Norovirus-Associated Gastroenteritis Vesikari Score and Pre-Existing Salivary IgA in Young Children from Rural South Africa.

Norovirus (NoV) is the leading cause of viral gastroenteritis, mostly affecting young children worldwide. However, limited data are available to determine the severity of norovirus-associated AGE (acute gastroenteritis) and to correlate it with the NoV-specific IgA antibodies' level. Between October 2019 and September 2021, two hundred stool samples were randomly collected from symptomatic cases for the vesikari score and NoV-specific IgA assessment in young children from rural South Africa. Additionally, one hundred saliva specimens were concomitantly sampled within the same cohort to evaluate the NoV-specific salivary IgA levels. In addition, 50 paired saliva and stool samples were simultaneously collected from asymptomatic children to serve as controls. NoV strains in stool samples were detected using real-time RT-PCR, amplified, and genotyped with RT-PCR and Sanger sequencing. ELISA using NoV VLP (virus-like particles) GII.4 as antigens was performed on the saliva specimens. Dehydrated children were predominantly those with NoV infections (65/74, 88%; p < 0.0001). NoV-positive infections were significantly associated with the severe diarrhea cases having a high vesikari score (55%, 33/60) when compared to the non-severe diarrheal score (29.3%, 41/140; p < 0.0308). NoV of the GII genogroup was mainly detected in severe diarrhea cases (50.9%, 30/59; p = 0.0036). The geometric means of the NoV-specific IgA level were higher in the asymptomatic NoV-infected group (0.286) as compared to the symptomatic group (0.174). This finding suggests that mucosal immunity may not protect the children from the NoV infection. However, the findings indicated the contribution of the pre-existing NoV-specific IgA immune response in reducing the severity of diarrheal disease. A high vesikari score of AGE associated with the NoV GII genogroup circulating in the study area underscores the need for an appropriate treatment of AGE based on the severity level of NoV-associated clinical symptoms in young children.

Microbiomes of Urine and the Prostate Are Linked to Human Prostate Cancer Risk Groups.

BACKGROUND: Bacteria play a suspected role in the development of several cancer types, and associations between the presence of particular bacteria and prostate cancer have been reported. OBJECTIVE: To provide improved characterisation of the prostate and urine microbiome and to investigate the prognostic potential of the bacteria present. DESIGN, SETTING, AND PARTICIPANTS: Microbiome profiles were interrogated in sample collections of patient urine (sediment microscopy: n = 318, 16S ribosomal amplicon sequencing: n = 46; and extracellular vesicle RNA-seq: n = 40) and cancer tissue (n = 204). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Microbiomes were assessed using anaerobic culture, population-level 16S analysis, RNA-seq, and whole genome DNA sequencing. RESULTS AND LIMITATIONS: We demonstrate an association between the presence of bacteria in urine sediments and higher D'Amico risk prostate cancer (discovery, n = 215 patients, p < 0.001; validation, n = 103, p < 0.001, χ2 test for trend). Characterisation of the bacterial community led to the (1) identification of four novel bacteria (Porphyromonas sp. nov., Varibaculum sp. nov., Peptoniphilus sp. nov., and Fenollaria sp. nov.) that were frequently found in patient urine, and (2) definition of a patient subgroup associated with metastasis development (p = 0.015, log-rank test). The presence of five specific anaerobic genera, which includes three of the novel isolates, was associated with cancer risk group, in urine sediment (p = 0.045, log-rank test), urine extracellular vesicles (p = 0.039), and cancer tissue (p = 0.035), with a meta-analysis hazard ratio for disease progression of 2.60 (95% confidence interval: 1.39-4.85; p = 0.003; Cox regression). A limitation is that functional links to cancer development are not yet established. CONCLUSIONS: This study characterises prostate and urine microbiomes, and indicates that specific anaerobic bacteria genera have prognostic potential. PATIENT SUMMARY: In this study, we investigated the presence of bacteria in patient urine and the prostate. We identified four novel bacteria and suggest a potential prognostic utility for the microbiome in prostate cancer.

The role of viral genomics in understanding COVID-19 outbreaks in long-term care facilities.

