Benzimidazole Resistance-Associated Mutations in the β-tubulin Gene of Hookworms: A Systematic Review

doi:10.21203/rs.3.rs-4922397/v1

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Benzimidazole Resistance-Associated Mutations in the β-tubulin Gene of Hookworms: A Systematic Review

https://doi.org/10.21203/rs.3.rs-4922397/v1

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There is a growing number of reports on the occurrence of benzimidazole resistance-associated single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various helminths of veterinary and public health concerns. However, a comprehensive analysis of their occurrence and their contributions to conferring benzimidazole resistance among hookworms has yet to be done. The objectives of this systematic review are to summarize and synthesize peer-reviewed evidence on the occurrence of these resistance-associated mutations in hookworms, document their geographical distribution, and assess their contributions to conferring phenotypic resistance. Three databases were systematically searched using specific keywords. Research that assessed the occurrence of benzimidazole resistance-associated SNPs in hookworms, papers that reported the geographical distribution of these SNPs, and studies that investigated the SNPs’ resistance-associated phenotypic effects were included in the review. Research that was not done in hookworms, papers not in the English language, and literature reviews and book chapters were excluded. Critical appraisal checklists were used to determine the risk of bias in the selected papers. Data were extracted from the selected studies and analyzed. PROSPERO Systematic Review Protocol Registration No.: CRD42024510924. A total of 29 studies were included and analyzed. Of these, four were conducted in a laboratory setting, eight described development and validation of SNP detection methods, and the remaining 17 involved field research. Seven SNP-induced amino acid substitutions at four loci were reported among several hookworm species: Q134H, F167Y, E198A, E198K, E198V, F200Y, and F200L. SNPs have been reported in isolates occurring in the United States, Canada, Brazil, Haiti, Australia, New Zealand, Kenya, Ghana, Mozambique, and Tanzania. No reports were found in Asia. E198A and F200L were reported in Ancylostoma ceylanicum with laboratory-induced resistance. F167Y and Q134H conferred resistance in A. caninum, as revealed by in vitro investigations and field assessments. There is insufficient peer-reviewed evidence to prove the association between SNP occurrence and resistance. Mutations in the β-tubulin isotype 1 gene confer benzimidazole resistance in Ancylostoma caninum and A. ceylanicum, but similar evidence is lacking for human hookworms. Understanding benzimidazole resistance through further research can better inform treatment, prevention and control strategies.

Ancylostoma

Drug Resistance

Necator

One Health

Hookworm infections in humans and animals remain a significant neglected tropical disease globally (Traub et al. 2021; Clements and Alene 2022). Necator amercanus and Ancylostoma duidenale are the most known causes of human infections in many endemic areas (Loukas et al. 2016). Ancylostoma ceylanicum is a zoonotic hookworm that is prevalent in the Asia-Pacific region (Colella et al. 2021a). Ancylostoma caninum is the most widely distributed hookworm among canines (Hawdon and Wise 2021). Other hookworm species that are known to cause significant infections in animals and potential zoonotic infections in humans include A. braziliense, A. tubaeforme and Uncinaria stenocephala (Hawdon and Wise 2021). Hookworm infections in humans are reported to have 32–79% prevalence globally and cause 800,000 to 4 million disability-adjusted life years lost (James et al. 2018; Stracke et al. 2020; Clements and Alene 2022). Meanwhile, A. caninum and A. ceylanicum are estimated to affect 23% and 24% of dogs in the Asian continent (Zibaei et al. 2020). Their wide distribution and significant infection pathology both in humans and animals have prompted the development of various control and elimination efforts in the public and veterinary health fronts (World Health Organization 2011; Dantas-Torres et al. 2020). Of which, the cornerstone has been pharmacological interventions aimed at reducing infection burden and prevent reinfections. This is achieved through preventive chemotherapy or Mass Drug Administration strategy in humans and routine veterinary deworming in animals using benzimidazole anthelmintics, like albendazole, mebendazole and fenbendazole (World Health Organization 2017; Dantas-Torres et al. 2020).

