BackgroundThe World Health Organization (WHO) has emphasised the need for innovative diagnostic tools to support the control and elimination of neglected tropical diseases (NTDs). Microscopy-based diagnostics, the current standard, rely on trained technicians for labour-intensive processes, posing logistical challenges in the low-resource settings where NTDs are most prevalent. This study describes the technical details of an artificial intelligence-powered digital pathology (AI-DP) platform designed to support large-scale deworming programs for two NTDs, alongside its analytical performance and user experience in laboratory and field settings.Methodology/principal findingsThe AI-DP platform integrates electronic data capture tools, whole-slide imaging scanners, onboard AI analysis, and result verification software to automate microscopy-based screening. Targeting soil-transmitted helminthiasis (STH) and intestinal schistosomiasis (SCH) as initial use cases, the system was deployed in Ethiopia and Uganda, scanning 951 Kato-Katz (KK) thick smears containing 43,919 verified helminth eggs. Using 5-fold cross-validation, precision/recall/average precision were 95.4%/91.7%/97.1% for Ascaris lumbricoides, 95.9%/86.7%/94.8% for Trichuris trichiura, 84.6%/86.6%/91.4% for hookworm, and 89.1%/79.1%/89.2% for Schistosoma mansoni. Feedback from 14 field users across 30 real-world scenarios indicated the AI-DP platform's improved usability, particularly in hardware portability and software interfaces, though the average scan time of 12.5 minutes per smear was identified as a limitation.Conclusions/significanceThe AI-DP platform demonstrates potential as a tool for efficient monitoring and evaluation of STH and SCH control programs by providing near-real-time data with quality controls. However, further validation studies are needed to assess its clinical diagnostic performance, field usability, and cost-effectiveness in large-scale STH and SCH deworming programs. Given that the platform also provides a pipeline for any microscopy-based diagnosis, its potential for other NTDs also needs further attention.

Soil-transmitted helminthiases are recognised by the World Health Organization as one of the 20 neglected tropical diseases, primarily affecting communities with socioeconomic disadvantages in tropical and subtropical regions. Of the four soil-transmitted helminths, Ascaris stands out as the most widespread, affecting more than 700 million people globally. Today, the diagnostic standard for ascariasis is based on microscopic examination of stool, which faces important limitations. Although serological diagnosis is a promising alternative, the current landscape of well-validated commercial serological diagnostics is sobering. An ELISA based on homogenate from Ascaris suum lung stage larvae (AsLungL3-ELISA) showed significant potential to inform human and veterinary prevention and control programs against ascariasis. Therefore, this study aimed to identify the immunogenic proteins in Ascaris lung stage larval homogenate and investigate the antibody response towards recombinantly expressed versions of these proteins. Given the potential of recombinant-based assays for both human and veterinary applications, the study encompasses experiments involving both humans and pigs. First, immuno-affinity purifications were coupled with liquid chromatography-tandem mass spectrometry, resulting in three lists of immunogenic proteins (for children, adults, and pigs). As a proof of concept, four promising immunogenic proteins (polyprotein ABA-1, paramyosin, apolipophorin and an S60 ribosomal protein) were recombinantly produced in Escherichia coli and the antibody response against these recombinants was evaluated using ELISA. While the results for pigs were inconclusive due to non-specific binding of antibodies, the findings for potential human serodiagnostic applications detecting IgG4 appeared promising. For both polyprotein ABA-1 and paramyosin, a notable difference in OD values was observed between children and adults who were AsLungL3-ELISA negative and positive. In conclusion, this study is a steppingstone towards the development of new serodiagnostic assays and demonstrates that recombinant protein production offers an efficient method to produce diagnostic Ascaris antigens without requiring pig studies.

