

[20] Furtado LFV, Alves WP, Moreira TB, Costa Junior LM, Miranda RRC, Rabelo ÉML. Standardization and application of the tetraprimer ARMS-PCR technique for screening of the E198A SNP in the β-tubulin gene of hookworm populations in Brazil. Vet Parasitol 2016; 224: 65-67.

[21] Gill BS. Anthelmintic resistance in India. Vet Parasitol 1996; 63(1): 173-176.

[22] Rajagopal A, Sabu L, Radhika R, Devada K, Aravindakshan T. High prevalence of the E198A polymorphism in isotype-1 β-tubulin gene of Haemonchus contortus populations in organized goat farms in Kerala, South India. Small Rumin Res 2020; 188: 106110.