More Than Meets the Eye: Associations of Vaginal Bacteria with Gram Stain Morphotypes Using Molecular Phylogenetic Analysis

Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than Mobiluncus (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than Mobiluncus cells in women with highest Nugent score (p<0.001). Prevotella and Porphyromonas spp. were significantly associated with the “Bacteroides morphotype,” whereas Bacteroides species were rare. Gram-negative rods designated Mobiluncus morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.     

Why did the invasive walking catfish cross the road? Terrestrial chemoreception described for the first time in a fish

Clarias batrachus (walking catfish) is an invasive species in Florida, renowned for its air-breathing and terrestrial locomotor capabilities. However, it is unknown how this species orients in terrestrial environments. Furthermore, while anecdotal life history information is widespread for this species in its nonnative range, little of this information exists in the literature. The goals of this study were to identify sensory modalities that C. batrachus use to orient on land, and to describe the natural history of this species in its nonnative range. Fish (n = 150) were collected from around Ruskin, FL, and housed in a greenhouse, where experiments took place. Individual catfish were placed in the center of a terrestrial arena and were exposed to nine treatments: two controls, L-alanine, quinine, allyl isothiocynate, sucrose, volatile hydrogen sulphide, pond water and aluminium foil. These fish exhibited significantly positive chemotaxis toward alanine and pond water, and negative chemotaxis away from volatile hydrogen sulphide, suggesting chemoreception – both through direct contact and through the air – is important to their terrestrial orientation. Additionally, 88 people from Florida wildlife-related Facebook groups who have personal observations of C. batrachus on land were interviewed for information regarding their terrestrial natural history. These data were combined with observations from 38 YouTube videos. C. batrachus appear to emerge most frequently during or just after heavy summer rains, particularly from stormwater drains in urban areas, where they may feed on terrestrial invertebrates. By better understanding the full life history of C. batrachus, we can improve management of this species.     

Feedback GAP: pragmatic,cluster-randomized trial of goal setting and action plans to increase the effectiveness of audit and feedback interventions in primary care

Background

Audit and feedback to physicians is a commonly used quality improvement strategy, but its optimal design is unknown. This trial tested the effects of a theory-informed worksheet to facilitate goal setting and action planning, appended to feedback reports on chronic disease management, compared to feedback reports provided without these worksheets.

Methods

A two-arm pragmatic cluster randomized trial was conducted, with allocation at the level of primary care clinics. Participants were family physicians who contributed data from their electronic medical records. The ‘usual feedback’ arm received feedback every six months for two years regarding the proportion of their patients meeting quality targets for diabetes and/or ischemic heart disease. The intervention arm received these same reports plus a worksheet designed to facilitate goal setting and action plan development in response to the feedback reports. Blood pressure (BP) and low-density lipoprotein cholesterol (LDL) values were compared after two years as the primary outcomes. Process outcomes measured the proportion of guideline-recommended actions (e.g., testing and prescribing) conducted within the appropriate timeframe. Intention-to-treat analysis was performed.

Results

Outcomes were similar across groups at baseline. Final analysis included 20 physicians from seven clinics and 1,832 patients in the intervention arm (15% loss to follow up) and 29 physicians from seven clinics and 2,223 patients in the usual feedback arm (10% loss to follow up). Ten of 20 physicians completed the worksheet at least once during the study. Mean BP was 128/72 in the feedback plus worksheet arm and 128/73 in the feedback alone arm, while LDL was 2.1 and 2.0, respectively. Thus, no significant differences were observed across groups in the primary outcomes, but mean haemoglobin A1c was lower in the feedback plus worksheet arm (7.2% versus 7.4%, p<0.001). Improvements in both arms were noted over time for one-half of the process outcomes.

Discussion

Appending a theory-informed goal setting and action planning worksheet to an externally produced audit and feedback intervention did not lead to improvements in patient outcomes. The results may be explained in part by passive dissemination of the worksheet leading to inadequate engagement with the intervention.

Trial registration

ClinicalTrials.gov NCT00996645

     

Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE)

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl₂ differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.     

