Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term nemepsins for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

An in vitro larval motility assay to determine anthelmintic sensitivity for human hookworm and Strongyloides species

With the implementation of programs to control lymphatic filariasis and soil-transmitted helminths using broad spectrum anthelmintics, including albendazole and ivermectin, there is a need to develop an in vitro assay for detection of drug resistance. This report describes an in vitro assay for measuring the effects of ivermectin and benzimidazoles on the motility of larvae of the hookworm species Ancylostoma ceylanicum, A. caninum, and Necator americanus, and Strongyloides species including Strongyloides stercoralis, and S. ratti. A dose-response relationship was demonstrated with each of the parasite species, with distinct differences observed between the various species. In pilot field testing of the assay with N. americanus larvae recovered from human fecal samples, a dose-response relationship was observed with ivermectin. While the assay has demonstrated the ability to determine drug responsiveness, its usefulness in resistance detection will require correlation with the clinical outcome among individuals infected with parasite strains showing different drug sensitivities.