The discovery of a novel series of compounds with single-dose efficacy against juvenile and adult Schistosoma species.

Treatment and control of schistosomiasis depends on a single drug, praziquantel, but this is not ideal for several reasons including lack of potency against the juvenile stage of the parasite, dose size, and risk of resistance. We have optimised the properties of a series of compounds we discovered through high throughput screening and have designed candidates for clinical development. The best compounds demonstrate clearance of both juvenile and adult S. mansoni worms in a mouse model of infection from a single oral dose of < 10 mg/kg. Several compounds in the series are predicted to treat schistosomiasis in humans across a range of species with a single oral dose of less than 5 mg/kg.

Antiprotozoal and antihelminthic properties of plants ingested by wild Japanese macaques (Macaca fuscata yakui) in Yakushima Island.

ETHNOPHARMACOLOGICAL RELEVANCE: Primates forage on a variety of plant parts to balance their dietary intake to meet requirements of energy, nutrition and maintenance, however the reason(s) leading them to ingest some plants which have no nutritional value and/or contain bioactive or even toxic secondary metabolites is recently gaining closer attention. The growing literature suggests that primates consume plants for medicinal purposes (self-medication) as well, particularly when infected with parasites and pathogens (bacteria, viruses, microbes). Interestingly, some of the plants they consume are also used by humans for similar purposes or may have potential uses for humans. MATERIALS AND METHODS: As part of a 16-month study of the parasite ecology of a sub-species of Japanese macaques (Macaca fuscata yakui) on the island of Yakushima, we surveyed their feeding habits and collected a subset of plants and plant parts observed being ingested by macaques. The ethnomedicinal value of these plants was surveyed and methanolic extracts of 45 plant parts were tested in vitro against important parasites of humans, including four protozoan parasites Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, and the trematode flatworm Schistosoma mansoni. Potential toxicity of the extracts was also assessed on mammalian cells. RESULTS: A wide range of ethnomedicinal uses in Asia for these plants is noted, with 37% associated with the treatment of parasites, pathogens and related symptoms. Additionally, the 45 extracts tested showed broad and significant activity against our test organisms. All extracts were active against T. b. rhodesiense. The majority (over 80%) inhibited the growth of P. falciparum and L. donovani. Half of the extracts also displayed antiprotozoal potential against T. cruzi while only several extracts were active against both larval and adult stages of S. mansoni. Cytotoxicity was generally low, although several extracts lacked specific toxicity to test parasites. CONCLUSIONS: Our results indicated a number of plants and their parts to have antiparasitic activity not previously reported in the ethnopharmacological literature. Enhanced understanding of the primate diets, particularly during periods of intensified parasite infection risk may help to further narrow down plants of interest for lead compound development. The study of animal self-medication is a complementary approach, with precedence, to drug discovery of new lead drug compounds against human parasitic diseases.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Adult Schistosoma mansoni.

An estimated 600 million people are affected by the helminth disease schistosomiasis caused by parasites of the genus Schistosoma. There is currently only one drug recommended for treating schistosomiasis, praziquantel (PZQ), which is effective against adult worms but not against the juvenile stage. In an attempt to identify improved drugs for treating the disease, we have carried out high throughput screening of a number of small molecule libraries with the aim of identifying lead compounds with balanced activity against all life stages of Schistosoma. A total of almost 300,000 compounds were screened using a high throughput assay based on motility of worm larvae and image analysis of assay plates. Hits were screened against juvenile and adult worms to identify broadly active compounds and against a mammalian cell line to assess cytotoxicity. A number of compounds were identified as promising leads for further chemical optimization.

Application of RNAi to Genomic Drug Target Validation in Schistosomes.

