Retrospective screening of routine respiratory samples revealed undetected community transmission and missed intervention opportunities for SARS-CoV-2 in the United Kingdom.

In the early phases of the SARS coronavirus type 2 (SARS-CoV-2) pandemic, testing focused on individuals fitting a strict case definition involving a limited set of symptoms together with an identified epidemiological risk, such as contact with an infected individual or travel to a high-risk area. To assess whether this impaired our ability to detect and control early introductions of the virus into the UK, we PCR-tested archival specimens collected on admission to a large UK teaching hospital who retrospectively were identified as having a clinical presentation compatible with COVID-19. In addition, we screened available archival specimens submitted for respiratory virus diagnosis, and dating back to early January 2020, for the presence of SARS-CoV-2 RNA. Our data provides evidence for widespread community circulation of SARS-CoV-2 in early February 2020 and into March that was undetected at the time due to restrictive case definitions informing testing policy. Genome sequence data showed that many of these early cases were infected with a distinct lineage of the virus. Sequences obtained from the first officially recorded case in Nottinghamshire - a traveller returning from Daegu, South Korea - also clustered with these early UK sequences suggesting acquisition of the virus occurred in the UK and not Daegu. Analysis of a larger sample of sequences obtained in the Nottinghamshire area revealed multiple viral introductions, mainly in late February and through March. These data highlight the importance of timely and extensive community testing to prevent future widespread transmission of the virus.

Microbiological culture versus 16S/18S rRNA gene PCR-sanger sequencing for infectious keratitis: a three-arm, diagnostic cross-sectional study.

Background: To compare the diagnostic performance of microbiological culture and 16S/18S rRNA gene polymerase chain reaction (PCR)-Sanger sequencing for infectious keratitis (IK) and to analyse the effect of clinical disease severity on test performance and inter-test concordance. Methods: This was a three-arm, diagnostic cross-sectional study. We included all eligible patients who presented with presumed bacterial/fungal keratitis to the Queen's Medical Centre, Nottingham, UK, between June 2021 and September 2022. All patients underwent simultaneous culture (either direct or indirect culture, or both) and 16S (pan-bacterial)/18S (pan-fungal) ribosomal RNA (rRNA) PCR-Sanger sequencing. The bacterial/fungal genus and species identified on culture were confirmed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Relevant clinical data were also collected to analyze for any potential clinico-microbiological correlation. Main outcome measures included the diagnostic yield, test accuracy (including sensitivity and specificity), and inter-test agreement [including percent agreement and Cohen's kappa (k)]. Results: A total of 81 patients (86 episodes of IK) were included in this study. All organisms identified were of bacterial origin. Diagnostic yields were similar among direct culture (52.3%), indirect culture (50.8%), and PCR (43.1%; p = 0.13). The addition of PCR enabled a positive diagnostic yield in 3 (9.7%) direct culture-negative cases. Based on composite reference standard, direct culture had the highest sensitivity (87.5%; 95% CI, 72.4-95.3%), followed by indirect culture (85.4%; 95% CI, 71.6-93.5%) and PCR (73.5%; 95% CI, 59.0-84.6%), with 100% specificity noted in all tests. Pairwise comparisons showed substantial agreement among the three tests (percent agreement = 81.8-86.2%, Cohen's k = 0.67-0.72). Clinico-microbiological correlation demonstrated higher culture-PCR concordance in cases with greater infection severity. Conclusions: This study highlights a similar diagnostic performance of direct culture, indirect culture and 16S rRNA PCR for bacterial keratitis, with substantial inter-test concordance. PCR serves as a useful diagnostic adjuvant to culture, particularly in culture-negative cases or those with lesser disease severity (where culture-PCR concordance is lower).

Clinical features, risk factors and outcomes of contact lens-related bacterial keratitis in Nottingham, UK: a 7-year study.

BACKGROUND/OBJECTIVES: To examine the clinical characteristics, risk factors and outcomes of contact lens-related bacterial keratitis (CLBK) in a large UK tertiary referral centre. SUBJECTS/METHODS: A retrospective analysis of all patients who presented to the Queen's Medical Centre, Nottingham, UK, with suspected CLBK between October 2015 to September 2022 (a 7-year period) was performed. Relevant data on demographic factors, CL wear behaviour, causes, clinical characteristics, and outcomes were analysed. RESULTS: We included 138 patients with CLBK; the mean age was 42.0 ± 17.8 years and 74 (53.6%) patients were male. Most CLBK were related to soft CL wear (94.5%), particularly monthly disposable (42.5%) and daily disposable (24.4%) CLs. Poor CL wear behaviour/hygiene was documented in 57.1% cases. Among the 64 (46.4%) microbiological-positive cases (n = 73 organisms), Pseudomonas aeruginosa (36, 49.3%) and Staphylococcus spp. (16, 21.9%) were most commonly identified. Six (4.3%) cases were polymicrobial. Most (97.0%) patients were successfully treated with topical antibiotics alone, with 80.6% achieving good final corrected-distance-visual-acuity (CDVA) of ≥ 0.30 logMAR. Poor visual outcome (final CDVA < 0.30 logMAR) was significantly associated with presenting CDVA < 0.6 logMAR (p = 0.002) and central ulcer (p = 0.004). Poor corneal healing (complete healing of > 30 days from initial presentation) was significantly associated with age > 50 years (p = 0.028), female gender (p = 0.020), and infiltrate size >3 mm (p = 0.031). CONCLUSIONS: Poor CL wear behaviour/hygiene is commonly observed in CLBK, highlighting the importance of improved counselling and awareness regarding CL use and hygiene. When presented early and managed appropriately, most patients are able to achieve good clinical outcomes with medical treatment alone.

Examining diabetic heel ulcers through an ecological lens: microbial community dynamics associated with healing and infection.

PURPOSE: While some micro-organisms, such as Staphylococcus aureus, are clearly implicated in causing tissue damage in diabetic foot ulcers (DFUs), our knowledge of the contribution of the entire microbiome to clinical outcomes is limited. We profiled the microbiome of a longitudinal sample series of 28 people with diabetes and DFUs of the heel in an attempt to better characterize the relationship between healing, infection and the microbiome. METHODOLOGY: In total, 237 samples were analysed from 28 DFUs, collected at fortnightly intervals for 6 months or until healing. Microbiome profiles were generated by 16S rRNA gene sequence analysis, supplemented by targeted nanopore sequencing.Result/Key findings. DFUs which failed to heal during the study period (20/28, 71.4 %) were more likely to be persistently colonized with a heterogeneous community of micro-organisms including anaerobes and Enterobacteriaceae (log-likelihood ratio 9.56, P=0.008). During clinically apparent infection, a reduction in the diversity of micro-organisms in a DFU was often observed due to expansion of one or two taxa, with recovery in diversity at resolution. Modelling of the predicted species interactions in a single DFU with high diversity indicated that networks of metabolic interactions may exist that contribute to the formation of stable communities. CONCLUSION: Longitudinal profiling is an essential tool for improving our understanding of the microbiology of chronic wounds, as community dynamics associated with clinical events can only be identified by examining changes over multiple time points. The development of complex communities, particularly involving Enterobacteriaceae and strict anaerobes, may be contributing to poor outcomes in DFUs and requires further investigation.