Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.

Performance evaluation of the Viasure PCR assay for the diagnosis of monkeypox: A multicentre study.

BACKGROUND: Monkeypox virus (MPXV) is the causative agent of the 2022 monkeypox global outbreak. Rapid detection of MPXV infection is essential to inform patient management and public health response. Currently, there is a lack of established real-time PCR assays to support a rapid diagnosis of monkeypox. OBJECTIVES: To evaluate the performance characteristics of the Viasure MPXV PCR assay in three London teaching hospitals. STUDY DESIGN: Prospectively collected paired patient swabs from matched or unmatched anatomical sites were evaluated by the reference laboratory and Viasure MPXV PCR assays. A subset of samples were also tested for HSV, VZV, and/or Treponema pallidum DNA. RESULTS: 217 paired samples were evaluated. 91.2% of the paired swabs generated concordant results whilst 8.8% generated discordant results. The accuracy, diagnostic sensitivity, diagnostic specificity, positive predictive value, negative predictive value, likelihood ratio positive, and likelihood ratio negative of the Viasure PCR assay across the hospitals were 93.2 - 96.3%, 90.0 - 100%, 88.2 - 100%, 94.9 - 100%, 87.9 - 100%, 8.50 - 14.41, and 0.00 - 0.10 respectively. MPXV co-infections with HSV were detected in two patients. Five patients were negative for monkeypox but positive for herpes or chickenpox. CONCLUSIONS: The Viasure MPXV PCR assay demonstrated excellent performance characteristics, was easy to use, and is fit for routine diagnostic purpose. Where implemented, the assay would allow rapid and accurate laboratory diagnosis of MPXV infections and support a timely management of monkeypox. To reduce the risk of false negative detections, vesicular lesions from any anatomical site should be preferentially and optimally sampled.

Psychometric assessment of the Cardiac Depression Scale Short Form in cardiac outpatients.

BACKGROUND: Depression is common in patients with cardiovascular disease and is a risk marker for increased mortality. The valid and reliable detection of depression is fundamental to the appropriate management of these patients. AIM: The aim of this study was to evaluate the psychometric characteristics of the Cardiac Depression Scale Short Form 1 (DS-SF1) and the Cardiac Depression Scale Short Form 2 (DS-SF2) for screening cardiac outpatients in clinical settings. METHODS: Adult cardiac outpatients attending a cardiovascular clinic completed the Cardiac Depression Scale (CDS), two versions of the DS-SF (DS-SF1 and DS-SF2) and the Physical Health Questionnaire 2 (PHQ2-Y/N) prior to their cardiac consultation. RESULTS: Data from 326 patients (224 men; mean±SD age 66.25±14.39 years) were analysed. The DS-SF1 (mean score 16.28±5.70) had good construct validity with the CDS ( r=0.77; p<0.0001), adequate convergence with the PHQ2-Y/N ( r=0.59; p<0.0001) and good internal consistency (α=0.73). The DS-SF2 (mean score 15.80±6.80) had a better construct validity with the CDS ( r=0.84; p<0.0001) and the PHQ2-Y/N ( r=0.69; p<0.0001) and better internal consistency (α=0.82). The DS-SF2 showed strong criterion validity with the CDS with a DS-SF2 ⩾15 cut-point yielding 90% sensitivity and 73% specificity (area under the curve 0.92) for detecting depression (CDS ⩾95). CONCLUSION: These findings confirm the excellent psychometric properties of the DS-SF2 as an ideal tool for screening depression in cardiac patients in clinical practice. The DS-SF2 should be regarded as the definitive version of the DS-SF.

Harnessing the genome: development of a hierarchical typing scheme for meticillin-resistant Staphylococcus aureus.

A major barrier to using genome sequencing in medical microbiology is the ability to interpret the data. New schemes that provide information about the importance of sequence variation in both clinical and public health settings are required. Meticillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen that is being observed with increasing frequency in community settings. Better tools are needed to improve our understanding of its transmissibility and micro-epidemiology in order to develop effective interventions. Using DNA microarray technology we identified a set of 20 binary targets whose presence or absence could be determined by PCR, producing a PCR binary typing scheme (PCR-BT). This was combined with multi-locus sequence type-based, sequence nucleotide polymorphism typing to form a hierarchical typing scheme. When applied to a set of epidemiologically unrelated isolates, a high degree of concordance was observed with PFGE (98.8 %). The scheme was able to detect the presence or absence of an outbreak strain in eight out of nine outbreak investigations, demonstrating epidemiological concordance. PCR-BT was better than PFGE at distinguishing between outbreak strains, particularly where epidemic MRSA-15 was involved. The method developed here is a rapid, digital typing scheme for S. aureus for use in both micro- and macro-epidemiological investigations that has the advantage of being suitable for use in routine diagnostic laboratories. The targets are defined and therefore the types can be defined by any platform capable of detecting the sequences used, including whole genome sequencing.