Design and validation of Dolosigranulum pigrum specific PCR primers using the bacterial core genome.

Dolosigranulum pigrum-a lactic acid bacterium that is increasingly recognized as an important member of the nasal microbiome. Currently, there are limited rapid and low-cost options for confirming D. pigrum isolates and detecting D. pigrum in clinical specimens. Here we describe the design and validation of a novel PCR assay targeting D. pigrum that is both sensitive and specific. We designed a PCR assay targeting murJ, a single-copy core species gene identified through the analysis of 21 D. pigrum whole genome sequences. The assay achieved 100% sensitivity and 100% specificity against D. pigrum and diverse bacterial isolates and an overall 91.1% sensitivity and 100% specificity using nasal swabs, detecting D. pigrum at a threshold of 1.0 × 104 D. pigrum 16S rRNA gene copies per swab. This assay adds a reliable and rapid D. pigrum detection tool to the microbiome researcher toolkit investigating the role of generalist and specialist bacteria in the nasal environment.

Staphylococcal Communities on Skin Are Associated with Atopic Dermatitis and Disease Severity.

The skin microbiota of atopic dermatitis (AD) patients is characterized by increased Staphylococcus aureus colonization, which exacerbates disease symptoms and has been linked to reduced bacterial diversity. Skin bacterial communities in AD patients have mostly been described at family and genus levels, while species-level characterization has been limited. In this study, we investigated the role of the bacteria belonging to the Staphylococcus genus using targeted sequencing of the tuf gene with genus-specific primers. We compared staphylococcal communities on lesional and non-lesional skin of AD patients, as well as AD patients with healthy controls, and determined the absolute abundance of bacteria present at each site. We observed that the staphylococcal community, bacterial alpha diversity, and bacterial densities were similar on lesional and non-lesional skin, whereas AD severity was associated with significant changes in staphylococcal composition. Increased S. aureus, Staphylococcus capitis, and Staphylococcus lugdunensis abundances were correlated with increased severity. Conversely, Staphylococcus hominis abundance was negatively correlated with severity. Furthermore, S. hominis relative abundance was reduced on AD skin compared to healthy skin. In conclusion, various staphylococcal species appear to be important for skin health.

Alteration of Bacterial Communities in Anterior Nares and Skin Sites of Patients Undergoing Arthroplasty Surgery: Analysis by 16S rRNA and Staphylococcal-Specific tuf Gene Sequencing.

The aim was to study alterations of bacterial communities in patients undergoing hip or knee arthroplasty to assess the impact of chlorhexidine gluconate soap decolonisation and systemic antibiotic prophylaxis. A Swedish multicentre, prospective collection of samples obtained from elective arthroplasty patients (n = 83) by swabbing anterior nares, skin sites in the groin and the site of planned surgery, before and after arthroplasty surgery, was analysed by 16S rRNA (V3-V4) gene sequencing and a complementary targeted tuf gene sequencing approach to comprehensively characterise alterations in staphylococcal communities. Significant reductions in alpha diversity was detected for both bacterial (p = 0.04) and staphylococcal (p = 0.03) groin communities after arthroplasty surgery with significant reductions in relative Corynebacterium (p = 0.001) abundance and Staphylococcus hominis (p = 0.01) relative staphylococcal abundance. In nares, significant reductions occurred for Staphylococcus hominis (p = 0.02), Staphylococcus haemolyticus (p = 0.02), and Staphylococcus pasteuri (p = 0.003) relative to other staphylococci. Staphylococcus aureus colonised 35% of anterior nares before and 26% after arthroplasty surgery. Staphylococcus epidermidis was the most abundant staphylococcal species at all sampling sites. No bacterial genus or staphylococcal species increased significantly after arthroplasty surgery. Application of a targeted tuf gene sequencing approach provided auxiliary staphylococcal community profiles and allowed species-level characterisation directly from low biomass clinical samples.

The Epidome - a species-specific approach to assess the population structure and heterogeneity of Staphylococcus epidermidis colonization and infection.

