MicroRNA Expression and Intestinal Permeability in Children Living in a Slum Area of Bangladesh

Introduction: MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression. Changes in miRNA expression have been reported in a number of intestinal diseases, in both tissue samples and readily accessible specimens like stools. Pathogenic infections, diet, toxins, and other environmental factors are believed to influence miRNA expression. However, modulation of miRNAs in humans is yet to be thoroughly investigated. In this study, we examined the expression levels of two human miRNAs (miRNA-122 and miRNA-21) in stool samples of a group of Bangladeshi children who had an altered/increased intestinal permeability (IIP).Methods: Stool samples were collected from children with IIP (L:M > 0.09) and normal intestinal permeability (NIP) (L:M ≤ 0.09). Quantitative PCR was performed to quantify the levels of miRNA-122 and miR-21 in stools. Commercial ELISA kits were used to measure gut inflammatory markers Calprotectin and REG1B. Serum samples were tested using Human Bio-Plex Pro Assays to quantify IL-1β, IL-2, IL-5, IL-10, IL-13, IFN-γ, and TNF-α. Total nucleic acid extracted from stool specimens were used to determine gut pathogens using TaqMan Array Card (TAC) system real-time polymerase chain reaction.Results: The expression levels of miRNA-122 (fold change 11.6; p < 0.001, 95% CI: 6.14–11.01) and miR-21 (fold change 10; p < 0.001, 95% CI: 5.05–10.78) in stool were upregulated in children with IIP than in children with normal intestinal permeability (NIP). Significant correlations were observed between stool levels of miR-122 and miR-21 and the inflammatory cytokines IL-1β, IL-2, IFN-γ, and TNF-α (p < 0.05). Children with IIP were frequently infected with rotavirus, Campylobacter jejuni, Bacteroides fragilis, adenovirus, norovirus, astrovirus, and various Escherichia coli strains (ETEC_STh, ETEC_STp, EAEC_aaiC, EAEC_aatA) (p < 0.001). miR-122 significantly correlated with the fecal inflammatory biomarkers REG1B (p = 0.015) and Calprotectin (p = 0.030), however miR-21 did not show any correlation with these fecal biomarkers.

Establishment of a miRNA profile in paediatric HIV-1 patients and its potential as a biomarker for effectiveness of the combined antiretroviral therapy

AbstractmiRNAs have been extensively studied in pathological conditions, including viral infections, such as those provoked by HIV-1. Several cellular and circulating miRNAs are altered during HIV-1 infection, with either beneficial effects on host defenses or enhanced virus infectivity. Blood samples were collected in sterile EDTA tubes and plasma was separated and stored, as were PBMCs. RNA was isolated and reverse-transcribed. Finally, the miRNA gene expression profile was assessed using TaqMan Array Human microRNA Card A v2.0. A comprehensive statistical analysis was performed on the results obtained. This is the first study on miRNAs in HIV-1 paediatric patients, and a miRNA profile differentiating patients starting combination antiretroviral therapy (cART) at different times after HIV-1 diagnosis was established. Thirty-four miRNAs were observed to have different expression levels between the control group and the cART group. The data indicates the need to start cART as soon as possible after the establishment of HIV-1 infection to assure the best outcome possible. Finally, the selected 34 miRNAs may be used as biomarkers for prognosis and assessing therapy effectiveness. However, more research must be conducted to establish adequate quantitative correlations.

