Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool

Background : 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. Methodology and principal findings : 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 RNA later and were stored at 4 degrees 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. Conclusions : 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

The supervisory role of applying governance mechanisms to reduce the effects of the Covid-19 pandemic in the public shareholding insurance companies listed on the Palestine Stock Exchange

The study aimed to identify the supervisory role of applying governance mechanisms to reduce the effects of the Covid-19 pandemic in the public shareholding insurance companies listed on the Palestine Stock Exchange. Governance Committees (72) working in insurance companies listed on the Palestine Stock Exchange, numbering (5) companies, and a comprehensive inventory method was used due to the small size of the study population. The results of the study concluded that the boards of directors of the insurance companies listed on the Palestine Exchange are aware of the limits of their role and responsibility in providing control over management, and applying effective governance mechanisms to manage the state of uncertainty resulting from the Covid-19 pandemic crisis. The insurance companies listed on the Palestine Exchange also disclose the role of Governance mechanisms in contributing to the development of the necessary measures to reduce the effects of the pandemic, and the managements of these companies adopt the application of governance mechanisms in light of the Covid-19 pandemic, which need to provide the necessary support from stakeholders. The study recommended that the public shareholding insurance companies listed on the Palestine Exchange should disclose the effects caused by the Covid-19 pandemic on the directions and operations of these companies, and the measures they have taken to address the negative effects resulting from the pandemic that affected their financial and administrative performance. Companies and their boards of directors to develop appropriate mechanisms for corporate governance practices through the participation of many stakeholders and corporate departments to counter the negative effects of the COVID-19 pandemic

Comprehensive evaluation of stool-based diagnostic methods and benzimidazole resistance markers to assess drug efficacy and detect the emergence of anthelmintic resistance : a Starworms study protocol

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 FECPAK(G2)) 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 beta-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 beta-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

Seeing revolution non-linearly: www.filmingrevolution.org

Filming Revolution, launched in 2015, is an online interactive data base documentary tracing the strands and strains of independent (mostly) documentary filmmaking in Egypt since the revolution. Consisting of edited interviews with 30 filmmakers, archivists, activists, and artists based in Egypt, the website is organised by the themes that emerged from the material, allowing the viewer to engage in an unlimited set of “curated dialogues” about issues related to filmmaking in Egypt since 2011. With its constellatory interactive design, Filming Revolution creates as much as documents a community of makers, as it attempts to grapple with approaches to filmmaking in the wake of such momentous historical events. The non-hierarchical polysemous structure of the project is meant to echo the rhizomatic, open-ended aspect of the revolution and its aftermath, in yet another affirmation and instantiation of contemporary civil revolution as a non-linear, ever-unfolding, on-going, event

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Background: Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. // Methods: We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung's disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. // Findings: We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung's disease) from 264 hospitals (89 in high-income countries, 166 in middle-income countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in low-income countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. // Interpretation: Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between low-income, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Molecular diagnosis of human soil-transmitted helminths and monitoring for benzimidazole resistance-associated polymorphisms

