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Background To work towards reaching the WHO goal of eliminating soil-transmitted helminth (STH) infections as a public health problem, the total number of children receiving anthelmintic drugs has strongly increased over the past few years. However, as drug pressure levels rise, the development of anthelmintic drug resistance (AR) is more and more likely to appear. Currently, any global surveillance system to monitor drug efficacy and the emergence of possible AR is lacking. Consequently, it remains unclear to what extent the efficacy of drugs may have dropped and whether AR is already present. The overall aim of this study is to recommend the best diagnostic methods to monitor drug efficacy and molecular markers to assess the emergence of AR in STH control programs. Methods A series of drug efficacy trials will be performed in four STH endemic countries with varying drug pressure (Ethiopia and Brazil: low drug pressure, Lao PDR: moderate drug pressure and Tanzania: high drug pressure). These trials are designed to assess the efficacy of a single oral dose of 400 mg albendazole (ALB) against STH infections in school-aged children (SAC) by microscopic (duplicate Kato-Katz thick smear, Mini-FLOTAC and FECPAKG2) and molecular stool-based diagnostic methods (quantitative PCR (qPCR)). Data will be collected on the cost of the materials used, as well as the time required to prepare and examine stool samples for the different diagnostic methods. Following qPCR, DNA samples will also be submitted for pyrosequencing to assess the presence and prevalence of single nucleotide polymorphisms (SNPs) in the β-tubulin gene. These SNPs are known to be linked to AR in animal STHs. Discussion The results obtained by these trials will provide robust evidence regarding the cost-efficiency and diagnostic performance of the different stool-based diagnostic methods for the assessment of drug efficacy in control programs. The assessment of associations between the frequency of SNPs in the β-tubulin gene and the history of drug pressure and drug efficacy will allow the validation of these SNPs as a marker for AR in human STHs. Trial registration The trial was retrospectively registered the 7th of March 2018 on Clinicaltrials.gov (ID: NCT03465488)., Author summary Soil-transmitted helminths (STHs) affect 1.4 billion people worldwide and cause significant morbidity when the intensity of infection is high. Currently, these infections are controlled in school-aged children by preventive chemotherapy with the benzimidazole drugs albendazole (ALB) or mebendazole (MEB). However, for the success of these control programs, it is essential to keep track of the efficacy of these drugs and to screen parasite populations for a possible rise of anthelmintic resistance (AR). In this light, a series of trials will be performed to assess the efficacy of ALB treatment against STH in four endemic countries with varying drug pressure. Both microscopic and molecular stool-based diagnostic methods will be used to evaluate drug efficacy. DNA samples will also be analysed for the presence and prevalence of mutations in a gene that was previously linked to AR in animal STHs. The results of these trials will provide evidence on the efficacy of ALB, help select the optimal diagnostic method to assess drug efficacy and provide information regarding the usefulness of genetic markers for AR detection in human STHs.

Background : Standard diagnosis of human soil-transmitted helminth (STH) infections is based on the microscopic detection of helminth eggs in stool and supports programmatic decision making in control programs. However, the current standard diagnostic techniques still show a number of limitations. Recently, the FECPAK(G2) method was developed to detect helminth infections and asses drug efficacy in sheep or cattle. It includes a device that takes digital images of helminth eggs that have been concentrated into one microscopic field of view and stores these images online for future evaluation. The goal of this study was to introduce a standard operating procedure (SOP) for the detection and quantification of human STH eggs using the FECPAK(G2) and to optimize 2 crucial steps of the protocol, namely the sedimentation step (aimed at separating sinking eggs from floating debris) and the accumulation step (aimed at concentrating the eggs by flotation). Methodology/Principal findings : A total of 55 stool samples from naturally infected children were used from 4 different geographical areas (Ethiopia, Laos, Tanzania and Brazil). The results showed that Trichuris eggs generally moved slower than eggs of the other two STH species during both sedimentation in water in the FECPAK(G2) sedimenter as during accumulation in flotation solution in the FECPAK(G2) cassettes. The highest number of eggs were present in the slurry of the sedimenter after overnight sedimentation (Ascaris: 95.7%, Trichuris: 89.8% and hookworm: 94.2% of the eggs). A minimum of 24 minutes were needed to ensure the accumulation of at least 80% of the eggs from all three STH species in the FECPAKG2 cassette (Ascaris: 96.1%; Trichuris: 88.2% and hookworm: 87.6%). Conclusions/Significance : This study introduces for the first time a SOP for the FECPAK method. Different aspects of the method for diagnosing human STH infections were optimized. Our study forms the basis for a thorough and objective evaluation of the system as a diagnostic tool that could be implemented in STH control programs.