Pharmacological interventions that underpin helminth infection control programs and the recent reports of lowered drug efficacy has raised concerns regarding the emergence of resistance among soil-transmitted helminths (STH), including hookworms (Freeman et al. 2019; Tinkler 2020; Colella et al. 2021b). This concern is rooted in what had happened in livestock nematodes that had developed benzimidazole resistance due to repeated drug administration over protracted periods (Von Samson-Himmelstjerna et al. 2007; Dilks et al. 2020; O’Halloran 2021). Among veterinary helminths, benzimidazole resistance is conferred by single nucleotide polymorphisms (SNPs) that occur in the β-tubulin isotype 1 gene (Furtado et al. 2016b). Specifically, canonical SNPs include F167Y TTC, TTT/Phenylalanine/PHE ◊ TAC, TAT/Tyrosine/TYR), E198A (GAG, GAA/Glutamic acid/GLU ◊ GCG, GCA/Alanine/ALA), F200Y (TTC, TTT/Phenylalanine/PHE ◊ TAC, TAT/Tyrosine/TYR) (Von Samson-Himmelstjerna et al. 2007). These mutations alter the amino acid and structural constitution of the β-tubulin protein which in turn adversely affects the binding efficacy of the benzimidazole drug (Lacey and Gill 1994; Furtado et al. 2016b). The occurrence and potential widespread distribution of these SNPs in helminths of public and veterinary health concern could undermine current control, elimination, and prevention efforts which can result in persistent infections with dire pathological consequences to human and animal health.

Reports of the occurrence of benzimidazole resistance-associated mutations among different hookworm species have been slowly growing since the advent of molecular techniques that enable the assessment of parasite genes. Hence, this systematic review aims to summarize and synthesize peer-reviewed evidence of their occurrence in multiple hookworm species, document their geographical distribution, and assess their contributions to conferring resistance through evaluating available phenotypic evidence. Based on current available evidence, we provide proof that SNPs in the β-tubulin isotype 1 gene confer phenotypic benzimidazole resistance in several hookworm species but not all. Moreover, several research gaps need further research attention, including ascertaining the precise mechanism underlying benzimidazole resistance in human hookworms.

Research Questions

The current systematic review was conducted according to the recommendations of the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2020 guidelines (Page et al. 2021). The systematic review protocol was submitted for registration in PROSPERO (https://www.crd.york.ac.uk/prospero/) (ID No. CRD42024510924). This review aimed to answer three main research questions:

1. What are the SNPs associated with benzimidazole resistance that have been reported among hookworms in the peer-reviewed literature?

2. What is the geographic distribution of these SNPs?

3. What are the resistance-related phenotypic effects of these reported SNPs?

Search Strategy

Literature searches were done in MEDLINE via PubMed, Scopus, and Science Direct [Accessed on 8 to 9 February 2023]. The search terms used were as follows: “Hookworm AND Benzimidazole Resistance AND SNPs”; “Ancylostoma AND Benzimidazole Resistance AND SNPs”; “Necator AND Benzimidazole Resistance AND SNPs”; “Ancylostoma duodenale AND Benzimidazole Resistance AND SNPs”; “Ancylostoma caninum AND Benzimidazole Resistance AND SNPs”; “Ancylostoma ceylanicum AND Benzimidazole Resistance AND SNPs”; “Ancylostoma braziliense AND Benzimidazole Resistance AND SNPs”; “Ancylostoma tubaeforme AND Benzimidazole Resistance AND SNPs”; “Necator americanus AND Benzimidazole Resistance AND SNPs”; and “Uncinaria stenocephala AND Benzimidazole Resistance AND SNPs.” No publication date restriction was used. The results of each search were obtained and recorded by exporting the RIS (Research Information Systems) file from the databases.

Study Selection, Screening, and Eligibility

The RIS files were imported to Mendeley Citation Manager (www.mendeley.com) [Accessed 9 to 11 February 2024]. The first author did the duplicate removal, screening, and selection of imported records using the same platform. The record’s title and abstract were utilized for the initial screening process. Records that did not have research titles and abstracts were removed (e.g., indexes). The full texts of the records were retrieved for the subsequent analysis. The inclusion criteria used were as follows: 1.) research that assessed the occurrence of SNPs associated with benzimidazole resistance in hookworms regardless of study design; and/or 2.) research that reported the geographical distribution of these SNPs; and/or 3.) research that reported the resistance-related phenotypic effects of these SNPs. From the included studies, 1.) research that were not done in hookworms, 2.) papers not in the English language, and 3.) literature reviews and book chapters were excluded. The study selection, screening, and eligibility assessment were reviewed by the second and third authors. Disputes were resolved through consensus by all authors. The screening process is depicted in Figure 1.