Concerns over the possibility of resistance developing to praziquantel (PZQ), has stimulated efforts to develop new drugs for schistosomiasis. In addition to the development of improved whole organism screens, the success of RNA interference (RNAi) in schistosomes offers great promise for the identification of potential drug targets to initiate drug discovery. In this study we set out to contribute to RNAi based validation of putative drug targets. Initially a list of 24 target candidates was compiled based on the identification of putative essential genes in schistosomes orthologous of C. elegans essential genes. Knockdown of Calmodulin (Smp_026560.2) (Sm-Calm), that topped this list, produced a phenotype characterised by waves of contraction in adult worms but no phenotype in schistosomula. Knockdown of the atypical Protein Kinase C (Smp_096310) (Sm-aPKC) resulted in loss of viability in both schistosomula and adults and led us to focus our attention on other kinase genes that were identified in the above list and through whole organism screening of known kinase inhibitor sets followed by chemogenomic evaluation. RNAi knockdown of these kinase genes failed to affect adult worm viability but, like Sm-aPKC, knockdown of Polo-like kinase 1, Sm-PLK1 (Smp_009600) and p38-MAPK, Sm-MAPK p38 (Smp_133020) resulted in an increased mortality of schistosomula after 2-3 weeks, an effect more marked in the presence of human red blood cells (hRBC). For Sm-PLK-1 the same effects were seen with the specific inhibitor, BI2536, which also affected viable egg production in adult worms. For Sm-PLK-1 and Sm-aPKC the in vitro effects were reflected in lower recoveries in vivo. We conclude that the use of RNAi combined with culture with hRBC is a reliable method for evaluating genes important for larval development. However, in view of the slow manifestation of the effects of Sm-aPKC knockdown in adults and the lack of effects of Sm-PLK-1 and Sm-MAPK p38 on adult viability, these kinases may not represent suitable drug targets.

Do shoes reduce hookworm infection in school-aged children on Pemba Island, Zanzibar? A pragmatic trial.

BACKGROUND: A non-blinded, cluster randomised controlled trial to test whether footwear reduces prevalence and intensity of hookworm infection in school-aged children on Pemba Island, Zanzibar. METHODS: Six schools were randomised to receive shoes and standard care (deworming with albendazole and health education) and six control schools to receive standard care only. Children provided a stool sample to assess prevalence and intensity of infection with soil transmitted helminthiases (Ascaris lumbricoides, Trichuris trichiura and hookworm). Shoes were then distributed to pupils in the intervention schools; deworming took place as part of the government's mass drug administration programme and a further round of stool samples were collected six months later. RESULTS: Nine hundred and fifteen children were traced at follow-up (1056 at baseline). As many children wore shoes in the control arm as the intervention arm. There was no difference in hookworm prevalence (23.4% for intervention schools, 21.3% for control schools, p=0.48), and no difference in mean hookworm infection in eggs/gram of stool (18, 1-36 in intervention schools, 18, 7-29 in control schools, p=0.23). Shoe-wearing increased across all schools, from 47.4 to 82.4%. If a child wore shoes at the end of the study, the relative risk of hookworm infection was 0.7 (CI 0.53-0.91). CONCLUSION: Due to contamination, the trial could not conclude that shoes were protective against hookworm infection but the intervention led to behavioural change, and observational data suggest that shoes are protective against hookworm. This trial is registered at ClinicalTrials.gov, NCT01869127.

Orally active antischistosomal early leads identified from the open access malaria box.

BACKGROUND: Worldwide hundreds of millions of schistosomiasis patients rely on treatment with a single drug, praziquantel. Therapeutic limitations and the threat of praziquantel resistance underline the need to discover and develop next generation drugs. METHODOLOGY: We studied the antischistosomal properties of the Medicines for Malaria Venture (MMV) malaria box containing 200 diverse drug-like and 200 probe-like compounds with confirmed in vitro activity against Plasmodium falciparum. Compounds were tested against schistosomula and adult Schistosoma mansoni in vitro. Based on in vitro performance, available pharmacokinetic profiles and toxicity data, selected compounds were investigated in vivo. PRINCIPAL FINDINGS: Promising antischistosomal activity (IC50: 1.4-9.5 µM) was observed for 34 compounds against schistosomula. Three compounds presented IC50 values between 0.8 and 1.3 µM against adult S. mansoni. Two promising early leads were identified, namely a N,N'-diarylurea and a 2,3-dianilinoquinoxaline. Treatment of S. mansoni infected mice with a single oral 400 mg/kg dose of these drugs resulted in significant worm burden reductions of 52.5% and 40.8%, respectively. CONCLUSIONS/SIGNIFICANCE: The two candidates identified by investigating the MMV malaria box are characterized by good pharmacokinetic profiles, low cytotoxic potential and easy chemistry and therefore offer an excellent starting point for antischistosomal drug discovery and development.