Neurocysticercosis, the central nervous system's localised form of cysticercosis, is considered to be the leading cause of epilepsy in the developing world. In Europe, the disease is mainly imported and affects both immigrants and travellers. However, autochthonous cases of cysticercosis in low-endemic countries could also originate from Taenia solium carriers (migrants or travellers) who acquired taeniasis overseas. Management of cysticercosis is a challenge for European healthcare providers as they are often hardly aware of this infection and have little familiarity in managing this disease. This study provides a summary of recommendations concerning screening, diagnosis and management of cysticercosis and T. solium taeniasis in Europe drawn up by nine experts in migrant health and imported diseases with experience in cysticercosis and T. solium taeniasis.

Standard diagnosis of human soil-transmitted helminth (STH) infections is based on the microscopic detection of helminth eggs in stool and supports programmatic decision making in control programs. However, the current standard diagnostic techniques still show a number of limitations. Recently, the FECPAKG2 method was developed to detect helminth infections and asses drug efficacy in sheep or cattle. It includes a device that takes digital images of helminth eggs that have been concentrated into one microscopic field of view and stores these images online for future evaluation. The goal of this study was to introduce a standard operating procedure (SOP) for the detection and quantification of human STH eggs using the FECPAKG2 and to optimize 2 crucial steps of the protocol, namely the sedimentation step (aimed at separating sinking eggs from floating debris) and the accumulation step (aimed at concentrating the eggs by flotation).; A total of 55 stool samples from naturally infected children were used from 4 different geographical areas (Ethiopia, Laos, Tanzania and Brazil). The results showed that Trichuris eggs generally moved slower than eggs of the other two STH species during both sedimentation in water in the FECPAKG2 sedimenter as during accumulation in flotation solution in the FECPAKG2 cassettes. The highest number of eggs were present in the slurry of the sedimenter after overnight sedimentation (Ascaris: 95.7%, Trichuris: 89.8% and hookworm: 94.2% of the eggs). A minimum of 24 minutes were needed to ensure the accumulation of at least 80% of the eggs from all three STH species in the FECPAKG2 cassette (Ascaris: 96.1%; Trichuris: 88.2% and hookworm: 87.6%).; This study introduces for the first time a SOP for the FECPAKG2 method. Different aspects of the method for diagnosing human STH infections were optimized. Our study forms the basis for a thorough and objective evaluation of the system as a diagnostic tool that could be implemented in STH control programs.

Mycotoxins, secondary metabolites produced by moulds, mainly Aspergillus spp., Fusarium spp. and Penicillium spp., are common contaminants of food and feed. The aim of this project was to evaluate: (i) the potential endocrine disruptor effects of fumonisin B1 (FB1), beauvericin (BEA), deoxynivalenol (DON) and zearalenone (ZEA) metabolites ?-zearalenol (?-ZEA) and ?zearalenol (?ZEA), alone and combined, using a bovine granulosa cell (GC) in vitro model and (ii) the individual and combined effects of FB1 and BEA on the intestinal barrier using Caco-2 cells cultured in vitro on semipermeable inserts. The results obtained indicated that FB1 alone at all tested doses (0; 0.5; 1; 1.5; 3; 6 ?M) had no effects on GC proliferation and progesterone (P4) production. In the presence of ?ZEA at30 ng/mL (0.094 ?M), FB1 at 30 ng/mL (0.042 ?M) showed a stimulatory effect on GC numbers. Cell proliferation decreased after exposure to ?ZEA alone at 5.0 mg/mL (15.6 ?M) and FB1 with ?-ZEA and ?ZEA at the same concentration. Regarding steroid production, FB1 at 30 ng/mL (0.042 ?M ) and 100 ng/mL (0.13 ?M amplified the inhibitory effect of ?ZEA at 30 ng/mL (0.094 ?M) on estradiol (E2) production, while FB1 alone increased (P

