Bruno Levecke

Assessment of the anthelmintic efficacy of albendazole in school children in seven countries where soil-transmitted helminths are endemic.

Background The three major soil-transmitted helminths (STH) Ascaris lumbricoides, Trichuris trichiura and Necator americanus/Ancylostoma duodenale are among the most widespread parasites worldwide. Despite the global expansion of preventive anthelmintic treatment, standard operating procedures to monitor anthelmintic drug efficacy are lacking. The objective of this study, therefore, was to define the efficacy of a single 400 milligram dose of albendazole (ALB) against these three STH using a standardized protocol. Methodology/Principal Findings Seven trials were undertaken among school children in Brazil, Cameroon, Cambodia, Ethiopia, India, Tanzania and Vietnam. Efficacy was assessed by the Cure Rate (CR) and the Fecal Egg Count Reduction (FECR) using the McMaster egg counting technique to determine fecal egg counts (FEC). Overall, the highest CRs were observed for A. lumbricoides (98.2%) followed by hookworms (87.8%) and T. trichiura (46.6%). There was considerable variation in the CR for the three parasites across trials (country), by age or the pre-intervention FEC (pre-treatment). The latter is probably the most important as it had a considerable effect on the CR of all three STH. Therapeutic efficacies, as reflected by the FECRs, were very high for A. lumbricoides (99.5%) and hookworms (94.8%) but significantly lower for T. trichiura (50.8%), and were affected to different extents among the 3 species by the pre-intervention FEC counts and trial (country), but not by sex or age. Conclusions/Significance Our findings suggest that a FECR (based on arithmetic means) of >95% for A. lumbricoides and >90% for hookworms should be the expected minimum in all future surveys, and that therapeutic efficacy below this level following a single dose of ALB should be viewed with concern in light of potential drug resistance. A standard threshold for efficacy against T. trichiura has yet to be established, as a single-dose of ALB is unlikely to be satisfactory for this parasite. Trial Registration ClinicalTrials.gov NCT01087099

Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers

Blastocystis is an enteric protist and one of the most frequently reported parasitic infections in humans and a variety of animal hosts. It has also been reported in numerous parasite surveys of animals in zoological gardens and in particular in non-human primate species. PCR-based methods capable of the direct detection of Blastocystis in faeces were used to detect Blastocystis from various hosts, including non-human primates, Australian native fauna, elephants and giraffes, as well as their keepers from a Western Australian zoo. Additional faecal samples were also collected from elephants and giraffes from four other zoos in Amsterdam (The Netherlands), Antwerp (Belgium), Melbourne and Werribee (Australia). Information regarding the general health and lifestyle of the human volunteers were obtained by questionnaire. Overall, 42% and 63% of animals and zoo-keepers sampled from the Western Australian zoo were positive for Blastocystis, respectively. The occurrence of Blastocystis in elephants and giraffes from other cities was similar. This is the first report of Blastocystis found in the elephant, giraffe, quokka, southern hairy nosed wombat and western grey kangaroo. Three novel and what appear to be highly host-specific subtypes (STs) of Blastocystis in the elephant, giraffe and quokka are also described. These findings indicate that further exploration of the genetic diversity of Blastocystis is crucial. Most zoo-keepers at the Perth Zoo were harbouring Blastocystis. Four of these zoo-keeper isolates were identical to the isolates from the southern hairy nosed wombat and five primate species.

Giardia and other intestinal parasites in different dog populations in Northern Belgium.

