Unravelling the metabolic impact of SBS-associated microbial dysbiosis: Insights from the piglet short bowel syndrome model

peer-reviewedLiver disease is a major source of morbidity and mortality in children with short bowel syndrome (SBS). SBS-associated microbial dysbiosis has recently been implicated in the development of SBS-associated liver disease (SBS-ALD), however the pathological implications of this association have not been explored. In this study high-throughput sequencing of colonic content from the well-validated piglet SBS-ALD model was examined to determine alterations in microbial communities, and concurrent metabolic alterations identified in urine samples via targeted mass spectrometry approaches (GC-MS, LC-MS, FIA-MS) further uncovered impacts of microbial disturbance on metabolic outcomes in SBS-ALD. Multi-variate analyses were performed to elucidate contributing SBS-ALD microbe and metabolite panels and to identify microbe-metabolite interactions. A unique SBS-ALD microbe panel was clearest at the genus level, with discriminating bacteria predominantly from the Firmicutes and Bacteroidetes phyla. The SBS-ALD metabolome included important alterations in the microbial metabolism of amino acids and the mitochondrial metabolism of branched chain amino acids. Correlation analysis defined microbe-metabolite clustering patterns unique to SBS-ALD and identified a metabolite panel that correlates with dysbiosis of the gut microbiome in SBS

Effect of Formulation Variables on the Stability of a Live, Rotavirus (RV3-BB) Vaccine Candidate using in vitro Gastric Digestion Models to Mimic Oral Delivery

In this work, two different in vitro gastric digestion models were used to evaluate the stability of a live attenuated rotavirus vaccine candidate (RV3-BB) under conditions designed to mimic oral delivery in infants. First, a forced-degradation model was established at low pH to assess the buffering capacity of formulation excipients and to screen for RV3-BB stabilizers. Second, a sequential-addition model was implemented to examine RV3-BB stability under conditions more representative of oral administration to infants. RV3-BB rapidly inactivated at < pH 5.0 (37 °C, 1 h) as measured by an infectivity RT-qPCR assay. Pre-neutralization with varying volumes of infant formula (Enfamil®) or antacid (Mylanta®) conferred partial to full protection of RV3-BB. Excipients with sufficient buffering capacity to minimize acidic pH inactivation of RV3-BB were identified (e.g., succinate, acetate, adipate), however, they concomitantly destabilized RV3-BB in accelerated storage stability studies. Both effects were concentration dependent, thus excipient optimization was required to design candidate RV3-BB formulations which minimize acid-induced viral inactivation during oral delivery while not destabilizing the vaccine during long-term 2–8 °C storage. Finally, a statistical Design -of-Experiments (DOE) study examining RV3-BB stability in the in vitro sequential-addition model identified key formulation parameters likely affecting RV3-BB stability during in vivo oral delivery

Formulation development of a live attenuated human rotavirus (RV3-BB) vaccine candidate for use in low- and middle-income countries

Formulation development was performed with the live, attenuated, human neonatal rotavirus vaccine candidate (RV3-BB) with three main objectives to facilitate use in low- and middle- income countries including (1) a liquid, 2–8°C stable vaccine, (2) no necessity for pre-neutralization of gastric acid prior to oral administration of a small-volume dose, and (3) a low-cost vaccine dosage form. Implementation of a high-throughput RT-qPCR viral infectivity assay for RV3-BB, which correlated well with traditional FFA assays in terms of monitoring RV3-BB stability profiles, enabled more rapid and comprehensive formulation development studies. A wide variety of different classes and types of pharmaceutical excipients were screened for their ability to stabilize RV3-BB during exposure to elevated temperatures, freeze-thaw and agitation stresses. Sucrose (50–60% w/v), PEG-3350, and a solution pH of 7.8 were selected as promising stabilizers. Using a combination of an in vitro gastric digestion model (to mimic oral delivery conditions) and accelerated storage stability studies, several buffering agents (e.g., succinate, adipate and acetate at ~200 to 400 mM) were shown to protect RV3-BB under acidic conditions, and at the same time, minimize virus destabilization during storage. Several optimized RV3-BB candidate formulations were identified based on negligible viral infectivity losses during storage at 2–8°C and −20°C for up to 12 months, as well as by relative stability comparisons at 15°C and 25°C (up to 12 and 3 months, respectively). These RV3-BB stability results are discussed in the context of stability profiles of other rotavirus serotypes as well as future RV3-BB formulation development activities

