Searching for new therapeutic options for the uncommon pathogen Mycobacterium chimaera: an open drug discovery approach.

BACKGROUND: Mycobacterium chimaera is a slowly growing non-tuberculous mycobacterium associated with outbreaks of fatal infections in patients after cardiac surgery, and it is increasingly being detected in patients with chronic lung conditions. M chimaera can cause disseminated disease, osteomyelitis, and chronic skin or soft-tissue infections. We aimed to find new inhibitory compounds and drug repurposing opportunities for M chimaera, as current therapeutic options often result in poor outcomes. METHODS: In an open drug discovery approach, we screened the Medicines for Malaria Venture (MMV) Pathogen Box to assess the in-vitro antimicrobial drug susceptibility of M chimaera compared with the antimicrobial drug susceptibility of the slowly growing, major human pathogen Mycobacterium tuberculosis, and the rapidly growing Mycobacterium abscessus reference strains. Compounds identified from an initial resazurin microtitre cell viability assay screen were further characterised by determining the minimum inhibitory concentration (MIC) of MMV Pathogen Box compounds against M chimaera; and the MICs of a panel of 20 drugs commonly used to treat mycobacterial infections against M tuberculosis, M abscessus, and M chimaera. We also assessed the time-kill kinetics of doxycycline, clarithromycin, ethambutol, and rifabutin against M chimaera. FINDINGS: M chimaera was inhibited by 21 (5%) of 400 compounds in the Pathogen Box. Ten compounds were active against all three mycobacteria. MMV675968, with activity against slowly growing mycobacteria that probably targets folate metabolism, had a mean MIC of 2·22 µM (0·80 µg/mL) against M chimaera. Antimicrobial susceptibility testing showed that oxazolidinones such as linezolid (mean MIC 3·13 µg/mL) were active against M chimaera and that bedaquiline was the most potent compound (mean MIC 0·02 µg/mL). Doxycycline, a broad-spectrum antimicrobial drug with excellent tissue penetration properties, also inhibited M chimaera with a mean MIC of 6·25 µg/mL. INTERPRETATION: Molecular diagnostics present an opportunity for more effective, targeted drug therapies-treating bacterial infections at the species level. Using an open drug discovery platform, we identified compounds that inhibit the newly recognised pathogen M chimaera. The existing evidence base is poor and the option for expensive drug discovery is improbable; therefore, we have also found options for drug repurposing. Future in-vivo efficacy studies will reveal whether these findings result in new, targeted treatment regimens for M chimaera. FUNDING: Wellcome Trust, National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), and the University of Sussex Junior Research Associate scheme.

A Systematic Review and Meta-Analysis of Inpatient Mortality Associated With Nosocomial and Community COVID-19 Exposes the Vulnerability of Immunosuppressed Adults.

Background: Little is known about the mortality of hospital-acquired (nosocomial) COVID-19 infection globally. We investigated the risk of mortality and critical care admission in hospitalised adults with nosocomial COVID-19, relative to adults requiring hospitalisation due to community-acquired infection. Methods: We systematically reviewed the peer-reviewed and pre-print literature from 1/1/2020 to 9/2/2021 without language restriction for studies reporting outcomes of nosocomial and community-acquired COVID-19. We performed a random effects meta-analysis (MA) to estimate the 1) relative risk of death and 2) critical care admission, stratifying studies by patient cohort characteristics and nosocomial case definition. Results: 21 studies were included in the primary MA, describing 8,251 admissions across 8 countries during the first wave, comprising 1513 probable or definite nosocomial COVID-19, and 6738 community-acquired cases. Across all studies, the risk of mortality was 1.3 times greater in patients with nosocomial infection, compared to community-acquired (95% CI: 1.005 to 1.683). Rates of critical care admission were similar between groups (Relative Risk, RR=0.74, 95% CI: 0.50 to 1.08). Immunosuppressed patients diagnosed with nosocomial COVID-19 were twice as likely to die in hospital as those admitted with community-acquired infection (RR=2.14, 95% CI: 1.76 to 2.61). Conclusions: Adults who acquire SARS-CoV-2 whilst already hospitalised are at greater risk of mortality compared to patients admitted following community-acquired infection; this finding is largely driven by a substantially increased risk of death in individuals with malignancy or who had undergone transplantation. These findings inform public health and infection control policy and argue for individualised clinical interventions to combat the threat of nosocomial COVID-19, particularly for immunosuppressed groups. Systematic Review Registration: PROSPERO CRD42021249023.