The diagnosis of soil-transmitted helminths (STHs; Ascaris, Trichuris and hookworms) is traditionally based on the demonstration of eggs in stool using microscopic techniques. While molecular techniques are more appropriate to speciate STH species they are seldom applied. In this study we speciated STH eggs from stool using molecular techniques to gain insights into the distribution of both human and animal STH species in the human host.We speciated 207 STH egg isolates from stool collected during the baseline survey of six drug efficacy trials conducted in Brazil, Cambodia, Cameroon, Ethiopia, Tanzania and Vietnam applying a PCR - restriction fragment length polymorphisms based approach.DNA of Ascaris was detected in 71 (34.3%) samples, of which all were identified as the human roundworm Ascaris lumbricoides. In 87 (42.0%) samples, DNA of Trichuris spp. was found and further speciation demonstrated the presence of the human Trichuris trichiura (100%) and the canine Trichuris vulpis (n=7; 8.0%; in Cameroon only). Hookworms were identified in 104 (50.2%) samples, with Necator americanus (n=73; 70.2%) being the predominant species followed by Ancylostoma duodenale (n=40; 38.5%).Our study indicates that STH infections in humans are predominantly caused by human STH species. They also suggest that zoonotic transmission occurs on a local scale.

Beauvericin (BEA) is a mycotoxin produced by Fusarium species, frequently occurring in cereal grains in combination with fumonisin B1 (FB1) and deoxynivalenol (DON). The aim of this study was to evaluate the in vitro toxic effects of BEA alone and combined with FB1 or DON on human intestinal Caco-2 cells cultured on semi-permeable inserts (Caloni et al., 2012). Caco-2 cells were treated for 24 h with BEA (1.5 µM) alone and combined with FB1 (1.5 µM) or DON (3.5 µM) on both apical (Ap) and basolateral (Bl) sides. Barrier impairment was assessed by measuring the trans-epithelial electrical resistance (TEER) after 1 h, 2 h and 24 h of treatment. At the end of the experiment, the culture medium was collected for interleukin-8 (IL-8) determination. The results indicate that TEER was not significantly affected by Ap or Bl exposure to BEA and FB1 alone, whereas a significant decrease (P, International Journal of Health, Animal Science and Food Safety, Vol 3, No 1s (2016)

Arithmetic mean egg per gram (EPG), egg-reduction rates (ERR) and extended ERRs for the four different treatments against T. trichiura, A. lumbricoides and hookworms infections. (DOCX 17 kb)

Fumonisin B1 (FB1) and beauvericin (BEA) are fusariotoxins found to co-exist in food and feed commodities. The aim of this study is to evaluate the individual and combined effects of FB1 and BEA on bovine granulosa cell proliferation and steroid production. Granulosa cells (GC) from small bovine follicles (1-5 mm) were cultured for 48 hours in 10% fetal bovine serum followed by 48 hours in a serum-free medium containing 500 ng/ml of testosterone (as an estradiol precursor), 30 ng/ml of FSH and 30 ng/ml of IGF-I with and without FB1 (3 µM) and BEA (3 µM). At the end of the experiment, the numbers of GC were determined using a Coulter counter (Beckman Coulter, USA) and concentrations of progesterone and estradiol in the culture medium were determined by radioimmunoassay. FB1 and BEA, both individually and in combination, showed an inhibitory effect (P < 0.05) on GC proliferation. The number of GC decreased by 17.7%, 54.2% and 61.0% after exposure to FB1 (3 µM), BEA (3 µM) and FB1 (3 µM) plus BEA (3 µM) respectively. FB1 (3 µM) had no effect (P > 0.05) on estradiol and progesterone production, whereas BEA (3 µM), both alone and in combination with FB1 (3 µM), was found to decrease (P < 0.001) the production of both steroids drastically. In conclusion, this in vitro study indicates that FB1 and BEA, both individually and in combination, may affect GC proliferation to different extents and shows the drastic inhibitory effects of BEA on steroid production., International Journal of Health, Animal Science and Food Safety, Vol 2, No 1s (2015): Proceeding of Veterinary and Animal Science Days

objective To assess the accuracy of Haemoglobin Colour Scale (HCS) in identifying severely anaemic [haemoglobin (Hb)

Background In low-incidence countries, most tuberculosis (TB) cases occur among migrants and are caused by reactivation of latent tuberculosis infection (LTBI) acquired in the country of origin. Diagnosis and treatment of LTBI are rarely implemented to reduce the burden of TB in immigrants, partly because the cost-effectiveness profile of this intervention is uncertain. The objective of this research is to perform a review of the literature to assess the cost-effectiveness of LTBI diagnosis and treatment strategies in migrants. Methods Scoping review of economic evaluations on LTBI screening strategies for migrants was carried out in Medline. Results Nine studies met the inclusion criteria. LTBI screening was cost-effective according to seven studies. Findings of four studies support interferon gamma release assay as the most cost-effective test for LTBI screening in migrants. Two studies found that LTBI screening is cost-effective only if carried out in immigrants who are contacts of active TB cases. Discussion and Conclusions Our findings support the cost-effectiveness of LTBI diagnostic and treatment strategies in migrants especially if they are focused on young subjects from high incidence countries. These strategies could represent and adjunctive and synergistic tool to achieve the ambitious aim of TB elimination. Electronic supplementary material The online version of this article (doi:10.1186/s12913-015-1045-3) contains supplementary material, which is available to authorized users.

