A 7-year review of clinical characteristics, predisposing factors and outcomes of post-keratoplasty infectious keratitis: the Nottingham infectious keratitis study.

Background/objectives: Post-keratoplasty infectious keratitis (PKIK) is a unique sight-threatening clinical entity which often poses significant therapeutic challenges. This study aimed to examine the clinical presentation, risk factors, management, and clinical outcomes of PKIK. Methods: This was a retrospective study of all patients who presented to the Queen's Medical Centre, Nottingham, with PKIK between September 2015 and August 2022 (a 7-year period). Relevant data on types of keratoplasty, clinical presentations, causative microorganisms, management, and outcome were analyzed. Results: Forty-nine PKIK cases, including four cases of interface infectious keratitis, were identified during the study period. The most common graft indications for PKP, DALK and EK were failed grafts (9, 37.5%), keratoconus (6, 54.5%) and Fuchs endothelial corneal dystrophy (FECD; 8, 57.1%), respectively. Staphylococcus spp. were the most commonly identified organisms (15, 50.0%). Bullous keratopathy (18, 36.7%), ocular surface disease (18, 36.7%), and broken/loose sutures (15, 30.6%) were the most common risk factors. Concurrent use of topical steroids was identified in 25 (51.0%) cases. Of 31 functioning grafts at presentation, 12 (38.7%) grafts failed at final follow-up with 15 (48.4%) patients retaining a CDVA of ≥1.0 logMAR. The overall estimated 5-year survival rate post-PKIK was 55.9% (95% CI, 35.9%-75.9%), with DALK having the highest survival rate [63.6% (95% CI, 28.9%-98.3%)], followed by EK [57.1% (95% CI, 20.4%-93.8%)] and PKP [52.7% (95% CI, 25.1%-80.3%)], though no statistical difference was observed (p=0.48). Conclusions: PKIK represents an important cause of IK and graft failure. Bullous keratopathy, OSD and suture-related complications are the commonest risk factors, highlighting the potential benefit of prophylactic topical antibiotics (for unhealthy ocular surface) and early suture removal (where possible) in reducing the risk of PKIK. Graft survival may be higher in lamellar keratoplasty following PKIK but larger studies are required to elucidate this observation.

Multidrug-Resistant ESBL-Producing E. coli in Clinical Samples from the UK.

Globally, cephalosporin therapy failure is a serious problem for infection control. One causative agent of cephalosporin-resistant infections is multidrug-resistant (MDR) E. coli producing extended-spectrum ß-lactamases (ESBLs) and/or plasmid-encoded AmpC (pAmpC) ß-lactamases. We evaluated the occurrence of ESBL/pAmpC genetic determinants in phenotypically MDR E. coli isolated from clinical samples of blood, faeces, ear effusion, urine and sputum from a UK hospital. Phenotypic resistance profiling for 18 antibiotics (from seven classes) showed that 32/35 isolates were MDR, with resistance to 4-16 of the tested antibiotics. Of the isolates, 97.1% showed resistance to ampicillin, 71.4% showed resistance to co-amoxiclav, cefotaxime, ceftazidime and ceftiofur, and 68.5% showed resistance to cefquinome. blaCTX-M, blaTEM and blaOXA-1 genes were detected in 23, 13 and 12 strains, respectively, and Intl1 was detected in 17 isolates. The most common subtypes among the definite sequence types were CTX-M-15 (40%) and TEM-1 (75%). No E. coli isolates carried pAmpC genes. Significant correlations were seen between CTX-M carriage and cefotaxime, ceftiofur, aztreonam, ceftazidime and cefquinome resistance; between blaCTX-M, blaTEM and blaOXA-1 carriage and ciprofloxacin resistance; and between Intl1 carriage and trimethoprim/sulfamethoxazole resistance. Thus, MDR phenotypes may be conferred by a relatively small number of genes. The level and pattern of antibiotic resistance highlight the need for better antibiotic therapy guidelines, including reduced use and improved surveillance.

Psychrobacter piechaudii shunt infection: first report of human infection.

Psychrobacter piechaudii is a recently described species of Gram-negative bacteria in the Moraxellaceae family. No cases of human infection due to this species have been described before. We report the case of an ex-premature infant girl with hydrocephalus secondary to intraventricular haemorrhage who underwent multiple cerebrospinal fluid (CSF) shunt operations. She ultimately developed Psychrobacter piechaudii meningitis, presenting as ventriculoperitoneal shunt dysfunction and wound leak, which necessitated removal of the shunt, a period of external ventricular drainage and antibiotics. We found this organism to be sensitive to intravenous ceftazidime (50 mg/kg) and ciprofloxacin, and a 7-10 day treatment course prior to shunt re-insertion (and 3 week total course) was sufficient. The patient is well post-operatively. To the best of our knowledge, this is the first reported case of Psychrobacter piechaudii infection in a human.