Risk of Bias Assessment

Studies that passed the eligibility criteria were of three main types: papers that reported the development and validation of SNP detection methods, laboratory studies that assessed resistance in resistant isolates from the field or laboratory-induced resistant hookworms, and field reports that account the occurrence of the SNPs in naturally infected populations, both in humans and animals. Due to this diversity in study types, three bias assessment tools were used. Field studies were assessed using the JBI Critical Appraisal Checklist for Analytical Cross-Sectional Studies (Supplementary Figure 1), while studies reporting detection methods were evaluated via the JBI Critical Appraisal Checklist for Diagnostic Test Accuracy Studies

(Supplementary Figure 2). For laboratory studies, the JBI Critical Appraisal Checklist for Quasi-Experimental Studies was used (Supplementary Figure 3). The risk of bias assessment was reviewed by the second and the third authors. Disputes regarding dispositions were arbitered by the fourth and fifth author.

Data Extraction and Evidence Synthesis

Eligible studies were given study codes (Supplementary Table 1). The authors’ names, title of the paper, abstract, year of publication, hookworm species studied, study’s geographical location, SNPs found and their corresponding measure (e.g., SNP prevalence or allelic frequency), method of SNP detection, history of benzimidazole administration, and reported phenotypic effects were extracted and recorded in a spreadsheet. These information were summarized and tabularized. Descriptive statistics (i.e., counts and percentages) were used to describe, summarize, and synthesize the pertinent results of the studies. Moreover, QGIS version 3.28 (https://qgis.org/en/site/) was utilized to construct maps that depict the geographical distribution of the SNPs.

Characteristics of the included studies

The systematic search was able to record 379 reports from three different databases (Fig. 1). 92.3% (350/379) of these were removed due to duplication, lack of titles and abstract, and excluded due to ineligibility. Of the 29 studies considered eligible, 13.8% were laboratory experiments that assessed resistance in resistant hookworms from the field and laboratory-induced isolates (Table 1). 27.6% were studies that reported the development and standardization of SNP detection methods (Table 2), and the remaining 58.6% were field studies aimed at assessing the occurrence of SNPs in various endemic loci (Table 3). The studies included in this systematic review were from 12 countries (Fig. 2). Of these, the most represented were the United States with 11 studies (37.9%) and Brazil with 9 studies (31.0%). Most of the studies in the former were done in A. caninum, while A. ceylanicum, A. caninum, A. braziliense, and N. americanus were among the species assessed in the latter. All 29 studies were deemed to have an acceptable level of bias risk (Supplementary Figs. 1, 2 and 3).

SNPs Associated with Benzimidazole Resistance in Hookworms

Seven SNP variations that occur in four amino acid locations were reported in the studies included (Tables 1, 2 and 3). In position 134, Glutamine is substituted with Histidine (CAA /Q/GLU ◊ CAT/H/HIS). Phenylalanine at position 167 was replaced with Tyrosine (TTC, TTT/F/PHE ◊ TAC, TAT/ Y/TYR). Three allelic variations were reported at position 198: E198A (GAG, GAA/Glutamic acid/GLU ◊ GCG, GCA/Alanine/ALA), E198K (GAG, GAA/Glutamic acid/GLU ◊ AAG/AAA/Lysine/LYS), and E198V (GAG, GAA/Glutamic acid/GLU ◊ GTA,GTG/Valine/VAL). Similarly, there are also two reported variants for Phenylalanine at position 200, where it may be replaced by Tyrosine (TTC, TTT/F/PHE ◊ TAC, TAT/Tyrosine/Y/TYR) or Leucine (TTC, TTT/F/PHE ◊ TTA/L/LEU). All the canonical benzimidazole resistance-associated mutations—F167Y, E198A, and F200Y—have been reported in N. americanus. Numerous papers have noted that F167Y is prevalent among A. caninum. E198A occurred in A. braziliense, A. caninum and N. americanus isolates from Brazil and N. americanus from Ghana. Recent studies using novel techniques, like deep amplicon sequencing, have uncovered various SNP variations at position 198: E198K in A. caninum and E198V in U. stenocephala. Sequencing of laboratory-induced albendazole-resistant A. ceylanicum revealed the emergence of E198A and F200L mutations. No mutations have been reported in A. tubaeforme.

