Capsular Typing Method for Streptococcus agalactiae Using Whole-Genome Sequence Data.

Group B streptococcus (GBS) capsular serotypes are major determinants of virulence and affect potential vaccine coverage. Here we report a whole-genome-sequencing-based method for GBS serotype assignment. This method shows strong agreement (kappa of 0.92) with conventional methods and increased serotype assignment (100%) to all 10 capsular types

A comparison of liquid and solid culture for determining relapse and durable cure in phase III TB trials for new regimens

Supported by the Global Alliance for TB Drug Development with support from the Bill & Melinda Gates Foundation, the Medical Research Council (MC_UU_12023/27), the European and Developing Countries Clinical Trials Partnership (grant IP.2007.32011.011), the US Agency for International Development, the UK Department for International Development, the Directorate General for International Cooperation of the Netherlands, Irish Aid, the Australia Department of Foreign Affairs and Trade and National Institutes of Health, AIDS Clinical Trials Group and by grants from the National Institute of Allergy and Infectious Diseases (NIAID) (UM1AI068634, UM1 AI068636 and UM1AI106701) and by NIAID grants to the University of KwaZulu Natal, South Africa, AIDS Clinical Trials Group (ACTG) site 31422 (1U01AI069469); to the Perinatal HIV Research Unit, Chris Hani Baragwanath Hospital, South Africa, ACTG site 12301 (1U01AI069453); and to the Durban International Clinical Trials Unit, South Africa, ACTG site 11201 (1U01AI069426); Bayer Healthcare for the donation of moxifloxacin; and Sanofi for the donation of rifampin. Additional grants were from Chief Scientist Office, Scottish Government, British Society of Antimicrobial Chemotherapy.Background:  Tuberculosis kills more people than any other infectious disease, and new regimens are essential. The primary endpoint for confirmatory phase III trials for new regimens is a composite outcome that includes bacteriological treatment failure and relapse. Culture methodology is critical to the primary trial outcome. Patients in clinical trials can have positive cultures after treatment ends that may not necessarily indicate relapse, which was ascribed previously to laboratory cross-contamination or breakdown of old lesions. Löwenstein-Jensen (LJ) medium was the previous standard in clinical trials, but almost all current and future trials will use the Mycobacteria Growth Indicator Tube (MGIT) system due to its simplicity and consistency of use, which will affect phase III trial results. LJ was used for the definition of the primary endpoint in the REMoxTB trial, but every culture was also inoculated in parallel into the MGIT system. The data from this trial, therefore, provide a unique opportunity to investigate and compare the incidence of false ‘isolated positives’ in liquid and solid media and their potential impact on the primary efficacy results. Methods:  All post-treatment positive cultures were reviewed in the REMoxTB clinical trial. Logistic regression models were used to model the incidence of isolated positive cultures on MGIT and LJ. Results:  A total of 12,209 sputum samples were available from 1652 patients; cultures were more often positive on MGIT than LJ. In 1322 patients with a favourable trial outcome, 126 (9.5%) had cultures that were positive in MGIT compared to 34 (2.6%) patients with positive cultures on LJ. Among patients with a favourable outcome, the incidence of isolated positives on MGIT differed by study laboratory (p < 0.0001) with 21.9% of these coming from one laboratory investigating only 4.9% of patients. No other baseline factors predicted isolated positives on MGIT after adjusting for laboratory. There was evidence of clustering of isolated positive cultures in some patients even after adjusting for laboratory, p < 0.0001. The incidence of isolated positives on MGIT did not differ by treatment arm (p = 0.845, unadjusted). Compared to negative MGIT cultures, positive MGIT cultures were more likely to be associated with higher grade TB symptoms reported within 7 days either side of sputum collection in patients with an unfavourable primary outcome (p < 0.0001) but not in patients with a favourable outcome (p = 0.481). Conclusions:  Laboratory cross-contamination was a likely cause of isolated positive MGIT cultures which were clustered in some laboratories. Certain patients had repeated positive MGIT cultures that did not meet the definition of a relapse. This pattern was too common to be explained by cross-contamination only, suggesting that host factors were also responsible. We conclude that MGIT can replace LJ in phase III TB trials, but there are implications for the definition of the primary outcome and patient management in trials in such settings. Most importantly, the methodologies differ in the incidence of isolated positives and in their capacity for capturing non-tuberculosis mycobacteria. It emphasises the importance of effective medical monitoring after treatment ends and consideration of clinical signs and symptoms for determining treatment failure and relapse.Publisher PDFPeer reviewe

Afternoon exercise is more efficacious than morning exercise at improving blood glucose levels in individuals with type 2 diabetes : a randomised crossover trial