Background Kato-Katz is a widely used method for the diagnosis of soil-transmitted helminth infection. Fecal samples cannot be preserved, and hence, should be processed on the day of collection and examined under a microscope within 60 min of slide preparation. Mini-FLOTAC is a technique that allows examining fixed fecal samples. We assessed the performance of Mini-FLOTAC using formalin-fixed stool samples compared to Kato-Katz and determined the dynamics of prevalence and intensity estimates of soil-transmitted helminth infection over a 31-day time period. Methodology The study was carried out in late 2013 on Pemba Island, Tanzania. Forty-one children were enrolled and stool samples were subjected on the day of collection to a single Kato-Katz thick smear and Mini-FLOTAC examination; 12 aliquots of stool were fixed in 5% formalin and subsequently examined by Mini-FLOTAC up to 31 days after collection. Principal Findings The combined results from Kato-Katz and Mini-FLOTAC revealed that 100% of children were positive for Trichuris trichiura, 85% for Ascaris lumbricoides, and 54% for hookworm. Kato-Katz and Mini-FLOTAC techniques found similar prevalence estimates for A. lumbricoides (85% versus 76%), T. trichiura (98% versus 100%), and hookworm (42% versus 51%). The mean eggs per gram of stool (EPG) according to Kato-Katz and Mini-FLOTAC was 12,075 and 11,679 for A. lumbricoides, 1,074 and 1,592 for T. trichiura, and 255 and 220 for hookworm, respectively. The mean EPG from day 1 to 31 of fixation was stable for A. lumbricoides and T. trichiura, but gradually declined for hookworm, starting at day 15. Conclusions/Significance The findings of our study suggest that for a qualitative diagnosis of soil-transmitted helminth infection, stool samples can be fixed in 5% formalin for at least 30 days. However, for an accurate quantitative diagnosis of hookworm, we suggest a limit of 15 days of preservation. Our results have direct implication for integrating soil-transmitted helminthiasis into transmission assessment surveys for lymphatic filariasis., Author Summary Soil-transmitted helminths are parasitic worms (hookworm, roundworm, and whipworm) that affect hundreds of millions of people. Kato-Katz is the most widely used technique for the diagnosis of soil-transmitted helminth infection. It requires the collection, processing, and microscopic examination of stool samples within the same day. In remote areas, laboratories are often far away from where stool samples are being collected, which makes it difficult to examine the samples on the same day. Mini-FLOTAC is an alternative to Kato-Katz technique that allows analysis of fixed stool samples several days after collection. We assessed the accuracy of the Mini-FLOTAC with fixed stool samples. The study was carried out in late 2013 on Pemba Island and 41 children participated. Fresh stool samples were first examined by Kato-Katz and then fixed in formalin and examined by Mini-FLOTAC (12 examinations within one month). We found that for a qualitative diagnosis (presence or absence of infection) stool samples can be fixed for 31 days. However, for an accurate quantitative diagnosis of hookworm eggs, stool samples should not be stored for more than 15 days, as egg counts decline. Our results have implications for integrating soil-transmitted helminth surveys with other neglected tropical diseases.