Performance and safety of praziquantel for treatment of intestinal schistosomiasis in infants and preschool children.

BACKGROUND: In 2012 the WHO formally recognised that infants and preschool children are at significant risk of schistosomiasis and qualify for treatment with praziquantel (PZQ). Targeted surveys determining both the performance and safety of this drug are now needed in endemic areas. We have formally assessed parasitological cure and putative side-effects in a prospective cohort of Schistosoma mansoni-infected children (aged 5 months-7 years old) in lakeshore settings of Uganda. METHODOLOGY/PRINCIPAL FINDINGS: From a total of 369 children found to be egg-patent for intestinal schistosomiasis, 305 were followed-up three to four weeks after PZQ treatment and infection status re-assessed. Separately, a previously tested side-effect questionnaire was employed before and 24 hours after PZQ treatment to assess incidence and amelioration of symptoms in young children and their mothers. While the overall observed parasitological cure was 56.4%, a significant difference was found between a sub-set of children who had a history of multiple PZQ treatments (between one and four in an 18 month period), where cure rate was 41.7%, and those who had never received treatment (cure rate was 77·6%). PZQ proved to be safe, with only mild reported side effects which cleared within a month after treatment. Prevalence of reported symptoms was significantly lower in children than in mothers, and fewer side-effects were reported upon subsequent rounds of PZQ treatment. CONCLUSION/SIGNIFICANCE: Our findings show that PZQ treatment of young children resulted in satisfactory cure rates, and marked reduction in egg-output, with only mild and transient reported side-effects. However, the cure rate is clearly lower in younger children and those with history of previous treatment. Cure rate, but not egg reduction rate, was also lower in children with heavier pre-intervention infection intensity. With chemotherapy now recommended as a long-term strategy for disease control in young children, research into optimising the periodicity of targeted treatment strategies is now crucial.

Whole organism high-content screening by label-free, image-based Bayesian classification for parasitic diseases.

Sole reliance on one drug, Praziquantel, for treatment and control of schistosomiasis raises concerns about development of widespread resistance, prompting renewed interest in the discovery of new anthelmintics. To discover new leads we designed an automated label-free, high content-based, high throughput screen (HTS) to assess drug-induced effects on in vitro cultured larvae (schistosomula) using bright-field imaging. Automatic image analysis and Bayesian prediction models define morphological damage, hit/non-hit prediction and larval phenotype characterization. Motility was also assessed from time-lapse images. In screening a 10,041 compound library the HTS correctly detected 99.8% of the hits scored visually. A proportion of these larval hits were also active in an adult worm ex-vivo screen and are the subject of ongoing studies. The method allows, for the first time, screening of large compound collections against schistosomes and the methods are adaptable to other whole organism and cell-based screening by morphology and motility phenotyping.

Soil-transmitted helminthiasis in rural south-west China: prevalence, intensity and risk factor analysis.