The occurrence of SNPs was determined and reported in varying manners. The detection techniques employed included ARMS-PCR, PCR-RFLP, real-time PCR, pyrosequencing, Sanger sequencing, and next generation sequencing. This variety in techniques resulted in different measures of SNP occurrence. For instance, F200Y SNP prevalence could be as low as 0.8% among A. caninum from Brazil (through ARMS-PCR) up to 2.3% of hookworm samples assessed in Kenya (through pyrosequencing) (Diawara et al. 2013a; Furtado et al. 2014). Through real-time PCR and Sanger sequencing, differences in the occurrence of these SNPs from pre- to post-treatment assessments were also noted in Ghana (Orr et al. 2019). SNP occurrence was also measured using allele frequencies, especially by studies that utilized deep amplicon sequencing technology. Susceptible and resistant A. caninum isolates from the United States exhibited allelic frequencies that were significantly increased in the latter (1.2 to 8.8% vs. 12.7 to 99.7%) (Jimenez Castro et al. 2019). Also, deep amplicon sequencing of N. americanus samples from Ghana showed 3.15% and 3.13% allelic frequencies for E198A and F200Y, respectively (George et al. 2022). These results show that varying approaches in assessing the occurrence of resistance-associated SNPs entail varying methods of measuring their emergence. These differences in results may impair the epidemiological assessment of SNP occurrence (i.e., reporting of SNP prevalence vs. allelic frequency). However, high throughput techniques, like deep amplicon sequencing, provide a more sensitive SNP detection method through simultaneous sequencing of pooled samples.

Table 1

Laboratory studies that assessed Benzimidazole-resistance associated SNPs in resistant hookworm isolates from the field and laboratory-induced resistant isolates.
Year Published	Author	Country	Hookworm Species	SNPs reported	Method of detection	History of drug administration	Phenotypic effects
2019	Furtado et al. (2019)	Brazil	Ancylostoma ceylanicum	No SNPs found	RFLP-PCR	Albendazole	Benzimidazole resistance was induced but no SNPs were found among resistant hookworms.
2019	Kitchen et al. (2019)	United States	Ancylostoma caninum	F167Y	Sanger sequencing	Fenbendazole	The multidrug-resistant isolate had increased Thiabendazole IC₅₀ and IC₉₀. F167Y was gene edited into C. elegans and conferred similar resistance profiles.
2022	Medeiros et al. (2022)	Brazil	Ancylostoma ceylanicum	E198A: Gradual increase in proportion as the dose increased. F200L: 12% of resistant hookworms	Sanger sequencing	Albendazole	Benzimidazole resistance was induced. E198A and F200L were found among resistant hookworms
2024	McKean et al. ( 2024)	United States	Ancylostoma caninum	Q134H F167Y	Deep Amplicon Sequencing	Thiabendazole	The resistant isolate had increased IC₅₀ and RR compared to previously described resistant isolates.
SNPs = Single nucleotide polymorphism RFLP-PCR = Restriction Fragment Length Polymorphism- Polymerase Chain Reaction RR = Resistance Ratio IC₅₀ = Inhibitory Concentration 50% IC₉₅ = Inhibitory Concentration 95%

Table 2

Studies reporting the development and standardization of detection methods for resistance-associated SNPs.
Year Published	Author and reference	Country	Hookworm Species	SNPs Reported	Method of detection	History of drug administration	Phenotypic effects
2007	Schwenkenbecher et al. (2007)	United States	Ancylostoma caninum	F167Y: 0.24% allelic frequency F200Y: 0.05% allelic frequency	Real-time PCR	Mebendazole	Reduced egg reduction rates
2009	Schwenkenbecher and Kaplan (2009)	United States	Ancylostoma caninum	No SNPs found	Real-time PCR	50% of samples were treated with Fenbendazole	No report of effects
2013	Diawara et al. (2013b)	Tanzania and Haiti	Necator americanus	F200: Tanzania – 10% allelic frequency Haiti – 36% allelic frequency	Pyrosequencing	Tanzania – Mebendazole Haiti - Albendazole	No report of effects
2015	Furtado and Rabelo (2015)	Brazil	Ancylostoma caninum	No SNPs found	ARMS-PCR	No treatment history reported	No report of effects
2016	Furtado et al. (2016)	Brazil	Ancylostoma caninum	No SNPs found	ARMS PCR	No treatment history reported	No report of effects
2016	Rashwan et al. (2016)	Sri Lanka	Necator americanus	No SNPs found	Smart amplification method	No treatment history reported	No report of effects
2018	Furtado et al. (2018)	Brazil	Ancylostoma braziliense	E198A: 1.2% SNP prevalence among the samples	RFLP-PCR	No treatment history reported	No report of effects
2021	Gandasegui et al. (2021)	Mozambique	Necator americanus	F167Y: 2.1–6.85% allelic frequency E198A: 1.05–17% allelic frequency F200Y: 1.15–8.3% allelic frequency	Pyrosequencing	Albendazole	No reports of effects
SNPs = Single nucleotide polymorphism ARMS-PCR = Amplification Refractory Mutation System-Polymerase Chain Reaction RFLP-PCR = Restriction Fragment Length Polymorphism- Polymerase Chain Reaction