Site specific incidence rate of virulence related genes of enteroaggregative Escherichia coli and association with enteric inflammation and growth in children

There is a lack of information highlighting the possible association between strain carrying genes of enteroaggregative Escherichia coli (EAEC) and environmental enteric dysfunction (EED) and on linear growth during childhood. Strain carrying genes of EAEC from stool samples collected from 1705 children enrolled in the MAL-ED birth cohort were detected by TaqMan Array Cards. We measured site-specific incidence rate by using Poisson regression models, identified the risk factors and estimated the associations of strain carrying genes of EAEC with the composite EED score and linear growth at 24 months of age. Overall highest incidence rate (43.3%) was found among children having infection with the aggR gene, which was the greatest in Tanzania (56.7%). Low maternal education, lack of improved floor, and ownership of domestic cattle were found to be risk factors for EAEC infection. In the multivariate models, after adjusting the potential covariates, strain carrying genes of EAEC showed strong positive associations with the EED scores and with poor linear growth at 24 months of age. Our analyses may lay the cornerstone for a prospective epidemiologic investigation for a potential vaccine development aimed at reducing the burden of EAEC infections and combat childhood malnutrition.

Pathogens detected using a syndromic molecular diagnostic platform in patients hospitalized with severe respiratory illness in South Africa in 2017

Background Pneumonia continues to be a leading cause of death globally; however, in >50% of cases, an etiological agent is not identified. We describe the use of a multi-pathogen platform, TaqMan array card (TAC) real-time PCR, for the detection of pathogens in patients hospitalized with severe respiratory illness (SRI). Methods We conducted prospective hospital-based surveillance for SRI among patients at two sentinel sites in South Africa between January and December 2017. Patients were included in this study if a blood specimen and at least one respiratory specimen (naso- and oro-pharyngeal (NP/OP) swabs and/or sputum) were available for testing. We tested respiratory specimens for 21 respiratory pathogens and blood samples for nine bacteria using TAC. Pathogen detection was compared by age group and HIV status using the chi-squared test. Results During 2017, 956 patients were enrolled in SRI surveillance, and of these, 637 (67%) patients were included in this study (637 blood, 487 NP/OP and 411 sputum specimens tested). At least one pathogen was detected in 83% (527/637) of patients. Common pathogens detected included H. influenzae (225/637; 35%), S. pneumoniae (224/637; 35%), rhinovirus (144/637; 23%), S. aureus (129/637; 20%), K. pneumoniae (85/637; 13%), M. tuberculosis (75/637; 12%), and respiratory syncytial virus (57/637; 9%). Multiple pathogens (≥2) were co-detected in 57% (364/637) of patients. Conclusion While use of a multi-pathogen platform was useful in the detection of a pathogen in the majority of the patients, pathogen co-detections were common and would need clinical assessment for usefulness in individual-level treatment and management decisions.

Characterization of enteric disease in children using a low-cost specimen preservation method

BACKGROUND: Diarrhea is a leading cause of death in children under five. Molecular methods exist for the rapid detection of enteric pathogens; however, the logistical costs of storing stool specimens limit applicability. We sought to demonstrate that dried filter paper specimen preservation can identify diarrheal diseases causing significant morbidity among children in resource constrained countries. METHODS: A sub-study was nested into cholera surveillance in Cameroon. Enrollment criteria included: enrollment between 8/1/16 - 10/1/18; age < 18 years; a stool specimen; ≥ three loose stools within 24hours with the presence of dehydration and/or blood. 7227 persons were enrolled, for which 2746 met enrollment criteria and 337 were included in this analysis using the enteric TaqMan Array Card. Bacterial pathogens were compared to severity of diarrhea, age and sex, among other variables. RESULTS: 107 were ETEC positive of which: 40.2% (N=43) LT-STh, 19.6% (N=21) LT-STp; and 49.5% (53) LT-only. Major CFs were present in 43.9% of ETEC-positives. 96 were positive for Shigella, of which 14 (14.6%) reported dysentery. Model-derived quantitative cutoffs identified 116 (34.4%) with one highly diarrhea-associated pathogen and 16 (4.7%) with ≥ two. Shigellae and rotavirus were most strongly associated with diarrhea in children with mixed infections. CONCLUSION: Dried filter paper preserved specimens eliminate the need for frozen stool specimens and will facilitate enteric surveillance and contribute to the understanding of disease burden, which is needed to guide vaccine development and introduction. This study confirms Rotavirus, Shigella and ETEC as major contributors to pediatric diarrheal disease in two regions of Cameroon.

