Deep profiling of potential substrate atlas of porcine epidemic diarrhea virus 3C-like protease.

Coronavirus (CoV) 3C-like protease (3CLpro) is essential for viral replication and is involved in immune escape by proteolyzing host proteins. Deep profiling the 3CLpro substrates in the host proteome extends our understanding of viral pathogenesis and facilitates antiviral drug discovery. Here, 3CLpro from porcine epidemic diarrhea virus (PEDV), an enteropathogenic CoV, was used as a model which to identify the potential 3CLpro cleavage motifs in all porcine proteins. We characterized the selectivity of PEDV 3CLpro at sites P5-P4'. We then compiled the 3CLpro substrate preferences into a position-specific scoring matrix and developed a 3CLpro profiling strategy to delineate the protein substrate landscape of CoV 3CLpro. We identified 1,398 potential targets in the porcine proteome containing at least one putative cleavage site and experimentally validated the reliability of the substrate degradome. The PEDV 3CLpro-targeted pathways are involved in mRNA processing, translation, and key effectors of autophagy and the immune system. We also demonstrated that PEDV 3CLpro suppresses the type 1 interferon (IFN-I) cascade via the proteolysis of multiple signaling adaptors in the retinoic acid-inducible gene I (RIG-I) signaling pathway. Our composite method is reproducible and accurate, with an unprecedented depth of coverage for substrate motifs. The 3CLpro substrate degradome establishes a comprehensive substrate atlas that will accelerate the investigation of CoV pathogenicity and the development of anti-CoV drugs.IMPORTANCECoronaviruses (CoVs) are major pathogens that infect humans and animals. The 3C-like protease (3CLpro) encoded by CoV not only cleaves the CoV polyproteins but also degrades host proteins and is considered an attractive target for the development of anti-CoV drugs. However, the comprehensive characterization of an atlas of CoV 3CLpro substrates is a long-standing challenge. Using porcine epidemic diarrhea virus (PEDV) 3CLpro as a model, we developed a method that accurately predicts the substrates of 3CLpro and comprehensively maps the substrate degradome of PEDV 3CLpro. Interestingly, we found that 3CLpro may simultaneously degrade multiple molecules responsible for a specific function. For instance, it cleaves at least four adaptors in the RIG-I signaling pathway to suppress type 1 interferon production. These findings highlight the complexity of the 3CLpro substrate degradome and provide new insights to facilitate the development of anti-CoV drugs.

Prevalence of irritable bowel syndrome and functional dyspepsia after acute gastroenteritis: systematic review and meta-analysis.

OBJECTIVE: Disorders of gut-brain interaction may arise after acute gastroenteritis. Data on the influence of pathogen type on the risk of postinfection IBS (PI-IBS), as on postinfection functional dyspepsia (PI-FD), are limited. We conducted a systematic review and meta-analysis to determine prevalence of PI-IBS or PI-FD after acute gastroenteritis. DESIGN: We included observational studies recruiting ≥50 adults and reporting prevalence of IBS or FD after acute gastroenteritis with ≥3-month follow-up. A random effects model was used to estimate prevalence and ORs with 95% CIs. RESULTS: In total, 47 studies (28 170 subjects) were eligible. Overall prevalence of PI-IBS and PI-FD were 14.5% and 12.7%, respectively. IBS persisted in 39.8% of subjects in the long-term (>5 years follow-up) after diagnosis. Individuals experiencing acute gastroenteritis had a significantly higher odds of IBS (OR 4.3) and FD (OR 3.0) than non-exposed controls. PI-IBS was most associated with parasites (prevalence 30.1%), but in only two studies, followed by bacteria (18.3%) and viruses (10.7%). In available studies, Campylobacter was associated with the highest PI-IBS prevalence (20.7%) whereas Proteobacteria and SARS-CoV-2 yielded the highest odds for PI-IBS (both OR 5.4). Prevalence of PI-FD was 10.0% for SARS-CoV-2 and 13.6% for bacteria (Enterobacteriaceae 19.4%). CONCLUSION: In a large systematic review and meta-analysis, 14.5% of individuals experiencing acute gastroenteritis developed PI-IBS and 12.7% PI-FD, with greater than fourfold increased odds for IBS and threefold for FD. Proinflammatory microbes, including Proteobacteria and subcategories, and SARS-CoV-2, may be associated with the development of PI-IBS and PI-FD.