Table 3

Field studies uncovered by the systematic review that assessed and reported benzimidazole resistance-associated SNPs in different hookworm species.
Year Published	Author and reference	Country	Hookworm Species	SNPs Reported	Method of detection	History of drug administration	Phenotypic effects
2004	Albonico et al. (2004)	Tanzania	Necator amerincanus, Ancylostoma duodenale	No SNPs found	Sanger sequencing	Mebendazole	No report of effects
2013	Diawara et al. (2013a)	Haiti, Kenya, Panama	No species identified	Kenya: F167Y – 2.3% SNP prevalence E198A – No SNPs found F200Y – No SNPs found Haiti: No SNPs found Panama: No SNPs found	Pyrosequencing	Albendazole	Kenya: 89.9% ERR using McMaster and 96.8% using Kato-Katz Haiti: 98.6% ERR using McMaster Panama: 99.8% ERRusing Kato-Katz
2014	Furtado et al. (2014)	Brazil	Ancylostoma caninum	F200Y: 0.8% SNP prevalence E198A: No SNPs found	ARMS-PCR and Sanger sequencing	No treatment history reported	No report of effects
2015	Furtado and Rabelo (2015)	Brazil	Ancylostoma caninum	F167Y: No SNPs found	ARMS-PCR	No treatment history reported	No report of effects
2018	Zuccherato et al. (2018)	Brazil	Necator americanus	E198A: 1.4% SNP prevalence among the eggs assessed F200Y: 1.1% SNP prevalence among the eggs assessed	RFLP-PCR	No treatment history reported	No report of effects
2019	Orr et al. (2019)	Ghana	Necator americanus	F167Y: Pre MDA- 1.7% SNP prevalence Post MDA – 5% SNP prevalence F200Y: Pre MDA – 41.9% Post MDA – 52% E198A: 29% of F200Y mutants	Real-time PCR and Sanger sequencing	Albendazole	No report of effects
2019	Jimenez-Castro et al. (2019)	United States	Ancylostoma caninum	F167Y: Susceptible isolates − 1.2 to 8.8% SNP frequencies Resistant isolates − 12.7 to 99.7% SNP frequencies E198A: No SNPs found F200Y: No SNPs found	Deep Amplicon Sequencing	Fenbendazole	EHA revealed greater resistance ratios (IC₅₀ Resistant/IC₅₀ Susceptible ) among worms with the SNPs upon exposure to Fenbendazole
2020	Furtado et al. (2020)	Brazil	Necator americanus	No SNPs found	ARMS-PCR and Sanger sequencing	No treatment history reported	No report of effects
2021	Jimenez-Casrtro et al. (2021)	United States	Ancylostoma caninum	F167Y: 99% of the samples sequenced	Deep Amplicon Sequencing	Febantel-Pyrantel	EHA revealed increasing IC₅₀ and IC₉₅ in resistant isolates
2022	George et al. (2022)	Ghana	Necator americanus	F167Y: No SNPs found E198A: 3.15% allelic frequency of amplicons F200Y: 3.13% frequency of amplicons Other SNPs reported	Deep Amplicon Sequencing	Albendazole	The SNPs decreased the burden of infection: 700 ± 1026 to 126 ± 143 egg per gram.
2022	Grau-Pujol et al. (2022)	Mozambique	Necator americanus	F167Y: Pre MDA – 16.7% SNP prevalence Post MDA – No SNPs found E198A: Pre MDA – 10.9 to 21.4% Post MDA – 15.2 to 16.3% F200Y: Pre MDA – 10.4 to 49% Post MDA – 10.9 to 24.3%	Pyrosequencing	Albendazole	CR: Kato-katz − 82.5% qPCR − 53.8% ERR: Kato-katz − 30.5%
2023	Leutenegger et al. (2023)	United States	Ancylostoma caninum	F167Y: 0.02 to 10.6% SNP prevalence	Real-time PCR and Sanger Sequencing	No treatment history reported	No report of effects
2023	Venkatesan et al. (2023)	United States	Ancylostoma caninum	F167Y: 49.7% SNP prevalence Q134H: 31.1% SNP prevalence	Deep Amplicon Sequencing	No treatment history reported	The Q134H mutation was edited in C. elegans. Similar resistance level to F167Y was observed.
2023	Stocker et al. (2023)	Australia and New Zealand	Ancylostoma caninum and Uncinaria stenocephala	A. caninum: E198K – 0.36% allelic frequency U. stenocpehala: E198V – 2 .8% allelic frequency	Deep Amplicon Sequencing	No treatment history reported	No report of effects
2023	Evason et al. (2023)	United States and Canada	Ancylostoma caninum	F167Y: 0.05% of the samples had SNPs	Real-time PCR	No treatment history reported	No report of effects
2024	Leutenegger et al. (2024)	United States and Canada	Ancylostoma caninum	F167Y: United States – 11.3% SNP prevalence Canada – 3.6 to 7.7% SNP prevalence	Real-time PCR	No treatment history reported	No report of effects
2024	Stocker et al. (2024)	United States	Ancylostoma braziliense, Ancylostoma tubaeforme, Uncinaria stenocephala	No SNPs found	Deep Amplicon Sequencing	No treatment history reported	No report of effects
SNPs = Single nucleotide polymorphism ARMS-PCR = Amplification Refractory Mutation System-Polymerase Chain Reaction RFLP-PCR = Restriction Fragment Length Polymorphism- Polymerase Chain Reaction MDA = Mass Drug Administration EHA = Egg Hatch Assay IC₅₀ = Inhibitory Concentration 50% IC₉₅ = Inhibitory Concentration 95%