TaqMan Array card testing of participant-collected stool smears to determine the pathogen-specific epidemiology of travelers’ diarrhea

Background: We assessed the compliance with self-collection of stool smears on Whatman® FTA® Elute Card (FTA Card) and detection of travelers’ diarrhea (TD) associated pathogens using a quantitative PCR assay (customized TaqMan® array card [TAC]), in a prospective, observational cohort of travelers. Methods: Enrolled travelers documented symptoms on a travel diary and collected an FTA Card during a diarrheal episode, or at the end of travel if they remained asymptomatic. TAC testing was performed on FTA Cards from TD cases and 1:1 matched asymptomatic controls and 1:1 matched loose stool cases that did not meet TD criteria. Odds ratios (OR) were used to determine the association between detected pathogens and TD. Results: 484 of 2456 (19.7%) travelers completed an illness diary and met TD criteria, and 257 (53.1%) collected an FTA Card during the TD episode. FTA Cards were stored for a median of 2 years at room temperature (IQR: 1-4 years) before extraction and testing. The overall TAC detection rate in TD cases was 58.8% (95%CI: 52.5-64.8). Enterotoxigenic E. coli was the most common pathogen in TD cases (26.8%) and 3.5% of samples were positive for norovirus. The odds of detecting TD-associated pathogens in 231 matched cases and asymptomatic controls was 5.4 (95% CI: 3.6-8.1) and 2.0 (95% CI:1.1-3.7) in 121 matched TD and loose stool cases (p &lt; 0.05). Enteroaggregative E coli was the most common pathogen detected in asymptomatic controls and loose stool cases. Detection of diarrheagenic E coli, Shigella/enteroinvasive E coli (EIEC), and Campylobacter spp. was significantly associated with TD. Conclusions: FTA Cards are a useful adjunct to traditional stool collection methods for evaluating the pathogen-specific epidemiology of TD in austere environments. Qualitative detection of pathogens was associated with TD. Measures to improve compliance and quality of FTA Card collection with decreased storage duration may further optimize detection.

Green Banana mixed Diet is Beneficial in the Management of Childhood Persistent Diarrhea: an Open, Randomized-Controlled Trial (Preprint)

BACKGROUND Persistent diarrhea (PD), diarrhea for ≥14 days, accounts for 36%-56% of all diarrhea-related death. Studies demonstrated that green banana helps in the resolution of diarrhea. OBJECTIVE We assessed whether a lactose-free diet rice suji (RS) or rice suji containing green banana (GB-RS), improves outcome compared with 3/4th strength rice suji (3/4th RS) in 6 to 35 months old children having PD in the icddr,b Dhaka hospital. METHODS In this open-labeled, randomized, controlled trial, children with PD not improving with milk suji (the initial diet) received any of the three study diets: GB-RS, RS, 3/4th RS. The primary outcome was the percentage of children who recovered from diarrhea by day 5 with an intention to treat analysis. RESULTS From 1st December 2017 to 31st August 2019, we randomly allocated 45 children to each group. By day 5, 66 children recovered from PD, of whom 26 (58%), 14 (31%), 26 (58%) received GB-RS, RS, and 3/4th RS respectively. Significantly more resolution of PD occurred in GB-RS and 3/4th RS groups compared to RS group [relative risk (RR) 1⸱71, 95% CI 1⸱19-2⸱60; p=0⸱019]. Two (10%) children died, one each in RS group and GB-RS group. TaqMan array card (TAC) identified EAEC, rotavirus, norovirus, EPEC, astrovirus, and Campylobacter as the major pathogens associated with PD. CONCLUSIONS GB-RS and 3/4th RS are efficacious in the management of PD in young children. We also demonstrated a wide range of entero-pathogen associated with PD by TAC which needs further study to find a causal link. CLINICALTRIAL ClinicalTrials.gov, ID: NCT03366740 Registered on 8th December 2017 INTERNATIONAL REGISTERED REPORT RR2-10.2196/15759