Undetected carriage explains apparent Staphylococcus aureus acquisition in a non-outbreak healthcare setting.

OBJECTIVES: Previous studies have been unable to identify patient or staff reservoirs for the majority of the nosocomial S. aureus acquisitions which occur in the presence of good infection control practice. We set out to establish the extent to which undetected pre-existing carriage explains apparent nosocomial S. aureus acquisition. METHODS: Over two years elective cardiothoracic admissions were screened for S. aureus carriage before and during hospital admission. Routine screening (nose/groin/wound sampling), was supplemented by sampling additional body sites (axilla/throat/rectum) and culture-based methods optimised to detect fastidious phenotypes (small colony variants, cell wall deficient variants) and molecular identification by PCR. RESULTS: 35% of participants (53/151) were S. aureus carriers according to routine pre-healthcare screening; increasing to 42% (63/151) when additional body sites and enhanced cultures were employed. 71% (5/7) of apparent acquisitions were explained by pre-existing carriage using augmented measures. Enhanced culture identified a minority of colonised individuals (3/151 including 1 MRSA carrier) who were undetected by routine and additional screening cultures. 4/14 (29%) participants who became culture-negative during admission had S. aureus genomic material detected at discharge. CONCLUSIONS: Conventional sampling under-estimates carriage of S. aureus and this explains the majority of apparent S. aureus acquisitions among elective cardiothoracic patients.

Von Willebrand factor (vWF): marker of endothelial damage and thrombotic risk in COVID-19?

BACKGROUND: COVID-19 infection is characterised, among other features, by a prothrombotic state with high rate of venous thromboembolism (VTE), D-dimer, and fibrinogen levels. Clinical observations have also highlighted that these patients have elevated von Willebrand factor (vWF) and factor VIIIc. METHODS: 24 consecutive COVID-19 positive patients were selected from the intensive care unit (ICU) or the high acuity ward of Brighton and Sussex University Hospitals NHS Trust. RESULTS: The rate of VTE was 25% and mortality rate was 16.7%. Fibrinogen and D-Dimers were elevated, 7.9 (1.6) g/L and 2.4 (2.02) ug/ml respectively. Factor VIIIc and von vWF antigen levels were both extremely elevated at 279 (148) u/dL and 350 (131) % respectively, which are comparable to levels seen in ICU patients with severe sepsis. vWF levels were significantly higher in patients that died (p=0.017) and showed a positive correlation with age. There was a statistically significant association between COVID-19 disease and non-O blood group (p=0.02); 80% (4/5) of COVID-19 patients with VTE were blood group A. CONCLUSION: Very high levels of vWF and factor VIIIc are common in COVID-19 patients, comparable to levels in severely septic non-COVID ICU patients. This could contribute to the hypercoagulable state and increased VTE rate in COVID-19. Further studies are needed to evaluate the use of vWF for stratifying thrombotic risk in COVID-19 and to determine if elevated vWF is contributing to disease pathogenesis.

Spontaneously Occurring Small-Colony Variants of Staphylococcus aureus Show Enhanced Clearance by THP-1 Macrophages.

Staphylococcus aureus is a common cause of chronic and relapsing infection, especially when the ability of the immune system to sterilize a focus of infection is compromised (e.g., because of a foreign body or in the cystic fibrosis lung). Chronic infections are associated with slow-growing colony phenotypes of S. aureus on solid media termed small-colony variants (SCVs). Stable SCVs show characteristic mutations in the electron transport chain that convey resistance to antibiotics, particularly aminoglycosides. This can be used to identify SCVs from within mixed-colony phenotype populations of S. aureus. More recently, populations of SCVs that rapidly revert to a "wild-type" (WT) colony phenotype, in the absence of selection pressure, have also been described. In laboratory studies, SCVs accumulate through prolonged infection of non-professional phagocytes and may represent an adaptation to the intracellular environment. However, data from phagocytic cells are lacking. In this study, we mapped SCV and WT colony populations in axenic growth of multiple well-characterized methicillin-sensitive and methicillin-resistant S. aureus strains. We identified SCVs populations on solid media both in the presence and absence of gentamicin. We generated stable SCVs from Newman strain S. aureus, and infected human macrophages with WT S. aureus (Newman, 8325-4) and their SCV counterparts (SCV3, I10) to examine intracellular formation and survival of SCVs. We show that SCVs arise spontaneously during axenic growth, and that the ratio of SCV:WT morphology differs between strains. Exposure to the intracellular environment of human macrophages did not increase formation of SCVs over 5 days and macrophages were able to clear stable SCV bacteria more effectively than their WT counterparts.