Geographical distribution of Benzimidazole resistance associated SNPs

Among the countries represented in this systematic review, SNPs were most frequently reported in the United States, where F167Y and Q134H were prevalent among A. caninum isolates in different parts of the country (Fig. 2A and Fig. 3A) (Jimenez Castro et al. 2021; Venkatesan et al. 2023). Similarly, F167 was also prevalent in N. americanus from African countries like Ghana and Mozambique (Orr et al. 2019; Grau-Pujol et al. 2022) (Fig. 3B). Meanwhile, F200Y was reported in Kenya and Tanzania in the same hookworm species (Diawara et al. 2013a, b). The same SNP has also been documented in A. caninum from Brazil, and N. americanus from Haiti (Diawara et al. 2013a; Furtado et al. 2014). The only study in the Asia-Pacific region that reported SNPs were from Australia and New Zealand, where novel mutations in position 198 were observed in A. caninum and U. stenocephala (Stocker et al. 2023). Interestingly, no research reports resistance-associated SNPs in Asia. One study developed an isothermal SNP detection technique that utilized samples from Sri Lanka but none of these had the resistance-associated mutations (Rashwan et al. 2016). These results indicate that there is no apparent geographic predilection in the occurrence of the SNPs associated with benzimidazole resistance.

Phenotypic Effects of SNPs

The link between benzimidazole resistance and the SNPs were evaluated and reported in numerous ways. In laboratory studies, induced resistance in A. ceylanicum was initially not linked with any SNPs (Furtado et al. 2019). However, utilization of a different sequencing approach revealed the association of E198A to resistance and the emergence of F200L (Medeiros et al. 2022). In this study, increasing concentrations of albendazole correlated well with the occurrence of E198A heterozygous and homozygote mutated hookworms. Among A. caninum, in vitro techniques, like Egg Hatch Assay (EHA) and Larva Development Assay (LDA), together with molecular methods have determined the pivotal role of F167Y and Q134H. In a study of multidrug resistant canine hookworms from the United States, EHA of these isolates revealed that the IC₅₀ were increased by several folds when compared susceptible controls and genotyping revealed the presence of F167Y in all isolates with resistant phenotypes (Jimenez Castro et al. 2019). A novel SNP-induced amino acid substitution that contribute to drug resistance has been reported in A. caninum from the same country. Q134H was caused markedly increased IC₉₅ against Fenbendazole (Venkatesan et al. 2023). Moreover, C. elegans that was genetically modified to contain the mutation resulted in enhanced viability against benzimidazole exposure (Kitchen et al. 2019; Venkatesan et al. 2023). In field studies of human hookworms, fecal ERR (egg reduction rates) were utilized in determining resistance. The results from most of the included studies, however, have not provided a clear causality between the occurrence of the SNPs and phenotypic benzimidazole resistance among human hookworms. For instance, all the canonical resistance-associated SNPs have been reported to be present in hookworms yet the ERR remained satisfactory post albendazole administration (Diawara et al. 2013a). In another report, reduced ERR in N. americanus was observed but the F167Y, E198A and F200L SNPs occurred at varying levels pre- and post-treatment (Grau-Pujol et al. 2022). Unfortunately, there are no laboratory studies among human hookworms that could provide further insight into benzimidazole resistance. The aforementioned evidence points out that benzimidazole resistance is caused by F167Y and Q134H in A. caninum. Laboratory results show the abundance of E198A and F200L in induced resistance in A. ceylanicum. However, this etiologic association of resistance has yet to be thoroughly examined in human hookworms, N. americanus and A. duodenale.

