Smallpox vaccination induces a substantial increase in commensal skin bacteria that promote pathology and influence the host response.

Interactions between pathogens, host microbiota and the immune system influence many physiological and pathological processes. In the 20th century, widespread dermal vaccination with vaccinia virus (VACV) led to the eradication of smallpox but how VACV interacts with the microbiota and whether this influences the efficacy of vaccination are largely unknown. Here we report that intradermal vaccination with VACV induces a large increase in the number of commensal bacteria in infected tissue, which enhance recruitment of inflammatory cells, promote tissue damage and influence the host response. Treatment of vaccinated specific-pathogen-free (SPF) mice with antibiotic, or infection of genetically-matched germ-free (GF) animals caused smaller lesions without alteration in virus titre. Tissue damage correlated with enhanced neutrophil and T cell infiltration and levels of pro-inflammatory tissue cytokines and chemokines. One month after vaccination, GF and both groups of SPF mice had equal numbers of VACV-specific CD8+ T cells and were protected from disease induced by VACV challenge, despite lower levels of VACV-neutralising antibodies observed in GF animals. Thus, skin microbiota may provide an adjuvant-like stimulus during vaccination with VACV and influence the host response to vaccination.

Secreted hepatitis B virus splice variants differ by HBV genotype and across phases of chronic hepatitis B infection.

Chronic hepatitis B (CHB) is characterized by progression through different phases of hepatitis B virus (HBV) infection and disease. Although not necessary for HBV replication, there is increasing evidence that HBV splice variants are associated with liver disease progression and pathogenesis. However, there have been no studies till date on the frequency or diversity of splice variants for different HBV genotypes across the phases of CHB. Next generation sequencing data from 404 patient samples of HBV genotype A, B, C or D in Phase I, Phase II or Phase IV of CHB was analysed for HBV splice variants using an in house bioinformatics pipeline. HBV splice variants differed in frequency and type by genotype and phase of natural history. Splice variant Sp1 was the most frequently detected (206/404, 51% of patients), followed by Sp13 (151/404 37% of patients). The frequency of variants was generally highest in Phase II (123/165, 75% of patients), a phase typically associated with enhanced immune activation, followed by Phase I (69/99, 70% of patients). Splice variants were associated with reduced hepatitis B e antigen (HBeAg) levels and statistically reduced likelihood of achieving HBsAg loss (functional cure) in Phase II patients for Sp1 and Sp13 (p = .0014 and .0156, respectively). The frequency of HBV splice variants in patient serum differed markedly by HBV genotype and phase of CHB natural history. The increased levels of HBV splice variants detected in CHB phase II patients compared with the higher replicative Phase I in particular warrants further investigation.

Analysis of Hepatitis B Virus Haplotype Diversity Detects Striking Sequence Conservation Across Genotypes and Chronic Disease Phase.

BACKGROUND AND AIMS: We conducted haplotype analysis of complete hepatitis B virus (HBV) genomes following deep sequencing from 368 patients across multiple phases of chronic hepatitis B (CHB) infection from four major genotypes (A-D), analyzing 4,110 haplotypes to identify viral variants associated with treatment outcome and disease progression. APPROACH AND RESULTS: Between 18.2% and 41.8% of nucleotides and between 5.9% and 34.3% of amino acids were 100% conserved in all genotypes and phases examined, depending on the region analyzed. Hepatitis B e antigen (HBeAg) loss by week 192 was associated with different haplotype populations at baseline. Haplotype populations differed across the HBV genome and CHB history, this being most pronounced in the precore/core gene. Mean number of haplotypes (frequency) per patient was higher in immune-active, HBeAg-positive chronic hepatitis phase 2 (11.8) and HBeAg-negative chronic hepatitis phase 4 (16.2) compared to subjects in the "immune-tolerant," HBeAg-positive chronic infection phase 1 (4.3, P< 0.0001). Haplotype frequency was lowest in genotype B (6.2, P< 0.0001) compared to the other genotypes (A = 11.8, C = 11.8, D = 13.6). Haplotype genetic diversity increased over the course of CHB history, being lowest in phase 1, increasing in phase 2, and highest in phase 4 in all genotypes except genotype C. HBeAg loss by week 192 of tenofovir therapy was associated with different haplotype populations at baseline. CONCLUSIONS: Despite a degree of HBV haplotype diversity and heterogeneity across the phases of CHB natural history, highly conserved sequences in key genes and regulatory regions were identified in multiple HBV genotypes that should be further investigated as targets for antiviral therapies and predictors of treatment response.