Soil-transmitted helminths (STHs) are a major burden on human health in developing countries. Ascaris lumbricoides, Trichuris trichiura and the hookworms, Necator americanus and Ancylostoma duodenale are the most prevalent STHs, infecting more than 1.5 billion people worldwide. School-age children are the most at risk of heavy infection with STHs and of developing severe morbidity, leading to malnourishment, with cognitive and educational deficits. Infection with STHs can complicate pregnancy, placing both mothers and children at higher risk of severe mortality.Large-scale treatment with benzimidazole (BZ) drugs, albendazole (ABZ) or mebendazole (MBZ), is the major control strategy to control STH infections in mass drug administration (MDA) programs. Intensive reliance on two drugs of the same anthelmintic class with suboptimal efficacy greatly increases the possibility that BZ resistance may develop. In veterinary parasites, intensive use of the same anthelmintics has led to the emergence of BZ resistance which is caused by a single nucleotide polymorphism (SNP) at codon position 200, 167, or 198 in the β-tubulin isotype 1 gene. These SNPs have also been correlated with poor response to BZ treatment in human T. trichiura.Diagnosis of STHs has traditionally relied on stool microscopy, which has a number of deficiencies. PCR-based methods have achieved critical advances in the detection of several parasitic infections and have been applied for sensitive and specific detection of STHs and monitoring for BZ resistance-associated SNPs. However, the requirement for expensive equipment, highly skilled personnel and a well-equipped laboratory make their implementation difficult in low-resource or field settings. Rapid, sensitive and cost-effective diagnostic tools are crucial for detection of the STH infections and monitoring for BZ resistance before resistance reaches clinically significant levels.Isothermal molecular methods, such as loop-mediated isothermal amplification (LAMP) are unique technologies that have emerged as a promising approach for diagnosis of viral, fungal, bacterial, and parasitic diseases. SmartAmp2 (Smart amplification process) is a specific type of LAMP, with unique asymmetrical primer design for detection of SNPs or DNA targets under isothermal conditions.New genotyping assays were developed based on the SmartAmp2 method, to screen for β-tubulin SNPs in T. trichiura, N. americanus and A. lumbricoides. The assays showed high sensitivity and specificity in field samples and also demonstrated high tolerance to inhibitors in fecal samples. Following this, colorimetric STH diagnostic assays were developed based on the SmartAmp2 method for the detection of STHs. Rapid and sensitive assays were developed to identify the STHs in field samples. The assays showed high specificity and demonstrated high tolerance to inhibitors in fecal samples. The visual inspection of the results allowed the assay readout with the naked eye, eliminating the need for costly sophisticated equipment. In conclusion, novel diagnostic assays were developed with great potential to be implemented in resource limited settings for the detection of STH infections and for screening for BZ resistance-associated SNPs.Les géohelminthes jouent un rôle important dans la santé humaine des pays en voie de développement. Ascaris lumbricoides, Trichuris trichiura et les ankylostomes Necator americanus et Ancylostoma duodenale sont les infections parasitaires les plus répandues et sont transmises par le sol et par contamination faeco-oral. Elles affectent plus de 1.5 milliard de personnes mondialement. Les enfants sont les plus à risque de développer de graves infections et une morbidité importante, prédisposant à la malnutrition et aux déficits cognitifs et éducationnels. Ces géohelminthes peuvent provoquer des complications pendant une grossesse et augmentent le risque de mortalité chez la mère et l'enfant.Le traitement à grande échelle avec les benzimidazoles (BZ), soit l'albendazole et le mébendazole, est la stratégie de lutte la plus répandue dans les programmes d'administration de médicament massive contre ces infections parasitaires. L'utilisation intensive de ces deux chimiothérapies de la même famille d'anthelminthiques et d'efficacité sous-optimale, augmente considérablement le potentiel de résistance aux BZ. Cette utilisation intensive d'anthelminthiques similaires en parasitologie vétérinaire a provoqué l'émergence de la résistance aux BZ. Cette résistance est causée par un polymorphisme du nucléotide simple (SNP) localisé aux positions de codon 200, 167 et 198 sur le gène de la β-tubuline. Ces SNP sont aussi associés à la faible efficacité du traitement contre T. trichiura par BZ chez l'homme. Le diagnostic usuel des géohelminthes se fait par microscopie des selles ; une technique ayant plusieurs lacunes. Des méthodes basées sur la PCR ont permis des percés majeures au dépistage des infections parasitaires et peuvent s'appliquer à la détection spécifique et sensible des infections par géohelminthes, ainsi qu'à l'évaluation de la résistance aux BZ associée aux SNP. Toutefois, ceci requiert de l'équipement dispendieux, du personnel hautement qualifié et un laboratoire bien équipé dont la mise en place est inapplicable aux études sur le terrain ou par manque de ressources. Il est crucial de développer des diagnostics rapides, sensibles et peu coûteux afin de dépister les infections par géohelminthes, et de surveiller la résistance aux BZ associée aux SNP avant que les taux de résistance n'atteignent des niveaux cliniques critiques.Des méthodes moléculaires isothermiques, tel que la technique LAMP (loop-mediated isothemal amplification), sont des technologies uniques et émergentes ayant un potentiel prometteur pour diagnostiquer les maladies virales, fongiques, bactériennes et parasitaires. Le SmartAmp2 est une technique de LAMP spécifique avec un modèle d'amorce asymétrique unique pour la détection de SNP ou d'ADN cible.Des nouvelles techniques de génotypage ont été développées selon la méthode du SmartAmp2 pour filtrer la présence de SNP associés à la résistance aux BZ dans la β-tubuline chez T. trichiura, N. americanus et A. lumbricoides. Ces tests démontrent une grande sensibilité et spécificité avec des échantillons collectés sur le terrain et démontrent aussi une bonne tolérance aux inhibiteurs de PCR dans des échantillons fécaux. Par la suite, des tests diagnostiques colorimétriques ont été développés selon la méthode SmartAmp2 pour la détection des géohelminthes. Des tests rapides et sensibles ont été développés pour identifier ces infections lors d'échantillonnage sur le terrain. Ils ont démontrés une haute spécificité et une grande tolérance aux inhibiteurs avec des échantillons fécaux. L'inspection visuelle des résultats permet de lire les résultats à œil nu, éliminant le besoin d'équipement sophistiqué. En conclusion, de nouveaux outils diagnostiques ont été développés. Ils possèdent un fort potentiel d'utilisation pour dépister les infections aux géohelminthes et les SNP associés à la résistance aux BZ dans un environnement disposant de peu de ressources