Impact of a school-based water and hygiene intervention on child health and school attendance in Addis Ababa, Ethiopia: a cluster-randomised controlled trial.

BACKGROUND: School-based water, sanitation and hygiene (WASH) may improve the health and attendance of schoolchildren, particularly post-menarcheal girls, but existing evidence is mixed. We examined the impact of an urban school-based WASH programme (Project WISE) on child health and attendance. METHODS: The WISE cluster-randomised trial, conducted in 60 public primary schools in Addis Ababa, Ethiopia over one academic year, enrolled 2-4 randomly selected classes per school (~ 100 pupils) from grades 2 to 8 (aged 7-16) in an 'open cohort'. Schools were assigned 1:1 by stratified randomisation to receive the intervention during the 2021/2022 or the 2022/2023 academic year (waitlist control). The intervention included improvements to drinking water storage, filtration and access, handwashing stations and behaviour change promotion. Planned sanitation improvements were not realised. At four unannounced classroom visits post-intervention (March-June 2022), enumerators recorded primary outcomes of roll-call absence, and pupil-reported respiratory illness and diarrhoea in the past 7 days among pupils present. Analysis was by intention-to-treat. RESULTS: Of 83 eligible schools, 60 were randomly selected and assigned. In total, 6229 eligible pupils were enrolled (median per school 101.5; IQR 94-112), 5987 enrolled at study initiation (23rd November-22nd December 2021) and the remaining 242 during follow-up. Data were available on roll-call absence for 6166 pupils (99.0%), and pupil-reported illness for 6145 pupils (98.6%). We observed a 16% relative reduction in odds of pupil-reported respiratory illness in the past 7 days during follow-up in intervention vs. control schools (aOR 0.84; 95% CI 0.71-1.00; p = 0.046). There was no evidence of effect on pupil-reported diarrhoea in the past 7 days (aOR 1.15; 95% CI 0.84-1.59; p = 0.39) nor roll-call absence (aOR 1.07; 95% 0.83-1.38; p = 0.59). There was a small increase in menstrual care self-efficacy (aMD 3.32 on 0-100 scale; 95% CI 0.05-6.59), and no evidence of effects on other secondary outcomes. CONCLUSIONS: This large-scale intervention to improve school WASH conditions city-wide had a borderline impact on pupil-reported respiratory illness but no effect on diarrhoeal disease nor pupil absence. Future research should establish relationships between WASH-related illness, absence and other educational outcomes. TRIAL REGISTRATION: ClinicalTrials.gov, number NCT05024890.

High frequency of antimicrobial resistance in Salmonella and Escherichia coli causing diarrheal diseases at the Yirimadio community health facility, Mali.

BACKGROUND: Diarrhoea is a public health problem, especially in developing countries where it is the second leading cause of child mortality. In Low Income Countries like in Mali, self-medication and inappropriate use of antibiotics due to the scarcity of complementary diagnostic systems can lead to the development of multidrug-resistant bacteria causing diarrhoea. The objective of this work was to determine the microorganisms responsible for diarrhoea in children under 15 years of age and to characterize their sensitivity to a panel of antibiotics used in a peri-urban community in Mali. The study involved outpatient children visiting the Yirimadio Community Health Centre and diagnosed with diarrhoea. Stool samples from those patients were collected and analysed by conventional stools culture and the susceptibility to antibiotics of detected bacteria was determined by the disc diffusion method in an agar medium. RESULT: Overall, 554 patients were included. Children under the age of 3 years accounted for 88.8% (492 of 554) of our study population. Two bacterial species were isolated in this study, Escherichia coli 31.8% (176 of 554) and Salmonella 2.9% (16 of 554). In the 176, E. coli strains resistance to amoxicillin and to cotrimoxazole was seen in 93.8% (165 of 176) and 92.6% ( 163 of 176), respectively. The ESBL resistance phenotype accounted for 39,8% (70 of 176) of E. coli. Sixteen (16) strains of Salmonella were found, of which one strain (6.3%) was resistant to amoxicillin and to amoxicillin + clavulanic acid. Another one was resistant to chloramphenicol (6.3%). Two strains of Salmonella were resistant to cotrimoxazole (12.5%) and two others were resistant to cefoxitin (12.5%). CONCLUSIONS: The data suggest that E. coli is frequently involved in diarrhoea in children under 3 years of age in this peri-urban setting of Bamako, Mali, with a high rate of resistance to amoxicillin and cotrimoxazole, the most widely used antibiotics in the management of diarrhoea in this setting.