Weight shapes the intestinal microbiome in preterm infants: results of a prospective observational study.

BACKGROUND: The intestinal microbiome in preterm infants differs markedly from term infants. It is unclear whether the microbiome develops over time according to infant specific factors. METHODS: We analysed (clinical) metadata - to identify the main factors influencing the microbiome composition development - and the first meconium and faecal samples til the 4th week via 16 S rRNA amplican sequencing. RESULTS: We included 41 infants (gestational age 25-30 weeks; birth weight 430-990 g. Birth via Caesarean section (CS) was associated with placental insufficiency during pregnancy and lower BW. In meconium samples and in samples from weeks 2 and 3 the abundance of Escherichia and Bacteroides (maternal faecal representatives) were associated with vaginal delivery while Staphylococcus (skin microbiome representative) was associated with CS. Secondly, irrespective of the week of sampling or the mode of birth, a transition was observed as children children gradually increased in weight from a microbiome dominated by Staphylococcus (Bacilli) towards a microbiome dominated by Enterobacteriaceae (Gammaproteobacteria). CONCLUSIONS: Our data show that the mode of delivery affects the meconium microbiome composition. They also suggest that the weight of the infant at the time of sampling is a better predictor for the stage of progression of the intestinal microbiome development/maturation than postconceptional age as it less confounded by various infant-specific factors.

Relative abundance of the Prevotella genus within the human gut microbiota of elderly volunteers determines the inter-individual responses to dietary supplementation with wheat bran arabinoxylan-oligosaccharides.

BACKGROUND: The human colon is colonised by a dense microbial community whose species composition and metabolism are linked to health and disease. The main energy sources for colonic bacteria are dietary polysaccharides and oligosaccharides. These play a major role in modulating gut microbial composition and metabolism, which in turn can impact on health outcomes. RESULTS: We investigated the influence of wheat bran arabinoxylan oligosaccharides (AXOS) and maltodextrin supplements in modulating the composition of the colonic microbiota and metabolites in healthy adults over the age of 60. Male and female volunteers, (n = 21, mean BMI 25.2 ± 0.7 kg/m2) participated in the double-blind, cross over supplement study. Faecal samples were collected for analysis of microbiota, short chain fatty acids levels and calprotectin. Blood samples were collected to measure glucose, cholesterol and triglycerides levels. There was no change in these markers nor in calprotectin levels in response to the supplements. Both supplements were well-tolerated by the volunteers. Microbiota analysis across the whole volunteer cohort revealed a significant increase in the proportional abundance of faecal Bifidobacterium species (P ≤ 0.01) in response to AXOS, but not maltodextrin, supplementation. There was considerable inter-individual variation in the other bacterial taxa that responded, with a clear stratification of volunteers as either Prevotella-plus (n = 8; > 0.1% proportional abundance) or Prevotella-minus (n = 13; ≤0.1% proportional abundance) subjects founded on baseline sample profiles. There was a significant increase in the proportional abundance of both faecal Bifidobacterium (P ≤ 0.01) and Prevotella species (P ≤ 0.01) in Prevotella-plus volunteers during AXOS supplementation, while Prevotella and Bacteroides relative abundances showed an inverse relationship. Proportional abundance of 26 OTUs, including bifidobacteria and Anaerostipes hadrus, differed significantly between baseline samples of Prevotella-plus compared to Prevotella-minus individuals. CONCLUSIONS: The wheat bran AXOS supplementation was bifidogenic and resulted in changes in human gut microbiota composition that depended on the initial microbiota profile, specifically the presence or absence of Prevotella spp. as a major component of the microbiota. Our data therefore suggest that initial profiling of individuals through gut microbiota analysis should be considered important when contemplating nutritional interventions that rely on prebiotics. TRIAL REGISTRATION: Clinical trial registration number: NCT02693782 . Registered 29 February 2016 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT02693782?term=NCT02693782&rank=1.