Background: Copro-parasitological diagnosis is still a challenge in management of helminth infections at individualand community levels in resource-limited settings.The aim of our study was to compare the performance of three quantitative techniques: Kato-Katz, McMaster andMini-FLOTAC methids. The study was carried out in Oran, Northern Argentina.Methods: 200 schoolchildren were enrolled to provide a single stool sample, which was tested for helminthinfections with Kato-Katz, McMaster and Mini-FLOTAC methods. The Mini-FLOTAC was performed with two flotationsolutions (FS2 saturated saline and FS7 zinc sulphate). Preparation and reading time for each of the three methodswas calculated both when processing single and multiple samples.Results: Out of 193 schoolchildren examined, 40% were positive for any helminth infection by any method; themost prevalent was Hymenolepis nana (23%) followed by Ascaris lumbricoides (17%) and a third group of lessprevalent helminths: Enterobius vermicularis, Trichuris trichiura and hookworms (11% all together). Mini-FLOTAC FS2was more sensitive than FS7 for H. nana (93% vs 78%) and for other helminths (85% vs 80%), whereas FS7 was moresensitive for A. lumbricoides (87% vs 61%). Kato-Katz method was more sensitive than McMaster method for A.lumbricoides (84% vs 48%) and for other helminths (48% vs 43%) except for H. nana (49% vs 61%). As for eggcounts, Mini-FLOTAC FS2 reported 904 eggs per gram of faeces (EPG) for H. nana (vs 457 with McMaster and 111with Kato-Katz) and 1177 EPG for A. lumbricoides (vs 1315 with Kato-Katz and 995 with McMaster); FS2 detected thehighest EPG for both H.nana and A.lumbricoides (904 vs 568 and 1177 vs 643 respectively), the differences werenot statistically significant. The technique feasibility was calculated: Kato-Katz mean time was 48 minutes/sample,Mini-FLOTAC 13 minutes/sample and McMaster 7 minutes/sample. However, especially for Kato-Katz and Mini-FLOTAC,the mean time (min/sample) decreased significantly when processing multiple samples.Conclusions: Mini-FLOTAC is a promising technique for helminth diagnosis, it is more sensitive than Kato-Katz andMcMaster for H. nana and as sensitive as Kato-Katz and more sensitive than McMaster for A. lumbricoides identification.Egg counts differences although relevant, did not reach statistical significance. Fil: Barda, Beatrice. San Raffaele Hospital; Italia Fil: Cajal, Pamela. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Villagran, Eliana. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Cimino, Ruben. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Juarez, Marisa. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Krolewiecki, Alejandro Javier. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina Fil: Rinaldi, Laura. Universidad de Napoles Federico III; Italia Fil: Cringoli, Giuseppe. Universidad de Napoles Federico III; Italia Fil: Burioni, Roberto. San Raffaele Hospital; Italia Fil: Albonico, Marco. Ivo de Carneri Foundation; Italia