Fecal microbiota transplantation alleviates mild-moderate COVID-19 associated diarrhoea and depression symptoms: A prospective study of a randomized, double-blind clinical trial.

Currently, the emergence of the endemic Coronavirus disease (COVID-19) situation still poses a serious threat to public health. However, it remains elusive about the role of fecal microbiota transplantation in treating COVID-19. We performed a randomized, double-blind, placebo-controlled clinical trial enrolling a cohort of 40 COVID-19 patients with mild-moderate symptoms. Our results showed that fecal microbiota transplantation provided an amelioration in diarrhoea (p = 0.026) of digestive system and depression (p = 0.006) of neuropsychiatric-related symptom in COVID-19 patients, respectively. Meanwhile, we found that the number of patients with diarrhoea decreased from 19 to 0 on day 7 after fecal microbiota transplantation treatment, and it was statistically changed compared to the placebo group (p = 0.047). Of note, the serum concentration of aspartate aminotransferase-to-alanine aminotransferase ratio (AST/ALT, fecal microbiota transplantation, pre vs. post: 0.966 vs. 0.817), a biomarker for predicting long COVID-19, was significantly reduced by fecal microbiota transplantation. In all, our study supports that fecal microbiota transplantation could be a novel therapeutic strategy for COVID-19 patients with diarrhoea and depressive symptoms, which is potentially valuable in ameliorating long COVID-19 symptoms.

LINC02605 involved in paediatric Mycoplasma pneumoniae pneumonia complicated with diarrhoea via miR-539-5p/CXCL1 axis.

BACKGROUND: To investigate the involvement of LINC02605 in the progression of paediatric Mycoplasma pneumoniae pneumonia (MPP). METHODS: One hundred and thirty-two children with MPP (90 simple MPP and 42 MPP + diarrhoea) were enrolled, and their plasma was collected for detection of LINC026505 expression. CCK-8 kit and commercial apoptosis kit were introduced to determine cell growth and apoptosis. In silico prediction analyses were conducted to predict the downstream miRNA for LINC02605, following verification by dual luciferase reporter assay. The lipid-associated membrane proteins (LAMPs) were used to treat A549 and Coca-2 cells. RESULTS: LIN02605 was highly expressed in the MPP, especially in MPP complicated with diarrhoea. LINC02605 downregulation in A549 cells correlated with significant suppression of cell apoptosis rate and growth inhibition rate in vitro. Introduction of miR-539-5p inhibited luciferase activity in a reporter system containing the wild-type LINC02605 and CXCL1. After stimulation with LAMPs, overexpression of LINC02605 and CXCL1 and inhibition of miR-539-5p were found. miR-539-5p and CXCL1 knockdown resulted in a rescue effect on the LINC02605-inhibited cell apoptosis. LAMPs induced IL-1ß in intestinal epithelial cells and IL-1ß induced LINC02605 expression in A549 cells. CONCLUSIONS: LINC02605 was upregulated in MPP and miR-539-5p was a target for LINC02605. LINC02605 may be involved in the crosstalk between the gastrointestinal tract and the respiratory tract.

Determinants of diarrheal disease in children undergoing complementary feeding in a low-income urban setting in Kenya.

OBJECTIVES: This study aimed to identify and characterise the determinants influencing the occurrence of diarrheal diseases in children aged 6-24 months undergoing complementary feeding within a low-income urban community in Kenya. METHODS: This study followed a cross-sectional design and recruited caregivers of children aged 6-24 months from 302 households. The dependent variable was the 2-week diarrhoea prevalence among children, with independent variables including sociodemographic characteristics, child immunisation and feeding status, and water and sanitation facilities. Data analysis was performed using SPSS. Descriptive statistics and logistic regression analyses were used to assess associations between independent variables and the occurrence of diarrheal diseases. RESULTS: The majority of caregivers were female (n = 282, 93.4%), aged 25-34 years (n = 156, 51.7%), had attained secondary school education (n = 154, 51%), were unemployed (n = 162, 53.6%), and earned Ksh 10,000 (USD 100) or less. 296 (98%) indexed children were fully vaccinated against rotavirus. Most households used improved drinking water sources (n = 272, 90.1%). Most caregivers did not regularly wash their hands with soap and water (n = 225, 74.5%). The 2-week diarrhoea prevalence among children was 34.1% (103/302), with 69.9% (72/103) of these cases seeking care at a healthcare facility. Logistic regression analysis revealed that children of caregivers earning Ksh 20,000 and below (aOR = 2.9[1.3-6.5], p = 0.01), and those from households using unimproved sanitation facilities (aOR = 1.9 [CI 1-3.4], p = 0.042), had significantly higher odds of having diarrhoea. CONCLUSION: The study found a high prevalence of diarrhoea in Kenyan children aged 6-24 months, with caregiver income and household sanitation facilities significantly impacting the occurrence of the disease. The study suggests integrated approaches, including education, income generation, hygiene, and improved nutrition, to address the burden of diarrheal disease.