Childhood cancers pose a significant public health challenge in sub-Saharan Africa (SSA) due to complex interlinked socioeconomic factors that contribute to delayed diagnoses, limited treatment options, and unacceptable survival rates. This systematic review aims to present insights into the barriers to receiving timely diagnosis and treatment of childhood cancers in SSA. A systematic literature search was conducted for published full-text articles from January 2000 to June 2024 in SSA across three databases: PubMed, Google Scholar, and Web of Science. We used a qualitative synthesis approach to examine the barriers and reasons for delays in seeking, reaching, or receiving quality care. We identified 12 studies, of which 10 focused on all types of childhood cancer, retinoblastoma, and leukemia. When exploring barriers contributing to delays, eight key themes emerged from the data. The first theme was health service barriers, which included: poor cancer care and health system delays; lack of diagnostic and therapeutic services; and lack of childhood cancer policy. The second theme was healthcare provider-related barriers, which included inadequate knowledge of diagnosis and characterization; service burden and lack of training in cancer screening and diagnosis. The third to eighth themes were limited understanding of childhood cancer among the population (theme 3), financial challenges/household poverty (theme 4), emotional and psychological challenges (theme 5), poor healthcare-seeking behavior (theme 6), stigma and discrimination (theme 7), and limited social support and cultural beliefs (theme 8). Barriers that contributed to the delay in the timely diagnosis and treatment of childhood cancer were healthcare provider-related barriers, limited understanding of childhood cancer, financial challenges/household poverty, emotional and psychological challenges, poor healthcare-seeking behavior, stigma and discrimination, and limited social support and cultural beliefs. Therefore, targeted education campaigns, community outreach programs, and policy changes to mitigate these barriers and improve outcomes for children with cancer are important.

Background:Effective infection prevention and control (IPC) and antimicrobial stewardship programs (ASPs) are essential components of hospitals. This study aims to evaluate the status of IPC and ASPs at Jimma University Medical Center (JUMC), a tertiary care hospital, in Southwest Oromia, Ethiopia. Methods:A facility-based cross-sectional study was conducted in two parts. In part one, data on the core components of IPC were collected using the WHO Infection Prevention and Control Assessment Framework (IPCAF) tool. The hospital's IPC level was determined based on the total scores for the core components. In part two, ASP assessment tool was used to gather data on the core elements of ASPs, and the hospital's ASP status was determined based on the total scores. Results:The overall IPC scores of JUMC was 455, corresponding to an intermediate level. The highest scores were observed for the IPC program (70), monitoring and feedback of IPC practices (67.5), and multimodal strategies (65). The lowest scores were for hospital-acquired infections (HAIs) surveillance (25) and IPC education and training (50). Regarding ASPs, the overall score was 51.2%, indicating a moderate level of ASP. Significant gaps were found in ASP core elements; education (0%), tracking antibiotic use (27.3%) and hospital leadership commitment (42.9%). Conclusions:The IPC and ASP programs at JUMC are performing at intermediate and moderate levels, respectively. Attention is required to strengthen HAIs surveillance, tracking of antibiotic uses, and education and training. The overlap between IPC and ASPs presents an opportunity for future collaborative efforts to enhance both programs.

Schistosomiasis and soil-transmitted helminth (STH) infections occurring during pregnancy may pose adverse health consequences to the mother and the developing baby. This study aims to determine the prevalence of Schistosoma mansoni and STHs, and their association with adverse birth outcomes among pregnant women in Jimma Town. A cross-sectional study involving 314 pregnant women was conducted in Jimma Town, Southwest Ethiopia. The pregnant women were recruited from selected public health facilities during their antenatal care (ANC) visits from August to December 2021. Data on demographic characteristics and factors associated with S. mansoni and STH infections were collected using a pretested questionnaire. Moreover, during the third trimester, stool specimen of each pregnant woman was examined using Kato-Katz technique, and hemoglobin was measured using a HemoCue analyzer. Data on adverse birth outcomes were collected during delivery. Data were analyzed using STATA-MP_12 (StataCorp., TX, USA). The overall prevalence of intestinal helminthic infections was 26.1