Thrombotic risk in COVID-19: a case series and case-control study.

BACKGROUND: A possible association between COVID-19 infection and thrombosis, either as a direct consequence of the virus or as a complication of inflammation, is emerging in the literature. Data on the incidence of venous thromboembolism (VTE) are extremely limited. METHODS: We describe three cases of thromboembolism refractory to heparin treatment, the incidence of VTE in an inpatient cohort, and a case-control study to identify risk factors associated with VTE. RESULTS: We identified 274 confirmed (208) or probable (66) COVID-19 patients. 21 (7.7%) were diagnosed with VTE. D-dimer was elevated in both cases (confirmed VTE) and controls (no confirmed VTE) but higher levels were seen in confirmed VTE cases (4.1 vs 1.2 µg/mL, p<0.001). CONCLUSION: Incidence of VTE is high in patients hospitalised with COVID-19. Urgent clinical trials are needed to evaluate the role of anticoagulation in COVID-19. Monitoring of D-dimer and anti-factor Xa levels may be beneficial in guiding management.

Inability to sustain intraphagolysosomal killing of Staphylococcus aureus predisposes to bacterial persistence in macrophages.

Macrophages are critical effectors of the early innate response to bacteria in tissues. Phagocytosis and killing of bacteria are interrelated functions essential for bacterial clearance but the rate-limiting step when macrophages are challenged with large numbers of the major medical pathogen Staphylococcus aureus is unknown. We show that macrophages have a finite capacity for intracellular killing and fail to match sustained phagocytosis with sustained microbial killing when exposed to large inocula of S. aureus (Newman, SH1000 and USA300 strains). S. aureus ingestion by macrophages is associated with a rapid decline in bacterial viability immediately after phagocytosis. However, not all bacteria are killed in the phagolysosome, and we demonstrate reduced acidification of the phagolysosome, associated with failure of phagolysosomal maturation and reduced activation of cathepsin D. This results in accumulation of viable intracellular bacteria in macrophages. We show macrophages fail to engage apoptosis-associated bacterial killing. Ultittop mately macrophages with viable bacteria undergo cell lysis, and viable bacteria are released and can be internalized by other macrophages. We show that cycles of lysis and reuptake maintain a pool of viable intracellular bacteria over time when killing is overwhelmed and demonstrate intracellular persistence in alveolar macrophages in the lungs in a murine model.

Laboratory sample turnaround times: do they cause delays in the ED?

OBJECTIVES: Blood tests are requested for approximately 50% of patients attending the emergency department (ED). The time taken to obtain the results is perceived as a common reason for delay. The objective of this study was therefore to investigate the turnaround time (TAT) for blood results and whether this affects patient length of stay (LOS) and to identify potential areas for improvement. METHODS: A time-in-motion study was performed at the ED of the John Radcliffe Hospital (JRH), Oxford, UK. The duration of each of the stages leading up to receipt of 101 biochemistry and haematology results was recorded, along with the corresponding patient's LOS. RESULTS: The findings reveal that the mean time for haematology results to become available was 1 hour 6 minutes (95% CI: 29 minutes to 2 hours 13 minutes), while biochemistry samples took 1 hour 42 minutes (95% CI: 1 hour 1 minute to 4 hours 21 minutes), with some positive correlation noted with the patient LOS, but no significant variation between different days or shifts. CONCLUSIONS: With the fastest 10% of samples being reported within 35 minutes (haematology) and 1 hour 5 minutes (biochemistry) of request, our study showed that delays can be attributable to laboratory TAT. Given the limited ability to further improve laboratory processes, the solutions to improving TAT need to come from a collaborative and integrated approach that includes strategies before samples reach the laboratory and downstream review of results.