Only few studies in rural China have explored the epidemiology of intestinal helminth infections and identified risk factors for transmission. The study was carried out in Simao and Mengla counties, where single fecal samples were collected from 317 school-aged children and from 94 inhabitants of a single village. Fecal specimens were examined with the Kato-Katz thick smear method and examined for helminth eggs. Data regarding socio-demographic and behavioral risk factors were collected using questionnaires. In Simao County the overall soil-transmitted helminthes (STH) prevalence was 40.2% (2.7, 5.4 and 35.7% for ascariasis, trichuriasis and hookworm infection, respectively). The STH infection rates were significantly higher in Mengla County, with an overall prevalence of 68.3% (19.0, 34.6 and 47.3% for ascariasis, trichuriasis and hookworm infection, respectively). Females were less likely to be infected with Trichuris trichiura (OR 0.29; 95% CI 0.15-0.56) and with hookworms (OR 0.55; 95% CI 0.33-0.93) than males. Hookworm infections were more prevalent among those 12 years of age or older (OR 2.9; 95% CI 1.2-7.1). Children of mothers with educational attainment of secondary school or higher had a protective effect against T. trichiura (OR 0.18; 95% CI 0.06-0.54) and hookworm (OR 0.21; 95% CI 0.09-0.51) infections. In the village survey, hookworm was the most prevalent species (62.8%) with infection seen in those 50 years of age and older. Based on recommended intervention strategies by the World Health Organization, Simao County should opt for school-based deworming once each year, while Mengla County should implement a similar strategy biannually, but should include the elderly population.

Immune system development during early childhood in tropical Latin America: evidence for the age-dependent down regulation of the innate immune response.

The immune response that develops in early childhood underlies the development of inflammatory diseases such as asthma and there are few data from tropical Latin America (LA). This study investigated the effects of age on the development of immunity during the first 5 years of life by comparing innate and adaptive immune responses in Ecuadorian children aged 6-9 months, 22-26 months, and 48-60 months. Percentages of naïve CD4+ T cells declined with age while those of memory CD4(+) and CD8(+) T cells increased indicating active development of the immune system throughout the first five years. Young infants had greater innate immune responses to TLR agonists compared to older children while regulatory responses including SEB-induced IL-10 and percentages of FoxP3(+) T-regulatory cells decreased with age. Enhanced innate immunity in early life may be important for host defense against pathogens but may increase the risk of immunopathology.

Integrated dataset of screening hits against multiple neglected disease pathogens.

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.

Comparison of microscopy and Alamar blue reduction in a larval based assay for schistosome drug screening.

BACKGROUND: In view of the current widespread use of and reliance on a single schistosomicide, praziquantel, there is a pressing need to discover and develop alternative drugs for schistosomiasis. One approach to this is to develop High Throughput in vitro whole organism screens (HTS) to identify hits amongst large compound libraries. METHODOLOGY/PRINCIPAL FINDINGS: We have been carrying out low throughput (24-well plate) in vitro testing based on microscopic evaluation of killing of ex-vivo adult S. mansoni worms using selected compound collections mainly provided through the WHO-TDR Helminth Drug Initiative. To increase throughput, we introduced a similar but higher throughput 96-well primary in vitro assay using the schistosomula stage which can be readily produced in vitro in large quantities. In addition to morphological readout of viability we have investigated using fluorometric determination of the reduction of Alamar blue (AB), a redox indicator of enzyme activity widely used in whole organism screening. A panel of 7 known schistosome active compounds including praziquantel, produced diverse effects on larval morphology within 3 days of culture although only two induced marked larval death within 7 days. The AB assay was very effective in detecting these lethal compounds but proved more inconsistent in detecting compounds which damaged but did not kill. The utility of the AB assay in detecting compounds which cause severe morbidity and/or death of schistosomula was confirmed in testing a panel of compounds previously selected in library screening as having activity against the adult worms. Furthermore, in prospective library screening, the AB assay was able to detect all compounds which induced killing and also the majority of compounds designated as hits based on morphological changes. CONCLUSION: We conclude that an HTS combining AB readout and image-based analysis would provide an efficient and stringent primary assay for schistosome drug discovery.

Praziquantel analogs with activity against juvenile Schistosoma mansoni.