Direct and indirect costs of acute diarrhea in children under five years of age in Indonesia: Health facilities and community survey

Background: Diarrhea remains a major cause of child morbidity and mortality in low- and middle-income countries. Reliable data on the economic burden of diarrhea is required to support the selection of appropriate health intervention programs. This study aimed to estimate the costs of acute diarrhea in children under five years of age in Indonesia, a large middle-income country with a substantial diarrheal burden. Methods: Direct medical cost data were extracted retrospectively for 1050 children under five years of age with acute diarrhea receiving inpatient care across 45 health facilities in seven Indonesian provinces during 2017–2020. Direct medical costs for children treated in outpatient settings were estimated by collecting unit costs associated with standard diarrhea case management in children. A structured interview of 240 caregivers of inpatients was also conducted retrospectively to estimate direct non-medical costs as well as indirect costs from caregiver income loss. Results: The weighted average direct medical cost for treatment of acute diarrhea as an inpatient and outpatient across health facility types was US$99.8 (SD±$56.8)(35% room costs, 29% professional fees, 26% medication costs, 10% diagnostic costs) and US$7.6 (SD±$4.3) (34% diagnostic costs, 28% medication costs, 27% professional fees, 10% registration fees), respectively. The average direct non-medical household cost for an acute diarrheal admission was US$4.90 and the indirect cost was US$9.90. Conclusion: There is a significant economic burden associated with acute diarrhea in children in Indonesia. This study, based on a wide variety of health care settings and geographical regions, provides data to inform the economic evaluation of rotavirus vaccines and other diarrheal prevention programs. Funding: This work was supported by a research grant from the Murdoch Children's Research Institute (MCRI) and PATH; and the Indonesian Technical Advisory Group on Immunization (ITAGI)

Developing a manufacturing process to deliver a cost effective and stable liquid human rotavirus vaccine

Despite solid evidence of the success of rotavirus vaccines in saving children from fatal gastroenteritis, more than 82 million infants worldwide still lack access to a rotavirus vaccine. The main barriers to global rotavirus vaccine coverage include cost, manufacturing capacity and suboptimal efficacy in low- and lower-middle income countries. One vaccine candidate with the potential to address the latter is based on the novel, naturally attenuated RV3 strain of rotavirus, RV3-BB vaccine administered in a birth dose strategy had a vaccine efficacy against severe rotavirus gastroenteritis of 94% at 12 months of age in infants in Indonesia. To further develop this vaccine candidate, a well-documented and low-cost manufacturing process is required. A target fully loaded cost of goods (COGs) of ≤$3.50 per course of three doses was set based on predicted market requirements. COGs modelling was leveraged to develop a process using Vero cells in cell factories reaching high titers, reducing or replacing expensive reagents and shortening process time to maximise output. Stable candidate liquid formulations were developed allowing two-year storage at 2–8 °C. In addition, the formulation potentially renders needless the pretreatment of vaccinees with antacid to ensure adequate gastric acid neutralization for routine oral vaccination. As a result, the formulation allows small volume dosing and reduction of supply chain costs. A dose ranging study is currently underway in Malawi that will inform the final clinical dose required. At a clinical dose of ≤6.3 log10 FFU, the COGs target of ≤$3.50 per three dose course was met. At a clinical dose of 6.5 log10 FFU, the final manufacturing process resulted in a COGs that is substantially lower than the current average market price, 2.44 USD per dose. The manufacturing and formulation processes were transferred to BioFarma in Indonesia to enable future RV3-BB vaccine production

The impact of the rotavirus vaccine on diarrhoea, five years following national introduction in Fiji.