HBV variants are common in the 'immune-tolerant' phase of chronic hepatitis B.

Nucleos(t)ide analogues (NUC) treatment prevents progression of liver fibrosis in subjects with chronic hepatitis B (CHB). However, risk of hepatocellular carcinoma (HCC) persists despite viral suppression. Specific HBV variants have been associated with adverse outcomes, including HCC; however, the frequency of these variants during the seemingly benign immunotolerant (IT) phase is unknown. Next-generation sequencing and detailed virological characterization on a cohort of treatment-naïve IT subjects were performed to determine the frequency of clinically relevant viral variants. Samples from 97 subjects (genotype B/C 55%/45%, median HBV-DNA 8.5 log10 IU/mL, median HBsAg 4.8 log10  IU/mL, median HBeAg 3.6 log10  PEIU/mL) were analysed. Despite subjects being in the IT phase, clinically relevant HBV variants were common at baseline, particularly in the basal core promoter (BCP, overlaps the hepatitis B X (HBx) gene), precore and PreS regions. BCP/HBx variants were independently associated with lower baseline HBeAg, HBsAg and HBV-DNA titres. Precore variants were independently associated with higher baseline ALT. Increased viral diversity was associated with increased age and lower HBV-DNA, HBsAg and HBeAg levels. Low-level (<5%) drug resistance-associated amino acid substitutions in the HBV reverse transcriptase were detected in 9 (9%) subjects at pre-treatment but were not associated with reduced antiviral activity. Future studies should evaluate whether the detection of HBV variant during IT CHB is predictive of progression to immune clearance and poor prognosis, and whether early initiation of antiviral therapy during IT CHB to prevent the selection of HBV variants is clinically beneficial.

Multi-Compartment Profiling of Bacterial and Host Metabolites Identifies Intestinal Dysbiosis and Its Functional Consequences in the Critically Ill Child.

OBJECTIVES: Adverse physiology and antibiotic exposure devastate the intestinal microbiome in critical illness. Time and cost implications limit the immediate clinical potential of microbial sequencing to identify or treat intestinal dysbiosis. Here, we examined whether metabolic profiling is a feasible method of monitoring intestinal dysbiosis in critically ill children. DESIGN: Prospective multicenter cohort study. SETTING: Three U.K.-based PICUs. PATIENTS: Mechanically ventilated critically ill (n = 60) and age-matched healthy children (n = 55). INTERVENTIONS: Collection of urine and fecal samples in children admitted to the PICU. A single fecal and urine sample was collected in healthy controls. MEASUREMENTS AND MAIN RESULTS: Untargeted and targeted metabolic profiling using 1H-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry or urine and fecal samples. This was integrated with analysis of fecal bacterial 16S ribosomal RNA profiles and clinical disease severity indicators. We observed separation of global urinary and fecal metabolic profiles in critically ill compared with healthy children. Urinary excretion of mammalian-microbial co-metabolites hippurate, 4-cresol sulphate, and formate were reduced in critical illness compared with healthy children. Reduced fecal excretion of short-chain fatty acids (including butyrate, propionate, and acetate) were observed in the patient cohort, demonstrating that these metabolites also distinguished between critical illness and health. Dysregulation of intestinal bile metabolism was evidenced by increased primary and reduced secondary fecal bile acid excretion. Fecal butyrate correlated with days free of intensive care at 30 days (r = 0.38; p = 0.03), while urinary formate correlated inversely with vasopressor requirement (r = -0.2; p = 0.037). CONCLUSIONS: Disruption to the functional activity of the intestinal microbiome may result in worsening organ failure in the critically ill child. Profiling of bacterial metabolites in fecal and urine samples may support identification and treatment of intestinal dysbiosis in critical illness.

Variation of Gut Mucosal Microbiome With Anti-Saccharomyces cerevisiae Antibody Status in Pediatric Crohn Disease.