The objectives of this study were to obtain data on the prevalence of intestinal parasites in different dog populations in northern Belgium, to estimate the zoonotic risk associated with these infections and to identify potential risk factors. Between 2004 and 2007 a total of 1159 faecal samples were collected from 451 household dogs, 357 dogs from breeding kennels and 351 dogs with gastrointestinal disorders. The samples from dogs with gastrointestinal disorders were sent to the diagnostic Laboratory for Parasitology at Ghent University by veterinary practitioners. In household dogs the prevalence of intestinal parasites was relatively low. Giardia was the most commonly found parasite (9.3%, CI 5.5-13.1), followed by Toxocara canis (4.4%, CI 2.7-6.8). Much higher infection rates were observed in kennel dogs, especially for Giardia spp. (43.9%, CI 37.8-50.0); T. canis (26.3%, CI 21.8-31.2) and Cystoisospora spp. (26.3%, CI 21.8-31.2). Also in dogs with gastrointestinal problems, Giardia spp. (18.1%, CI 13.1-23.1), Cystoisospora spp. (8.8%, CI 6.1-12.3) and T. canis (7.4%, CI 4.9-10.7) were the most frequently detected parasites. In all dog populations pups were more frequently infected with Cystoisospora (P<0.0001 to P<0.05), Giardia (P<0.001 to P<0.05), and T. canis (P<0.0001 to P<0.001) than adult dogs, except for T. canis in household dogs, where this correlation was not significant. A significant association of anthelmintic treatment with T. canis infections was only observed within the household population. Household dogs with a higher number of treatments per year were more frequently infected with T. canis (P<0.05). There was a significant difference between the different breeding kennels for the occurrence of Cystoisospora, Giardia and T. canis (P<0.0001) and large kennels harboured relatively more infected animals than smaller breeding facilities (P<0.05). However, this was not significant for Giardia spp. Breed and gender did not affect the risk of an infection in any of the study populations. Toxocara and Giardia present a zoonotic risk, especially in household dogs, where the majority of Giardia positive samples (80%) belonged to the zoonotic assemblage A. In kennel dogs and clinically affected dogs the host-specific Giardia assemblages C and D were most prevalent (94% and 80%, respectively).

Is anthelmintic resistance a concern for the control of human soil-transmitted helminths?

The major human soil-transmitted helminths (STH), Ascaris lumbricoides, hookworms (Necator americanus and Ancylostoma duodenale) and Trichuris trichiura have a marked impact on human health in many parts of the world. Current efforts to control these parasites rely predominantly on periodic mass administration of anthelmintic drugs to school age children and other at-risk groups. After many years of use of these same drugs for controlling roundworms in livestock, high levels of resistance have developed, threatening the sustainability of these livestock industries in some locations. Hence, the question arises as to whether this is likely to also occur in the human STH, thereby threatening our ability to control these parasites. This is particularly important because of the recent increase in mass control programmes, relying almost exclusively on benzimidazole anthelmintics. It will be important to ensure that resistance is detected as it emerges in order to allow the implementation of mitigation strategies, such as use of drug combinations, to ensure that the effectiveness of the few existing anthelmintic drugs is preserved. In this review we address these issues by firstly examining the efficacy of anthelmintics against the human STH, and assessing whether there are any indications to date that resistance has emerged. We then consider the factors that influence the effect of current drug-use patterns in selecting for resistant parasite populations. We describe the tools currently available for resistance monitoring (field-based coprological methods), and those under development (in vitro bioassays and molecular tests), and highlight confounding factors that need to be taken into account when interpreting such resistance-monitoring data. We then highlight means to ensure that the currently available tools are used correctly, particularly with regard to study design, and we set appropriate drug-efficacy thresholds. Finally, we make recommendations for monitoring drug efficacy in the field, as components of control programmes, in order to maximise the ability to detect drug resistance, and if it arises to change control strategy and prevent the spread of resistance.

A comparison of the sensitivity and fecal egg counts of the McMaster egg counting and Kato-Katz thick smear methods for soil-transmitted helminths

Background The Kato-Katz thick smear (Kato-Katz) is the diagnostic method recommended for monitoring large-scale treatment programs implemented for the control of soil-transmitted helminths (STH) in public health, yet it is difficult to standardize. A promising alternative is the McMaster egg counting method (McMaster), commonly used in veterinary parasitology, but rarely so for the detection of STH in human stool. Methodology/Principal Findings The Kato-Katz and McMaster methods were compared for the detection of STH in 1,543 subjects resident in five countries across Africa, Asia and South America. The consistency of the performance of both methods in different trials, the validity of the fixed multiplication factor employed in the Kato-Katz method and the accuracy of these methods for estimating ‘true’ drug efficacies were assessed. The Kato-Katz method detected significantly more Ascaris lumbricoides infections (88.1% vs. 75.6%, p<0.001), whereas the difference in sensitivity between the two methods was non-significant for hookworm (78.3% vs. 72.4%) and Trichuris trichiura (82.6% vs. 80.3%). The sensitivity of the methods varied significantly across trials and magnitude of fecal egg counts (FEC). Quantitative comparison revealed a significant correlation (Rs >0.32) in FEC between both methods, and indicated no significant difference in FEC, except for A. lumbricoides, where the Kato-Katz resulted in significantly higher FEC (14,197 eggs per gram of stool (EPG) vs. 5,982 EPG). For the Kato-Katz, the fixed multiplication factor resulted in significantly higher FEC than the multiplication factor adjusted for mass of feces examined for A. lumbricoides (16,538 EPG vs. 15,396 EPG) and T. trichiura (1,490 EPG vs. 1,363 EPG), but not for hookworm. The McMaster provided more accurate efficacy results (absolute difference to ‘true’ drug efficacy: 1.7% vs. 4.5%). Conclusions/Significance The McMaster is an alternative method for monitoring large-scale treatment programs. It is a robust (accurate multiplication factor) and accurate (reliable efficacy results) method, which can be easily standardized.