This systematic review reports that there are seven SNP-induced resistance associated mutations among different species of hookworms: Q134H, F167Y, E198A, E198K, E198V, F200Y and F200L. Both F167Y and Q134H are widespread among A. caninum in the United States. Similarly, SNPs have also been reported in Brazil in South America, Haiti in the Caribbeans, and Kenya, Ghana, Mozambique and Tanzania in the African continent. There are no reports of these mutations in Asia. Laboratory evidence shows that E198A and F200L is linked with induced resistance in A. ceylanicum. Field evidence and in vitro assays affirm the relationship of F167Y and Q134H in conferring resistance in A. caninum. The association between resistance and the occurrence of SNPs in human hookworms has yet to be sufficiently confirmed and thus needs further research. The results of this systematic review have several implications.

Laboratory experiments using Caenorhabditis elegans has confirmed that mutations occurring in β-tubulin genes (i.e., F167Y, E198A, E198V, E198L, and F200Y) has resulted in resistance against Albendazole and Fenbendazole (Dilks et al., 2020). However, this novel study found that some of these mutations may result in fitness and survivability issues. Particularly, the E198L mutation which showed lower competitive fitness which means that worms with this mutation may not survive will in the field (Dilks et al., 2020). This mutation has not been reported in hookworms. Moreover, another laboratory assessment supports the aforementioned results by showing that C. elegans β-tubulin isotype1 is target of resistance-associated mutations, but β-tubulin isotype 2 may also be susceptible to such changes (Pallotto et al., 2022). Therefore, there is necessary biological evidence to support that benzimidazole resistance is due to mutations in the β-tubulin genes in helminths.

There is sufficient in vivo and field evidence to support that benzimidazole resistance is conferred by SNPs in the β-tubulin isotype 1 gene of several but not all hookworm species. For instance, field and laboratory studies show that resistance among A. caninum in the United States is linked to F167Y and Q134H (Kitchen et al. 2019; Venkatesan et al. 2023). Likewise, the association of E198A and F200L with laboratory-induced resistance in A. ceylanicum has also been established (Medeiros et al. 2022). However, for this zoonotic hookworm, no study in the peer-reviewed literature has assessed the occurrence of resistance conferring mutations in field isolates. Linking specific SNPs to benzimidazole resistance in human hookworms has yet to be substantiated by sufficient research evidence. This may potentially be due to the fact that no reports of benzimidazole resistance have been confirmed among human STH. Reports of reduced post-MDA egg reduction rates has been published but proof of its association with specific SNPs remains elusive (Diawara et al. 2013a; Orr et al. 2019; Grau-Pujol et al. 2022). There could be several methods that can be used to determine the precise mechanism of benzimidazole resistance in human hookworms. Laboratory studies that induce benzimidazole resistance in infected animal models may be utilized, as was the case for A. ceylanicum (Furtado et al. 2019; Medeiros et al. 2022). Subsequent analysis on resistant isolates can be done in the genome level (Doyle et al. 2022) or in the β-tubulin gene level through high throughput sequencing. Similarly, in vitro analysis through recombinant expression of wild-type and mutated β-tubulins, and their subsequent functional analysis can also provide insights into the molecular underpinnings of resistance (Liu et al. 2018). In silico modelling through docking studies with mutated β-tubulins and benzimidazole drug ligands may also provide illuminating insights into this research gap (Jones et al. 2022). Indeed, guidelines regarding confirming resistance in human STH are currently needed. While recommendations in assessing drug efficacy is available (World Health Organization 2013), precise criteria and procedures in ascertaining resistance has yet to be promulgated unlike in veterinary nematodes (Geurden et al. 2022; Beugnet et al. 2022; Kaplan et al. 2023).