Understanding pre-hospital disease management of fever and diarrhoea in children-Care pathways in rural Tanzania.

OBJECTIVE: Many children in sub-Saharan Africa die from infectious diseases like malaria, pneumonia, and diarrhoea that can be prevented by early diagnosis, effective and targeted treatment. This study aimed to gain insights into case management practices by parents before they present their children to hospital. METHODS: We conducted a cross-sectional study among 332 parents attending a district hospital with their under-fives symptomatic with fever and/or diarrhoea between November 2019 and July 2020 in rural Tanzania. Timely and targeted treatment was defined as seeking health care within 24 h of fever onset, and continued fluid intake in case of diarrhoea. RESULTS: The main admission diagnoses were acute respiratory infections (61.8%), malaria (25.3%), diarrhoea (18.4%) and suspected sepsis (8.1%). The majority of children (91%) received treatment prior to admission, mostly antipyretics (75.6%), local herbal medicines (26.8%), and antibiotics (17.8%)-half of them without prescription from a clinician. For diarrhoea, the use of oral rehydration solution was rare (9.0%), although perceived as easily accessible and affordable. 49.4% of the parents presented their children directly to the hospital, 23.2% went to a pharmacy/drug shop and 19.3% to a primary health facility first. Malaria symptoms began mostly 3 days before the hospital visit; only 25.4% of febrile children visited any health facility within 24 h of disease onset. Prior use of local herbal medicine (AOR = 3.2; 95% CI 1.4-7.3), visiting the pharmacy (adjusted Odds Ratio [AOR] = 3.1; 95% confidence interval [CI]: 1.0-9.8), the dispensary being the nearest health facility (AOR = 3.0; 95% CI: 1.5-6.2), and financial difficulties (AOR = 2.2; 95% CI 1.1-4.5) were associated with delayed treatment. CONCLUSION: This study suggests that antipyretics and antibiotics dispensed at pharmacies/drug shops, as well as use of local herbal medicines, delay early diagnosis and treatment, which can be life-threatening. Pharmacies/drug shops could be integrated as key focal points for sensitising community members on how to respond to paediatric illnesses and encourage the use of oral rehydration solutions.

Non-viral pathogens of infectious diarrhoea post-allogeneic stem cell transplantation are associated with graft-versus-host disease.

Infectious diarrhoea is common post-allogeneic haematopoietic stem-cell transplantation (alloHSCT). While the epidemiology of Clostridioides difficile infection (CDI) post-alloHSCT has been described, the impact of other diarrhoeal pathogens is uncertain. We reviewed all alloHSCT between 2017 and 2022 at a single large transplant centre; 374 patients were identified and included. The 1-year incidence of infectious diarrhoea was 23%, divided into viral (13/374, 3%), CDI (65/374, 17%) and other bacterial infections (16/374, 4%). There was a significant association between infectious diarrhoea within 1 year post-transplant and the occurrence of severe acute lower gastrointestinal graft-versus-host disease (GVHD, OR = 4.64, 95% CI 2.57-8.38, p < 0.001) and inferior GVHD-free, relapse-free survival on analysis adjusted for age, donor type, stem cell source and T-cell depletion (aHR = 1.64, 95% CI = 1.18-2.27, p = 0.003). When the classes of infectious diarrhoea were compared to no infection, bacterial (OR = 6.38, 95% CI 1.90-21.40, p = 0.003), CDI (OR = 3.80, 95% CI 1.91-7.53, p < 0.001) and multiple infections (OR = 11.16, 95% CI 2.84-43.92, p < 0.001) were all independently associated with a higher risk of severe GI GVHD. Conversely, viral infections were not (OR = 2.98, 95% CI 0.57-15.43, p = 0.20). Non-viral infectious diarrhoea is significantly associated with the development of GVHD. Research to examine whether the prevention of infectious diarrhoea via infection control measures or modulation of the microbiome reduces the incidence of GVHD is needed.