Trends in paediatric and adult bloodstream infections at a Ghanaian referral hospital: a retrospective study

Background

Bloodstream infections (BSI) are life-threatening emergencies. Identification of the common pathogens and their susceptibility patterns is necessary for timely empirical intervention.

Methods

We conducted a 4-year retrospective analysis of blood cultures from all patients excluding neonates at the Korle-Bu Teaching hospital, Ghana, from January 2010 through December 2013. Laboratory report data were used to determine BSI, blood culture contamination, pathogen profile, and antimicrobial resistance patterns.

Results

Overall, 3633 (23.16 %) out of 15,683 blood cultures were positive for various organisms. Pathogen-positive cultures accounted for 1451 (9.3 %, 95 % CI 8.5–9.8 %). Infants recorded the highest true blood culture positivity (20.9 %, n = 226/1083), followed by the elderly (13.3 %, n = 80/601), children (8.9 %, n = 708/8000) and adults (7.2 %, n = 437/6000) (p = 0.001 for Marascuilo’s post hoc). Overall occurrence of BSI declined with increasing age-group (p = 0.001) but the type of isolates did not vary with age except for Citrobacter, Escherichia coli, Klebsiella, Salmonella, and Enterococcus species. Gram negative bacteria predominated in our study (59.8 %, n = 867/1451), but the commonest bacterial isolate was Staphylococcus aureus (21.9 %, n = 318/1451)—and this trend run through the various age-groups. From 2010 to 2013, we observed a significant trend of yearly increase in the frequency of BSI caused by cephalosporin-resistant enterobacteria (Chi square for trend, p = 0.001). Meropenem maintained high susceptibility among all Gram-negative organisms ranging from 96 to 100 %. Among Staphylococcus aureus, susceptibility to cloxacillin was 76.6 %.

Conclusion

Our study shows a significantly high blood culture positivity in infants as compared to children, adults and the elderly. There was a preponderance of S. aureus and Gram-negative bacteria across all age-groups. Meropenem was the most active antibiotic for Gram-negative bacteria. Cloxacillin remains a very useful anti-staphylococcal agent.

     

Statistical tests for taxonomic distinctiveness from observations of monophyly

The observation of monophyly for a specified set of genealogical lineages is often used to place the lineages into a distinctive taxonomic entity. However, it is sometimes possible that monophyly of the lineages can occur by chance as an outcome of the random branching of lineages within a single taxon. Thus, especially for small samples, an observation of monophyly for a set of lineages--even if strongly supported statistically--does not necessarily indicate that the lineages are from a distinctive group. Here I develop a test of the null hypothesis that monophyly is a chance outcome of random branching. I also compute the sample size required so that the probability of chance occurrence of monophyly of a specified set of lineages lies below a prescribed tolerance. Under the null model of random branching, the probability that monophyly of the lineages in an index group occurs by chance is substantial if the sample is highly asymmetric, that is, if only a few of the sampled lineages are from the index group, or if only a few lineages are external to the group. If sample sizes are similar inside and outside the group of interest, however, chance occurrence of monophyly can be rejected at stringent significance levels (P < 10(-5)) even for quite small samples (approximately 20 total lineages). For a fixed total sample size, rejection of the null hypothesis of random branching in a single taxon occurs at the most stringent level if samples of nearly equal size inside and outside the index group--with a slightly greater size within the index group--are used. Similar results apply, with smaller sample sizes needed, when reciprocal monophyly of two groups, rather than monophyly of a single group, is of interest. The results suggest minimal sample sizes required for inferences to be made about taxonomic distinctiveness from observations of monophyly.     

Vaccination with SARS-CoV-2 variants of concern protects mice from challenge with wild-type virus

Vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against Coronavirus Disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild-type SARS-CoV-2 and from variants B.1.1.7, B.1.351, and P.1 for their immunogenicity and protective effect in vivo against challenge with wild-type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7-vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild-type SARS-CoV-2 in a mouse model.

This study explores the immune response induced by wild type and variant SARS-CoV-2 spike proteins, and the protection that these immune responses provide against challenge with wild type virus in the mouse model.