Multilocus genotyping of Cryptosporidium and Giardia in non-outbreak related cases of diarrhoea in human patients in Belgium

Stool samples from Belgian patients suffering from abdominal pain and/or diarrhoea were examined for Cryptosporidium and Giardia. Cryptosporidium-positive samples were genotyped using the 70 kDa heat shock protein and the 60 kDa glycoprotein (GP60) genes: C. hominis was identified in 54.2% and C. parvum in 45.8% of the samples. Sequencing at the GP60 locus indicated that subgenotype IbA10G2 of C. hominis and subgenotype IIaA15G2R1 of C. parvum were the most prevalent, although several other subgenotypes were identified. For Giardia, sequencing at the beta-giardin, triose phosphate isomerase (TPI) and glutamate dehydrogenase (GDH) genes revealed assemblage B as the most prevalent (74.4%) in human patients. A high degree of heterogeneity was found, especially on the beta-giardin gene, and to a lesser extent on the GDH gene. Furthermore, using a novel species-specific PCR based on the TPI gene, mixed infections with both assemblage A and B were detected in a large number (32.4%) of human patients, which might have important epidemiological implications.

A Public Health Response against Strongyloides stercoralis: Time to Look at Soil-Transmitted Helminthiasis in Full

Strongyloides stercoralis infections have a worldwide distribution with a global burden in terms of prevalence and morbidity that is largely ignored. A public health response against soil-transmitted helminth (STH) infections should broaden the strategy to include S. stercoralis and overcome the epidemiological, diagnostic, and therapeutic challenges that this parasite poses in comparison to Ascaris lumbricoides, Trichuris trichiura, and hookworms. The relatively poor sensitivity of single stool evaluations, which is further lowered when quantitative techniques aimed at detecting eggs are used, also complicates morbidity evaluations and adequate drug efficacy measurements, since S. stercoralis is eliminated in stools in a larval stage. Specific stool techniques for the detection of larvae of S. stercoralis, like Baermanns and Kogas agar plate, despite superiority over direct techniques are still suboptimal. New serologies using recombinant antigens and molecular-based techniques offer new hopes in those areas. The use of ivermectin rather than benzimidazoles for its treatment and the need to have curative regimens rather than lowering the parasite burden are also unique for S. stercoralis in comparison to the other STH due to its life cycle, which allows reproduction and amplification of the worm burden within the human host. The potential impact on STH of the benzimidazoles/ivermectin combinations, already used for control/elimination of lymphatic filariasis, should be further evaluated in public health settings. While waiting for more effective single-dose drug regimens and new sensitive diagnostics, the evidence and the tools already available warrant the planning of a common platform for STH and S. stercoralis control.

Molecular characterisation of Giardia duodenalis in captive non-human primates reveals mixed assemblage A and B infections and novel polymorphisms.