Background: The soil-transmitted helminth Strongyloides stercoralis is the most neglected of all neglected helminth infections. It is known to occur worldwide, more prominently in tropical and subtropical resource-poor areas. Environmental and socio-economic factors such as inadequate sanitation contribute to high infection numbers. Today’s estimates for the number of people infected with S. stercoralis globally are cited to be around 30-100 million people, however based on insufficient studies. Compared to the three widely known STHs, namely Ascaris lumbricoides, hookworm and Trichuris trichiura, in most countries, still only fragmentary information is available about the prevalence of S. stercoralis. A main factor contributing to this knowledge gap is the lack of high sensitivity diagnostic methods. The most commonly applied diagnostic methods for helminths have a very low sensitivity for detection of S. stercoralis. There are more appropriate diagnostic methods, yet they are not routinely applied. Furthermore these methods rely on multiple sampling for sensitivity increase, mainly because the irregularities with which S. stercoralis larvae are excreted in low-intensity infections. With Ivermectin, a safe and highly efficacious drug is available for the treatment of S. stercoralis, yet it is not routinely used in helminth control programs. Cambodia, located in Southeast Asia, provides ideal conditions not only for STH-infections, but also other helminthic and protozoan infections. However, there is still only little data available on S. stercoralis. In the rural communities, where people mainly rely on subsistence farming, there is a constant exposure to risk factors for STH-infection. Furthermore, specific details about the parasite, its biology and genetics, as well as transmission dynamics remain poorly understood. E.g., excreted larvae are detected in diagnostic tests, however, excretion dynamics are poorly investigated. Another aspect important for control is the possible zoonotic potential of S. stercoralis. Strongyloides infects animals and humans. If and how frequent infection between animals and humans occurs is unknown. Aims and objectives: The work presented in this PhD was divided into three sections. The first section of the work consisted in examining today’s literature to draw a more accurate picture of the global prevalence, diagnosis and risk factors for S. stercoralis. Furthermore we aimed at analysing information about the treatments available for S. stercoralis today. The second section focused on S. stercoralis in communities in an endemic setting. We aimed of assessing co-infections, risk factors and transmission dynamics. The third section focused on the individual level of infection with S. stercoralis. We aimed to assess the excretion pattern in individuals before treatment and the impact of treatment, we genotyped larvae collected from humans and animals, to get more insight into possible transmission between humans and animals, and we validated a new PCR assay for the detection of S. stercoralis. Methods: For the first section a literature search was conducted including all available studies reporting on the prevalence of S. stercoralis in communities and from hospitals. The data was combined and used for a meta-analysis to give prevalence estimates per country. Further were possible risk factors analysed with the help of a meta-analysis. A second review was conducted collecting information on the treatment of strongyloidiasis. Pooled cure rates were established for all drugs included in a meta-analysis. The second and third section of this work were based on field work conducted in Kandal, Preah Vihear and Takeo province in Cambodia. To assess multiparasitism in humans and animals, a community-based survey was conducted. Inhabitants of rural villages were enrolled, and stool samples collected. The samples were analysed with several diagnostic methods: Kato Katz, Baermann, Koga Agar plate culture, formalin-ethyl acetate concentration method and PCR. Animals samples were analysed with the flotation method and PCR. The studies reported in the third section were undertaken in the framework of our large-scale community-based surveys in Preah Vihear and Takeo province. For the study on the excretion pattern of S. stercoralis larvae, we used a modified Baermann method. By weighing the amount stool used, we established the larvae per gram stool that were excreted. For the genotyping of single worms, the worms were collected either from the Baermann or Koga Agar plate culture and fixed in 70% ethanol for subsequent molecular analysis. For the collection of S. stercoralis from animals, a veterinarian was appointed. This enabled us to collect the stool directly from the animals. Results and conclusions: In total, 354 studies were included in the S. stercoralis prevalence review. Almost two thirds (63.3%) of these studies applied low sensitivity diagnostic methods. Serology was only used in 9.9% of the studies. Only two countries report 40 or more studies, namely Brazil (43 studies) and Thailand (40 studies). For many countries, potentially endemic for S. stercoralis, no data is available, e.g. in sub-saharan Africa where we could identify studies only in 20 (43.5%) of the 46 countries. We conclude that the prevalence of S. stercoralis is still underreported. We demonstrated that there are still many gaps and “black spots” on the world map remaining today. Diagnostic methods most commonly applied in studies on STH infections today are not appropriate for the detection of S. stercoralis, which is one of the main factors for the underreporting of the prevalence. We included 68 studies for the review on treatment. Efficacies of 14 different drugs were reported. The four drugs tested the most include Ivermectin (in 30 studies), Albendazole (in 23 studies), Thiabendazole (in 21 studies) and Mebendazole (in 5 studies). A meta-analysis revealed the best pooled cure rate for Ivermectin with 0.97 (95% BCI: 0.94 – 1.00) and Thiabendazole with 0.95 (95% BCI: 0.92 – 1.00). We conclude that Ivermectin is the best available treatment. Our studies further confirmed the effectiveness and safety in application of ivermectin. As ivermectin already has been applied in mass drug administration (MDA) programmes, for instance in the treatment of lymphatic filariasis, inclusion of ivermectin in helminth control MDA-programmes is an option to be considered in endemic areas. Other potential candidates we report for treatment of S. stercoralis need to be further evaluated, as the heterogeneity of available data (diagnostic methods applied, reports of adverse events) makes it difficult to assess and compare the established cure rates. Our field survey conducted in rural villages in Preah Vihear province, Cambodia, revealed that intestinal multiparasitism was very frequent, in humans as in animals. From 218 humans examined, 191 (87.2%) had at least one intestinal parasitic infection. We could document 14 different parasitic species; eight helminthic and six protozoan parasites. In humans, most frequently diagnosed were hookworms (63.3%), Entamoeba spp. (27.1%), S. stercoralis (24.3%), Giardia duodenalis (22.0%) and Blastocystis hominis (18.4%). In the 94 dogs sampled, the most common infections diagnosed were hookworm (80.8%), Spirometra spp. (21.3%) and Strongyloides spp. (14.9%). Altogether, we detected eleven parasitic species (eight helminhts and three protozoans). Seventy-seven (81.9%) had at least one infection diagnosed. In 76 pigs, 74 (97.4%) had at least one infection diagnosed. Of the twelve detected parasitic infection (eight helminths and four protozoan), the most frequent were Isospora suis (75.0%), Oesophagostomum spp. (73.7%) and Entamoeba spp. (31.6%). The molecular analysis of hookworms revealed that 50.8% of the hookworm infected humans are due to A. ceylanicum. In the dogs positive for hookworm, 90.0% were infected with A ceylanicum. In conclusion, we could demonstrate the high prevalence of intestinal multiparasitism in rural villages in Cambodia, not only in humans but also in domestic animals. We documented that infection with A. ceylanicum is rampant in humans in rural villages, and we showed the potential for zoonotic transmission between dogs and humans. We argue that for prevention and control, integrated approaches are needed, including not only the humans but the animals as well. Our studies focusing on the individual level of strongyloidiasis infection, we analysed the excretion pattern of S. stercoralis larvae. Thirty-nine positive cases were followed for seven days prior to ivermectin treatment. Larvae per gram stool (LPG) were calculated. The highest and lowest average density per person observed were 151.2 LPG (range: 28.8 – 410.6 LPG) and 0.03 LPG (range 0 – 0.07 LPG), respectively. Local maxima estimation technique did not detect a pattern of excretion, indicating that larvae excretion is random and does not follow a cyclic pattern. High-intensity infections (>10 LPG every day) were correctly diagnosed S. stercoralis positive every day. In low-intensity infections, patients did not excrete larvae for up to two consecutive days. They would have been wrongly diagnosed as infection-free in a one day examination. Those results underline that low-intensity infections can easily be missed if only single stool samples are analysed, as the individuals can have excretion-free days. This further emphasizes the importance of analysing samples of the same individual on consecutive days to increase sensitivity of the diagnostic methods and to subsequently better estimate the true prevalence We evaluated the performance of real-time monoplex PCR for detection of S. stercoralis. We compared the PCR assay with the results of coprological diagnostic methods (Baermann and Koga Agar plate culture). We estimated an overall sensitivity and specificity of 61.0% and 92.7%, respectively. The cycle-treshold (ct) values indicate that PCR most often failed to detect infection in low-intensity infections. We conclude that it is a suitable option for individual diagnosis, but is difficult to apply in field settings. We performed a molecular analysis of single S. stercoralis worms. We sequenced 269 individual worms collected from 29 individuals. We could identify three different genotypes of Strongyloides in our study population, co-existing sympatrically within the same host. Yet, we were not able to detect heterozygous worms, which would indicate that there is no interbreeding. With our large-scale studies conducted in Cambodia, we could demonstrate the presence of high infection rates of S. stercoralis, never been reported at that high level in Cambodia. It shows that applying good sensitivity diagnostic methods on consecutive days gives prevalence rates that are a closer to the true prevalence and considerably higher than what has been previously reported. We conclude that this is true for most S. stercoralis endemic settings and hence, the true prevalence is likely to be higher than the numbers reported in studies which apply low sensitivity diagnostic methods.