Background We aimed to gain insights into the role of known single nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 of the ß-tubulin gene as markers for possible benzimidazole resistance in human soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura , Necator americanus and Ancylostsoma duodenale ). Methods Firstly, we determined the analytical performance of our PCR/pyrosequencing assays. Secondly, we applied them on stool samples collected during clinical trials in Ethiopia, Lao PDR, and Pemba Island (Tanzania) to assess any associations between the presence/ratio of mutant (MT): wild type (WT) SNPs and drug pressure history, individual drug response and time of sampling (baseline vs. follow-up sample). Principal findings Overall, the limit of blank of our in-house PCR/pyrosequencing assays to detect MT SNPs was non-zero (∼3.5%), and hence the limit of detection for MT SNPs was relatively high (2% – 7%). The assays systematically overestimated the true underlying ratio of MT:WT SNPS within sample, but we derived functions for more accurate estimates. The assays were more precise when the ratio MT:WT SNPs was high (>5%). No PCR amplicon was observed in 25% of the samples subjected to PCR. In the remaining samples, the presence of MT SNPs in codon 200 was detected in half of the analysed Trichuris samples, the proportion of the analysed samples containing MT SNPs did not exceed 14% for all other codons and STH species. Associations between drug pressure history, individual drug response and time of sampling, were not consistent across all codons and STHs. Conclusion We could not provide compelling evidence for the role of the known SNPs in the ß-tubulin gene as markers for benzimidazole resistance. Our study also highlights that there is a need to assess the diagnostic performance of any assays in order to readily interpret and compare results. Further research should therefore also focus on genes other than the ß-tubulin genes. Author summary Although large-sale deworming programs are reducing the morbidity caused by intestinal worms, widespread treatment of large populations for a long period of time may trigger drug resistance. An early detection of DNA mutations that may give rise to resistant worm population is therefore important. We evaluated the analytical performance of in-house assays to detect DNA mutations that are known to cause resistant intestinal worms of animals. Subsequently, we applied these assays on stool samples to verify (i) whether the mutations are more prevalent in areas were large proportions of children have been dewormed for a longer period, (ii) a poor individual drug response can be explained by higher frequency of the mutations. Our results indicate that comprehensive evaluation of the analytical performance of the genotyping tests was required to readily interpret the results. We did not find any compelling evidence that the presence of mutations was associated with either drug pressure or poor individual drug responses. This suggests that it is warranted to explore other mutations than those documented in animal worms. ### Competing Interest Statement The authors have declared no competing interest.

Background Soil-transmitted helminth (STH) control programs currently lack evidence-based recommendations for cost-efficient survey designs for monitoring and evaluation. Here, we present a framework to provide evidence-based recommendations, using a case study of therapeutic drug efficacy monitoring based on the examination of helminth eggs in stool. Methods We performed an in-depth analysis of the operational costs to process one stool sample for three diagnostic methods (Kato-Katz, Mini-FLOTAC and FECPAK G2 ). Next, we performed simulations to determine the probability of detecting a truly reduced therapeutic efficacy for different scenarios of STH species ( Ascaris lumbricoides , Trichuris trichiura and hookworms), pre-treatment infection levels, survey design (screen and select ( SS ); screen, select and retest ( SSR ) and no selection ( NS )) and number of subjects enrolled (100–5,000). Finally, we integrated the outcome of the cost assessment into the simulation study to estimate the total survey costs and determined the most cost-efficient survey design. Principal findings Kato-Katz allowed for both the highest sample throughput and the lowest cost per test, while FECPAK G2 required both the most laboratory time and was the most expensive. Counting of eggs accounted for 23% (FECPAK G2 ) or ≥80% (Kato-Katz and Mini-FLOTAC) of the total time-to-result. NS survey designs in combination with Kato-Katz were the most cost-efficient to assess therapeutic drug efficacy in all scenarios of STH species and endemicity. Conclusions/significance We confirm that Kato-Katz is the fecal egg counting method of choice for monitoring therapeutic drug efficacy, but that the survey design currently recommended by WHO ( SS ) should be updated. Our generic framework, which captures laboratory time and material costs, can be used to further support cost-efficient choices for other important surveys informing STH control programs. In addition, it can be used to explore the value of alternative diagnostic techniques, like automated egg counting, which may further reduce operational costs. Trial Registration ClinicalTrials.gov NCT03465488

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.

