Novel insights in the fecal egg count reduction test for monitoring drug efficacy against soil-transmitted helminths in large-scale treatment programs

Background

The fecal egg count reduction test (FECRT) is recommended to monitor drug efficacy against soil-transmitted helminths (STHs) in public health. However, the impact of factors inherent to study design (sample size and detection limit of the fecal egg count (FEC) method) and host-parasite interactions (mean baseline FEC and aggregation of FEC across host population) on the reliability of FECRT is poorly understood.

Methodology/Principal Findings

A simulation study was performed in which FECRT was assessed under varying conditions of the aforementioned factors. Classification trees were built to explore critical values for these factors required to obtain conclusive FECRT results. The outcome of this analysis was subsequently validated on five efficacy trials across Africa, Asia, and Latin America. Unsatisfactory (<85.0%) sensitivity and specificity results to detect reduced efficacy were found if sample sizes were small (<10) or if sample sizes were moderate (10–49) combined with highly aggregated FEC (k<0.25). FECRT remained inconclusive under any evaluated condition for drug efficacies ranging from 87.5% to 92.5% for a reduced-efficacy-threshold of 90% and from 92.5% to 97.5% for a threshold of 95%. The most discriminatory study design required 200 subjects independent of STH status (including subjects who are not excreting eggs). For this sample size, the detection limit of the FEC method and the level of aggregation of the FEC did not affect the interpretation of the FECRT. Only for a threshold of 90%, mean baseline FEC <150 eggs per gram of stool led to a reduced discriminatory power.

Conclusions/Significance

This study confirms that the interpretation of FECRT is affected by a complex interplay of factors inherent to both study design and host-parasite interactions. The results also highlight that revision of the current World Health Organization guidelines to monitor drug efficacy is indicated. We, therefore, propose novel guidelines to support future monitoring programs.     

A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility

Background

Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods.

Methodology/Principal Findings

Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC₅₀ values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus.

Conclusions/Significance

Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.     

Correlation between discharged worms and fecal egg counts in human clonorchiasis

Background

Stool examination by counting eggs per gram of feces (EPGs) is the best method to estimate worm burden of Clonorchis sinensis in infected humans. The present study investigated a correlation between EPGs and worm burden in human clonorchiasis.

Methods and Findings

A total of 60 residents, 50 egg-positive and 10 egg-negative, in Sancheong-gun, Korea, participated in this worm collection trial in 2006–2009. They were diagnosed by egg positivity in feces using the Kato-Katz method. After administration of praziquantel, they were purged with cathartics on the next day, and then discharged adult worms were collected from their feces. Their EPGs ranged from 0 to 65,544. Adult worms of C. sinensis were collected from 17 egg-positive cases, and the number of worms ranged from 1 to 114 in each individual. A positive correlation between EPGs and numbers of worms was demonstrated (r = 0.681, P<0.001). Worm recovery rates were 9.7% in cases of EPGs 1–1,000 and 73.7% in those of EPGs over 1,000. No worms were detected from egg-negative subjects. Maximum egg count per worm per day was roughly estimated 3,770 in a subject with EPGs 2,664 and 106 collected worms.

Conclusions

The numbers of the worms are significantly correlated with the egg counts in human clonorchiasis. It is estimated that at least 110 worms are infected in a human body with EPGs around 3,000, and egg productivity of a worm per day is around 4,000.     

Bacteria stimulate hatching of yellow fever mosquito eggs

Background

Aedes aegypti Linnaeus is a peridomestic mosquito that lays desiccation-resistant eggs in water-filled human-made containers. Previous investigations connected egg hatching with declining dissolved oxygen (DO) that is associated with bacterial growth. However, past studies failed to uncouple DO from other potential stimulatory factors and they contained little quantitative information about the microbial community; consequently, a direct role for bacteria or compounds associated with bacteria in stimulating egg hatching cannot be dismissed.

Methodology/Principal Findings

Environmental factors stimulating hatch of Ae. aegypti eggs were investigated using non-sterile and sterile white oak leaf (WOL) infusions and a bacterial culture composed of a mix of 14 species originally isolated from bamboo leaf infusion. In WOL infusion with active microbes, 92.4% of eggs hatched in 2-h at an average DO concentration of 2.4 ppm. A 24-h old bacterial culture with a DO concentration of 0.73 ppm also stimulated 95.2% of eggs hatch within 1-h. In contrast, only 4.0% of eggs hatched in sterile infusion, whose DO averaged 7.4 ppm. Effects of bacteria were uncoupled from DO by exposing eggs to bacterial cells suspended in NaCl solution. Over a 4-h exposure period, 93.8% of eggs hatched while DO concentration changed minimally from 7.62 to 7.50 ppm. Removal of bacteria by ultra-filtration and cell-free filtrate resulted in only 52.0% of eggs hatching after 4-h at an average DO concentration of 5.5 ppm.