We reviewed all genomic epidemiology studies on COVID-19 in long-term care facilities (LTCFs) that had been published to date. We found that staff and residents were usually infected with identical, or near identical, SARS-CoV-2 genomes. Outbreaks usually involved one predominant cluster, and the same lineages persisted in LTCFs despite infection control measures. Outbreaks were most commonly due to single or few introductions followed by a spread rather than a series of seeding events from the community into LTCFs. The sequencing of samples taken consecutively from the same individuals at the same facilities showed the persistence of the same genome sequence, indicating that the sequencing technique was robust over time. When combined with local epidemiology, genomics allowed probable transmission sources to be better characterised. The transmission between LTCFs was detected in multiple studies. The mortality rate among residents was high in all facilities, regardless of the lineage. Bioinformatics methods were inadequate in a third of the studies reviewed, and reproducing the analyses was difficult because sequencing data were not available in many facilities.

Large-scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management.

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organizations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1565 positive samples (172 per 100 000 population) from 1376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6 % of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. In total, 1035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically distinct UK lineages were detected demonstrating local evolution, at a rate of ~2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a discrete sublineage associated with six care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients, indicating infection control measures were effective; and found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves.

CoronaHiT: high-throughput sequencing of SARS-CoV-2 genomes.

We present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.

Genetic characterisation of Norovirus strains in outpatient children from rural communities of Vhembe district/South Africa, 2014-2015.

BACKGROUND: Norovirus (NoV) is now the most common cause of both outbreaks and sporadic non-bacterial gastroenteritis worldwide. However, data supporting the role of NoV in diarrheal disease are limited in the African continent. OBJECTIVES: This study investigates the distribution of NoV genotypes circulating in outpatient children from rural communities of Vhembe district/South Africa. STUDY DESIGN: Stool specimens were collected from children under five years of age with diarrhea, and controls without diarrhea, between July 2014 and April 2015. NoV-positive samples, detected previously by Realtime PCR, were analysed using conventional RT-PCR targeting the partial capsid and polymerase genes. Nucleotide sequencing methods were performed to genotype the strains. RESULTS: The sequence analyses demonstrated multiple NoV genotypes including GI.4 (13.8%), GI.5 (6.9%), GII.14 (6.9%), GII.4 (31%), GII.6 (3.4%), GII.P15 (3.4%), GII.P21 (3.4%) and GII.Pe (31%). The most prevalent NoV genotypes were GII.4 Sydney 2012 variants (n=7) among the capsid genotypes, GII.Pe (n=9) among the polymerase genotypes and GII.Pe/GII.4 Sydney 2012 (n=8) putative recombinants among the RdRp/Capsid genotypes. Two unassigned GII.4 variants were found. CONCLUSIONS: The findings highlighted NoV genetic diversity and revealed continuous pandemic spread and predominance of GII.Pe/GII.4 Sydney 2012, indicative of increased NoV activity. An unusual RdRp genotype GII.P15 and two unassigned GII.4 variants were also identified from rural settings of the Vhembe district/South Africa. NoV surveillance is warranted to help to inform investigations into NoV evolution and disease burden, and to support on-going vaccine development programmes.

Norovirus prevalence and estimated viral load in symptomatic and asymptomatic children from rural communities of Vhembe district, South Africa.

BACKGROUND: Human Norovirus (NoV) is recognized as a major etiological agent of sporadic acute gastroenteritis worldwide. OBJECTIVES: This study describes the clinical features associated with Human NoV occurrence in children and determines the prevalence and estimated viral burden of NoV in symptomatic and asymptomatic children in rural South Africa. STUDY DESIGN: Between July 2014 and April 2015, outpatient children under 5 years of age from rural communities of Vhembe district, South Africa, were enrolled for the study. A total of 303 stool specimens were collected from those with diarrhea (n=253) and without (n=50) diarrhea. NoVs were identified using real-time one-step RT-PCR. RESULTS: One hundred and four (41.1%) NoVs were detected (62[59.6%] GII, 16[15.4%] GI, and 26[25%] mixed GI/GII) in cases and 18 (36%) including 9(50%) GII, 2(11.1%) GI and 7(38.9%) mixed GI/GII in controls. NoV detection rates in symptomatic and asymptomatic children (OR=1.24; 95% CI 0.66â¿¿2.33) were not significantly different. Comparison of the median CT values for NoV in symptomatic and asymptomatic children revealed significant statistical difference of estimated GII viral load from both groups, with a much higher viral burden in symptomatic children. CONCLUSIONS: Though not proven predictive of diarrhea disease in this study, the high detection rate of NoV reflects the substantial exposure of children from rural communities to enteric pathogens possibly due to poor sanitation and hygiene practices. The results suggest that the difference between asymptomatic and symptomatic children with NoV may be at the level of the viral load of NoV genogroups involved.