SmartAmp2 genotyping results of <i>Trichuris trichiura</i> samples at codon 200.

<p>SmartAmp2 amplification of F200Y (T<b>T</b>C>T<b>A</b>C) using wild type (WT) and mutant type (MT)-primer sets. Left, center, and right panels show assay results for homozygous WT (WT/WT), mixed (WT/MT), and homozygous MT (MT/MT) pooled samples, respectively. dR, difference of relative fluorescence unit.</p

Isothermal diagnostic assays for the detection of soil-transmitted helminths based on the SmartAmp2 method

Abstract Background Diagnosis of soil-transmitted helminths (STHs) has traditionally relied on stool microscopy, which has a number of critical deficiencies. Molecular diagnostics are powerful tools to identify closely related species, but the requirement for costly equipment makes their implementation difficult in low-resource or field settings. Rapid, sensitive and cost-effective diagnostic tools are crucial for accurate estimation of STH infection intensity in MDA programmes in which the goal is to reduce morbidity following repeated rounds of chemotherapy. Results In this study, colourimetric isothermal assays were developed using SmartAmp2 primer sets and reagents in loop-mediated amplification (LAMP) assays. Species-specific primer sets, designed on a specific target sequence in the β-tubulin gene, were used to identify Necator americanus, Trichuris trichiura and Ascaris lumbricoides. After initial optimization on control plasmids and genomic DNA from adult worms, assays were evaluated on field samples. Assays showed high sensitivity and demonstrated high tolerance to inhibitors in spiked faecal samples. Rapid and sensitive colourimetric assays were successfully developed to identify the STHs in field samples using hydroxy napthol blue (HNB) dye. Conclusions Rapid and simple colourimetric diagnostic assays, using the SmartAmp2 method, were developed, with the potential to be applied in the field for detection of STH infections and the estimation of response to treatment. However, further validation on large numbers of field samples is needed

SmartAmp2 primer design for β-tubulin SNP detection in <i>Trichuris trichiura</i>.

<p>Partial sequence of the β-tubulin isotype 1 gene carrying (<b>A</b>) SNP 198C (<b>B</b>) SNP 200A as well as the sequences of primers used for the SmartAmp2 assay for the two SNP positions. The locations of SNPs indicated in bold. BP (boost primer) was used as discrimination primer to target E198A (G<b>A</b>A>G<b>C</b>A) and F200Y (T<b>T</b>C>T<b>A</b>C) SNPs. The folding primer (FP) has a specific sequence (CCTATATATATATAGG) at the 5’ end to allow self-annealing hairpin formation.</p