Conclusions/Significance

Collectively, the results provide compelling evidence that bacteria or water-soluble compounds secreted by bacteria, not just low DO concentration, stimulate hatching of Ae. aegypti eggs. However, the specific cues involved remain to be identified. These research findings contribute new insight into an important aspect of the oviposition biology of Ae. aegypti, a virus vector of global importance, providing the basis for a new paradigm of environmental factors involved in egg hatching.     

Field evaluation of anthelmintic drug sensitivity using in vitro egg hatch and larval motility assays with Necator americanus recovered from human clinical isolates

A field-applicable assay for testing anthelmintic sensitivity is required to monitor for anthelmintic resistance. We undertook a study to evaluate the ability of three in vitro assay systems to define drug sensitivity of clinical isolates of the human hookworm parasite Necator americanus recovered from children resident in a village in Madang Province, Papua New Guinea. The assays entailed observation of drug effects on egg hatch (EHA), larval development (LDA), and motility of infective stage larvae (LMA). The egg hatch assay proved the best method for assessing the response to benzimidazole anthelmintics, while the larval motility assay was suitable for assessing the response to ivermectin. The performance of the larval development assay was unsatisfactory on account of interference caused by contaminating bacteria. A simple protocol was developed whereby stool samples were subdivided and used for immediate egg recovery, as well as for faecal culture, in order to provide eggs and infective larvae, respectively, for use in the egg hatch assay and larval motility assay systems. While the assays proved effective in quantifying drug sensitivity in larvae of the drug-susceptible hookworms examined in this study, their ability to indicate drug resistance in larval or adult hookworms remains to be determined.     

Effect of restricted preen-gland access on maternal self maintenance and reproductive investment in mallards

Background

As egg production and offspring care are costly, females should invest resources adaptively into their eggs to optimize current offspring quality and their own lifetime reproductive success. Parasite infections can influence maternal investment decisions due to their multiple negative physiological effects. The act of preening – applying oils with anti-microbial properties to feathers – is thought to be a means by which birds combat pathogens and parasites, but little is known of how preening during the reproductive period (and its expected disease-protecting effects) influences maternal investment decisions at the level of the egg.

Methodology/Principal Findings

Here, we experimentally prevented female mallards (Anas platyrhynchos) from accessing their preen gland during breeding and monitored female immunoresponsiveness (e.g., plasma lysozyme concentration) as well as some egg traits linked to offspring quality (e.g., egg mass, yolk carotenoid content, and albumen lysozyme levels). Females with no access to their preen gland showed an increase in plasma lysozyme level compared to control, normally preening females. In addition, preen-gland-restricted females laid significantly lighter eggs and deposited higher carotenoid concentrations in the yolk compared to control females. Albumen lysozyme activity did not differ significantly between eggs laid by females with or without preen gland access.

Conclusion/Significance

Our results establish a new link between an important avian self-maintenance behaviour and aspects of maternal health and reproduction. We suggest that higher yolk carotenoid levels in eggs laid by preen-gland-restricted females may serve to boost health of offspring that would hatch in a comparatively microbe-rich environment.     

Cure of hookworm infection with a cysteine protease inhibitor

Background

Hookworm disease is a major global health problem and principal among a number of soil-transmitted helminthiases (STHs) for the chronic disability inflicted that impacts both personal and societal productivity. Mass drug administration most often employs single-dose therapy with just two drugs of the same chemical class to which resistance is a growing concern. New chemical entities with the appropriate single-dose efficacy are needed.

