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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.

[...]Target #6 aims to achieve universal access to basic sanitation and hygiene in STH-endemic areas. [...]we identified some opportunities to improve existing diagnostic technologies. [...]our experience with the A. lumbricoides copro-antigen ABA-1 [11] points toward the same complexity as for the non-stool-based approaches, namely that (i) stool biomarkers are not yet identified for all STHs; (ii) multiplexing is possible only after (iii) sensitivity; and (iv) specificity for each biomarker and for each infection is fully optimized. [...]v) the relationship between antigens and morbidity is yet to be determined (if possible at all); and (vi) additional processing steps might be required (bead beating) which affect the user-friendliness. [...]it is unlikely that such copro-antigen technologies will become available within the time frame of the 2030 roadmap.

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.

This study compared the clinical sensitivity and the time-to-result of an individual testing (IT) and a cascaded pooled testing approach (CPT; a positive test result in a pooled sample triggers examination of smaller-sized pools or individual samples) for assessing the prevalence and the intensity of Schistosoma haematobium infection. We also compared the sensitivity of the CPT in detecting S. haematobium infection when deploying urine filtration microscopy (UFM) vs. urine reagent strips (URS), and testing 10 mL vs. 15 mL of urine. Between October 2021 and April 2022, S. haematobium eggs were counted in urine samples collected from school-aged children living in the Afar and Gambella Regional States of Ethiopia. Urine samples were collected at baseline (n = 1,288), and one month after administration of praziquantel (n = 118). All urine samples were processed through both an IT and a CPT approach (pools of 5, 10, 20, and 40 individual samples), deploying UFM (10 mL) and URS (10 mL). In addition, 15 mL urine was processed through the CPT deploying UFM. At baseline, the prevalence of S. haematobium infection estimated when using UFM and deploying a CPT approach was significantly lower (17.3%) compared to an IT approach (31.5%). The clinical sensitivity of the CPT in detecting S. haematobium eggs was 51.7%. The sensitivity increased significantly as a function of increasing log transformed urine egg counts (UECs) of the individual samples (OR 2.71, 95%CI 1.63 - 4.52). The sensitivity was comparable when the amount of urine examined was 10 mL (51.7%) vs. 15 ml (50.8%), and when UFM was used for testing vs. URS (51.5%). The mean log UECs estimated following the CPT approach was lower compared to the estimate by the IT (p <0.001). UECs of the individual samples estimated using the IT and CPT approaches were moderately correlated (r = 0.59 when 10 mL and 15 mL urine was examined after pooling). CPT reduced the time needed for processing urine samples and testing for S. haematobium infection by 29% with UFM and by 27.7% with URS. CPT based on UFM and URS techniques may help to rapidly identify areas with higher prevalence of S. haematobium infection (hotspots) in a population. However, the performance of this approach in estimating the prevalence of infection may be compromised, particularly in endemic areas with low intensity infection.

The World Health Organization emphasizes the importance of integrated monitoring and evaluation in neglected tropical disease (NTD) control programs. Serological assays offer a potential solution for integrated diagnosis of NTDs, particularly for those requiring mass drug administration (MDA) as primary control and elimination strategy. This scoping review aims (i) to provide an overview of assays using serum or plasma to detect infections with soil-transmitted helminths (STHs) in both humans and animals, (ii) to examine the methodologies used in this research field and (iii) to discuss advancements in serological diagnosis of STHs to guide prevention and control programs in veterinary and human medicine. We conducted a systematic search in the Ovid MEDLINE, Embase and Cochrane Library databases, supplemented by a Google search using predefined keywords to identify commercially available serological assays. Additionally, we performed a patent search through Espacenet. We identified 85 relevant literature records spanning over 50 years, with a notable increased interest in serological assay development in recent years. Most of the research efforts concentrated on diagnosing Ascaris infections in both humans and pigs, primarily using ELISA and western blot technologies. Almost all records targeted antibodies as analytes, employing proteins and peptides as analyte detection agents. Approximately 60% of sample sets described pertained to human samples. No commercially available tests for Trichuris or hookworms were identified, while for Ascaris, there are at least seven different ELISAs on the market. While a substantial number of assays are employed in epidemiological research, the current state of serological diagnosis for guiding STH prevention and control programs is limited. Only two assays designed for pigs are used to inform efficient deworming practices in pig populations. Regarding human diagnosis, none of the existing assays has undergone extensive large-scale validation or integration into routine diagnostics for MDA programs.

Background Large-scale deworming programs have been successful in reducing the burden of disease due to soil-transmitted helminth (STH; Ascaris lumbricloides, Trichuris trichiura and hookworm) infections, but re-infection in absence of other measures is unavoidable. We assessed the role of nail contamination as a source of infection with the goal to evaluate the potential of nail clipping as a simple measure to further reduce STH-attributable morbidity. Methods A cross-sectional study was conducted in Jimma Town (Ethiopia). Both stool samples and clipped nails were collected from 600 schoolchildren and microscopically screened for the presence of STHs. We also interviewed the children to gain insights into their hygiene practices. Subsequently, we explored any associations between infection, nail contamination and personal hygiene. Results Any STH infections were observed in 24.3% of the children (A. lumbricoides: 18.5%; T. trichiura: 9.8%; hookworm: 0.5%). The intensity of the infections was mainly low, only in a few cases a moderate-to-heavy intensity infection was observed (A. lumbricoides: 4.3%; T. trichiura: 0.2%). Other helminth species observed were Schistosoma mansoni (5.0%), Hymenolepis nana (2.7%), Taenia spp. and Enterobius vermicularis (<1.0%). The analysis of the nail material revealed the presence of A. lumbricoides (1.7%), Taenia spp. (1.0%), T. trichiura (0.5%), E. vermicularis (0.5%) and H. nana (0.2%). The odds of infection with any STH increased as the frequency of trimming decreased. The odds of nail contamination with any STH and A. lumbricoides were higher for younger children. Conclusions The presence of helminth eggs under the nails of children highlights a poor personal hygiene. The association between any STH infection and frequency of nail trimming needs to be explored in an intervention study. The recent prevalence of any STH infections indicated that scaling down of the frequency of deworming is justified but that STH is still a public health problem.