Six amide and four urea derivatives of praziquantel were synthesized and tested for antischistosomal activity against juvenile and adults stages of Schistosoma mansoni in infected mice. Only one of these had significant activity against adult worms, but, unlike praziquantel, six of these had low to modest activity against juvenile worms. A praziquantel ketone derivative had the best combination of activity against juveniles and adults, but it had no effect on the motility of adult S. mansoni in ex vivo culture. Cytochrome P450 metabolic stability data support the hypothesis that the major trans-cyclohexanol metabolite of praziquantel plays an important role in the antischistosomal activity of this drug.

Early helminth infections are inversely related to anemia, malnutrition, and malaria and are not associated with inflammation in 6- to 23-month-old Zanzibari children.

Helminths aggravate anemia and malnutrition among school children. We studied this association in a cross-sectional study of 6- to 23-month-old Zanzibari children (N = 2322) and a sub-sample of 690 children matched on age and helminth infection status. Ascaris, hookworm, and Trichuris infections were diagnosed along with recent fever, malaria infection, mid-upper arm circumference (MUAC) and hemoglobin concentration (Hb). Alpha-1-acid glycoprotein (AGP), C-reactive protein (CRP), height, and weight were measured in the sub-sample. Infected children had higher Hb (beta = 5.44 g/L, P < 0.001) and MUAC-for-age Z score (beta = 0.30 Z, P < 0.001) compared with uninfected children after adjusting for covariates. Although helminths were not associated with inflammation, their association with Hb or MUAC-for-age Z score was modified by inflammation. Malaria-infected children were less likely to be infected with helminths (adjusted odds ratios 0.63 [95% confidence interval: 0.49, 0.81]). Non-anemic, better nourished, or non-malaria-infected children may be more exploratory of their environments and therefore increase their exposure to soil-transmitted helminths.

Adjusting for the acute phase response is essential to interpret iron status indicators among young Zanzibari children prone to chronic malaria and helminth infections.

The extent to which the acute phase response (APR) influences iron status indicators in chronic infections is not well documented. We investigated this relationship using reported recent fever and 2 acute phase proteins (APP), C-reactive protein (CRP), and alpha-1-acid glycoprotein (AGP). In a sample of 690 children matched on age and helminth infection status at baseline, we measured plasma for AGP, CRP, ferritin, transferrin receptor (TfR), and erythropoietin (EPO) and whole blood for hemoglobin (Hb) concentration, zinc protoporphyrin (ZPP), and malaria parasite density, and we obtained maternal reports of recent fever. We then examined the influence of the APR on each iron status indicator using regression analysis with Hb as the outcome variable. Ferritin was inversely related to Hb in the APR-unadjusted model. Adjusting for the APR using reported recent fever alone was not sufficient to reverse the inverse Hb-ferritin relationship. However, using CRP and/or AGP resulted in the expected positive relationship. The best fit model included reported recent fever, AGP and CRP (R(2) = 0.241; P < 0.001). The best fit Hb-ZPP, Hb-TfR, and Hb-EPO models included reported recent fever and AGP but not CRP (R(2) = 0.253, 0.310, and 0.292, respectively; P < 0.001). ZPP, TfR, and EPO were minimally influenced by the APR, whereas ferritin was immensely affected. Reported recent fever alone cannot be used as a marker for the APR. Either AGP or CRP is useful for adjusting if only 1 APP can be measured. However, AGP best predicted the APR in this population.

Early exposure of infants to GI nematodes induces Th2 dominant immune responses which are unaffected by periodic anthelminthic treatment.