Background We previously demonstrated that serology holds promise as an alternative diagnostic tool to copromicroscopy to monitor and evaluate deworming programs targeting soil-transmitted helminths (STHs). Here we explored the dynamics of anti-Ascaris antibodies (Ab) and evaluated the Ab-isotype of choice to assess the longitudinal exposure to Ascaris in Ethiopian school children. Methodology Between October 2018 and February 2020, stool and blood samples were collected every four months from school children (4 to 6 years of age). Stool samples were analyzed by duplicate Kato-Katz to assess the presence and intensity of any STH infection. Plasma Ab-responses against the total extract of Ascaris suum lung third stage larvae were measured through in-house Ab-ELISA’s for seven different Ab-isotypes. Principal findings At baseline, 42.4% of the 66 children were excreting eggs of any STH, Trichuris (37.9%) being the most prevalent. The cumulative prevalence (proportion of children tested that positive at least once over the entire study period) was 56.1% for Trichuris and 31.8% for Ascaris. For Ascaris, re-infections were frequently observed, whereas for Trichuris, children often remained excreting eggs following drug administration. When measuring anti-Ascaris Ab-levels, the cumulative seroprevalence was generally higher (IgG4: 60.6%; IgG1: 50.0%; IgE: 36.4%). The individual anti-Ascaris IgG4 levels at baseline were positively associated with the fecal egg counts averaged over the study period, the rate of egg-appearance and the number of positive test results. There was no apparent cross-reactivity between the anti-Ascaris IgG4 Ab-ELISA and Trichuris. Conclusions/Significance We demonstrate that the children are exposed to STH before the age of four and that the exposure to Ascaris is underestimated when measured with copromicroscopy. Compared to other Ab-isotypes, IgG4 is the Ab-isotype of choice to measure Ascaris exposure in STH endemic settings. Finally, the results also highlight that measuring anti-Ascaris IgG4 levels holds promise as a tool to identify individuals at higher risk for continued exposure to this STH.

Background 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. Methods 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. Principal findings 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. Conclusions/Significance 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 The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding β-tubulin proteins, the principal targets of BZ drugs. Methodology and principal findings First, the β-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different β-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. Significance This study demonstrated the presence of at least seven β-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the β-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of β-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

The scientific community identified non stool-based biomarkers as the way forward to support soil-transmitted helminth (STH; Ascaris lumbricoides , Trichuris trichiura and the hookworms Ancylostoma duodenale and Necator americanus ) and schistosome ( S . mansoni and S . haematobium ) deworming programs. This support is needed in making the decision of whether or not to stop preventive chemotherapy intervention efforts and to ultimately transition towards a post-intervention surveillance phase. We applied a two-step micro-array approach to identify antigenic linear epitopes in the STH and S . mansoni proteomes. In a first experiment, we identified antigenic peptides by applying sera from 24 STH and/or S . mansoni infected Ethiopian children on a high-density peptide microarray containing 3.3 million peptides derived from the complete STH and S . mansoni proteomes. A second array experiment with 170,185 peptides that were recognized in the first array was designed to identify non-specific antibody reactivity by applying sera from 24 healthy individuals from Belgium (a non-endemic country). From this array testing cascade, several peptides were identified for STH but none of them appeared to be unique for one species. We therefore concluded that for STH, none of the peptides revealed to be sufficiently sensitive or species specific. For S . mansoni , some promising peptides were identified prompting future investigation. Based on these results, it is unlikely that linear epitopes would be highly useful in detecting species-specific antibody responses to STH in endemic communities. For S . mansoni , one particular peptide of the micro-exon gene 12 (MEG-12) protein deserves further research. In addition, this study emphasizes the need of well-characterized biobanks for biomarker discovery, particularly when the integration of multiple disease programs is envisioned.