It remains largely unknown where and how infections with soil-transmitted helminths (STHs; Ascaris, Trichuris, Necator and Ancylostoma) occur. We therefore aimed to identify possible sources of infection by assessing the environmental contamination in an STH-endemic area. We first performed a series of laboratory experiments designed to optimize a soil straining-flotation method to detect and quantify Ascaris and Trichuris eggs in soil, and to validate the diagnostic performance of the optimized method when followed by microscopy and qPCR. In a second phase, we applied this method to assess the level of STH contamination in 399 environmental samples collected from 10 school compounds, 50 households and 9 open markets in Jimma Town (Ethiopia). Subsequently, we explored associations between the environmental contamination and both the corresponding STH epidemiology at the level of the schools and the household characteristics. Finally, we assessed the knowledge, attitude and practice (KAP) towards STHs in school children. Our soil straining-flotation method has an analytical sensitivity of 50 eggs per 100 grams of soil and egg recovery rate of 36.0% (Ascaris) and 8.0% (Trichuris). The analysis of field samples with both microscopy and qPCR revealed the presence of 8 different helminth species of medical importance, including but not limited to the human STHs. There was a significant association between the environmental contamination and prevalence of any STH infections at the school level only. The KAP indicated a lack of knowledge and awareness of STHs. Our optimized straining-flotation method has a moderate diagnostic performance and revealed that life stages of helminths are ubiquitous in the environment, which might be due to the poor sanitary facilities at both the schools and the households, and a poor level of KAP towards STHs. Further research is required to gain more insights into the contribution of these life stages to transmission.

Background With the World Health Organization's (WHO) publication of the 2021-2030 neglected tropical diseases (NTDs) roadmap, the current gap in global diagnostics became painfully apparent. Improving existing diagnostic standards with state-of-the-art technology and artificial intelligence has the potential to close this gap. Methodology/Principal findings We prototyped an artificial intelligence-based digital pathology (AI-DP) device to explore automated scanning and detection of helminth eggs in stool prepared with the Kato-Katz (KK) technique, the current diagnostic standard for diagnosing soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura and hookworms) and Schistosoma mansoni (SCH) infections. First, we embedded a prototype whole slide imaging scanner into field studies in Cambodia, Ethiopia, Kenya and Tanzania. With the scanner, over 300 KK stool thick smears were scanned, resulting in total of 7,780 field-of-view (FOV) images containing 16,990 annotated helminth eggs (Ascaris: 8,600; Trichuris: 4,083; hookworms: 3,623; SCH: 684). Around 90% of the annotated eggs were used to train a deep learning-based object detection model. From an unseen test set of 752 FOV images containing 1,671 manually verified STH and SCH eggs (the remaining 10% of annotated eggs), our trained object detection model extracted and classified helminth eggs from co-infected FOV images in KK stool thick smears, achieving a weighted average precision (± standard deviation) of 94.9% ± 0.8% and a weighted average recall of 96.1% ± 2.1% across all four helminth egg species. Conclusions/Significance We present a proof-of-concept for an AI-DP device for automated scanning and detection of helminth eggs in KK stool thick smears. We identified obstacles that need to be addressed before the diagnostic performance can be evaluated against the target product profiles for both STH and SCH. Given that these obstacles are primarily associated with the required hardware and scanning methodology, opposed to the feasibility of AI-based results, we are hopeful that this research can support the 2030 NTDs road map and eventually other poverty-related diseases for which microscopy is the diagnostic standard.

A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNAlater and were stored at 4°C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs.

Monitoring and evaluation (M&E) is a key component of large-scale neglected tropical diseases (NTD) control programs. Diagnostic tests deployed in these M&E surveys are often imperfect, and it remains unclear how this affects the population-based program decision-making. We developed a 2-stage lot quality assurance sampling (LQAS) framework for decision-making that allows for both imperfect diagnostics and spatial heterogeneity of infections. We applied the framework to M&E of soil-transmitted helminth control programs as a case study. For this, we explored the impact of the diagnostic performance (sensitivity and specificity), spatial heterogeneity (intra-cluster correlation), and survey design on program decision-making around the prevalence decisions thresholds recommended by WHO (2%, 10%, 20% and 50%) and the associated total survey costs. The survey design currently recommended by WHO (5 clusters and 50 subjects per cluster) may lead to incorrect program decisions around the 2% and 10% prevalence thresholds, even when perfect diagnostic tests are deployed. To reduce the risk of incorrect decisions around the 2% prevalence threshold, including more clusters (≥10) and deploying highly specific diagnostic methods (≥98%) are the most-cost saving strategies when spatial heterogeneity is moderate-to-high (intra-cluster correlation >0.017). The higher cost and lower throughput of improved diagnostic tests are compensated by lower required sample sizes, though only when the cost per test is <6.50 US$ and sample throughput is ≥3 per hour. Our framework provides a means to assess and update M&E guidelines and guide product development choices for NTD. Using soil-transmitted helminths as a case study, we show that current M&E guidelines may severely fall short, particularly in low-endemic and post-control settings. Furthermore, specificity rather than sensitivity is a critical parameter to consider. When the geographical distribution of an NTD within a district is highly heterogeneous, sampling more clusters (≥10) may be required.