Methods and Findings

Using various life-cycle stages of the hookworm Ancylostoma ceylanicum in vitro and a hamster model of infection, we report the potent, dose-dependent cidal activities of the peptidyl cysteine protease inhibitors (CPIs) K11002 (4-mopholino-carbonyl-phenylalanyl-homophenylalanyl- vinyl sulfone phenyl) and K11777 (N-methylpiperazine-phenylalanyl-homophenylalanyl-vinylsulfone phenyl). The latter is in late pre-clinical testing for submission as an Investigational New Drug (IND) with the US Federal Drug Administration as an anti-chagasic. In vitro, K11002 killed hookworm eggs but was without activity against first-stage larvae. The reverse was true for K11777 with a larvicidal potency equal to that of the current anti-hookworm drug, albendazole (ABZ). Both CPIs produced morbidity in ex vivo adult hookworms with the activity of K11777 again being at least the equivalent of ABZ. Combinations of either CPI with ABZ enhanced morbidity compared to single compounds. Strikingly, oral treatment of infected hamsters with 100 mg/kg K11777 b.i.d. (i.e., a total daily dose of 200 mg/kg) for one day cured infection: a single 100 mg/kg treatment removed >90% of worms. Treatment also reversed the otherwise fatal decrease in blood hemoglobin levels and body weights of hosts. Consistent with its mechanism of action, K11777 decreased by >95% the resident CP activity in parasites harvested from hamsters 8 h post-treatment with a single 100 mg/kg oral dose.

Conclusion

A new, oral single-dose anthelmintic that is active in an animal model of hookworm infection and that possesses a distinct mechanism of action from current anthelmintics is discovered. The data highlight both the possibility of repurposing the anti-chagasic K11777 as a treatment for hookworm infection and the opportunity to further develop CPIs as a novel anthelmintic class to target hookworms and, possibly, other helminths.     

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

Cost analysis of various low pathogenic avian influenza surveillance systems in the Dutch egg layer sector

Background

As low pathogenic avian influenza viruses can mutate into high pathogenic viruses the Dutch poultry sector implemented a surveillance system for low pathogenic avian influenza (LPAI) based on blood samples. It has been suggested that egg yolk samples could be sampled instead of blood samples to survey egg layer farms. To support future decision making about AI surveillance economic criteria are important. Therefore a cost analysis is performed on systems that use either blood or eggs as sampled material.

Methodology/Principal Findings

The effectiveness of surveillance using egg or blood samples was evaluated using scenario tree models. Then an economic model was developed that calculates the total costs for eight surveillance systems that have equal effectiveness. The model considers costs for sampling, sample preparation, sample transport, testing, communication of test results and for the confirmation test on false positive results. The surveillance systems varied in sampled material (eggs or blood), sampling location (farm or packing station) and location of sample preparation (laboratory or packing station). It is shown that a hypothetical system in which eggs are sampled at the packing station and samples prepared in a laboratory had the lowest total costs (i.e. € 273,393) a year. Compared to this a hypothetical system in which eggs are sampled at the farm and samples prepared at a laboratory, and the currently implemented system in which blood is sampled at the farm and samples prepared at a laboratory have 6% and 39% higher costs respectively.

Conclusions/Significance

This study shows that surveillance for avian influenza on egg yolk samples can be done at lower costs than surveillance based on blood samples. The model can be used in future comparison of surveillance systems for different pathogens and hazards.     

QTL mapping of egg albumen quality in egg layers

Background

A fresh, good quality egg has a firm and gelatinous albumen that anchors the yolk and restricts growth of microbiological pathogens. As the egg ages, the gel-like structure collapses, resulting in thin and runny albumen. Occasionally thin albumen is found in a fresh egg, giving the impression of a low quality product. A mapping population consisting of 1599 F₂ hens from a cross between White Rock and Rhode Island Red lines was set up, to identify loci controlling albumen quality. The phenotype for albumen quality was evaluated by albumen height and in Haugh units (HU) measured on three consecutive eggs from each F₂ hen at the age of 40 weeks. For the fine-mapping analysis, albumen height and HU were used simultaneously to eliminate contribution of the egg size to the phenotype.

Results

Linkage analysis in a small population of seven half-sib families (668 F₂) with 162 microsatellite markers spread across 27 chromosomes revealed two genome-wide significant regions with additive effects for HU on chromosomes 7 and Z. In addition, two putative genome-wide quantitative trait loci (QTL) regions were identified on chromosomes 4 and 26. The QTL effects ranged from 2 to 4% of the phenotypic variance. The genome-wide significant QTL regions on chromosomes 7 and Z were selected for fine-mapping in the full set composed of 16 half-sib families. In addition, their existence was confirmed by an association analysis in an independent commercial Hy-Line pure line.