Giardia is frequently detected in stools of non-human primates (NHP). However, a molecular identification has been rarely applied to Giardia isolates from NHP, and the distribution of the zoonotic assemblages A and B remains unclear. Moreover, little is known about the genetic variability among the isolates, although this may contribute to the elucidation of the different transmission pathways, including the role of NHP as a reservoir for human giardiasis. Therefore, 258 Giardia samples from 31 NHP species housed in nine zoological gardens and one sanctuary in Belgium and The Netherlands were characterised based on an assemblage-specific PCR targeting the triose phosphate isomerase (tpi) gene to identify both assemblage A and B infections. In addition, a multi-locus sequencing approach based on the glutamate dehydrogenase, the tpi and the beta-giardin genes was used to examine both the genetic variability and the ability to allocate these isolates to different NHP groups. Overall, assemblage B was the most prevalent (78.6%), but mixed assemblage A and B infections occurred in 32.7% of the samples. Sequencing of the isolates revealed the presence of new polymorphisms for both assemblages and at the three loci examined. The majority of the assemblage B isolates could not be grouped into recently described sub-assemblages, particularly at the tpi gene. Isolates could only be allocated to a specific group when polymorphisms of the three loci were combined. The results confirm that NHP are a potential reservoir for zoonotic transmission and advocate the use of assemblage-specific primers in molecular epidemiological surveys, as mixed infections are likely to be underestimated. The high level of heterogeneity within assemblages indicates that a revised nomenclature of these sub-assemblages is needed, but points out the potency of a multi-locus sequencing approach to unravel the complex epidemiology of Giardia duodenalis.

Field Validity and Feasibility of Four Techniques for the Detection of Trichuris in Simians: A Model for Monitoring Drug Efficacy in Public Health?

BACKGROUND Soil-transmitted helminths, such as Trichuris trichiura, are of major concern in public health. Current efforts to control these helminth infections involve periodic mass treatment in endemic areas. Since these large-scale interventions are likely to intensify, monitoring the drug efficacy will become indispensible. However, studies comparing detection techniques based on sensitivity, fecal egg counts (FEC), feasibility for mass diagnosis and drug efficacy estimates are scarce. METHODOLOGY/PRINCIPAL FINDINGS In the present study, the ether-based concentration, the Parasep Solvent Free (SF), the McMaster and the FLOTAC techniques were compared based on both validity and feasibility for the detection of Trichuris eggs in 100 fecal samples of nonhuman primates. In addition, the drug efficacy estimates of quantitative techniques was examined using a statistical simulation. Trichuris eggs were found in 47% of the samples. FLOTAC was the most sensitive technique (100%), followed by the Parasep SF (83.0% [95% confidence interval (CI): 82.4-83.6%]) and the ether-based concentration technique (76.6% [95% CI: 75.8-77.3%]). McMaster was the least sensitive (61.7% [95% CI: 60.7-62.6%]) and failed to detect low FEC. The quantitative comparison revealed a positive correlation between the four techniques (Rs = 0.85-0.93; p<0.0001). However, the ether-based concentration technique and the Parasep SF detected significantly fewer eggs than both the McMaster and the FLOTAC (p<0.0083). Overall, the McMaster was the most feasible technique (3.9 min/sample for preparing, reading and cleaning of the apparatus), followed by the ether-based concentration technique (7.7 min/sample) and the FLOTAC (9.8 min/sample). Parasep SF was the least feasible (17.7 min/sample). The simulation revealed that the sensitivity is less important for monitoring drug efficacy and that both FLOTAC and McMaster were reliable estimators. CONCLUSIONS/SIGNIFICANCE The results of this study demonstrated that McMaster is a promising technique when making use of FEC to monitor drug efficacy in Trichuris.

Diversity and distribution of Blastocystis sp. subtypes in non-human primates.

Blastocystis SSU-rDNA sequence data from 317 captive and free-living non-human primates (NHPs) representing 30 genera of apes, Old and New World (OW and NW) monkeys and prosimians were analysed to investigate subtype (ST) and allele distribution among hosts. Excluding 20 mixed ST infections, 27% of the sequences belonged to ST1, 22% to ST2, 34% to ST3, 1% to ST4, 4% to ST5, 11% to ST8, <1% to ST13 and 1% to ST15. The study confirmed cryptic host specificity of ST1 and ST3; conversely, considerable overlap in ST2 alleles exists among humans and NHPs. Subtype distribution in humans and NHPs differs mainly in that ST4 is rarely reported in NHPs while ST5 and ST8 are both unusual in humans. This may be due to host specificity and/or the apparent geographically restricted range of some subtypes. While the distribution of ST1, ST2 and ST3 was independent of NHP group or geographical association, ST5 was seen only in apes and OW monkeys and ST8 primarily in arboreal NHPs and only in species native to Asia or South America.