Data availability The data analysed during the current study are available from the corresponding author on reasonable request. Funding The authors are supported by grants from Novo Nordisk Foundation (NNF14OC0011493 and NNF14OC0009941), Swedish Diabetes Foundation (DIA2015-052), Wenner-Gren Foundation, Swedish Research Council (2015-00165), Strategic Research Program in Diabetes at Karolinska Institutet (2009-1068), Stockholm County Council (SLL20150517 and SLL20170159) and Swedish Heart Lung Foundation (20150423).Peer reviewedPublisher PD

Inadequate Lopinavir Concentrations With Modified 8-hourly Lopinavir/Ritonavir 4:1 Dosing During Rifampicin-based Tuberculosis Treatment in Children Living With HIV

Background: Lopinavir/ritonavir plasma concentrations are profoundly reduced when co-administered with rifampicin. Super-boosting of lopinavir/ritonavir is limited by nonavailability of single-entity ritonavir, while double-dosing of co-formulated lopinavir/ritonavir given twice-daily produces suboptimal lopinavir concentrations in young children. We evaluated whether increased daily dosing with modified 8-hourly lopinavir/ritonavir 4:1 would maintain therapeutic plasma concentrations of lopinavir in children living with HIV receiving rifampicin-based antituberculosis treatment. // Methods: Children with HIV/tuberculosis coinfection weighing 3.0 to 19.9kg, on rifampicin-based antituberculosis treatment were commenced or switched to 8-hourly liquid lopinavir/ritonavir 4:1 with increased daily dosing using weight-band dosing approach. A standard twice-daily dosing of lopinavir/ritonavir was resumed 2 weeks after completing antituberculosis treatment. Plasma sampling was conducted during and 4 weeks after completing antituberculosis treatment. // Results: Of 20 children enrolled; 15, 1–7 years old, had pharmacokinetics sampling available for analysis. Lopinavir concentrations (median [range]) on 8-hourly lopinavir/ritonavir co-administered with rifampicin (n = 15; area under the curve0–24 55.32mg/h/L [0.30–398.7mg/h/L]; Cmax 3.04mg/L [0.03–18.6mg/L]; C8hr 0.90mg/L [0.01–13.7mg/L]) were lower than on standard dosing without rifampicin (n = 12; area under the curve24 121.63mg/h/L [2.56–487.3mg/h/L]; Cmax 9.45mg/L [0.39–26.4mg/L]; C12hr 3.03mg/L [0.01–17.7mg/L]). During and after rifampicin cotreatment, only 7 of 15 (44.7%) and 8 of 12 (66.7%) children, respectively, achieved targeted pre-dose lopinavir concentrations ≥1mg/L. // Conclusions: Modified 8-hourly dosing of lopinavir/ritonavir failed to achieve adequate lopinavir concentrations with concurrent antituberculosis treatment. The subtherapeutic lopinavir exposures on standard dosing after antituberculosis treatment are of concern and requires further evaluation

Spot sputum samples are at least as good as early morning samples for identifying Mycobacterium tuberculosis

Supported by the Global Alliance for TB Drug Development with support from the Bill and Melinda Gates Foundation, the European and Developing Countries Clinical Trials Partnership (Grant IP.2007.32011.011), US Agency for International Development, UK Department for International Development, Directorate General for International Cooperation of the Netherlands, Irish Aid, Australia Department of Foreign Affairs and Trade, National Institutes of Health, AIDS Clinical Trials Group. The study was also supported by grants from the National Institute of Allergy and Infectious Diseases (NIAID) (UM1AI068634, UM1 AI068636, and UM1AI106701) and by NIAID grants to the University of KwaZulu Natal, South Africa, AIDS Clinical Trials Group (ACTG) site 31422 (1U01AI069469); to the Perinatal HIV Research Unit, Chris Hani Baragwanath Hospital, South Africa, ACTG site 12301 (1U01AI069453); and to the Durban International Clinical Trials Unit, South Africa, ACTG site 11201 (1U01AI069426). Bayer Healthcare for donated moxifloxacin and Sanofi donated rifampin.Background:  The use of early morning sputum samples (EMS) to diagnose tuberculosis (TB) can result in treatment delay given the need for the patient to return to the clinic with the EMS, increasing the chance of patients being lost during their diagnostic workup. However, there is little evidence to support the superiority of EMS over spot sputum samples. In this new analysis of the REMoxTB study, we compare the diagnostic accuracy of EMS with spot samples for identifying Mycobacterium tuberculosis pre- and post-treatment. Methods:  Patients who were smear positive at screening were enrolled into the study. Paired sputum samples (one EMS and one spot) were collected at each trial visit pre- and post-treatment. Microscopy and culture on solid LJ and liquid MGIT media were performed on all samples; those missing corresponding paired results were excluded from the analyses. Results:  Data from 1115 pre- and 2995 post-treatment paired samples from 1931 patients enrolled in the REMoxTB study were analysed. Patients were recruited from South Africa (47%), East Africa (21%), India (20%), Asia (11%), and North America (1%); 70% were male, median age 31 years (IQR 24–41), 139 (7%) co-infected with HIV with a median CD4 cell count of 399 cells/μL (IQR 318–535). Pre-treatment spot samples had a higher yield of positive Ziehl–Neelsen smears (98% vs. 97%, P = 0.02) and LJ cultures (87% vs. 82%, P = 0.006) than EMS, but there was no difference for positivity by MGIT (93% vs. 95%, P = 0.18). Contaminated and false-positive MGIT were found more often with EMS rather than spot samples. Surprisingly, pre-treatment EMS had a higher smear grading and shorter time-to-positivity, by 1 day, than spot samples in MGIT culture (4.5 vs. 5.5 days, P < 0.001). There were no differences in time to positivity in pre-treatment LJ culture, or in post-treatment MGIT or LJ cultures. Comparing EMS and spot samples in those with unfavourable outcomes, there were no differences in smear or culture results, and positive results were not detected earlier in Kaplan–Meier analyses in either EMS or spot samples. Conclusions:  Our data do not support the hypothesis that EMS samples are superior to spot sputum samples in a clinical trial of patients with smear positive pulmonary TB. Observed small differences in mycobacterial burden are of uncertain significance and EMS samples do not detect post-treatment positives any sooner than spot samples.Publisher PDFPeer reviewe

Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics

Genomic Epidemiology of Complex, Multispecies, Plasmid-Borne bla KPC Carbapenemase in Enterobacterales in the United Kingdom from 2009 to 2014.

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the bla KPC family) is one of the most common transmissible carbapenem resistance mechanisms worldwide. The dissemination of bla KPC historically has been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the United Kingdom, bla KPC has represented a large-scale, persistent management challenge for some hospitals, particularly in North West England. The dissemination of bla KPC has evolved to be polyclonal and polyspecies, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole-genome sequencing of 604 bla KPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterization. We observed the dissemination of bla KPC (predominantly bla KPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 [97%] isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), bla KPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments among plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates), and IncR (252/604 isolates) replicons. The subset of reconstructed plasmid sequences (21 isolates, 77 plasmids) also highlighted modular exchange among non-bla KPC and bla KPC plasmids and the common presence of multiple replicons within bla KPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales for the implementation of adequate surveillance approaches and for control

The hospital microbiome project: meeting report for the UK science and innovation network UK-USA workshop ‘beating the superbugs: hospital microbiome studies for tackling antimicrobial resistance’, October 14th 2013

The UK Science and Innovation Network UK-USA workshop ‘Beating the Superbugs: Hospital Microbiome Studies for tackling Antimicrobial Resistance’ was held on October 14th 2013 at the UK Department of Health, London. The workshop was designed to promote US-UK collaboration on hospital microbiome studies to add a new facet to our collective understanding of antimicrobial resistance. The assembled researchers debated the importance of the hospital microbial community in transmission of disease and as a reservoir for antimicrobial resistance genes, and discussed methodologies, hypotheses, and priorities. A number of complementary approaches were explored, although the importance of the built environment microbiome in disease transmission was not universally accepted. Current whole genome epidemiological methods are being pioneered in the UK and the benefits of moving to community analysis are not necessarily obvious to the pioneers; however, rapid progress in other areas of microbiology suggest to some researchers that hospital microbiome studies will be exceptionally fruitful even in the short term. Collaborative studies will recombine different strengths to tackle the international problems of antimicrobial resistance and hospital and healthcare associated infections

Mannose-binding lectin genotypes: lack of association with susceptibility to thoracic empyema

<p>Abstract</p> <p>Background</p> <p>The role of the innate immune protein mannose-binding lectin (MBL) in host defence against severe respiratory infection remains controversial. Thoracic empyema is a suppurative lung infection that arises as a major complication of pneumonia and is associated with a significant mortality. Although the pathogenesis of thoracic empyema is poorly understood, genetic susceptibility loci for this condition have recently been identified. The possible role of MBL genotypic deficiency in susceptibility to thoracic empyema has not previously been reported.</p> <p>Methods</p> <p>To investigate this further we compared the frequencies of the six functional <it>MBL </it>polymorphisms in 170 European individuals with thoracic empyema and 225 healthy control individuals.</p> <p>Results</p> <p>No overall association was observed between MBL genotypic deficiency and susceptibility to thoracic empyema (2 × 2 Chi square = 0.02, <it>P </it>= 0.87). Furthermore, no association was seen between MBL deficiency and susceptibility to the Gram-positive or pneumococcal empyema subgroups. MBL genotypic deficiency did not associate with progression to death or requirement for surgery.</p> <p>Conclusions</p> <p>Our results suggest that MBL genotypic deficiency does not associate with susceptibility to thoracic empyema in humans.</p