OBJECTIVES: Crohn disease (CD) is a chronic relapsing condition possibly caused by a dysbiotic microbiome. Approximately 30% to 60% of patients with CD have anti-Saccharomyces cerevisiae antibody (ASCA), but any association with gut microbiota is unexplored. We hypothesized that ASCA positivity would predict a signature microbial status and clinical phenotype. METHODS: Ileocolonic mucosal biopsies were obtained from children with CD (n = 135), and controls without inflammatory bowel disease (n = 45). Comparison was made between ASCA status, microbial diversity, and clinical characteristics. RESULTS: ASCA was highly specific but poorly sensitive for the diagnosis of CD. In patients with CD, ASCA positivity was associated with older age (≥10 years), ileocolonic disease, and long-term risk of surgery. Microbial alpha and beta diversity were similar in patients with CD with or without ASCA, but significantly less when compared to noninflammatory bowel disease controls. Microbial richness was similar across all 3 groups. Fourteen bacterial species were associated with ASCA-positive patients with CD and 14 species with ASCA-negative patients (P < 0.05). After using a false discovery rate correction Ruminococcus torques and bacterium Yersinia enterocolitica 61 remained significantly associated with CD ASCA positivity (P = 0.0178), whereas Enterobacter cloacae and Faecalibacterium prausnitzii were significantly associated with CD ASCA negativity (P = 0.0178 and 0.0342). CONCLUSION: ASCA-positive and ASCA-negative patients with CD have significant differences in gut microbiome composition, which could possibly be influencing the phenotype of the disease.

Diagnostics for Yaws Eradication: Insights From Direct Next-Generation Sequencing of Cutaneous Strains of Treponema pallidum.

Background: Yaws-like chronic ulcers can be caused by Treponema pallidum subspecies pertenue, Haemophilus ducreyi, or other, still-undefined bacteria. To permit accurate evaluation of yaws elimination efforts, programmatic use of molecular diagnostics is required. The accuracy and sensitivity of current tools remain unclear because our understanding of T. pallidum diversity is limited by the low number of sequenced genomes. Methods: We tested samples from patients with suspected yaws collected in the Solomon Islands and Ghana. All samples were from patients whose lesions had previously tested negative using the Centers for Disease Control and Prevention (CDC) diagnostic assay in widespread use. However, some of these patients had positive serological assays for yaws on blood. We used direct whole-genome sequencing to identify T. pallidum subsp pertenue strains missed by the current assay. Results: From 45 Solomon Islands and 27 Ghanaian samples, 11 were positive for T. pallidum DNA using the species-wide quantitative polymerase chain reaction (PCR) assay, from which we obtained 6 previously undetected T. pallidum subsp pertenue whole-genome sequences. These show that Solomon Islands sequences represent distinct T. pallidum subsp pertenue clades. These isolates were invisible to the CDC diagnostic PCR assay, due to sequence variation in the primer binding site. Conclusions: Our data double the number of published T. pallidum subsp pertenue genomes. We show that Solomon Islands strains are undetectable by the PCR used in many studies and by health ministries. This assay is therefore not adequate for the eradication program. Next-generation genome sequence data are essential for these efforts.

Direct Whole-Genome Sequencing of Cutaneous Strains of Haemophilus ducreyi.

Haemophilus ducreyi, which causes chancroid, has emerged as a cause of pediatric skin disease. Isolation of H. ducreyi in low-income settings is challenging, limiting phylogenetic investigation. Next-generation sequencing demonstrates that cutaneous strains arise from class I and II H. ducreyi clades and that class II may represent a distinct subspecies.

Strategies for managing rival bacterial communities: Lessons from burying beetles.