Background Helminths and protozoa infections pose a great burden especially in developing countries, due to morbidity caused by both acute and chronic infection. The aim of our survey was to analyze the intestinal parasitic burden in communities from Mwanza region, Tanzania. Methods Subjects (n = 251) from four villages on the South of Lake Victoria have been analyzed for intestinal parasites with direct smear (DS), formol-ether concentration method (FECM) and the newly developed Mini-FLOTAC technique; urinary schistosomiasis was also assessed in a subsample (n = 151); symptoms were registered and correlation between clinic and infections was calculated by chi-squared test and logistical regression. Results Out of the subjects screened for intestinal and for urinary parasites, 87% (218/251) were found positive for any infection, 69% (174/251) carried a helminthic and 67% (167/251) a protozoan infection, almost half of them had a double or triple infection. The most common helminths were hookworms, followed by Schistosoma mansoni and Schistosoma haematobium. Among protozoa, the most common was Entamoeba coli followed by Entamoeba histolytica/dispar and Giardia intestinalis. Mini-FLOTAC detected a number of helminth infections (61.7%) higher than FECM (38.6%) and DS (17.9%). Some positive associations with abdominal symptoms were found and previous treatment was negatively correlated with infection. Conclusion Despite the limited size of the examined population the current study indicates a high prevalence of intestinal parasitic infection in Bukumbi area, Tanzania, and Mini-FLOTAC showed to be a promising diagnostic tool for helminth infections. This high parasitic burden calls for starting a regular deworming programme and other preventive interventions in schools and in the community.

Background Helminths and protozoa infections pose a great burden especially in developing, countries, due to morbidity caused both by acute and chronic infections. Data on distribution of intestinal parasitic infections among the native and expatriates populations in Himachal Pradesh are scarce. The aim of our survey was to analyze the intestinal parasitic burden in communities from Dharamsala, Kangra district, in clinical and public health settings. We also field-tested the mini- FLOTAC, an innovative diagnostic device. Methods Subjects referring to the Tibetan Delek Hospital for abdominal discomfort and all children of the Tibetan Primary School in Dharamsala were screened for intestinal parasitic infections with direct smear, formol-ether concentration (FEC) method and mini-FLOTAC, their clinical history was recorded, and correlations between clinical symptoms and infections analyzed. Results 152 subjects were screened for intestinal parasites, of which 72 subjects in the outpatients department (OPD) (36 expatriates and 36 natives) and 80 in the school. 60% of schoolchildren and 57% of OPD patients were found positive for any infection, the most represented were protozoa infections (50%), whereas helminthic infections accounted only for 13% and 20% in OPD patients and schoolchildren, respectively. The most prevalent among helminths was Ascaris lumbricoides (11%). Giardia intestinalis was more present among schoolchildren than the OPD patients (20% vs 6%) and E. histolytica / dispar was more prevalent among the OPD patients (42%) than the school children (23%). Correlations were found between nausea and loose or watery stools and parasitic infections, particularly in expatriates, whereas schoolchildren, despite being as infected as adults, were completely asymptomatic. Mini-FLOTAC detected higher number of helminth infections whereas FEC method was more accurate for the diagnosis of protozoa. Conclusions This study presents an accurate snapshot of intestinal parasitic infections in Dharamsala, and their high prevalence calls for more awareness and control measures. Mini-FLOTAC is a promising and simple technique for the diagnosis of helminth infections.

Background Soil-transmitted helminths and intestinal protozoa infection are widespread in developing countries, yet an accurate diagnosis is rarely performed. The aim of this study was to evaluate the recently developed mini–FLOTAC method and to compare with currently more widely used techniques for the diagnosis of intestinal parasitic infections in different settings. Methodology/Principal Findings The study was carried out in Dharamsala, Himachal Pradesh, India, and in Bukumbi, Tanzania. A total of 180 pupils from two primary schools had their stool analyzed (n = 80 in Dharamsala and n = 100 in Bukumbi) for intestinal parasitic infections with three diagnostic methods: direct fecal smear, formol-ether concentration method (FECM) and mini-FLOTAC. Overall, 72% of the pupils were positive for any intestinal parasitic infection, 24% carried dual infections and 11% three infections or more. The most frequently encountered intestinal parasites were Entamoeba coli, Entamoeba histolytica/dispar, Giardia intestinalis, hookworm, (and Schistosoma mansoni, in Tanzania). Statistically significant differences were found in the detection of parasitic infections among the three methods: mini-FLOTAC was the most sensitive method for helminth infections (90% mini-FLOTAC, 60% FECM, and 30% direct fecal smear), whereas FECM was most sensitive for intestinal protozoa infections (88% FECM, 70% direct fecal smear, and 68% mini-FLOTAC). Conclusion/Significance We present the first experiences with the mini-FLOTAC for the diagnosis of intestinal helminths and protozoa. Our results suggest that it is a valid, sensitive and potentially low-cost alternative technique that could be used in resource-limited settings — particularly for helminth diagnosis., Author Summary The mini-FLOTAC has been recently developed as a novel direct method for the diagnosis of intestinal parasitic infections. Mini-FLOTAC attempts to address the challenge of using modern technology matched with high sensitivity, affordability, and appropriateness of diagnosis in resource-limited settings where intestinal parasitic infections are widespread. We compared accuracy and feasibility of mini-FLOTAC with currently more widely used diagnostic methods, such as the direct fecal smear and the formol-ether concentration method. Our study was carried out in Dharamsala, India, and in Bukumbi, Tanzania in order to evaluate the methods under different field conditions and diverse parasitic infection profiles. Among 180 primary schoolchildren examined, 72% were positive for any intestinal parasitic infection. Mini-FLOTAC detected the highest number of helminth infections (90% sensitivity), whereas the formol-ether concentration was the most sensitive approach for intestinal protozoa infections (88% sensitivity). The logistic advantages of mini-FLOTAC are that the procedure does not require any centrifugation step or expensive equipment, it can be performed on fresh and fixed stool samples, and only requires 10–12 min of preparation before microscopic analysis. Our data suggest that mini-FLOTAC is a promising diagnostic tool for helminth diagnosis; therefore, follow-up studies in other settings are warranted.