Conclusions

We identified four chicken genomic regions that affect albumen quality. Our results also suggest that genes that affect albumen quality act both directly and indirectly through several different mechanisms. For instance, the QTL regions on both fine-mapped chromosomes 7 and Z overlapped with a previously reported QTL for eggshell quality, indicating that eggshell membranes may play a role in albumen quality.     

Correlative and Dynamic Imaging of the Hatching Biology of Schistosoma japonicum from Eggs Prepared by High Pressure Freezing

Background

Schistosome eggs must traverse tissues of the intestine or bladder to escape the human host and further the life cycle. Escape from host tissues is facilitated by secretion of immuno-reactive molecules by eggs and the formation of an intense strong granulomatous response by the host which acts to exclude the egg into gut or bladder lumens. Schistosome eggs hatch on contact with freshwater, but the mechanisms of activation and hatching are poorly understood. In view of the lack of knowledge of the behaviour of egg hatching in schistosomes, we undertook a detailed dynamic and correlative study of the hatching biology of Schistosoma japonicum.

Methodology/Principal Findings

Hatching eggs of S. japonicum were studied using correlative light and electron microscopy (EM). The hatching behaviour was recorded by video microscopy. EM preparative methods incorporating high pressure freezing and cryo-substitution were used to investigate ultrastructural features of the miracidium and extra-embryonic envelopes in pre-activated and activated eggs, and immediately after eggshell rupture. Lectin cytochemistry was performed on egg tissues to investigate subcellular location of specific carbohydrate groups.

Conclusions/Significance

The hatching of S. japonicum eggs is a striking phenomenon, whereby the larva is liberated explosively while still encapsulated within its sub-shell envelopes. The major alterations that occur in the egg during activation are scission of the outer envelope-eggshell boundary, autolysis of the cellular inner envelope, and likely hydration of abundant complex and simple polysaccharides in the lacunal space between the miracidial larva and surrounding envelopes. These observations on hatching provide insight into the dynamic activity of the eggs and the biology of schistosomes within the host.     

Physiological and biochemical characterization of egg extract of black widow spiders to uncover molecular basis of egg toxicity

Background

Black widow spider (L. tredecimguttatus) has toxic components not only in the venomous glands, but also in other parts of the body and its eggs. It is biologically important to investigate the molecular basis of the egg toxicity.

Results

In the present work, an aqueous extract was prepared from the eggs of the spider and characterized using multiple physiological and biochemical strategies. Gel electrophoresis and mass spectrometry demonstrated that the eggs are rich in high-molecular-mass proteins and the peptides below 5 kDa. The lyophilized extract of the eggs had a protein content of 34.22% and was shown to have a strong toxicity towards mammals and insects. When applied at a concentration of 0.25 mg/mL, the extract could completely block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations within 12.0 ± 1.5 min. Using whole-cell patch-clamp technique, the egg extract was demonstrated to be able to inhibit the voltage-activated Na⁺, K⁺ and Ca²⁺ currents in rat DRG neurons. In addition, the extract displayed activities of multiple hydrolases. Finally, the molecular basis of the egg toxicity was discussed.

Conclusions

The eggs of black widow spiders are rich in proteinous compounds particularly the high-molecular-mass proteins with different types of biological activity The neurotoxic and other active compounds in the eggs are believed to play important roles in the eggs’ toxic actions.     

Anticancer activities of six selected natural compounds of some Cameroonian medicinal plants

Background

Natural products are well recognized as sources of drugs in several human ailments. In the present work, we carried out a preliminary screening of six natural compounds, xanthone V₁ (1); 2-acetylfuro-1,4-naphthoquinone (2); physcion (3); bisvismiaquinone (4); vismiaquinone (5); 1,8-dihydroxy-3-geranyloxy-6-methylanthraquinone (6) against MiaPaCa-2 pancreatic and CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ADR5000. Compounds 1 and 2 were then tested in several other cancer cells and their possible mode of action were investigated.