Gastrointestinal adverse events and weight reduction in people with type 2 diabetes treated with tirzepatide in the SURPASS clinical trials.

AIMS: To evaluate gastrointestinal adverse events (AEs) and the impact of nausea, vomiting or diarrhoea (N/V/D) and any gastrointestinal (GI) AEs overall on weight change with tirzepatide across the SURPASS-1 to -5 clinical trials. MATERIALS AND METHODS: Participants with type 2 diabetes were randomized to receive once-weekly tirzepatide (5, 10 or 15 mg) or comparator (placebo, semaglutide 1 mg once weekly, or titrated daily basal insulins) as monotherapy or added on to background antihyperglycaemic medication(s). This post hoc analysis subdivided participants within each trial into subgroups that self-reported (yes/no) any N/V/D or GI AEs. Change from baseline in body weight at the primary timepoint was assessed within each trial and subgroup. Mediation analyses were conducted to evaluate the contribution of direct and indirect (mediated by N/V/D or GI AEs) effects of tirzepatide on weight change versus comparators. RESULTS: Across the SURPASS-1 to -5 trials (N = 6263), nausea (12%-24%), diarrhoea (12%-22%), and vomiting (2%-13%) were the most common GI AEs reported with tirzepatide; these were transient and of mild-to-moderate severity. Mean weight reduction at the primary timepoint with tirzepatide was consistent between participants who reported N/V/D (-6.2 to -14.9 kg) and those who did not report N/V/D (-6.2 to -13.3 kg). Mean weight reduction was significantly (P < 0.01) greater with tirzepatide compared with placebo, semaglutide 1 mg, and basal insulins within the N/V/D and GI AEs subgroups. Mediation analyses suggested minimal contribution (<6%) of N/V/D and GI AEs to the overall difference in weight change between tirzepatide and comparators. CONCLUSIONS: Superior weight reduction with tirzepatide versus comparators appears to be independent of reported N/V/D or GI AEs.

Recombinant porcine interferon delta 8 inhibits swine acute diarrhoea syndrome coronavirus infection in vitro and in vivo.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV), which originates from zoonotic transmission of bat coronaviruses in the HKU2 lineage, causes severe illness in pigs and carries a high risk of spreading to humans. At present, there are no licenced therapeutics for the treatment of SADS-CoV. In this study, we examined the effectiveness of recombinant porcine interferon delta 8 (IFN-δ8) against SADS-CoV both in vitro and in vivo. In vitro experiments showed that IFN-δ8 inhibited SADS-CoV proliferation in a concentration-dependent manner, with complete inhibition occurring at a concentration of 5 µg/mL. In vivo experiments demonstrated that two 50 µg/kg doses of IFN-δ8 injected intraperitoneally protected piglets against lethal challenge, blocked viral shedding, attenuated intestinal damage, and decreased the viral load in the jejunum and ileum. Further findings suggested that IFN-δ8 inhibited SADS-CoV infection by increasing the expression of IFN-stimulated genes. These results indicate that IFN-δ8 shows promise as a biological macromolecule drug against SADS-CoV infection.

Bile acid diarrhoea and metabolic changes after cholecystectomy: a prospective case-control study.

INTRODUCTION: Bile acid diarrhoea (BAD) can occur due to disruption to the enterohepatic circulation such as following cholecystectomy. However, the mechanism behind this is as yet unknown. The aim of this study was to determine the rate of post-cholecystectomy diarrhoea and to assess whether FGF19 within the gallbladder was associated with the development of BAD. METHODS: This was a prospective case-control study in which patients were assessed pre- and post- cholecystectomy (study group) and compared with patients also having laparoscopic surgery but not cholecystectomy (control group). Their bowel habits and a GIQLI questionnaire was performed to compare the pre- and post-operative condition of the two groups. Gallbladder tissue sample was tested for FGF19 and PPARα in the study group patients. A subset had serum lipid levels, FGF19 and C4 measurements. RESULTS: Gallbladder PPAR α was found to have a significant correlation with stool consistency, with the lower the PPARα concentration the higher the Bristol stool chart number (i.e. looser stool). There were no significant correlation when assessing the effect of gallbladder FGF19 concentration on bowel habit, stool consistency, lipid levels, BMI or smoking. The study group showed a significant increase in triglycerides post-operatively, however there were no changes in cholesterol, HDL and LDL levels. Correlation of the increased triglyceride levels with stool consistency and frequency showed no significant results DISCUSSION AND CONCLUSION: We did not find any direct evidence that FGF19 levels within the gallbladder impact the development of post-cholecystectomy diarrhoea. There was however a significant increase in triglycerides postoperatively. There was also no correlation of bowel habits with PPARα suggesting the observed rise is independent of this pathway. Further work is required particularly relating to the gut microbiome to further investigate this condition.