BACKGROUND: In 2012, Fiji became the first independent Pacific island country to introduce rotavirus vaccine. We describe the impact of rotavirus vaccine on all-cause diarrhoea admissions in all ages, and rotavirus diarrhoea in children <5 years of age. METHODS: An observational study was conducted retrospectively on all admissions to the public tertiary hospitals in Fiji (2007-2018) and prospectively on all rotavirus-positive diarrhoea admissions in children <5 years at two hospital sites (2006-2018, and 2010-2015), along with rotavirus diarrhoea outpatient presentations at one secondary public hospital (2010-2015). The impact of rotavirus vaccine was determined using incidence rate ratios (IRR) of all-cause diarrhoea admissions and rotavirus diarrhoea, comparing the pre-vaccine and post-vaccine periods. All-cause admissions were used as a control. Multiple imputation was used to impute missing stool samples. FINDINGS: All-cause diarrhoea admissions declined among all age groups except among infants ≤2 months old and adults ≥55 years. For children <5 years, all-cause diarrhoea admissions declined by 39% (IRR)=0•61, 95%CI; 0•57-0•65, p-value<0•001). There was an 81% (95%CI; 51-94%) reduction in mortality among all-cause diarrhoea admissions in children under <5 years. Rotavirus diarrhoea admissions at the largest hospital among children <5 years declined by 87% (IRR=0•13, 95%CI; 0•10-0•17, p-value<0•001). Among rotavirus diarrhoea outpatient presentations, the IRR was 0•39 (95%CI; 0•11, 1.21, p-value=0.077). INTERPRETATIONS: Morbidity and mortality due to rotavirus and all-cause diarrhoea in Fiji has declined in people aged 2 months to 54 years after the introduction of the RV vaccine. FUNDING: Supported by WHO and the Australian Government

Neonatal rotavirus vaccine (RV3-BB) immunogenicity and safety in a neonatal and infant administration schedule in Malawi: a randomised, double-blind, four-arm parallel group dose-ranging study.

BackgroundRotavirus vaccines reduce rotavirus-related deaths and hospitalisations but are less effective in high child mortality countries. The human RV3-BB neonatal G3P[6] rotavirus vaccine administered in a neonatal schedule was efficacious in reducing severe rotavirus gastroenteritis in Indonesia but had not yet been evaluated in African infants.MethodsWe did a phase 2, randomised, double-blind, parallel group dose-ranging study of three doses of oral RV3-BB rotavirus vaccine in infants in three primary health centres in Blantyre, Malawi. Healthy infants less than 6 days of age with a birthweight 2·5 to 4·0 kg were randomly assigned (1:1:1:1) into one of four treatment groups: neonatal vaccine group, which included high-titre (1·0 × 107 focus-forming unit [FFU] per mL), mid-titre (3·0 × 106 FFU per mL), or low-titre (1·0 × 106 FFU per mL); and infant vaccine group, which included high-titre (1·0 × 107 FFU per mL) using a computer generated code (block size of four), stratified by birth (singleton vs multiple). Neonates received their three doses at 0-5 days to 10 weeks and infants at 6-14 weeks. Investigators, participant families, and laboratory staff were masked to group allocation. Anti-rotavirus IgA seroconversion and vaccine take (IgA seroconversion and stool shedding) were evaluated. Safety was assessed in all participants who received at least one dose of vaccine or placebo. The primary outcome was the cumulative IgA seroconversion 4 weeks after three doses of RV3-BB in the neonatal schedule in the high-titre, mid-titre, and low-titre groups in the per protocol population, with its 95% CI. With the high-titre group as the active control group, we did a non-inferiority analysis of the proportion of participants with IgA seroconversion in the mid-titre and low-titre groups, using a non-inferiority margin of less than 20%. This trial is registered at ClinicalTrials.gov (NCT03483116).FindingsBetween Sept 17, 2018, and Jan 27, 2020, 711 participants recruited were randomly assigned into four treatment groups (neonatal schedule high titre n=178, mid titre n=179, low titre n=175, or infant schedule high titre n=179). In the neonatal schedule, cumulative IgA seroconversion 4 weeks after three doses of RV3-BB was observed in 79 (57%) of 139 participants in the high-titre group, 80 (57%) of 141 participants in the mid-titre group, and 57 (41%) of 138 participants in the low-titre group and at 18 weeks in 100 (72%) of 139 participants in the high-titre group, 96 (67%) of 143 participants in the mid-titre group, and 86 (62%) of 138 of participants in the low-titre. No difference in cumulative IgA seroconversion 4 weeks after three doses of RV3-BB was observed between high-titre and mid-titre groups in the neonatal schedule (difference in response rate 0·001 [95%CI -0·115 to 0·117]), fulfilling the criteria for non-inferiority. In the infant schedule group 82 (59%) of 139 participants had a cumulative IgA seroconversion 4 weeks after three doses of RV3-BB at 18 weeks. Cumulative vaccine take was detected in 483 (85%) of 565 participants at 18 weeks. Three doses of RV3-BB were well tolerated with no difference in adverse events among treatment groups: 67 (39%) of 170 participants had at least one adverse event in the high titre group, 68 (40%) of 172 participants had at least one adverse event in the mid titre group, and 69 (41%) of 169 participants had at least one adverse event in the low titre group.InterpretationRV3-BB was well tolerated and immunogenic when co-administered with Expanded Programme on Immunisation vaccines in a neonatal or infant schedule. A lower titre (mid-titre) vaccine generated similar IgA seroconversion to the high-titre vaccine presenting an opportunity to enhance manufacturing capacity and reduce costs. Neonatal administration of the RV3-BB vaccine has the potential to improve protection against rotavirus disease in children in a high-child mortality country in Africa.FundingBill & Melinda Gates Foundation, Australian Tropical Medicine Commercialisation Grant

