Control of human soil-transmitted helminths (STHs) relies on preventive chemotherapy of schoolchildren applying the benzimidazoles (BZ) albendazole or mebendazole. Anthelmintic resistance (AR) is a common problem in nematodes of veterinary importance but for human STHs, information on drug efficacy is limited and routine monitoring is rarely implemented. Herein, the efficacy of single dose albendazole (400 mg) was evaluated in 12 schools in the Huye district of Rwanda where Ascaris is the predominant STH. Ascaris eggs were detected by wet mount microscopy and the Mini-FLOTAC method to assess cure rate (CR) and faecal egg count reduction (FECR). Blood and faecal samples were analysed for co-infections with Plasmodium sp. and Giardia duodenalis, respectively. Ascaris positive samples collected before and after treatment were analysed for putatively BZ-resistance associated β-tubulin gene single nucleotide polymorphisms. The overall CR was 69.9% by Mini-FLOTAC and 88.6% by wet mount microscopy. The FECR was 75.4% and the 95% calculated confidence intervals were 50.4–87.8% using sample variance, 55.4–88.8% by bootstrapping, and 75.0–75.7% applying a Markov Chain Monte Carlo Bayesian approach. FECR varied widely between 0 and 96.8% for individual schools. No putative BZ-resistance associated polymorphisms were found in the four Ascaris β-tubulin isotype genes examined. Since FECRs <95% indicate reduced efficacy, these findings raise the suspicion of BZ resistance. In the absence of respective molecular evidence, heritable AR in the local Ascaris populations cannot be formally proven. However, since FECRs <95% indicate reduced efficacy, BZ resistance may be suspected which would be alarming and calls for further analyses and routine monitoring in preventive chemotherapy programs.

Control of helminth infections is a major task in livestock production to prevent health constraints and economic losses. However, resistance to established anthelmintic substances already impedes effective anthelmintic treatment in many regions worldwide. Thus, there is an obvious need for sensitive and reliable methods to assess the resistance status of at least the most important nematode populations. Several single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length β-tubulin isotype 1 and 2 and α-tubulin coding sequences of the cattle nematode Ostertagia ostertagi. Additionally, the Cooperia oncophora α-tubulin coding sequence was identified. Phylogenetic maximum-likelihood analysis revealed that both isotype 1 and 2 are orthologs to the Caenorhabditis elegans ben-1 gene which is also associated with BZ resistance upon mutation. In contrast, a Trichuris trichiura cDNA, postulated to be β-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans β-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like β-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in β-tubulin isotype 1 codons 167, 198 and 200 of C. oncophora and O. ostertagi. PCR-fragments representing either of the two alleles were combined in defined ratios to evaluate the pyrosequencing assay. The correlation between the given and the measured allele frequencies of the respective SNPs was very high. Subsequently laboratory isolates and field populations with known resistance status were analyzed. With the exception of codon 167 in Cooperia, increases of resistance associated alleles were detected for all codons in at least one of the phenotypically resistant population. Pyrosequencing provides a fast, inexpensive and sensitive alternative to conventional resistance detection methods.

BackgroundCutaneous dirofilariosis is a canine mosquito-borne zoonosis that can cause larva migrans disease in humans. Dirofilaria repens is considered an emerging pathogen occurring with high prevalence in Mediterranean areas and many parts of tropical Asia. In Hong Kong, a second species, Candidatus Dirofilaria hongkongensis, has been reported. The present study aimed to compare mitochondrial genomes from these parasites and to obtain population genetic information.Methods and FindingsComplete mitochondrial genomes were obtained by PCR and Sanger sequencing or ILLUMINA sequencing for four worms. Cytochrome oxidase subunit 1 sequences identified three as D. repens (all from Europe) and one as C. D. hongkongensis (from India). Mitochondrial genomes have the same organization as in other spirurid nematodes but a higher preference for thymine in the coding strand. Phylogenetic analysis was in contradiction to current taxonomy of the Onchocercidae but in agreement with a recent multi-locus phylogenetic analysis using both mitochondrial and nuclear markers. D. repens and C. D. hongkongensis sequences clustered together and were the common sister group to Dirofilaria immitis. Analysis of a 2.5 kb mitochondrial genome fragment from macrofilaria or canine blood samples from Europe (42), Thailand (2), India (1) and Vietnam (1) revealed only small genetic differences in the D. repens samples including all European and the Vietnam sample. The Indian C. D. hongkongensis and the two Thai samples formed separate clusters and differences were comparatively large.ConclusionGenetic differences between Dirofilaria spp. causing cutaneous disease can be considerable whereas D. repens itself was genetically quite homogenous. C. D. hongkongensis was identified for the first time from the Indian subcontinent. The full mitochondrial genome sequence strengthens the hypothesis that it represents an independent species and the Thai samples might represent another cryptic species, Candidatus Dirofilaria sp. ‘Thailand II’, or a quite divergent population of C. D. hongkongensis.

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles – only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.

Resistance to benzimidazoles (BZs) in trichostrongyloid nematodes is a worldwide problem for livestock production, particularly regarding small ruminants. Sensitive and reliable methods are required to assess anthelmintic resistance status. Currently available methods for BZ resistance detection can be divided into three main groups, in vivo (e.g. faecal egg count reduction test), in vitro (e.g. egg hatch assay) and molecular tests. Three single nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene of various nematode species correlate with BZ resistance. While PCR-based methods have been reported for the three most economically important nematodes of sheep, namely, Trichostrongylus, Haemonchus and Teladorsagia, pyrosequencing assays are so far only available for the latter two. Here, the design and evaluation of pyrosequencing assays for isotype-1 and isotype-2 β-tubulin genes of Trichostrongylus colubriformis are described. PCR fragments carrying the susceptible and corresponding resistant genotype were combined in defined ratios to evaluate assay sensitivity and linearity. The correlation between the given and the measured allele frequencies of the respective SNPs (codons F167Y, E198A and F200Y) was very high. Pyrosequencing assays for Haemonchus, Teladorsagia and Trichostrongylus were subsequently used for a BZ resistance survey, carried out in the three European countries, namely Ireland, Italy and Switzerland. Larval cultures obtained from field survey samples in 2012 and 2013 were used for pyrosequencing. The test was applied when the target species represented at least 10% of the sample. Trichostrongylus and Teladorsagia were detected in all countries' samples whereas Haemonchus was not detected in samples from Ireland. SNPs in isotype-1 associated with resistance were detected for all three species, with frequencies at codon F200Y far exceeding those at codons F167Y and E198A. Elevated SNP frequencies in isotype-2 of Trichostrongylus were only rarely detected. Farms with BZ resistance-associated SNP frequencies above 10% were most often found in Switzerland followed by Ireland and Italy.