Methodology/Findings

The tested compounds were previously isolated from the Cameroonian medicinal plants Vismia laurentii (1, 3, 4, 5 and 6) and Newbouldia laevis (2). The preliminary cytotoxicity results allowed the selection of xanthone V₁ and 2-acetylfuro-1,4-naphthoquinone, which were then tested on a panel of cancer cell lines. The study was also extended to the analysis of cell cycle distribution, apoptosis induction, caspase 3/7 activation and the anti-angiogenic properties of xanthone V₁ and 2-acetylfuro-1,4-naphthoquinone. IC₅₀ values around or below 4 µg/ml were obtained on 64.29% and 78.57% of the tested cancer cell lines for xanthone V₁ and 2-acetylfuro-1,4-naphthoquinone, respectively. The most sensitive cell lines (IC₅₀<1 µg/ml) were breast MCF-7 (to xanthone V₁), cervix HeLa and Caski (to xanthone V₁ and 2-acetylfuro-1,4-naphthoquinone), leukemia PF-382 and melanoma colo-38 (to 2-acetylfuro-1,4-naphthoquinone). The two compounds showed respectively, 65.8% and 59.6% inhibition of the growth of blood capillaries on the chorioallantoic membrane of quail eggs in the anti-angiogenic assay. Upon treatment with two fold IC₅₀ and after 72 h, the two compounds induced cell cycle arrest in S-phase, and also significant apoptosis in CCRF-CEM leukemia cells. Caspase 3/7 was activated by xanthone V₁.

Conclusions/Significance

The overall results of the present study provided evidence for the cytotoxicity of compounds xanthone V₁ and 2-acetylfuro-1,4-naphthoquinone, and bring supportive data for future investigations that will lead to their use in cancer therapy.     

Is Aboriginal Food Less Allergenic? Comparing IgE-Reactivity of Eggs from Modern and Ancient Chicken Breeds in a Cohort of Allergic Children

Background

Hen''s egg allergy ranks among the most frequent primary food allergies in children. We aimed to investigate sensitization profiles of egg allergic patients and compare in vitro IgE reactivities of eggs from ancient chicken breeds (Araucana and Maran) with those from conventional laying hen hybrids.

Methodology

Egg allergic children (n = 25) were subjected to skin prick test, double blind placebo controlled food challenge, and sensitization profiles to Gal d 1–5 were determined by allergen microarray. IgE binding and biological activity of eggs from different chicken breeds were investigated by immunoblot, ELISA, and mediator release assays.

Principal Findings

We found that Gal d 1 and Gal d 2 are generally major egg allergens, whereas Gal d 3–5 displayed high sensitization prevalence only in patients reacting to both, egg white and yolk. It seems that the onset of egg allergy is mediated by egg white allergens expanding to yolk sensitization in later stages of disease. Of note, egg white/yolk weight ratios were reduced in eggs from Auraucana and Maran chicken. As determined in IgE immunoblots and mass analysis, eggs from ancient chicken breeds did not differ in their protein composition. Similar IgE-binding was observed for all egg white preparations, while an elevated allergenicity was detected in egg yolk from Araucana chicken.

Conclusion/Significance

Our results on allergenicity and biological activity do not confirm the common assumption that aboriginal food might be less allergenic. Comprehensive diagnosis of egg allergy should distinguish between reactivity to hen''s egg white and yolk fractions to avoid unnecessary dietary restrictions to improve life quality of the allergic child and its family.     

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.     

Oviposition-Stimulant and Ovicidal Activities of Moringa oleifera Lectin on Aedes aegypti

Background

Natural insecticides against the vector mosquito Aedes aegypti have been the object of research due to their high level of eco-safety. The water-soluble Moringa oleifera lectin (WSMoL) is a larvicidal agent against A. aegypti. This work reports the effects of WSMoL on oviposition and egg hatching of A. aegypti.

Methodology/Principal Findings

WSMoL crude preparations (seed extract and 0–60 protein fraction), at 0.1 mg/mL protein concentration, did not affect oviposition, while A. aegypti gravid females laid their eggs preferentially (73%) in vessels containing isolated WSMoL (0.1 mg/mL), compared with vessels containing only distilled water (control). Volatile compounds were not detected in WSMoL preparation. The hatchability of fresh eggs deposited in the solutions in the oviposition assay was evaluated. The numbers of hatched larvae in seed extract, 0–60 protein fraction and WSMoL were 45±8.7 %, 20±11 % and 55±7.5 %, respectively, significantly (p<0.05) lower than in controls containing only distilled water (75–95%). Embryos were visualized inside fresh control eggs, but not within eggs that were laid and maintained in WSMoL solution. Ovicidal activity was also assessed using stored A. aegypti eggs. The protein concentrations able to reduce the hatching rate by 50% (EC50) were 0.32, 0.16 and 0.1 mg/mL for seed extract, 0–60 protein fraction and WSMoL, respectively. The absence of hatching of stored eggs treated with WSMoL at 0.3 mg/mL (EC99) after transfer to medium without lectin indicates that embryos within the eggs were killed by WSMoL. The reduction in hatching rate of A. aegypti was not linked to decrease in bacteria population.