The Adult Carbohydrate Perception Questionnaire Identifies Patients with Lactose or Fructose Intolerance Who Respond to Diet.

INTRODUCTION: There is no evidence that a positive breath test is a good predictor of the success of a carbohydrate-restricted diet. Our objective was to investigate whether patients in whom lactose intolerance (LIT) or fructose intolerance (FIT) is diagnosed by validated symptom measurement respond to diet. METHODS: Patients referred for evaluation of LIT or FIT underwent hydrogen/methane breath testing (malabsorption test) and symptom measurement with the adult Carbohydrate Perception Questionnaire (aCPQ, intolerance test) before and after 50 g lactose or 25 g fructose. Patients with a positive aCPQ received instructions on specific diets and supplements. Severity of abdominal pain, bloating, diarrhoea, flatulence, and nausea were measured using a visual analogue scale (VAS) before (VAS1, mm) and after (VAS2, mm) diet. The change in VAS for individual symptoms and overall symptoms after diet is expressed as deltaVAS (mm) and as change relative to VAS1 (%). RESULTS: Forty-one patients were included (23 LIT, 8 FIT, 10 LIT+FIT). Eight patients had negative breath tests (no malabsorption). After 2 months of diet, the overall VAS and the individual symptoms decreased (p < 0.001). Overall VAS1 and the VAS1 for individual symptoms correlated significantly with the decrease in deltaVAS (mm) after diet. Nineteen patients (46%) had total recovery, and additional 13 patients (32%) had improvement of >50%. Response to diet was independent of breath test results. CONCLUSION: This uncontrolled and unblinded study suggests that patients with carbohydrate intolerance diagnosed by aCPQ benefit significantly from diet, independent of the presence of malabsorption. Controlled studies are required to confirm these results in larger patient groups.

Diarrhoeagenic Escherichia coli associated with childhood diarrhoea in Osun state, Nigeria.

INTRODUCTION: Diarrhoea is a major public health concern in developing countries, usually exacerbated by poor water, sanitation and hygiene but its aetiology is under-studied, particularly away from capital cities. We identified diarrhoeagenic Escherichia coli (DEC) from stools collected in Ile-Ife and Ilesa, Osun state, Nigeria and determined their antibiotic resistance profiles. METHODS: Stool samples from 167 children with diarrhoea and 334 controls under the age of 5 years were cultured for Escherichia coli and Salmonella. Bacterial isolates were identified biochemically and DEC were identified by PCR. Antimicrobial susceptibility testing was by modified Kirby-Bauer disc diffusion method in accordance with the CLSI guidelines. Data were analyzed using Chi-square and Fisher's exact tests. RESULT: Diarrhoea infection is significantly high among children under 12 months (p = 0.002), caregivers without at least primary school education (p = 0.006), breastfeeding for under 6 months (p˂0.001), and caregivers who were siblings (p = 0.004). DEC was detected in 69(41.3%) cases but only 86(25.7%) controls (p < 0.001) and more commonly recovered during the wet season (p < 0.001). Enterotoxigenic E. coli (p = 0.031), enteropathogenic E. coli (p = 0.031) and Shiga-toxin-producing E. coli (p = 0.044) were recovered more commonly from cases than controls. DEC from patients with diarrhoea were commonly resistant to sulphonamides (91.3%), trimethoprim (82.6%), and ampicillin (78.3%) but were largely susceptible to quinolones and carbapenems (97.1%). CONCLUSION: Enteropathogenic, enterotoxigenic and Shiga toxin-producing E. coli are associated with diarrhoea in our setting, and show considerable resistance to first-line antimicrobials. Risk factors for DEC diarrhoea include infancy, inadequate breastfeeding and caregivers with education below primary school.

Prevalence of Cryptosporidium and Giardia infections in under-five children with diarrhoea in Blantyre, Malawi.