BACKGROUND: Although generally known as a human commensal, Staphylococcus epidermidis is also an opportunistic pathogen that can cause nosocomial infections related to foreign body materials and immunocompromized patients. Infections are often caused by multidrug-resistant (MDR) lineages that are difficult and costly to treat, and can have a major adverse impact on patients' quality of life. Heterogeneity is a common phenomenon in both carriage and infection, but present methodology for detection of this is laborious or expensive. In this study, we present a culture-independent method, labelled Epidome, based on an amplicon sequencing-approach to deliver information beyond species level on primary samples and to elucidate clonality, population structure and temporal stability or niche selection of S. epidermidis communities. RESULTS: Based on an assessment of > 800 genes from the S. epidermidis core genome, we identified genes with variable regions, which in combination facilitated the differentiation of phylogenetic clusters observed in silico, and allowed classification down to lineage level. A duplex PCR, combined with an amplicon sequencing protocol, and a downstream analysis pipeline were designed to provide subspecies information from primary samples. Additionally, a probe-based qPCR was designed to provide valuable absolute abundance quantification of S. epidermidis. The approach was validated on isolates representing skin commensals and on genomic mock communities with a sensitivity of < 10 copies/µL. The method was furthermore applied to a sample set of primary skin and nasal samples, revealing a high degree of heterogeneity in the S. epidermidis populations. Additionally, the qPCR showed a high degree of variation in absolute abundance of S. epidermidis. CONCLUSIONS: The Epidome method is designed for use on primary samples to obtain important information on S. epidermidis abundance and diversity beyond species-level to answer questions regarding the emergence and dissemination of nosocomial lineages, investigating clonality of S. epidermidis communities, population dynamics, and niche selection. Our targeted-sequencing method allows rapid differentiation and identification of clinically important nosocomial lineages in low-biomass samples such as skin samples.

eHealth: Disease activity measures are related to the faecal gut microbiota in adult patients with ulcerative colitis.

BACKGROUND/AIM: Microbial dysbiosis in inflammatory bowel disease (IBD) is poorly understood. Faecal samples collected for the purposes of microbiota analysis are not yet a part of everyday clinical practice. To explore associations between faecal microbiota and disease activity measures in adult IBD patients, for the purpose of possibly integrating microbiota measures in an existing IBD eHealth application for disease-monitoring. METHODS: We collected faecal samples from adult IBD patients for one year while they were home-monitoring for disease activity, using faecal calprotectin (FC) and the Simple Clinical Colitis Activity Index (SCCAI). Faecal samples were analysed in two different ways: commercially available test consisting of 54 pre-determined bacterial markers (DNA test) and 16S rRNA gene sequencing (16S-seq). Univariable linear mixed effect models were fitted to predict disease scores using normalised relative abundances as fixed effects. RESULTS: Seventy-eight IBD patients provided a total of 288 faecal samples for microbiota analysis. Two hundred and thirty-four of the samples were from patients with ulcerative colitis (UC). Peptostreptococcus anaerobius was found to correlate significantly with increasing FC, while an additional 24 genera were found to be associated with FC and/or SCCAI (16S-seq). Bacterial markers (DNA test) for Proteobacteria, Shigella spp. and Escherichia spp., were significantly correlated with increasing FC measures, while another 14 markers were found to be associated with FC and/or SCCAI. CONCLUSIONS: In patients with UC, results of both methods are associated with disease activity, correlating significantly with Peptostretococcus anaerobius (16S-seq) and with Proteobacteria, Shigella spp. and Escherichia spp. (DNA test).

Anaesthesia care providers employed in humanitarian settings by Médecins Sans Frontières: a retrospective observational study of 173 084 surgical cases over 10 years.

