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.

Horizontal and Vertical Transmission of Powassan Virus by the Invasive Asian Longhorned Tick, Haemaphysalis longicornis, Under Laboratory Conditions.

The Asian longhorned tick, Haemaphysalis longicornis, is an ixodid tick native to East Asia that was first detected in North America outside a port of entry in 2017. This invasive species has since been detected in 17 states. As the invasive range of the tick continues to expand, the vector competence of H. longicornis for pathogens native to North America must be assessed. Here, we evaluate the vector competence of H. longicornis for Powassan virus (POWV) under laboratory conditions. POWV is a North American tick-borne flavivirus that is typically transmitted through the bite of Ixodes species ticks. The invasive range of H. longicornis is expected to overlap heavily with the geographic range of Ixodes scapularis and POWV cases, highlighting the potential for this invasive tick species to amplify POWV transmission in natural foci should the native tick vectors and H. longicornis share similar hosts. In these studies, adult female H. longicornis ticks were infected with POWV via anal pore microinjection. Viral RNA and infectious virions were detected in tick tissues via q-RT-PCR and focus-forming assay, respectively. POWV-injected female ticks were infested on mice, and virus was transmitted to mice during tick feeding, as shown by clinical signs of disease and seroconversion in the tick-exposed mice, as well as the detection of viral RNA in various mouse tissues. A POWV-injected female tick transmitted virus to her larval progeny, indicating that H. longicornis can vertically transmit POWV. These naturally-infected larval ticks were also able to transmit POWV to the mouse on which they fed and to the nymphal stage after molting, further demonstrating that H. longicornis can transmit POWV in the horizontal and transstadial modes. Larval and nymphal ticks were also orally infected with POWV while feeding on viremic mice. Additionally, this study provides the first report of POWV neuropathology based on a natural tick transmission model of POWV. Together, our results suggest that the invasive H. longicornis tick is a competent vector of POWV. These findings underline the growing danger this tick may pose to human health in the United States. Additional scholarship on the tick's biology, ecology, and pathogen transmission dynamics in nature will be important towards understanding the full public health impact of this invasive species.

Tracking and responding to an outbreak of tuberculosis using MIRU-VNTR genotyping and whole genome sequencing as epidemiological tools.

Background: We describe an outbreak that contributed to a near doubling of the incidence of tuberculosis in Southampton, UK. We examine the importance of 24 locus mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) genotyping in its identification and management and the role of whole genome sequencing (WGS) in tracing the spread of the strain. Methods: Outbreak cases were defined as those diagnosed between January and December 2011 with indistinguishable 24 locus-MIRU-VNTR genotypes or, cases linked epidemiologically. A cluster questionnaire was administered by TB nurses to identify contacts and social settings. Results: Overall, 25 patients fulfilled the case definition. No cases with this MIRU-VNTR genotype had been detected in the UK previously. Connections were found between all cases through household contacts or social venues including a football club, Internet cafe and barber's shop. Public health actions included extended contact tracing, venue screening and TB awareness-raising. The outbreak resulted in a high rate of transmission and high incidence of clinical disease among contacts. Conclusions: This outbreak illustrates the value of combining active case-finding with prospective MIRU-VNTR genotyping to identify settings to undertake public health action. In addition WGS revealed that the VNTR-defined cluster was a single outbreak and that active TB transmission not reactivation was responsible for this outbreak in non-UK born individuals.

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.

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.

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.

Deep-seated resistance in relapsed paratyphoid fever.

We describe a case of relapsed paratyphoid fever in which the isolate had reduced susceptibility to ciprofloxacin due to a rare mutation within the gyrA gene. 18fluorodeoxyglucose positron emission tomography scanning identified deep-seated infection including unsuspected aortitis and highlights the utility of novel imaging techniques to improve our understanding and treatment of this disease.