Cystic echinococcosis (CE), also called hydatid disease (HD) is a parasitic disease caused by larval stage of Echinococcus granulosus and is considered as a food-borne zoonotic, neglected tropical disease (NTD). Backyard slaughtering and inadequate veterinary services are major risk factors for high prevalence of the disease in developing countries. Here, we determined the prevalence of HD in the cattle slaughtered in the municipal abattoir of Jimma Town, Southwest Ethiopia by an abattoir based cross-sectional study, which included 389 randomly selected cattle. The source of cattle, age, sex, breed, the body condition and any acute illness were assessed in ante-mortem investigations. Moreover, during post-mortem examination, vital organs suspected for hydatid cysts (HCs) were carefully examined by close inspection, digital palpation and sharp incision. Fertility and viability were tested for all HCs containing hydatid fluid. The overall prevalence of HC among the cattle was 52.9% (206/389). Lungs were the most affected organ 70.9% (146/206), and 29.6%, 62.1%, and 8.7% of the HCs were fertile, sterile and calcified, respectively. The HC abundance and viability had statistically significant association with the organs affected, and fertile HCs were most common at lung. Collectively, our study revealed that CE is highly prevalent in slaughtered cattle at the Jimma area, Ethiopia and lungs are the major affected organs. An integrated prevention and control strategies from various sectors through 'One Health' approach are essential to tackle the problem, and proper awareness must be built up among people about the devastating effects of the disease.

Background Albendazole (ALB) is administered annually to millions of children through global deworming programs targeting soil-transmitted helminths (STHs: Ascaris lumbricoides, Trichuris trichiura and hookworms, Necator americanus and Ancylostoma duodenale). However, due to the lack of large individual patient datasets collected using standardized protocols and the application of population-based statistical methods, little is known about factors that may affect individual responses to treatment. Methodology/Principal findings We re-analyzed 645 individual patient data from three standardized clinical trials designed to assess the efficacy of a single 400 mg oral dose of ALB against STHs in schoolchildren from different study sites, each with varying history of drug pressure based on duration of mass drug administration programs: Ethiopia, low; Lao People’s Democratic Republic (PDR), moderate; Pemba Island (Tanzania), high. Using a Bayesian statistical modelling approach to estimate individual responses (individual egg reduction rates, ERRi), we found that efficacy was lower in Pemba Island, particularly for T. trichiura. For this STH, the proportion of participants with a satisfactory response (ERRi ≥50%), was 65% in Ethiopia, 61% in Lao PDR but only 29% in Pemba Island. There was a significant correlation between ERRi and infection intensity prior to drug administration (ERRi decreasing as a function of increasing infection intensity). Individual age and sex also affected the drug response, but these were of negligible clinical significance and not consistent across STHs and study sites. Conclusions/Significance We found decreased efficacy of ALB against all the STHs analyzed in Pemba Island (Tanzania), an area with high drug pressure. This does not indicate causality, as this association may also be partially explained by differences in infection intensity prior to drug administration. Notwithstanding, our results indicate that without alternative treatment regimens, program targets will not be achievable on Pemba Island because of inadequate efficacy of ALB. Trial registration The study was registered on Clinicaltrials.gov (ID: NCT03465488) on March 7, 2018.

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p=0.023) and the number of eggs per gram (epg) counts (p<0.001). This report demonstrates that urinary 2-MPC can be considered an A. lumbricoides-specific biomarker that can be used to monitor infection intensity.

Background 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. Methods and principal findings 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. Conclusions/Significance 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.

Introduction: Soil-transmitted helminthiasis (STH) remains a major public health problem in school children in Ethiopia. Although direct wet mount microscopy (DWMM) is the means to diagnose parasitic diseases in health care facilities in Ethiopia, it remains unclear what its diagnostic performance is for STH. Methodology: A cross-sectional study was performed in Jimma Town (Ethiopia) and included 600 children from 10 primary schools. The diagnostic sensitivity of DWMM was compared to a composite reference standard (CRS) consisting of Kato-Katz, McMaster and Mini-FLOTAC. We also explored the impact of intensity of infection (the highest faecal egg counts (FECs; expressed as eggs per gram of stool (EPG)) across the CRS) on the diagnostic sensitivity of DWMM. Results: Based on the CRS, there were 210 Ascaris (35.0%), 312 Trichuris (52.0%) and 102 hookworm cases (17.0%). The median intensity of infections equalled 2,057 EPG for Ascaris, 200 EPG for Trichuris and 110 EPG for hookworms. The sensitivity of DWMM was 73.8% for Ascaris, but was around 17% for both Trichuris and hookworms. The sensitivity significantly increased with intensity of STH. For Ascaris, the odds for detecting an infection intensity of 1,000 EPG was 6.2 times higher than detecting an infection of 100 EPG. For Trichuris and hookworms, these odds ratios were 7.1 and 14. Conclusions: The diagnostic sensitivity of DWMM is low for STH, but it is able to detect those subjects that are in the highest need of treatment, and hence contributes to the global goal to eliminate STH as a public health problem.