The role of bacteria in animal development, ecology and evolution is increasingly well understood, yet little is known of how animal behaviour affects bacterial communities. Animals that benefit from defending a key resource from microbial competitors are likely to evolve behaviours to control or manipulate the animal's associated external microbiota. We describe four possible mechanisms by which animals could gain a competitive edge by disrupting a rival bacterial community: "weeding," "seeding," "replanting" and "preserving." By combining detailed behavioural observations with molecular and bioinformatic analyses, we then test which of these mechanisms best explains how burying beetles, Nicrophorus vespilloides, manipulate the bacterial communities on their carcass breeding resource. Burying beetles are a suitable species to study how animals manage external microbiota because reproduction revolves around a small vertebrate carcass. Parents shave a carcass and apply antimicrobial exudates on its surface, shaping it into an edible nest for their offspring. We compared bacterial communities in mice carcasses that were either fresh, prepared by beetles or unprepared but buried underground for the same length of time. We also analysed bacterial communities in the burying beetle's gut, during and after breeding, to understand whether beetles could be "seeding" the carcass with particular microbes. We show that burying beetles do not "preserve" the carcass by reducing bacterial load, as is commonly supposed. Instead, our results suggest they "seed" the carcass with bacterial groups which are part of the Nicrophorus core microbiome. They may also "replant" other bacteria from the carcass gut onto the surface of their carrion nest. Both these processes may lead to the observed increase in bacterial load on the carcass surface in the presence of beetles. Beetles may also "weed" the bacterial community by eliminating some groups of bacteria on the carcass, perhaps through the production of antimicrobials themselves. Whether these alterations to the bacterial community are adaptive from the beetle's perspective, or are simply a by-product of the way in which the beetles prepare the carcass for reproduction, remains to be determined in future work. In general, our work suggests that animals might use more sophisticated techniques for attacking and disrupting rival microbial communities than is currently appreciated.

Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.

BACKGROUND: Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112-5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq. RESULTS: Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the Staphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (OTUs) from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The PacBio full-length bacterial 16S rRNA gene datasets generated 261 OTUs, which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index reported a higher diversity in the MiSeq dataset. CONCLUSION: The PacBio sequencing error rate is now in the same range of the previously widely used Roche 454 sequencing platform and current MiSeq platform. Species-level microbiome analysis revealed some inconsistencies between the full-length bacterial 16S rRNA gene capillary sequencing and PacBio sequencing.

Association between specific mucosa-associated microbiota in Crohn's disease at the time of resection and subsequent disease recurrence: a pilot study.

BACKGROUND AND AIM: Crohn's disease pathogenesis involves alterations in the gut microbiota. We characterized the mucosa-associated microbiota at the time of surgical resection and 6 months later to identify bacterial profiles associated with recurrence and remission. METHODS: Tissue samples were collected from surgical resection specimens in 12 Crohn's disease patients, and at 6 months postoperative colonoscopy from the neoterminal ileum and anastomosis. Endoscopic recurrence was assessed using the Rutgeerts score. Microbiota was characterized using microarray and 454 pyrosequencing. Longitudinal comparisons were made within patients, and cross-sectional comparisons made with colonoscopic biopsies from the terminal ileum and cecum of 10 healthy subjects. RESULTS: Microbiota of healthy subjects had high diversity and was dominated by the Firmicutes, Bacteroidetes, and Proteobacteria phyla. Biodiversity was lower in Crohn's disease patients at the time of surgery, increased after surgery, but still differed from healthy subjects. Crohn's disease patients with recurrent disease retained a microbiota favoring proteolytic-fueled fermentation and lactic acid-producing bacteria, including Enterococcus and Veillonella spp., while those maintaining remission demonstrated predominant saccharolytic Bacteroides, Prevotella, and Parabacteroides spp., and saccharolytic, butyrate-producing Firmicutes. CONCLUSION: In Crohn's disease, the mucosa-associated microbiota diversity is reduced at the time of surgery, but also differs between patients with different clinical outcomes at 6 months. These findings may provide prognostic information at the time of surgery, allowing identification of patients at increased risk of recurrence, and provide basis for a more targeted approach for therapeutic interventions after surgery.

Current knowledge and systematic review of viruses associated with Crohn's disease.

The aetiology of Crohn's disease (CD) is currently unknown. A viral trigger was proposed more than 40 years ago and has been the focus of many investigations. We summarised the current literature surrounding the association between viruses and CD and conducted a systematic review of all studies investigating this association quantitatively. Studies were identified by searching for 13 specific virus names or the general term 'virus' and 'Crohn's disease' in search engines PubMed and OVID. A total of 1315 studies were identified, of which 78 studies had a laboratory result. Of the 78, 46 case-control studies met all the inclusion criteria for forest plot analysis. The most common viruses studied were EBV, CMV and measles virus (MV). Forest plot analysis for each virus was carried out (fitted using random effects) and identified evidence of an association between CD and CMV (risk ratio [RR] 1.602, 95% confidence interval [CI] 1.069 to 2.400) with some suggestion that EBV may also be associated with CD (RR 1.366, 95% CI 0.996 to 1.873). However, there was evidence of large heterogeneity in the results from the identified studies for EBV. There was little evidence of an association with CD for MV, human herpes virus 6, human herpes virus 8, human simplex virus, varicella-zoster virus, mumps virus, Rubella virus, rotavirus, norovirus and adenovirus. There is still some question around whether CD is associated with the presence of a currently known virus.