OBJECTIVE: To describe the extent to which different categories of anaesthesia provider are used in humanitarian surgical projects and to explore the volume and nature of their surgical workload. DESIGN: Descriptive analysis using 10 years (2008-2017) of routine case-level data linked with routine programme-level data from surgical projects run exclusively by Médecins Sans Frontières-Operational Centre Brussels (MSF-OCB). SETTING: Projects were in contexts of natural disaster (ND, entire expatriate team deployed by MSF-OCB), active conflict (AC) and stable healthcare gaps (HG). In AC and HG settings, MSF-OCB support pre-existing local facilities. Hospital facilities ranged from basic health centres with surgical capabilities to tertiary referral centres. PARTICIPANTS: The full dataset included 178 814 surgical cases. These were categorised by most senior anaesthetic provider for the project, according to qualification: specialist physician anaesthesiologists, qualified nurse anaesthetists and uncertified anaesthesia providers. PRIMARY OUTCOME MEASURE: Volume and nature of surgical workload of different anaesthesia providers. RESULTS: Full routine data were available for 173 084 cases (96.8%): 2518 in ND, 42 225 in AC, 126 936 in HG. Anaesthesia was predominantly led by physician anaesthesiologists (100% in ND, 66% in AC and HG), then nurse anaesthetists (19% in AC and HG) or uncertified anaesthesia providers (15% in AC and HG). Across all settings and provider groups, patients were mostly healthy young adults (median age range 24-27 years), with predominantly females in HG contexts, and males in AC contexts. Overall intra-operative mortality was 0.2%. CONCLUSION: Our findings contribute to existing knowledge of the nature of anaesthetic provision in humanitarian settings, while demonstrating the value of high-quality, routine data collection at scale in this sector. Further evaluation of perioperative outcomes associated with different models of humanitarian anaesthetic provision is required.

Genome investigations show host adaptation and transmission of LA-MRSA CC398 from pigs into Danish healthcare institutions.

Over the last decade, an increasing number of infections with livestock-associated methicillin-resistant Staphylococcus aureus of clonal complex 398 (LA-MRSA CC398) in persons without contact to livestock has been registered in Denmark. These infections have been suggested to be the result of repeated spillover of random isolates from livestock into the community. However, other studies also found emerging sub-lineages spreading among humans. Based on genome-wide SNPs and genome-wide association studies (GWAS), we assessed the population structure and genomic content of Danish LA-MRSA CC398 isolates from healthcare-associated infections from 2014 to 2016 (n = 73) and compared these to isolates from pigs in Denmark from 2014 (n = 183). Phylogenetic analyses showed that most human isolates were closely related to and scattered among pig isolates showing that the majority of healthcare-associated infections are the result of repeated spillover from pig farms, even though cases of human-to-human transmission also were identified. GWAS revealed frequent loss of antimicrobial resistance genes and acquisition of human-specific virulence genes in the human isolates showing adaptation in response to changes in selective pressures in different host environments, which over time could lead to the emergence of LA-MRSA CC398 lineages more adapted to human colonization and transmission.

Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing.

Methylation of guanosine on position N7 (m7G) on internal RNA positions has been found in all domains of life and have been implicated in human disease. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications at nucleotide resolution. In our method, m7G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We detect positions with increased mutation rates in the reduced and control samples taking the possibility of sequencing/alignment error into account and use replicates to calculate statistical significance based on log likelihood ratio tests. We show that m7G-MaP-seq efficiently detects known m7G modifications in rRNA with mutational rates up to 25% and we map a previously uncharacterised evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA. Furthermore, we identify m7G modifications in budding yeast, human and arabidopsis tRNAs and demonstrate that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA, including human Let-7e. Likewise, high sequencing depth m7G-MaP-seq analysis of mRNA from E. coli or yeast cells did not identify any internal m7G modifications.

Training non-physician anaesthetists in sub-Saharan Africa: a qualitative investigation of providers' perspectives.