Background Efforts to control soil-transmitted helminth (STH) infections have intensified over the past decade. Field-survey data on STH prevalence, infection intensity and drug efficacy is necessary to guide the implementation of control programs and should be of the best possible quality. Methodology During four clinical trials designed to evaluate the efficacy of albendazole against STHs in Brazil, Ethiopia, Lao PDR and Tanzania, quality control (QC) was performed on the duplicate Kato-Katz thick smears and the data entry. We analyzed datasets following QC on both fecal egg counts (FECs) and data entry, and compared the prevalence of any STH infection and moderate-to-heavy intensity (MHI) infections and the drug efficacy against STH infections. Results Across the four study sites, a total of 450 out of 4,830 (9.3%) Kato-Katz thick smears were re-examined. Discrepancies in FECs varied from ~3% (hookworms) to ~6.5% (Ascaris lumbricoides and Trichuris trichiura). The difference in STH prevalence and prevalence of MHI infections using the datasets with and without QC of the FECs did not exceed 0.3%, except for hookworm infections in Tanzania, where we noted a 2.2 percentage point increase in MHI infections (pre-QC: 1.6% vs. post-QC: 3.8%). There was a 100% agreement in the classification of drug efficacy of albendazole against STH between the two datasets. In total, 201 of the 28,980 (0.65%) data entries that were made to digitize the FECs were different between both data-entry clerks. Nevertheless, the overall prevalence of STH, the prevalence of MHI infections and the classification of drug efficacy remained largely unaffected. Conclusion/significance In these trials, where staff was informed that QC would take place, minimal changes in study outcomes were reported following QC on FECs or data entry. Nevertheless, imposing QC did reduce the number of errors. Therefore, application of QC together with proper training of the personnel and the availability of clear standard operating procedures is expected to support higher data quality.

The World Health Organization (WHO) has defined moderate-to-heavy intensity (M&HI) infections with soil-transmitted helminths (Ascaris lumbricoides, Trichuris trichiura and the two hookworms, Ancylostoma duodenale and Necator americanus) based on specific values of eggs per gram of stool, as measured by the Kato-Katz method. There are a variety of novel microscopy and DNA-based methods but it remains unclear whether applying current WHO thresholds on to these methods allows for a reliable classification of M&HI infections. We evaluated both WHO and method-specific thresholds for classifying the M&HI infections for novel microscopic (FECPAKG2, McMaster and Mini-FLOTAC) and DNA-based (qPCR) diagnostic methods. For this, we determined method-specific thresholds that best classified M&HI infections (defined by Kato-Katz and WHO thresholds; reference method) in two multi-country drug efficacy studies. Subsequently, we verified whether applying these method-specific thresholds improved the agreement in classifying M&HI infections compared to the reference method. When we applied the WHO thresholds, the new microscopic methods mainly misclassified M&HI as low intensity, and to a lesser extent low intensity infection as M&HI. For FECPAKG2, applying the method-specific thresholds significantly improved the agreement for Ascaris (moderate → substantial), Trichuris and hookworms (fair → moderate). For Mini-FLOTAC, a significantly improved agreement was observed for hookworms only (fair → moderate). For the other STHs, the agreement was almost perfect and remained unchanged. For McMaster, the method-specific thresholds revealed a fair to a substantial agreement but did not significantly improve the agreement. For qPCR, the method-specific thresholds based on genome equivalents per ml of DNA moderately agreed with the reference method for hookworm and Trichuris infections. For Ascaris, there was a substantial agreement. We defined method-specific thresholds that improved the classification of M&HI infections. Validation studies are required before they can be recommended for general use in assessing M&HI infections in programmatic settings.