BACKGROUND: Diarrhoeal diseases are common among children in low- and middle-income countries and are major causes of morbidity and mortality. Cryptosporidium and Giardia are considered to be the main parasitic causes of diarrhoea in children. The aim of the present study was to determine the prevalence and associated factors of Cryptosporidium and Giardia infection in children under five years of age presenting at two health centres (Ndirande and Limbe) in Blantyre, Malawi. METHODS: This cross-sectional study was performed from February to July 2019 and included 972 children under 5 years of age with diarrhoea. Stool samples were immediately tested after collection at enrolment with a rapid diagnostic test for Cryptosporidium and Giardia infection. Descriptive statistics were used to assess the prevalence of these protozoan parasitic infections, and differences in the basic demographic and anthroponotic variables (between children with diarrhoea and parasite infection, being either Cryptosporidium and Giardia or both versus children with diarrhoea but no RDT confirmed parasite infection) were assessed. Their association with Cryptosporidium and Giardia infection was analysed using simple logistic regressions. RESULTS: Of the children recruited, 88 (9.1%) tested positive for Cryptosporidium and 184 (18.9%) for Giardia. Children with only a Giardia infection or a coinfection (of both parasites) were significantly older (mean age 24-26 months) compared to children with only a Cryptosporidium infection (mean age 13 months) or no parasitic infection (mean age 14 months). No significant differences were found with respect to gender, body temperature, stunting or wasting between the different groups of children with moderate to severe diarrhoea. Children attending the Ndirande health centre had almost two times higher odds of testing positive for both infections than those attending Limbe health centre. CONCLUSION: Cryptosporidium and Giardia infections are highly prevalent in children < 5 years with moderate to severe diarrhoea attending the Limbe and Ndirande health centres in Blantyre, Malawi.

Development and validation of a novel 7α-hydroxy-4-cholesten-3-one (C4) liquid chromatography tandem mass spectrometry method and its utility to assess pre-analytical stability.

OBJECTIVES: 7α-Hydroxy-4-cholesten-3-one (C4) is the common intermediary of both primary bile acids. C4 is recommended by the British Society of Gastroenterology for the investigation of bile acid diarrhoea (BAD) in patients with chronic diarrhoea. This project aimed to develop and validate an assay to quantitate C4 in serum and assess the stability of C4 in unseparated blood. METHODS: Accuracy was underpinned by calibrating to quantitative nuclear magnetic resonance analysis. C4 was analysed in a 96-well plate format with a deuterated C4 internal standard and liquid-liquid extraction. Validation followed the 2018 Food and Drug Administration guidelines. To assess C4 stability, healthy volunteers (n=12) donated 8 fasted samples each. Samples were incubated at 20 °C for up to 72 h and retrieved, centrifuged, aliquoted and frozen for storage at different time points prior to C4 analysis. RESULTS: The C4 method demonstrated excellent analytical performance and passed all validation criteria. The method was found to be accurate, precise, free from matrix effects and interference. After 72 h of delayed sample separation, C4 concentration gradually declined by up to 14 % from baseline. However, the change was not significant for up to 12 h. CONCLUSIONS: We present a robust method of analysing serum C4, offering a convenient alternative to 75SeHCAT for BAD investigation. C4 was found to decline in unseparated blood over time; however, after 12 h the mean change was <5 % from baseline. Our results suggest C4 is suitable for collection from both primary and secondary care prior to gastroenterology referral.

The effect of the implementation of the international code of marketing of breast-milk substitutes on child mortality in Ghana and Tanzania.

OBJECTIVE: The International Code of Marketing of Breast-Milk Substitutes is an important instrument to protect and promote appropriate infant and young child feeding and the safe use of commercial milk formulas. Ghana and Tanzania implemented the Code into national legislation in 2000 and 1994, respectively. We aimed to estimate the effects of the Code implementation on child mortality (CM) in both countries. SETTING: The countries analysed were Ghana and Tanzania. PARTICIPANTS: For CM and HIV rates, data from the Institute for Health Metrics and Evaluation from up to 2019 were used. Data for income and skilled birth rates were retrieved from the World Bank, for fertility from the World Population Prospects, for vaccination from the Global Health Observatory and for employment from the International Labour Organization. DESIGN: We used the synthetic control group method and performed placebo tests to assess statistical inference. The primary outcomes were CM by lower respiratory infections, mainly pneumonia, and diarrhoea and the secondary outcome was overall CM. RESULTS: One-sided inference tests showed statistically significant treatment effects for child deaths by lower respiratory infections in Ghana (P = 0·0476) and Tanzania (P = 0·0476) and for diarrhoea in Tanzania (P = 0·0476). More restrictive two-sided inference tests showed a statistically significant treatment effect for child deaths by lower respiratory infections in Ghana (P = 0·0476). No statistically significant results were found for overall CM. CONCLUSION: The results suggest that the implementation of the Code in both countries had a potentially beneficial effect on CM due to infectious diseases; however, further research is needed to corroborate these findings.