We have previously shown a reduction in anaemia and wasting malnutrition in infants <3 years old in Pemba Island, Zanzibar, following repeated anthelminthic treatment for the endemic gastrointestinal (GI) nematodes Ascaris lumbricoides, hookworm and Trichuris trichiura. In view of the low intensity of worm infections in this age group, this was unexpected, and it was proposed that immune responses to the worms rather than their direct effects may play a significant role in morbidity in infants and that anthelminthic treatment may alleviate such effects. Therefore, the primary aims of this study were to characterise the immune response to initial/early GI nematode infections in infants and the effects of anthelminthic treatment on such immune responses. The frequency and levels of Th1/Th2 cytokines (IL-5, IL-13, IFN-gamma and IL-10) induced by the worms were evaluated in 666 infants aged 6-24 months using the Whole Blood Assay. Ascaris and hookworm antigens induced predominantly Th2 cytokine responses, and levels of IL-5 and IL-13 were significantly correlated. The frequencies and levels of responses were higher for both Ascaris positive and hookworm positive infants compared with worm negative individuals, but very few infants made Trichuris-specific cytokine responses. Infants treated every 3 months with mebendazole showed a significantly lower prevalence of infection compared with placebo-treated controls at one year following baseline. At follow-up, cytokine responses to Ascaris and hookworm antigens, which remained Th2 biased, were increased compared with baseline but were not significantly affected by treatment. However, blood eosinophil levels, which were elevated in worm-infected children, were significantly lower in treated children. Thus the effect of deworming in this age group on anaemia and wasting malnutrition, which were replicated in this study, could not be explained by modification of cytokine responses but may be related to eosinophil function.

Chronic intestinal nematode infection exacerbates experimental Schistosoma mansoni infection.

Mixed-parasite infections are common in many parts of the world, but little is known of the effects of concomitant parasite infections on the immune response or on disease progression. We have investigated the in vivo effects of a chronic gastrointestinal nematode infection on the infectivity and development of the immune response against the common trematode helminth Schistosoma mansoni. The data show that mice carrying an established chronic Trichuris muris infection and coinfected with S. mansoni, had significantly higher S. mansoni worm burdens than mice without coinfection. The increase in S. mansoni worm burden was accompanied by a higher egg burden in the liver. Kinetic analysis of S. mansoni establishment indicate reduced trapping of S. mansoni larvae during skin-to-lung migration, with T. muris-induced alterations in lung cytokine expression and inflammatory foci surrounding lung-stage schistosomula, suggesting that the immunomodulatory effects of chronic T. muris infection elicited at the gut mucosal surface extend to other organs and perhaps specifically to other mucosal surfaces. The data show that a preexisting chronic gastrointestinal nematode infection facilitates the survival and migration of S. mansoni schistosomula to the portal system, and as a result, increases the egg burden and associated pathology of S. mansoni infection.

Meclonazepam analogues as potential new antihelmintic agents.

New analogues of the potent antihelmintic meclonazepam were prepared and evaluated against Schistosoma mansoni. The biological data suggests substitution at positions 2 and 4 of meclonazepam could provide promising analogues for prophylactic and therapeutic activity against S. mansoni.

Characterization of beta-tubulin genes in hookworms and investigation of resistance-associated mutations using real-time PCR.

Human hookworms (Ancylostoma duodenale, Necator americanus) are a major cause of malnutrition and anemia, particularly in children, and high worm burdens can lead to stunted growth and mental retardation. Mass drug administration (MDA) with benzimidazole (BZ) anthelmintics has the potential to greatly reduce morbidity and infection prevalence. However, such treatment strategies may apply significant selection pressure on resistance alleles. In several Strongylid parasites of livestock, resistance to BZ drugs is associated with single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype-1 gene at codons 167 and 200. As an initial investigation into the possible development of BZ resistance in hookworms, we have cloned and sequenced the beta-tubulin isotype-1 genes of the canine hookworm Ancylostoma caninum and the two human hookworm species A. duodenale and N. americanus. The genomic sequences are highly conserved as evidenced by a similar structure of exons and introns; the 10 exons are of the same length in all three species and code for the same amino acids. The genomic sequences were then used to develop a real-time PCR assay for detecting polymorphisms in codons 167 and 200 in all three species. Hookworm specimens previously obtained from Pemba Island school children who had demonstrated a reduced response to treatment with mebendazole were then examined using the real-time PCR assay. None of the samples revealed significant levels of polymorphisms at these loci. If BZ resistance is present in the hookworm populations examined, the results do not support the hypothesis that changes in codons 167 and 200 of beta-tubulin isotype-1 are responsible for any resistance.