Aetiology and incidence of diarrhoea requiring hospitalisation in children under 5 years of age in 28 low-income and middle-income countries: findings from the Global Pediatric Diarrhea Surveillance network.

Introduction: Diarrhoea remains a leading cause of child morbidity and mortality. Systematically collected and analysed data on the aetiology of hospitalised diarrhoea in low-income and middle-income countries are needed to prioritise interventions. Methods: We established the Global Pediatric Diarrhea Surveillance network, in which children under 5 years hospitalised with diarrhoea were enrolled at 33 sentinel surveillance hospitals in 28 low-income and middle-income countries. Randomly selected stool specimens were tested by quantitative PCR for 16 causes of diarrhoea. We estimated pathogen-specific attributable burdens of diarrhoeal hospitalisations and deaths. We incorporated country-level incidence to estimate the number of pathogen-specific deaths on a global scale. Results: During 2017–2018, 29 502 diarrhoea hospitalisations were enrolled, of which 5465 were randomly selected and tested. Rotavirus was the leading cause of diarrhoea requiring hospitalisation (attributable fraction (AF) 33.3%; 95% CI 27.7 to 40.3), followed by Shigella (9.7%; 95% CI 7.7 to 11.6), norovirus (6.5%; 95% CI 5.4 to 7.6) and adenovirus 40/41 (5.5%; 95% CI 4.4 to 6.7). Rotavirus was the leading cause of hospitalised diarrhoea in all regions except the Americas, where the leading aetiologies were Shigella (19.2%; 95% CI 11.4 to 28.1) and norovirus (22.2%; 95% CI 17.5 to 27.9) in Central and South America, respectively. The proportion of hospitalisations attributable to rotavirus was approximately 50% lower in sites that had introduced rotavirus vaccine (AF 20.8%; 95% CI 18.0 to 24.1) compared with sites that had not (42.1%; 95% CI 33.2 to 53.4). Globally, we estimated 208 009 annual rotavirus-attributable deaths (95% CI 169 561 to 259 216), 62 853 Shigella-attributable deaths (95% CI 48 656 to 78 805), 36 922 adenovirus 40/41-attributable deaths (95% CI 28 469 to 46 672) and 35 914 norovirus-attributable deaths (95% CI 27 258 to 46 516). Conclusions: Despite the substantial impact of rotavirus vaccine introduction, rotavirus remained the leading cause of paediatric diarrhoea hospitalisations. Improving the efficacy and coverage of rotavirus vaccination and prioritising interventions against Shigella, norovirus and adenovirus could further reduce diarrhoea morbidity and mortality

Anthropometric Measures in Children with Renal Failure

Anthropometric and body composition assessments provide important information about the nutritional status of dialysis patients. A nutritional assessment by a trained dietitian is therefore recommended for patients diagnosed with renal disease because they are frequently in poor nutritional health. Knowledge of the nutritional health of patients with renal disease is necessary to prescribe and monitor appropriate clinical and nutrition therapies. Anthropometrical methods occupy a meaningful role in assessing the nutritional status of patients with renal disease, are cost-effective screening techniques for describing body size, and they are well suited for identifying levels of body composition, nutritional status, or risk for disease. However, children with renal disease present special problems for anthropometry, including decreased functional status and increased comorbidity, which challenge nutrition assessment methodology. Measures of weight, stature, calf circumference, arm circumference, and triceps and subscapular skinfolds have therefore been reported for dialysis patients, who tend to be shorter, lighter, and have less adipose tissue than healthy persons of the same age. Other techniques to assess body composition are dual energy X-ray absorptiometry (DEXA), bioelectrical impedance, total body water (TBW), and prediction equations. However, irrespective of the technique to be used, all are validated in healthy individuals based on the assumption that body composition parameters are relatively static, which isn’t the case in renal patients. Anthropometric measurements should be an integral component of the routine care of the child and adolescent with renal disease