Dataset Information

Rapid Genotyping of ?-tubulin Polymorphisms in Trichuris trichiura and Ascaris lumbricoides.

ABSTRACT:

Background

The benzimidazole (BZ) anthelmintics, albendazole (ABZ) and mebendazole (MBZ) are the most common drugs used for treatment of soil-transmitted helminths (STHs). Their intensive use increases the possibility that BZ resistance may develop. In veterinary nematodes, BZ resistance is caused by a single nucleotide polymorphism (SNP) in the ?-tubulin isotype 1 gene at codon position 200, 167 or 198, and these SNPs have also been correlated with poor response of human Trichuris trichiura to BZ treatment. It is important to be able to investigate the presence of resistance-associated SNPs in STHs before resistance becomes clinically established.

Methods

The objective of this study was to develop new genotyping assays to screen for the presence of ?-tubulin SNPs in T. trichiura and Ascaris lumbricoides. Rapid, simple and accurate genotyping assays were developed based on the SmartAmp2 method. Primer sets were optimized and selected to distinguish the SNP-variant genotypes. After initial optimization on control plasmids, the feasibility of the assay was assessed in field samples from Haiti and Panama. Finally, spiked fecal samples were assessed to determine the tolerance of Aac polymerase to fecal inhibitors.

Findings

Rapid SNP genotyping assays were developed to target ?-tubulin polymorphisms in T. trichiura and A. lumbricoides. The assays showed high sensitivity and specificity in field samples and also demonstrated high tolerance to PCR inhibitors in fecal samples.

Conclusion

These assays proved to be robust and efficient with the potential to be used as field tools for monitoring SNPs that could be associated with BZ resistance. However, further work is needed to validate the assays on large numbers of field samples before and after treatment.

SUBMITTER: Rashwan N

PROVIDER: S-EPMC5230752 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Publications

Rapid Genotyping of β-tubulin Polymorphisms in Trichuris trichiura and Ascaris lumbricoides.

Rashwan Nour N Scott Marilyn M Prichard Roger R

PLoS neglected tropical diseases 20170112 1

<h4>Background</h4>The benzimidazole (BZ) anthelmintics, albendazole (ABZ) and mebendazole (MBZ) are the most common drugs used for treatment of soil-transmitted helminths (STHs). Their intensive use increases the possibility that BZ resistance may develop. In veterinary nematodes, BZ resistance is caused by a single nucleotide polymorphism (SNP) in the β-tubulin isotype 1 gene at codon position 200, 167 or 198, and these SNPs have also been correlated with poor response of human Trichuris trich ...[more]

PMID: 28081124

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Project description:BACKGROUND: The soil-transmitted helminths (STH) Ascaris lumbricoides and Trichuris trichiura are gastrointestinal parasites causing many disabilities to humans, particularly children. The benzimidazole (BZ) drugs, albendazole (ALB) and mebendazole (MBZ), are commonly used for mass treatment for STH. Unfortunately, there is concern that increased use of anthelmintics could select for resistant populations of these human parasites. In veterinary parasites, and lately in filarial nematodes, a single amino acid substitution from phenylalanine to tyrosine, known to be associated with benzimidazole resistance, has been found in parasite beta-tubulin at position 200. We have developed pyrosequencer assays for codon 200 (TTC or TAC) in A. lumbricoides and T. trichiura to screen for this single nucleotide polymorphism (SNP). METHOD AND FINDINGS: Pyrosequencing assays were developed and evaluated for detecting the TTC or TAC SNP at codon 200 in beta-tubulin in A. lumbricoides and T. trichiura. Genomic DNA from individual worms, eggs isolated from individual adult worms or from fecal samples with known treatment history and origin, were sequenced at beta-tubulin by pyrosequencing, and genotypes were confirmed by conventional sequencing. The assays were applied to adult worms from a benzimidazole-naïve population in Kenya. Following this, these assays were applied to individual worms and pooled eggs from people in East Africa (Uganda and Zanzibar) and Central America (Panama) where mass anthelmintic drug programs had been implemented. All A. lumbricoides samples were TTC. However, we found 0.4% homozygous TAC/TAC in T. trichiura worms from non-treated people in Kenya, and 63% of T. trichiura egg pools from treated people in Panama contained only TAC. CONCLUSION: Although the codon 200 TAC SNP was not found in any of the A. lumbricoides samples analyzed, a rapid genotyping assay has been developed that can be used to examine larger populations of this parasite and to monitor for possible benzimidazole resistance development. The TAC SNP at codon 200, associated with benzimidazole resistance in other nematodes, does occur in T. trichiura, and a rapid assay has been developed to allow populations of this parasite to be monitored for the frequency of this SNP. Sample sizes were small, anthelmintic efficacy was not assessed, and treated and non-treated samples were from different locations, so these frequencies cannot be extrapolated to other populations of T. trichiura or to a conclusion about resistance to treatment. The occurrence of the TAC SNP at codon 200 of beta-tubulin in T. trichiura may explain why benzimidazole anthelmintics are not always highly effective against this species of STH. These assays will be useful in assessing appropriate treatment in areas of high T. trichiura prevalence and in monitoring for possible resistance development in these STH.