Using 16s rRNA sequencing to characterize the microbiome of tropical cutaneous ulcer disease: insights into the microbial landscape and implications for diagnosis and treatment.

Cutaneous ulcers are common in yaws-endemic areas. Although often attributed to 'Treponema pallidum subsp. pertenue' and Haemophilus ducreyi, quantitative PCR has highlighted a significant proportion of these ulcers are negative for both pathogens and are considered idiopathic. This is a retrospective analysis utilising existing 16S rRNA sequencing data from two independent yaws studies that took place in Ghana and the Solomon Islands. We characterized bacterial diversity in 38 samples to identify potential causative agents for idiopathic cutaneous ulcers. We identified a diverse bacterial profile, including Arcanobacterium haemolyticum, Campylobacter concisus, Corynebacterium diphtheriae, Staphylococcus spp. and Streptococcus pyogenes, consistent with findings from previous cutaneous ulcer microbiome studies. No single bacterial species was universally present across all samples. The most prevalent bacterium, Campylobacter ureolyticus, appeared in 42% of samples, suggesting a multifactorial aetiology for cutaneous ulcers in yaws-endemic areas. This study emphasizes the need for a nuanced understanding of potential causative agents. The findings prompt further exploration into the intricate microbial interactions contributing to idiopathic yaw-like ulcers, guiding future research toward comprehensive diagnostic and therapeutic strategies.

TLR4, IL10RA, and NOD2 mutation in paediatric Crohn's disease patients: an association with Mycobacterium avium subspecies paratuberculosis and TLR4 and IL10RA expression.

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in the pathogenesis of Crohn's disease (CD). The role of CD susceptibility genes in association with these microbes is not known. Sixty-two early onset paediatric CD patients and 46 controls with known MAP status were analysed for an association with 34 single nucleotide polymorphisms (SNPs) from 18 CD susceptibility genes. Functional studies on peripheral blood mononuclear cells (PBMCs) were conducted on 17 CD patients with known CD mutations to assess IL-6, IL-10, and TNF-α expression upon stimulation with MAP precipitated protein derivative (PPD) and lipopolysaccharide (LPS). In addition, surface expression of IL10R and TLR4 on resting B cells, NK cells, T cells, and monocytes was assessed. A mutation in TLR4 (rs4986790) and IL10RA (rs22291130) was significantly associated with MAP-positive CD patients compared to MAP-negative CD patients (27.6 vs. 6.1 %, p = 0.021, and 62.1 vs. 33.3 %, p = 0.024, respectively). PPD and LPS significantly increased IL-6, IL-10, and TNF-α production in PBMCs. IL-10 and TNF-α production were significantly lower in a subgroup of CD patients (5/12) with a known NOD2 mutation. Receptor for IL-10 was significantly higher expressed on NK cells (CD56low) and on NK T cells harbouring a NOD2 mutations compared to wildtype cells (p = 0.031 and 0.005, respectively). TLR4 was significantly higher expressed on NK cells (CD56high) harbouring a NOD2 mutations compared to wildtype cells (p = 0.038).

Optimization of high-throughput 16S rRNA gene amplicon sequencing: an assessment of PCR pooling, mastermix use and contamination.