OBJECTIVES: To explore the views of non-physician anaesthesia providers (NPAPs) and their colleagues regarding the effectiveness of NPAP training programmes in three contrasting sub-Saharan African countries. DESIGN: This was a qualitative exploratory descriptive study. Semistructured interviews were conducted online, recorded, transcribed and analysed thematically using NVivo. SETTING: Participants' homes or workplaces in Sierra Leone, Somaliland and Uganda. PARTICIPANTS: 15 NPAPs, physician anaesthetists and surgeons working in the countries concerned. RESULTS: Three major themes were identified: (1) discrepancy between urban training and rural practice, (2) prominent development of attitudes outside the curricular set during training, including approaches to learning and clinical responsibility and (3) the importance of interprofessional relationships developed during training for later practice. CONCLUSIONS: Anaesthesia providers in different cadres and very different country contexts in sub-Saharan Africa describe common themes in training which appear to be significant for their later practice. Not all these issues are explicitly planned for in current training programmes, although they are important in the view of providers. Subsequent programme development should consider these themes with a view to enhancing the safety and quality of anaesthesia practice in this context.

Strengthening the Anesthesia Workforce in Low- and Middle-Income Countries.

The majority of the world's population lacks access to safe, timely, and affordable surgical care. Although there is a health workforce crisis across the board in the poorest countries in the world, anesthesia is disproportionally affected. This article explores some of the key issues that must be tackled to strengthen the anesthesia workforce in low- and lower-middle-income countries. First, we need to increase the overall number of safe anesthesia providers to match a huge burden of disease, particularly in the poorest countries in the world and in remote and rural areas. Through using a task-sharing model, an increase is required in both nonphysician anesthesia providers and anesthesia specialists. Second, there is a need to improve and support the competency of anesthesia providers overall. It is important to include a broad base of knowledge, skills, and attitudes required to manage complex and high-risk patients and to lead improvements in the quality of care. Third, there needs to be a concerted effort to encourage interprofessional skills and the aspects of working and learning together with colleagues in a complex surgical ecosystem. Finally, there has to be a focus on developing a workforce that is resilient to burnout and the challenges of an overwhelming clinical burden and very restricted resources. This is essential for anesthesia providers to stay healthy and effective and necessary to reduce the inevitable loss of human resources through migration and cessation of professional practice. It is vital to realize that all of these issues need to be tackled simultaneously, and none neglected, if a sustainable and scalable solution is to be achieved.

Integrating intensified case finding of tuberculosis into HIV care: an evaluation from rural Swaziland.

BACKGROUND: Swaziland has the highest HIV prevalence in the world and the highest estimated tuberculosis incidence rate in the world. An estimated 80% of TB patients are also infected with HIV. TB detection through intensified case finding (ICF) has yet to become a routine aspect of integrated tuberculosis and HIV care. The purpose of this study was to evaluate implementation of ICF for TB into routine integrated tuberculosis and HIV care at 16 community clinics and one district hospital in Swaziland. METHODS: Nurses and lay counsellors conducted ICF using a TB screening tool and patient pathway at all HIV service entry points in clinics and the hospital. The patient pathway had three-stages; screening, sputum smear diagnosis and TB treatment initiation. Outcomes and losses to follow up were monitored at each stage. Patient demographics, access, and service feasibility and effectiveness were compared at hospital and clinic sites. RESULTS: 1467 HIV patients at clinics and the hospital were screened over a 3 month period. Large losses to follow up occurred prior to the sputum diagnosis stage; only 47% (n = 172) of TB suspects provided a specimen. 28 cases of smear positive TB were diagnosed and 24 commenced treatment. People screened at clinics were significantly more likely to be female, older, and from rural or geographically remote areas (p < 0.001). There was no significant difference between the hospital and clinics sites in the proportion of all participants screened who were smear positive (x2 = 1.909; p = 0.16). The number needed to screen to detect one sputum positive TB case was 34 at clinics and 63 at the district hospital. CONCLUSIONS: ICF was operationally feasible and became established as a routine aspect of tuberculosis and HIV integrated care. ICF in community clinics was potentially more accessible to an underserved, rural population and was as effective as the hospital service in detecting smear positive TB.