Analysing the interlinkage between diarrhoea and drinking water faecal contamination in India.

Diarrhoea is one of the major waterborne diseases spread through the faecal-oral route causing over 10 million cases and over 1,000 deaths per year in India. This study critically evaluates the interlinkage between bacteriological water quality, i.e. faecal coliforms and diarrhoea cases for the three pre-pandemic years 2017, 2018 and 2019 based on multiple sources. With around 17% of households tap water connectivity as of August 2019, the majority of the Indian population depends on raw groundwater (GW) and surface water sources. For this, faecal coliform (FC) levels in surface and GW have been mapped at district levels using data from India's National Water Quality Monitoring Programme. Health Management Information System's data on diarrhoea have been used to understand the monthly and district-wise variation of diarrhoea. The trends of FC, diarrhoea inpatient cases, and diarrhoea inpatient rates have been discussed. The analysis showed issues associated with the reliability and usefulness of these datasets with 43% of total India districts with no reported FC values for the study period. This study reveals a clear gap in the interlinkage between diarrhoea and bacteriological water quality with the unavailability of granular water quality data as a major challenge.

Andrographolide induced cytotoxicity and cell cycle arrest in Giardia trophozoites.

Giardiasis is a prevalent parasitic diarrheal disease caused by Giardia lamblia, affecting people worldwide. Recently, the availability of several drugs for its treatment has highlighted issues such as multidrug resistance, limited effectiveness and undesirable side effects. Therefore, it is necessary to develop alternative new drugs and treatment strategies that can enhance therapeutic outcomes and effectively treat giardiasis. Natural compounds show promise in the search for more potent anti-giardial agents. Our investigation focused on the effect of Andrographolide (ADG), an active compound of the Andrographis paniculata plant, on Giardia lamblia, assessing trophozoite growth, morphological changes, cell cycle arrest, DNA damage and inhibition of gene expression associated with pathogenic factors. ADG demonstrated anti-Giardia activity almost equivalent to the reference drug metronidazole, with an IC50 value of 4.99 µM after 24 h of incubation. In cytotoxicity assessments and morphological examinations, it showed significant alterations in trophozoite shape and size and effectively hindered the adhesion of trophozoites. It also caused excessive ROS generation, DNA damage, cell cycle arrest and inhibited the gene expression related to pathogenesis. Our findings have revealed the anti-giardial efficacy of ADG, suggesting its potential as an agent against Giardia infections. This could offer a natural and low-risk treatment option for giardiasis, reducing the risk of side effects and drug resistance.

Prevalence of antibiotic use for childhood diarrhoea in Uganda after an ORS scale-up intervention: a repeated cross-sectional study.

BACKGROUND: Diarrhoea kills 500,000 children every year despite availability of cheap and effective treatment. In addition, a large number are inappropriately treated with antibiotics, which do not benefit the patient but can contribute to the development of antibiotic resistance. We investigated whether the prevalence of antibiotic use among children under the age of five with diarrhoea in Uganda changed following a national intervention to increase the use of oral rehydration salts (ORS), and whether any socioeconomic characteristics were associated with antibiotic use. METHODS: A cross-sectional survey was conducted among caregivers of children under the age of five and among private health care providers and drug sellers in Uganda in 2014. This was compared to a similar survey among private health care providers, and the national demographic and health survey in Uganda in 2016. Logistic regression was used to find associations between antibiotic use and socioeconomic characteristics, and chi-square test and independent sample t-test were used to find significant differences between groups. RESULTS: The prevalence of antibiotic use among children under the age of five with diarrhoea in Uganda decreased from 30.5% in 2014 to 20.0% (p < 0.001) in 2016. No associations between socioeconomic characteristics and the use of antibiotics were significant in both 2014 and 2016. CONCLUSIONS: The use of antibiotics in children with diarrhoeal disease decreased significantly in Uganda between 2014 and 2016. However, the extent of the contribution of the ORS scale-up programme to this decrease cannot be determined from this study.