16S rRNA gene sequencing is widely used to characterize human and environmental microbiomes. Sequencing at scale facilitates better powered studies but is limited by cost and time. We identified two areas in our 16S rRNA gene library preparation protocol where modifications could provide efficiency gains, including (1) pooling of multiple PCR amplifications per sample to reduce PCR drift and (2) manual preparation of mastermix to reduce liquid handling. Using nasal samples from healthy human participants and a serially diluted mock microbial community, we compared alpha and beta diversity, and compositional abundance where the PCR amplification was conducted in triplicate, duplicate or as a single reaction, and where manually prepared or premixed mastermix was used. One hundred and fifty-eight 16S rRNA gene sequencing libraries were prepared, including a replicate experiment. Comparing PCR pooling strategies, we found no significant difference in high-quality read counts and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate on principal coordinate analysis (PCoA) and non-metric dimensional scaling (NMDS) analysis. Choice of mastermix had no significant impact on high-quality read and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate in PCoA and NMDS analysis. Importantly, we observed contamination and variability of rare species (<0.01 %) across replicate experiments; the majority of contaminants were accounted for by removal of species present at <0.1 %, or were linked to reagents (including a primer stock). We demonstrate no requirement for pooling of PCR amplifications or manual preparation of PCR mastermix, resulting in a more efficient 16S rRNA gene PCR protocol.

Hepatitis B virus haplotype number at baseline is a predictive marker of functional cure during antiviral therapy for patients with genotypes A and D HBeAg-positive chronic hepatitis B.

BACKGROUNDS AND AIMS: We investigated associations between hepatitis B virus (HBV) genome-length haplotype number (HN) at baseline in subjects with HBeAg-positive chronic hepatitis B (CHB), and the likelihood of achieving functional cure during direct-acting antiviral therapy METHOD: We analysed 86 HBeAg-positive baseline samples from patients with HBV genotypes A and D who were enrolled in a Phase II trial of tenofovir disoproxil fumarate (TDF) to determine if HN was a biomarker of HBsAg loss during therapy. Findings were validated using baseline samples from 181 patients with HBV genotypes A and D from an independent clinical trial utilising TDF or tenofovir alafenamide therapy in HBeAg-positive CHB. RESULTS: In the HBeAg-positive test cohort, patients with genotypes A or D and ≤2 haplotypes had a minimum of 21-fold higher likelihood of achieving HBsAg loss on TDF. Baseline HN (p < 0.0001) was a stronger predictor of HBsAg loss on therapy than HBsAg titre (p = 0.03), HBeAg titre (p = 0.0002), or the presence of HBV basal core promoter (A1762T, p = 0.0379 and G1764A, p = 0.0176) or G1896A precore mutations (p = 0.0218). This finding was validated in the independent validation cohort. HN was statistically higher in patients with HBV genotypes B or C infection compared to genotypes A and D. CONCLUSION: Baseline HN ≤2 predicts which patients with HBV genotypes A or D will more likely progress to functional cure on current direct-acting antiviral therapy, with greater accuracy than current biomarkers including baseline HBsAg and HBeAg titre.

Expression profile of genes involved in pathogenesis of pediatric Crohn's disease.

BACKGROUND AND AIM: Expression profiling of genes specific to pediatric Crohn's Disease (CD) patients was performed to elucidate the molecular mechanisms underlying disease cause and pathogenesis at disease onset. METHODS: We used suppressive subtractive hybridization (SSH) and differential screening analysis to profile the mRNA expression patterns of children with CD and age- and sex-matched controls without inflammatory bowel disease (IBD). RESULTS: Sequence analysis of 1000 clones enriched by SSH identified 75 functionally annotated human genes, represented by 430 clones. The 75 genes have potential involvement in gene networks, such as antigen presentation, inflammation, infection mechanism, connective tissue development, cell cycle and cancer. Twenty-eight genes were previously described in association with CD, while 47 were new genes not previously reported in the context of IBD. Additionally, 29 of the 75 genes have been previously implicated in bacterial and viral infections. Quantitative real-time reverse transcription polymerase chain reaction performed on ileal-derived RNA from 13 CD and nine non-IBD patients confirmed the upregulation of extracellular matrix gene MMP2 (P = 0.001), and cell proliferation gene REG1A (P = 0.063) in our pediatric CD cohort. CONCLUSION: The retrieval of 28 genes previously reported in association with adult CD emphasizes the importance of these genes in the pediatric setting. The observed upregulation of REG1A and MMP2, and their known impact on cell proliferation and extracellular matrix remodeling, agrees with the clinical behavior of the disease. Moreover, the expressions of bacterial- and virus-related genes in our CD-patient tissues support the concept that microbial agents are important in the etiopathogenesis of CD.