Infection with Blastocystis spp. and its association with enteric infections and environmental enteric dysfunction among slum-dwelling malnourished adults in Bangladesh

Background Blastocystis spp. (Blastocystis) is a widely distributed gastrointestinal protist frequently reported in countries with tropical and sub-tropical climate. We sought to determine the factors associated with Blastocystis infection and investigate its role on biomarkers of intestinal health among slum-dwelling malnourished adults in Bangladesh. Methodology Total 524 malnourished adults with a body mass index ≤18.5 kg/m2 were included in this analysis. Presence of Blastocystis in feces was evaluated by TaqMan Array Card assays. Principal findings Blastocystis was tested positive in 78.6% of the participants. Prevalence of infection with atypical strains of enteropathogenic Escherichia coli (aEPEC) (56% vs. 38%, p<0.001), and Trichuris trichiura (28% vs. 15%, p-value = 0.02) was significantly greater in adults with Blastocystis, while Giardia intestinalis was significantly lower (8% vs. 14%, p-value = 0.04) in Blastocystis positive adults. Malnourished adults who were living in households with high crowding index (aOR = 2.18; 95% CI = 1.11, 4.65; p-value = 0.03), and infected with aEPEC (aOR = 2.14; 95% CI = 1.35, 3.44; p-value = 0.001) and Trichuris trichiura (aOR = 1.97; 95% CI = 1.08, 3.77; p = 0.03) were more likely to be infected with Blastocystis. A significant negative relationship was observed between Blastocystis and fecal concentrations of alpha-1 antitrypsin (β = -0.1; 95% CI = -1.7, -0.1; p-value<0.001) and Reg1B (β = -3.6; 95% CI = -6.9, -3.0; p-value = 0.03). Conclusions The study findings suggest that the presence of Blastocystis in human intestine influences gut health and may have potential pathogenic role in presence of other pathogens.

External Validation of a Mobile Clinical Decision Support System for Diarrhea Etiology Prediction in Children: A Multicenter Study in Bangladesh and Mali

Background: Diarrheal illness is a leading cause of antibiotic use for children in low- and middle-income countries. Determination of diarrhea etiology at the point-of-care without reliance on laboratory testing has the potential to reduce inappropriate antibiotic use. Methods: This prospective observational study aimed to develop and externally validate the accuracy of a mobile software application (App) for the prediction of viral-only etiology of acute diarrhea in children 0-59 months in Bangladesh and Mali. The App used previously derived and internally validated models using combinations of patient-intrinsic information (age, blood in stool, vomiting, breastfeeding status, and mid-upper arm circumference), pre-test odds using location-specific historical prevalence and recent patients, climate, and viral seasonality. Diarrhea etiology was determined with TaqMan Array Card using episode-specific attributable fraction (AFe) >0.5. Results: Of 302 children with acute diarrhea enrolled, 199 had etiologies above the AFe threshold. Viral-only pathogens were detected in 22% of patients in Mali and 63% in Bangladesh. Rotavirus was the most common pathogen detected (16% Mali; 60% Bangladesh). The viral seasonality model had an AUC of 0.754 (0.665-0.843) for the sites combined, with calibration-in-the-large=-0.393 (-0.455- -0.331) and calibration slope;=1.287 (1.207-1.367). By site, the pre-test odds model performed best in Mali with an AUC of 0.783 (0.705-0.86); the viral seasonality model performed best in Bangladesh with AUC 0.710 (0.595-0.825). Conclusion: The App accurately identified children with high likelihood of viral-only diarrhea etiology. Further studies to evaluate the Apps potential use in diagnostic and antimicrobial stewardship are underway.