Benzimidazole resistance presents a constraint in the control, elimination and prevention of hookworm infections in humans and companion animals. Resistance subverts pharmacological interventions that underpin most efforts to address infections, such as Mass Drug Administration using benzimidazole drugs in STH endemic areas (World Health Organization 2017). Similarly, it also hampers the veterinary deworming efforts that is aimed to abate infections and prevent reinfection in dogs and cats (Dantas-Torres et al. 2020). The emergence of four more SNPs, with the three canonical ones, in the β-tubulin isotype 1 gene of hookworms should cause some concerns both in public and veterinary health. The One Health implications of drug resistance among STH should also be kept in mind (Tinkler 2020; Ng’etich et al. 2023). The recent reports of widespread occurrence of these mutations in nation-wide studies, such as those conducted in the United States, should cause some alarm as it may relate to widespread occurrence of benzimidazole resistance. The lack of research in Asia is surprising since approximately a third of STH infections reside in this foci and most countries in this region currently have or have had MDA-based STH control programs (World Health Organization 2008, 2023; Mationg et al. 2021). This research gap should not preempt the notion that SNPs related to resistance are not present in this area. Resistance assessment needs attention in the region since reduced efficacy of benzimidazole drugs against hookworms from Cambodia has been recorded recently (Colella et al. 2021b). Further research that explores the molecular epidemiology of benzimidazole resistance mutations is currently needed to address this issue.

This systematic review has some limitations. The literature search was only conducted in three databases. This may have limited the number of studies assessed. However, Scopus, ScienceDirect and Medline via PubMed are trusted indexing services that house pertinent health research from reputable journals. Hence, the choice to limit the literature search in these databases has resulted in synthesis of results from good quality research, as proven by the fact that all eligible research has passed the risk of bias assessment. A meta-analysis was not pursued due the limited number of research, differences in the measurements of SNP occurrence and varying assays utilized to determine the phenotypic of effects of the mutations. Despite these limitations, the review was able to provide valuable insights regarding benzimidazole resistance in hookworms. Yet, several research gaps have arisen that require further research attention.

The systematic review revealed that seven benzimidazole resistance-associated SNPs at four amino acids loci have been reported among several hookworm species: Q134H, F167Y, E198A, E198K, E198V, F200Y and F200L. The F167Y and Q134H mutations are widespread in A. caninum in the United States. Similarly, SNPs have also been reported in Brazil, Haiti, Australia, New Zealand, Kenya, Ghana, Mozambique and Tanzania. There are no reports of these mutations in the Asian continent. Laboratory evidence shows that E198A and F200L are linked with induced resistance in A. ceylanicum. In vivo assays and field data affirm that F167Y and Q134H confer resistance in A. caninum. The association between resistance and the occurrence of SNPs in human hookworms has yet to be fully substantiated. Knowing the exact mechanism of resistance will provide insights on how to abate its emergence and can be used to optimize current mitigation efforts that are reliant on pharmacological interventions.

Supplementary Materials

Supplementary tables and figures are available at [DOI]

Acknowledgements

JCBT and AK would like to acknowledge the Postgraduate Scholarship for International Students of the Faculty of Medicine, Khon Kaen University for funding their doctoral studies. Also, MFH acknowledges Khon Kaen University Active Recruitment Scholarships for funding his master’s degree studies.

Author Contributions

JCBT: Conceptualization; Writing -Original Draft; Methodology; Formal Analysis. MFH: Methodology; Writing – Editing and Reviewing. AK: Methodology; Writing – Editing and Reviewing. SS and PS: Conceptualization; Writing – Editing and Reviewing; Supervision.

Data Availability

Data utilized in this systematic review can be accessed through requests to the corresponding author.

Competing Interest

The authors have no competing interests to declare.

Funding

This research did not receive any funding.

Ethics Statements

Ethics clearance is not required due to the nature of the study.

Consent for Publication

Not applicable.

Declaration of Generative AI in the writing process

This research did not utilize any large language models in the writing process.

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Benzimidazole Resistance-Associated Mutations in the β-tubulin Gene of Hookworms: A Systematic Review

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Characteristics of the included studies

SNPs Associated with Benzimidazole Resistance in Hookworms

Geographical distribution of Benzimidazole resistance associated SNPs

Phenotypic Effects of SNPs

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