Risk Factors, Clinical Outcomes, and Prognostic Factors of Bacterial Keratitis: The Nottingham Infectious Keratitis Study.

Background/Aim: To examine the risk factors, clinical characteristics, outcomes, and prognostic factors of bacterial keratitis (BK) in Nottingham, UK. Methods: This was a retrospective study of patients who presented to the Queen's Medical Centre, Nottingham, with suspected BK during 2015-2019. Relevant data, including the demographic factors, risk factors, clinical outcomes, and potential prognostic factors, were analysed. Results: A total of 283 patients (n = 283 eyes) were included; mean age was 54.4 ± 21.0 years and 50.9% were male. Of 283 cases, 128 (45.2%) cases were culture-positive. Relevant risk factors were identified in 96.5% patients, with ocular surface diseases (47.3%), contact lens wear (35.3%) and systemic immunosuppression (18.4%) being the most common factors. Contact lens wear was most commonly associated with P. aeruginosa whereas Staphylococci spp. were most commonly implicated in non-contact lens-related BK cases (p = 0.017). At presentation, culture-positive cases were associated with older age, worse presenting corrected-distance-visual-acuity (CDVA), use of topical corticosteroids, larger epithelial defect and infiltrate, central location and hypopyon (all p < 0.01), when compared to culture-negative cases. Hospitalisation was required in 57.2% patients, with a mean length of stay of 8.0 ± 8.3 days. Surgical intervention was required in 16.3% patients. Significant complications such as threatened/actual corneal perforation (8.8%), loss of perception of light vision (3.9%), and evisceration/enucleation (1.4%) were noted. Poor visual outcome (final corrected-distance-visual-acuity of <0.6 logMAR) and delayed corneal healing (>30 days from initial presentation) were significantly affected by age >50 years, infiltrate size >3 mm, and reduced presenting vision (all p < 0.05). Conclusion: BK represents a significant ocular morbidity in the UK, with ocular surface diseases, contact lens wear, and systemic immunosuppression being the main risk factors. Older age, large infiltrate, and poor presenting vision were predictive of poor visual outcome and delayed corneal healing, highlighting the importance of prevention and early intervention for BK.

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.

Comparative effects of viral-transport-medium heat inactivation upon downstream SARS-CoV-2 detection in patient samples.

Introduction. The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At the time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: (1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes. (2) Rapid time to result of <24 h is required in order to facilitate timely infection control measures.Hypothesis. Extraction-free sample processing would impact commercially available SARS-CoV-2 genome detection methods.Aim. We evaluated whether alternative commercially available kits provided sensitivity and accuracy of SARS-CoV-2 genome detection comparable to those used by regional National Healthcare Services (NHS).Methodology. We tested several detection methods and tested whether detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits.Results. Using purified RNA, we found the CerTest VIASURE kit to be comparable to the Altona RealStar system currently in use, and further showed that both diagnostic kits performed similarly in the BioRad CFX96 and Roche LightCycler 480 II machines. Additionally, both kits were comparable to a third alternative using a combination of Quantabio qScript one-step Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) mix and Centre for Disease Control and Prevention (CDC)-accredited N1 and N2 primer/probes when looking specifically at borderline samples. Importantly, when using the kits in an extraction-free protocol, following heat inactivation, we saw differing results, with the combined Quantabio-CDC assay showing superior accuracy and sensitivity. In particular, detection using the CDC N2 probe following the extraction-free protocol was highly correlated to results generated with the same probe following RNA extraction and reported clinically (n=127; R2=0.9259).Conclusion. Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat-inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply-chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.

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.

Comparison of culture based methods for the isolation of Clostridium difficile from stool samples in a research setting.

Effective isolation of Clostridium difficile from stool samples is important in the research setting, especially where low numbers of spores/vegetative cells may be present within a sample. In this study, three protocols for stool culture were investigated to find a sensitive, cost effective and timely method of C. difficile isolation. For the initial enrichment step, the effectiveness of two different rich media, cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared. For the comparison of four different, selective solid media; Cycloserine-cefoxitin fructose agar (CCFA), Cycloserine-cefoxitin egg yolk agar (CCEY), ChromID C. difficile and tryptone soy agar (TSA) with 5% sheep's blood with and without preceding broth enrichment were used. As a means to enable differentiation between C. difficile and other fecal flora, the effectiveness of the inclusion of a pH indictor (1% Neutral Red), was also evaluated. The data derived indicated that CCFB is more sensitive than CCMB-TAL, however, the latter had an improved recovery rate. A broth enrichment step had a reduced sensitivity over direct plating. ChromID C. difficile showed the best recovery rate whereas CCEY egg yolk agar was the most sensitive of the four. The addition of 1% Neutral Red did not show sufficient colour change when added to CCEY egg yolk agar to be used as a differential medium. For a low cost, timely and sensitive method of isolating C. difficile from stool samples we recommend direct plating onto CCEY egg yolk agar after heat shock.