Human Norovirus prevalence in Africa: a review of studies from 1990 to 2013.

OBJECTIVES: To assess the contribution of Human Norovirus to diarrhoeal diseases in Africa. METHODS: We conducted a systematic review of the PubMed and EMBASE databases for published articles of Human Norovirus in Africa between 1990 and 2013. Data were extracted from selected studies and analysed. RESULTS: A total of 208 eligible studies were identified, of which 55 (from 19 countries) met the inclusion criteria. Many cases were of sporadic gastroenteritis (70.9%) in children (82%), 65.4% of which were seen in an outpatient setting. Over half (59.4%) of the affected children were under 5 years of age. The pooled prevalence rate of Human NoV was 11% (95% CI 8-14%), and the meta-analysis indicated significant heterogeneity between the studies. However, the conditional negative binomial regression could not clearly find the factors affecting the Human NoV prevalence rates reported. A close relationship was found between Human Norovirus strains from environmental and clinical samples. CONCLUSION: Unreported sporadic gastroenteritis cases of Human Norovirus are common in Africa. Most are community-associated infections. Possible environmental transmission routes have been documented. Combined environmental and clinical studies are required for targeted actions to control transmission of Human Norovirus in Africa. Systematic surveillance of Human Norovirus is needed to measure the burden of Norovirus-induced gastroenteritis in Africa and support any requirements for vaccine development.

Determination of the attP and attB sites of phage CD27 from Clostridium difficile NCTC 12727.

The attP region of the Clostridium difficile phage CD27 was identified, located immediately downstream of the putative recombinase. The phage could integrate into two specific sites (attB) in the C. difficile genome, one of which was in an open reading frame encoding a putative ATPase of an ABC transporter and the other in an open reading frame encoding a putative ATPase of the flagella protein export apparatus. The prophage was capable of excision and formation of a circular molecule and phages were spontaneously released at a low frequency during growth. Infection and lysogeny of a C. difficile strain previously shown to be sensitive to CD27 were demonstrated, leading to a reduction in toxin production. Finally, a putative repressor was identified which is likely to be involved in maintaining lysogeny in these strains.

Evaluation of bacteriophage therapy to control Clostridium difficile and toxin production in an in vitro human colon model system.

Clostridium difficile is a leading cause of hospital-acquired diarrhoea and represents a major challenge for healthcare providers. Due to the decreasing efficacy and associated problems of antibiotic therapy there is a need for synergistic and alternative treatments. In this study we investigated the use of a specific bacteriophage, ΦCD27, in a human colon model of C. difficile infection. Our findings demonstrate a significant reduction in the burden of C. difficile cells and toxin production with phage treatment relative to an untreated control, with no detrimental effect on commensal bacterial populations. The results demonstrate the potential of phage therapy, and highlight the limitations of using phages that have lysogenic capacity.

Bacteriophage treatment significantly reduces viable Clostridium difficile and prevents toxin production in an in vitro model system.

Clostridium difficile is primarily a nosocomial pathogen, causing thousands of cases of antibiotic-associated diarrhoea in the UK each year. In this study, we used a batch fermentation model of a C. difficile colonised system to evaluate the potential of a prophylactic and a remedial bacteriophage treatment regime to control the pathogen. It is shown that the prophylaxis regime was effective at preventing the growth of C. difficile (p = <0.001) and precluded the production of detectable levels of toxins A and B. The remedial treatment regime caused a less profound and somewhat transient decrease in the number of viable C. difficile cells (p = <0.0001), but still resulted in a lower level of toxin production relative to the control. The numbers of commensal bacteria including total aerobes and anaerobes, Bifidobacterium sp., Bacteroides sp., Lactobacillus sp., total Clostridium sp., and Enterobacteriaceae were not significantly decreased by this therapy, whereas significant detrimental effects were observed with metronidazole treatment. Our study indicates that phage therapy has potential to be used for the control of C. difficile; it highlights the main benefits of this approach, and some future challenges.