Conclusions/Significance

WSMoL acted both as a chemical stimulant cue for ovipositing females and ovicidal agent at a given concentration. The oviposition-stimulant and ovicidal activities, combined with the previously reported larvicidal activity, make WSMoL a very interesting candidate in integrated A. aegypti control.     

Epidemiological and genetic data supporting the transmission of Ancylostoma ceylanicum among human and domestic animals

Background

Currently, information on species-specific hookworm infection is unavailable in Malaysia and is restricted worldwide due to limited application of molecular diagnostic tools. Given the importance of accurate identification of hookworms, this study was conducted as part of an ongoing molecular epidemiological investigation aimed at providing the first documented data on species-specific hookworm infection, associated risk factors and the role of domestic animals as reservoirs for hookworm infections in endemic communities of Malaysia.

Methods/Findings

A total of 634 human and 105 domestic canine and feline fecal samples were randomly collected. The overall prevalence of hookworm in humans and animals determined via microscopy was 9.1% (95% CI = 7.0–11.7%) and 61.9% (95% CI = 51.2–71.2%), respectively. Multivariate analysis indicated that participants without the provision of proper latrine systems (OR = 3.5; 95% CI = 1.53–8.00; p = 0.003), walking barefooted (OR = 5.6; 95% CI = 2.91–10.73; p<0.001) and in close contact with pets or livestock (OR = 2.9; 95% CI = 1.19–7.15; p = 0.009) were more likely to be infected with hookworms. Molecular analysis revealed that while most hookworm-positive individuals were infected with Necator americanus, Ancylostoma ceylanicum constituted 12.8% of single infections and 10.6% mixed infections with N. americanus. As for cats and dogs, 52.0% were positive for A. ceylanicum, 46.0% for Ancylostoma caninum and 2.0% for Ancylostoma braziliense and all were single infections.

Conclusion

This present study provided evidence based on the combination of epidemiological, conventional diagnostic and molecular tools that A. ceylanicum infection is common and that its transmission dynamic in endemic areas in Malaysia is heightened by the close contact of human and domestic animal (i.e., dogs and cats) populations.     

Low efficacy of single-dose albendazole and mebendazole against hookworm and effect on concomitant helminth infection in Lao PDR

Background

Albendazole and mebendazole are increasingly deployed for preventive chemotherapy targeting soil-transmitted helminth (STH) infections. We assessed the efficacy of single oral doses of albendazole (400 mg) and mebendazole (500 mg) for the treatment of hookworm infection in school-aged children in Lao PDR. Since Opisthorchis viverrini is co-endemic in our study setting, the effect of the two drugs could also be determined against this liver fluke.

Methodology

We conducted a randomized, open-label, two-arm trial. In total, 200 children infected with hookworm (determined by quadruplicate Kato-Katz thick smears derived from two stool samples) were randomly assigned to albendazole (n = 100) and mebendazole (n = 100). Cure rate (CR; percentage of children who became egg-negative after treatment), and egg reduction rate (ERR; reduction in the geometric mean fecal egg count at treatment follow-up compared to baseline) at 21–23 days posttreatment were used as primary outcome measures. Adverse events were monitored 3 hours post treatment.

Principal Findings

Single-dose albendazole and mebendazole resulted in CRs of 36.0% and 17.6% (odds ratio: 0.4; 95% confidence interval: 0.2–0.8; P = 0.01), and ERRs of 86.7% and 76.3%, respectively. In children co-infected with O. viverrini, albendazole and mebendazole showed low CRs (33.3% and 24.2%, respectively) and moderate ERRs (82.1% and 78.2%, respectively).

Conclusions/Significance

Both albendazole and mebendazole showed disappointing CRs against hookworm, but albendazole cured infection and reduced intensity of infection with a higher efficacy than mebendazole. Single-dose administrations showed an effect against O. viverrini, and hence it will be interesting to monitor potential ancillary benefits of a preventive chemotherapy strategy that targets STHs in areas where opisthorchiasis is co-endemic.

Clinical Trial Registration

Current Controlled Trials ISRCTN29126001     

Acidocalcisomes as calcium- and polyphosphate-storage compartments during embryogenesis of the insect Rhodnius prolixus Stahl

Background

The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus.

Methodology/Principal findings

Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H⁺-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5±0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs.

Conclusions

We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells.