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Here the authors provide an extension of their original FLOTAC protocol, describing the Mini-FLOTAC technique, optimized to perform diagnosis of helminth and protozoan infections in humans and animals where centrifugation may not be practical. This protocol is an extension to: Nat. Protoc.5, 503–515 (2010); doi: 10.1038/nprot.2009.235; published online 25 February 2010 The FLOTAC is a sensitive, accurate, and precise technique for the diagnosis of protozoan and helminth infections in humans and animals. However, it requires centrifugation, and hence might be out of reach in resource-constrained settings. As an extension of the original FLOTAC protocol, this protocol describes the Mini-FLOTAC technique, a logical evolution of FLOTAC conceived to perform multivalent, qualitative, and quantitative diagnosis of helminth and protozoan infections in human and animal feces, and urine. This has been found to be of most use in the processing of large numbers of samples with rapid laboratory workup, and for veterinary applications directly on-farm. In addition to the Mini-FLOTAC apparatus, we describe the use of the Fill-FLOTAC, a closed system used to facilitate the performance of the first four consecutive steps of the Mini-FLOTAC technique: fecal sample collection and weighing, homogenization, filtration, and filling of the Mini-FLOTAC chambers. Processing of an individual sample using this protocol requires ∼12 min.

Soil-transmitted helminth (STH) infections are the most widespread of the neglected tropical diseases, primarily affecting marginalized populations in low- and middle-income countries. More than one billion people are currently infected with STHs. For the control of these infections, the World Health Organization (WHO) recommends an integrated approach, which includes access to appropriate sanitation, hygiene education, and preventive chemotherapy (i.e., large-scale, periodic distribution of anthelmintic drugs). Since 2010, WHO has coordinated two large donations of benzimidazoles to endemic countries. Thus far, more than 3.3 billion benzimidazole tablets have been distributed in schools for the control of STH infections, resulting in an important reduction in STH-attributable morbidity in children, while additional tablets have been distributed for the control of lymphatic filariasis. This paper (i) summarizes the progress of global STH control between 2008 to 2018 (based on over 690 reports submitted by endemic countries to WHO); (ii) provides regional and country details on preventive chemotherapy coverage; and (iii) indicates the targets identified by WHO for the next decade and the tools that should be developed to attain these targets. The main message is that STH-attributable morbidity can be averted with evidence-informed program planning, implementation, and monitoring. Caution will still need to be exercised in stopping control programs to avoid any rebound of prevalence and loss of accrued morbidity gains. Over the next decade, with increased country leadership and multi-sector engagement, the goal of eliminating STH infections as a public health problem can be achieved.

Increasing anthelmintic resistance (AR) in livestock has stimulated growing efforts to monitor anthelmintic effectiveness (AE) on livestock farms. On-farm assessment of AE relies on measuring the reduction in faecal egg count (FEC) following treatment; and if conducted rigorously, qualifies as a formal FEC reduction test (FECRT) for AR. Substantial research effort has been devoted to designing robust protocols for the FECRT and its statistical interpretation; however, a wide range of factors other than AR can affect FEC reduction on farms. These are not always possible to control, and can affect the outcome and repeatability of AE measurements and confound the on-farm classification of AR using FECRT. This review considers confounders of FEC reduction, focusing on gastrointestinal nematodes of ruminants, including host and parasite physiology and demography; pharmacokinetic variation between drugs, parasites and hosts; and technical performance. Drug formulation and delivery, host condition and diet, and seasonal variation in parasite species composition, can all affect AE and hence observed FEC reduction. Causes of variation in FEC reduction should be attenuated, but this is not always possible. Regular monitoring of AE can indicate a need to improve anthelmintic administration practices, and detect AR early in its progression. Careful interpretation of FEC reduction, however, taking into account possible confounders, is essential before attributing reduced FEC reduction to AR. Understanding of confounders of FEC reduction will complement advances in FECRT design and interpretation to provide measures of anthelmintic efficacy that are both rigorous and accessible. L’augmentation de la résistance aux anthelminthiques (RA) chez le bétail a stimulé des efforts croissants pour mesurer l’efficacité des anthelminthiques (EA) dans les élevages. L’évaluation à la ferme de l’EA repose sur la mesure de la réduction du nombre d’œufs dans les selles (NOS) après le traitement ; si effectué rigoureusement, celui-ci est qualifié de test formel de réduction du NOS (TFRNOS) pour la RA. Des efforts de recherche substantiels ont été consacrés à la conception de protocoles robustes pour le TFRNOS et son interprétation statistique, mais un large éventail de facteurs autres que la RA peuvent affecter la réduction du NOS dans les exploitations. Ces facteurs ne sont pas toujours contrôlables et peuvent affecter le résultat et la répétabilité des mesures de l’EA et introduire de la confusion dans la classification à la ferme de la RA à l’aide de la TFRNOS. Cette revue examine les facteurs de confusion de la réduction du NOS, en se concentrant sur les nématodes gastro-intestinaux des ruminants, et considère la physiologie et la démographie de l’hôte et du parasite, la variation pharmacocinétique entre les médicaments, les parasites et les hôtes, et les performances techniques. La formulation et l’administration de médicaments, la condition et le régime alimentaire de l’hôte, ainsi que les variations saisonnières de la composition des espèces de parasites, peuvent tous affecter l’EA et donc la réduction du NOS observé. Les causes de variation de la réduction du NOS doivent être atténuées, mais cela n’est pas toujours possible. Une surveillance régulière de l’EA peut indiquer la nécessité d’améliorer les pratiques d’administration des anthelminthiques et de détecter la RA au début de sa progression. Une interprétation prudente de la réduction du NOS, cependant, en tenant compte des facteurs de confusion possibles, est essentielle avant d’attribuer la réduction du NOS à la RA. La compréhension des facteurs de confusion d’une moindre réduction du NOS complétera les progrès sur la conception et l’interprétation du TFRNOS, pour fournir des mesures de l’efficacité des anthelminthiques à la fois rigoureuses et accessibles.

Background An accurate understanding of the geographical distributions of both soil-transmitted helminths (STHs; Ascaris lumbricoides , Trichuris trichiura , and the hookworms Necator americanus and Ancylostoma duodenale ) and schistosomes (SCH; Schistosoma mansoni and S. haematobium ) is pivotal to be able to effectively design and implement mass drug administration (MDA) programmes. The objective of this study was to provide up-to-date data on the distribution of both STH and SCH in Ethiopia to inform the design of the national control program and to be able to efficiently achieve the 75% MDA coverage target set by the WHO. Methods Between 2013 and 2015, we assessed the distributions of STH and SCH infections in a nationwide survey covering 153,238 school-aged children (aged 5–15 years), from 625 woredas (districts), representing all nine Regional States and two City Administrations of Ethiopia. Nationwide disease maps were developed at the woreda level to enable recommendations on the design of the national MDA programme. Results The prevalence of any STH infection across the study population was 21.7%, with A. lumbricoides (12.8%) being the most prevalent STH, followed by hookworms (7.6%) and T. trichiura (5.9%). The prevalence for any SCH was 4.0% in areas where both SCH species were evaluated. Schistosoma mansoni was the most prevalent SCH (3.5 vs 0.3%). STHs were more prevalent in southwest Ethiopia, whereas SCH was found mostly in the west and northeast of the country. The prevalence of moderate-to-heavy intensity infections was 2.0% for STHs and 1.6% for SCH. For STH, a total of 251 woredas were classified as moderately ( n = 178) or highly endemic ( n = 73), and therefore qualify for an annual and biannual MDA program, respectively. For SCH, 67 woredas were classified as endemic and 8 as highly endemic, and hence they require every two years and annual MDA programme, respectively. Conclusions The results confirm that Ethiopia is endemic for both STHs and SCH, posing a significant public health problem. Following the WHO recommendations on mass drug administration, 18 and 14 million school-aged children are in need of MDA for STHs and SCH, respectively, based on the number of SACs that live on the eligible geographical areas.

Because the success of deworming programs targeting soil-transmitted helminths (STHs) is evaluated through the periodically assessment of prevalence and infection intensities, the use of the correct diagnostic method is of utmost importance. The STH community has recently published for each phase of a deworming program the minimal criteria that a potential diagnostic method needs to meet, the so-called target product profiles (TPPs). We compared the diagnostic performance of a single Kato-Katz (reference method) with that of other microscopy-based methods (duplicate Kato-Katz, Mini-FLOTAC and FECPAKG2) and one DNA-based method (qPCR) for the detection and quantification of STH infections in three drug efficacy trials in Ethiopia, Lao PDR, and Tanzania. Furthermore, we evaluated a selection of minimal diagnostic criteria of the TPPs. All diagnostic methods showed a clinical sensitivity of ≥90% for all STH infections of moderate-to-heavy intensities. For infections of very low intensity, only qPCR resulted in a sensitivity that was superior to a single Kato-Katz for all STHs. Compared to the reference method, both Mini-FLOTAC and FECPAKG2 resulted in significantly lower fecal egg counts for some STHs, leading to a substantial underestimation of the infection intensity. For qPCR, there was a positive significant correlation between the egg counts of a single Kato-Katz and the DNA concentration. Our results indicate that the diagnostic performance of a single Kato-Katz is underestimated by the community and that diagnostic specific thresholds to classify intensity of infection are warranted for Mini-FLOTAC, FECPAKG2 and qPCR. When we strictly apply the TPPs, Kato-Katz is the only microscopy-based method that meets the minimal diagnostic criteria for application in the planning, monitoring and evaluation phase of an STH program. qPCR is the only method that could be considered in the phase that aims to seek confirmation for cessation of program. ClinicalTrials.gov NCT03465488.

In this study, the prevalence and intensity of Schistosoma haematobium infection was determined among school-age children living in the Middle and Lower Awash Valley, Afar Regional State of Ethiopia. Between February and May 2014, urine samples were collected from 885 school-age children (5-16 years of age) from the Middle (n = 632; 4 villages) and Lower (n = 253; 3 villages) Awash Valley. All samples were processed using urine filtration to detect and quantify S. haematobium eggs. In addition, a subset of the urine samples was tested for hematuria using a urine dipstick (n = 556). The overall prevalence was 20.8% (95% Confidence Interval (CI) = 18.1%, 23.5%), based on urine filtration but the prevalence considerably varied across villages both in the Middle (from 12.5% to 37.0%) and Lower Awash Valley (from 0 to 5.3%). The overall mean urine egg count (UEC) among the infected children was 4.0 eggs/10 ml of urine (95% CI = 2.43, 5.52). The infection intensity varied from 0.4 eggs/10 ml of urine to 7.7 eggs/10 ml of urine in the Middle Awash Valley, and from 0 to 1.1 eggs/10 ml of urine in Lower Awash Valley. Age and sex were not associated with S. haematobium infection based on the multivariable logistic regression model. The prevalence of hematuria was 56.3% (95% CI = 52.2%, 60.4%) among a subset of the study participants (556) examined using the urine dipstick. The prevalence of hematuria also varies with villages from 8.3% to 93.2%. In conclusion, the prevalence of S. haematobium infection in the Middle Awash Valley was high and it varies across villages. Hence, children living in the present study villages of the Middle Awash Valley need to be treated with praziquantel to reduce morbidity and disrupt transmission.

Laboratory-based studies have highlighted that pooling stool and urine samples can reduce costs and diagnostic burden without a negative impact on the ability to estimate the intensity of soil-transmitted helminth (STH, Ascaris lumbricoides, Trichuris trichiura and hookworms) and schistosome infections (Schistosoma mansoni and S. haematobium). In this study, we compare individual and pooled stool examination strategies in a programmatic setting. Stool samples were collected from 2,650 children in 53 primary schools in Amhara Regional State, Ethiopia, during the national mapping of STHs and schistosome infections. Eggs of STHs and S. mansoni were quantified in both individual and pooled samples (pools were made from 10 individual samples) using a single Kato-Katz smear. A pooled diagnostic examination strategy provided comparable estimates of infection intensity with higher fecal egg count (expressed in eggs per gram of stool (EPG)) than those based on individual strategy (Ascaris: 45.1 EPG vs. 93.9, p = 0.03; Trichuris: 1.8 EPG vs. 2.1 EPG, p = 0.95; hookworms: 17.5 EPG vs. 28.5 EPG, p = 0.18; S. mansoni: 1.6 EPG vs. 3.4 EPG, p = 0.02), but had lower sensitivity (Ascaris: 90.0% vs. 55.0%; Trichuris: 91.7% vs. 16.7%; hookworms: 92.6% vs. 61.8%; S. mansoni: 100% vs. 51.7%, p < 0.001). A pooled approach resulted in a ~70% reduction in time required for sample testing, but reduced total operational costs by only ~11%. A pooled approach holds promise for the rapid assessment of intensity of helminth infections in a programmatic setting, but it is not major cost-saving strategy. Further investigation is required to determine when and how pooling can be utilized. Such work should also include validation of statistical methods to estimate prevalence based on pooling samples. Finally, the comparison of operational costs across different scenarios of national program management will help determine whether pooling is indeed worthwhile considering.

Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and A. ceylanicum), Strongyloides stercoralis and Schistosoma in human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used. A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing. We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs.

Giardia and Cryptosporidium are important causes of diarrhoea in Bangladesh. The high prevalence of both parasites in humans and cattle in rural Bangladesh and the common use of water ponds by village inhabitants and their animals suggest a potential for zoonotic transmission. Direct transmission of Giardia and Cryptosporidium between cattle and their handlers and indirect transmission through water ponds was investigated. Faecal/stool samples were collected from 623 calves and 125 calf handlers in a cross-sectional survey. In two villages, water samples were collected monthly from water ponds and faecal/stool samples were collected monthly from inhabitants and their cattle. Giardia cysts and Cryptosporidium oocysts were detected in water samples and in faecal/stool samples and positive samples were genotyped, to determine their human or animal origin. The prevalence of Giardia and Cryptosporidium in calves was 22% and 5% respectively. In calf handlers, the prevalence of Giardia and Cryptosporidium was 11.2% and 3.2% respectively. Both in the cross-sectional survey and in the longitudinal study in the villages, G. duodenalis assemblage E was most prevalent in calves, while in humans assemblage AII, BIII and BIV were found. In cattle, Cryptosporidium parvum, C. bovis and C. andersoni were identified, but no Cryptosporidium sequences were obtained from humans. Giardia and Cryptosporidium were detected in 14/24 and 12/24 water samples respectively. G. duodenalis assemblage E and BIV (-like), as well as C. andersoni and C. hominis were identified. Although the presence of Giardia and Cryptosporidium in both water ponds suggests that water-borne transmission of Giardia and Cryptosporidium is possible, the genotyping results indicate that there is no significant direct or indirect (water-borne) transmission of Giardia between cattle and people in this area of rural Bangladesh. No conclusions could be drawn for Cryptosporidium, because of the low number of sequences that were obtained from human and water samples.

Preventive chemotherapy (PC) with benzimidazole drugs is the backbone of soil-transmitted helminth (STH) control programs. Over the past decade, drug coverage has increased and with it, the possibility of developing anthelmintic resistance. It is therefore of utmost importance to monitor drug efficacy. Currently, a variety of novel diagnostic methods are available, but it remains unclear whether they can be used to monitor drug efficacy. In this study, we compared the efficacy of albendazole (ALB) measured by different diagnostic methods in a head-to-head comparison to the recommended single Kato-Katz. An ALB efficacy trial was performed in 3 different STH-endemic countries (Ethiopia, Lao PDR and Tanzania), each with a different PC-history. During these trials, stool samples were evaluated with Kato-Katz (single and duplicate), Mini-FLOTAC, FECPAKG2, and qPCR. The reduction rate in mean eggs per gram of stool (ERR) and mean genome equivalents / ml of DNA extract (GERR) were calculated to estimate drug efficacy. The results of the efficacy trials showed that none of the evaluated diagnostic methods could provide reduction rates that were equivalent to a single Kato-Katz for all STH. However, despite differences in clinical sensitivity and egg counts, they agreed in classifying efficacy according to World Health Organization (WHO) guidelines. This demonstrates that diagnostic methods for assessing drug efficacy should be validated with their intended-use in mind and that other factors like user-friendliness and costs will likely be important factors in driving the choice of diagnostics. In addition, ALB efficacy against STH infections was lower in sites with a longer history of PC. Yet, further research is needed to identify factors that contribute to this finding and to verify whether reduced efficacy can be associated with mutations in the β-tubulin gene that have previously been linked to anthelmintic resistance. ClinicalTrials.gov NCT03465488.