Development of the egg hatch assay for detection of anthelminthic resistance in human hookworms

Evidence of development and rapid spread of anthelminthic resistance in veterinary nematodes raises concern that the increasingly frequent treatments used in chemotherapy-based programmes to control human soil-transmitted helminths may select resistant worms. The aim of this study was to adapt, refine, and evaluate the Egg Hatch Assay (EHA) test, which has been used for veterinary nematodes, for field testing of benzimidazole (BZ) susceptibility/resistance in human hookworms. A second objective was to use this EHA to assess whether a population of worms resistant to mebendazole (MBZ) has built up in a sub-population of frequently treated children in Pemba Island. Stools from 470 school children enrolled in the first (Standard 1) and in the fifth (Standard 5) class were examined at baseline and at 21 days after treatment with 500 mg MBZ or placebo tablets. Standard 1 children had never received any MBZ treatment whilst Standard 5 children had received a total of 13 rounds of treatment. The EHA, involving culture of purified eggs with increasing drug concentrations showed that, for thiabendazole (TBZ), the mean ED50s (concentrations required to prevent 50% of the viable eggs from hatching) for all children at baseline were 0.079 μg/ml at 48 h and 0.120 μg/ml at 72 h (P<0.001). For MBZ, the mean ED50s for all children at baseline were 0.895 μg/ml at 48 h and 1.50 μg/ml at 72 h (P<0.001). For TBZ and for MBZ the ED50 from Standard 1 were similar to those from Standard 5 children both at 48 and at 72 h. At the follow-up for TBZ and for MBZ, there was no significant difference between the ED50 from children who had received MBZ and children treated with placebo. In Pemba, TBZ ED50 values of children non-exposed (Standard 1) and of children exposed (Standard 5) to MBZ treatment, and data from children treated with MBZ and placebo indicate that a drug-resistant worm population has not built up within treated individuals, and that periodic treatment has not yet selected for widespread BZ resistance, at least at the threshold detectable by the EHA in this study. However, ED50 values for strains isolated from Mafia island, an area never exposed to BZ treatment were lower than for Pemba, suggesting lowered sensitivity of hookworm eggs recovered from Pembian children towards BZ.