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.

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

ObjectivesPrevious 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.MethodsOver 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.Results35% 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.ConclusionsConventional sampling under-estimates carriage of S. aureus and this explains the majority of apparent S. aureus acquisitions among elective cardiothoracic patients.

Sampling practices determine the accuracy of blood culture in diagnosing bloodstream infection. The main acute hospital in this study introduced aerobic-only routine blood cultures aiming to increase the volume and number of aerobic samples. At the smaller acute site, aerobic–anaerobic pairs were sent routinely. Culture yield and sampling practices were compared at these two sites and it was found that anaerobic cultures increased the yield of pathogens including facultative anaerobes. Volume cultured and number of samples sent fell short of national recommendations. The aerobic-only policy did not result in more blood being cultured. Based on these findings, the main acute hospital is reintroducing aerobic–anaerobic pairs for routine culture.

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 mu g/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.

We read with interest your recent article by Fogarty et al.,1 in particular their conclusion that differences in thrombotic risk may contribute to ethnic disparities in mortality from coronavirus disease 2019 (COVID-19). This is especially important in the UK, where age-sex adjusted hospital death rates for COVID-19 are 2·17-times higher for people with ethnicity recorded as Black compared to those recorded as White, and 1·95 higher for those recorded as Asian.2 This excess mortality persists after adjustment for deprivation, body mass index (BMI), smoking and comorbidities,2 and despite correction for region, rural or urban living, deprivation, household composition, socioeconomic status and health.3 Similar data from the USA show that in 14 States, African-Americans represent 33% of hospitalisations for COVID-19, despite only making up 14% of the catchment population.4 Black ethnicity is a construct incorporating diverse populations of African descent. Studies from several communities labelled as 'Black', in particular African-Americans, imply a common increase in thrombotic risk, which may contribute to unexplained ethnic disparities in the UK and USA in COVID-19. Fogarty et al.1 propose that COVID-19 causes pulmonary intravascular coagulopathy. Our own study of patients hospitalised with COVID-19 found raised D-dimer levels and a high associated rate of venous thromboembolism (VTE; 7·7%).5 Another study of intensive care patients with COVID-19 also found raised D-dimer levels, without disseminated intravascular coagulation, and reported a 16·7% rate of pulmonary embolism (PE).6 Patients with COVID-19 acute respiratory distress syndrome (ARDS) had a sixfold increase in PE rates compared with matched patients with non-COVID-19 ARDS. The authors similarly concluded that thrombosis risk is not entirely explained by respiratory failure or critical illness per se, and may be associated with abnormal pulmonary microvascular thrombosis. Von Willebrand factor (VWF) and Factor VIII were also elevated in patients with COVID-19.6 A proposed model of pulmonary intravascular coagulopathy could depend on endothelial release of VWF, which mediates platelet aggregation, and prevents breakdown of circulating pro-thrombotic factor VIII. The Fogarty et al.1 article refers to increased thrombosis risk in African-Americans, who have a 67–104% higher age-sex adjusted rate of VTE than White Americans.7-9 Higher VTE risk in African-Americans is contributed to by BMI, hypertension, diabetes, kidney disease, anti-coagulation status and socioeconomic factors.7-9 Increased D-dimer levels have been demonstrated in African-Americans without VTE,10 which persist despite controlling for age, sex, VTE risk factors, medications and lifestyle.11 This suggests that African-Americans might have higher baseline clot formation and breakdown, even in the absence of detected VTE. African-Americans also have higher circulating levels of VWF, Factor VIII and fibrinogen.10, 12 Factor VIII and VWF are reported in covariate analyses to independently confer risk of VTE in African-Americans,7, 9 irrespective of ABO type, Factor VIII levels, hypertension, renal disease, recent surgery, diabetes, annual household income or alcohol use.13 Higher circulating VWF levels may be due to increased baseline production by endothelium, or reduced clearance. Along with elevated Factor VIII and D-dimer levels, these findings may imply that increased endothelial activation of the clotting cascade acts as a common pathway for VTE risk factors that are already known to occur at greater prevalence in African-Americans. Alternatively, differences in endothelial regulation of clot formation and breakdown may represent a distinct risk factor that is more common in this ethnic group, due to genetics or unrecognised environmental factors. Similar trends have been reported for Black people in the UK. One British centre described higher Factor VIII levels in Black patients with deep vein thrombosis (DVT) compared to White patients with DVT.14 Thrombin generation was increased in Black people with or without DVT compared to White people,14 suggesting that in both the UK and USA, some Black populations have higher thrombotic risk. If Black ethnicity and COVID-19 are both associated with increased VTE, Black people with COVID-19 may suffer from a combined thrombotic risk, which contributes to excess mortality. For Black people in the UK and USA, thrombotic risk would be one of multiple interacting biological and socioeconomic variables causing increased death from COVID-19. Interaction between Black ethnicity, thrombotic risk and mortality from COVID-19 could be mediated by traditional VTE risk factors such as BMI, diabetes and cardiovascular disease. Increased Factor VIII and VWF in some Black populations also imply a molecular mechanism for this. If COVID-19 does cause pulmonary intravascular coagulopathy with microvascular thrombosis, then VTE risk in Black people may confer increased vulnerability to COVID-19, even in the absence of detectable macrovascular thrombi such as PE. Thrombotic risk may not account for increased deaths from COVID-19 in British Asians. Excess mortality seems to particularly affect people from Pakistani, Bangladeshi and Indian backgrounds, even after correction for age, geography and socioeconomic variables.3 This current data do not appear to show significantly increased mortality in the British Chinese community, once corrected for age. Fogarty et al.1 discuss differences in coagulopathy between Caucasian and Chinese patients with COVID-19, and mention the lower rate of VTE in Chinese people in general, but there are limited data in South Asians regarding relative VTE risk. One large study reported reduced incidence of DVT in British Indians compared to White people,15 but there are no data on differences in clotting factors, including VWF and Factor VIII. Studies of ethnic disparities in outcomes from COVID-19 rely on crude distinctions between 'Black', 'Asian' and 'White'.2 This obscures ethnic variation within these groups, and specific subpopulations may have unique risk factors for both thrombosis and COVID-19. It is disappointing that there are little data on thrombotic risk in ethnic minority communities in countries outside the USA, given the extensive data on VTE in African-Americans. For COVID-19, research should prioritise multivariate analysis of risk factors for mortality in specific ethnic subgroups, with consideration of both socioeconomic and biological factors, particularly clotting factor levels. Investigations as to whether VWF and Factor VIII levels might independently correlate with outcome in COVID-19 are also urgently required. If certain ethnicities are at increased risk of thrombosis, this may have implications for thromboprophylaxis in COVID-19, such as full dose anti-coagulation for inpatients, or anti-platelet agents if not admitted to hospital. Roshan Ramasamy wrote the letter. Kate Milne, Deanne Bell, Simon Stoneham and Timothy Chevassut reviewed and revised the letter. There are no competing interests.

Staphylococcus aureusis 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 ofS. aureuson 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 ofS. 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-resistantS. aureusstrains. We identified SCVs populations on solid media both in the presence and absence of gentamicin. We generated stable SCVs from Newman strainS. aureus, and infected human macrophages with WTS. 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.

BACKGROUNDCOVID-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.METHODS24 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.RESULTSThe 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.CONCLUSIONVery 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.

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.

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.