Microbial Cell-Free DNA Identifies the Causative Pathogen in Infective Endocarditis and Remains Detectable Longer Than Conventional Blood Culture in Patients with Prior Antibiotic Therapy.

BACKGROUND: The diagnosis of infective endocarditis (IE) can be difficult, particularly if blood cultures fail to yield a pathogen. This study evaluates the potential utility of microbial cell-free DNA (mcfDNA) as a tool to identify the microbial etiology of IE. METHODS: Blood samples from patients with suspected IE were serially collected. mcfDNA was extracted from plasma and underwent next-generation sequencing. Reads were aligned against a library containing DNA sequences belonging to >1400 different pathogens. mcfDNA from organisms present above a statistical threshold were reported and quantified in molecules per milliliter (MPM). Additional mcfDNA was collected on each subject every 2-3 days for a total of 7 collections or until discharge. RESULTS: Of 30 enrolled patients with suspected IE, 23 had definite IE, 2 had possible IE, and IE was rejected in 5 patients by modified Duke Criteria. Only the 23 patients with definite IE were included for analysis. Both mcfDNA and blood cultures achieved a sensitivity of 87%. The median duration of positivity from antibiotic treatment initiation was estimated to be approximately 38.1 days for mcfDNA versus 3.7 days for blood culture (proportional odds, 2.952; P = .02771), using a semiparametric survival analysis. mcfDNA (log10) levels significantly declined (-0.3 MPM log10 units, 95% credible interval -0.45 to -0.14) after surgical source control was performed (pre- vs postprocedure, posterior probability >0.99). CONCLUSION: mcfDNA accurately identifies the microbial etiology of IE. Sequential mcfDNA levels may ultimately help to individualize therapy by estimating a patient's burden of infection and response to treatment.

Variation in Severe Acute Respiratory Syndrome Coronavirus 2 Bioaerosol Production in Exhaled Breath.

We developed a simple, noninvasive mask sampling method to quantify and sequence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from exhaled breath. We found substantial variation between individuals in SARS-CoV-2 copies exhaled over a 15-minute period, which moderately correlated with nasal swab viral load. Talking was associated with a median of 2 log10 greater exhaled viral copies. Exposure varies substantially between individuals but may be risk stratified by nasal swab viral load and whether the exposure involved conversation.

Microbial Cell-Free DNA Identifies Etiology of Bloodstream Infections, Persists Longer Than Conventional Blood Cultures, and Its Duration of Detection Is Associated With Metastatic Infection in Patients With Staphylococcus aureus and Gram-Negative Bacteremia.

BACKGROUND: Microbial cell-free DNA (mcfDNA) sequencing of plasma can identify the presence of a pathogen in a host. In this study, we evaluated the duration of pathogen detection by mcfDNA sequencing vs conventional blood culture in patients with bacteremia. METHODS: Blood samples from patients with culture-confirmed bloodstream infection were collected within 24 hours of the index positive blood culture and 48 to 72 hours thereafter. mcfDNA was extracted from plasma, and next-generation sequencing was applied. Reads were aligned against a curated pathogen database. Statistical significance was defined with Bonferroni adjustment for multiple comparisons (P < .0033). RESULTS: A total of 175 patients with Staphylococcus aureus bacteremia (n = 66), gram-negative bacteremia (n = 74), or noninfected controls (n = 35) were enrolled. The overall sensitivity of mcfDNA sequencing compared with index blood culture was 89.3% (125 of 140), and the specificity was 74.3%. Among patients with bacteremia, pathogen-specific mcfDNA remained detectable for significantly longer than conventional blood cultures (median 15 days vs 2 days; P < .0001). Each additional day of mcfDNA detection significantly increased the odds of metastatic infection (odds ratio, 2.89; 95% confidence interval, 1.53-5.46; P = .0011). CONCLUSIONS: Pathogen mcfDNA identified the bacterial etiology of bloodstream infection for a significantly longer interval than conventional cultures, and its duration of detection was associated with increased risk for metastatic infection. mcfDNA could play a role in the diagnosis of partially treated endovascular infections.

Safety of ACE-I and ARB medications in COVID-19: A retrospective cohort study of inpatients and outpatients in California.

INTRODUCTION: There is significant interest in the use of angiotensin converting enzyme inhibitors (ACE-I) and angiotensin II receptor blockers (ARB) in coronavirus disease 2019 (COVID-19) and concern over potential adverse effects since these medications upregulate the severe acute respiratory syndrome coronavirus 2 host cell entry receptor ACE2. Recent studies on ACE-I and ARB in COVID-19 were limited by excluding outpatients, excluding patients by age, analyzing ACE-I and ARB together, imputing missing data, and/or diagnosing COVID-19 by chest computed tomography without definitive reverse transcription polymerase chain reaction (RT-PCR), all of which are addressed here. METHODS: We performed a retrospective cohort study of 1023 COVID-19 patients diagnosed by RT-PCR at Stanford Hospital through April 8, 2020 with a minimum follow-up time of 14 days to investigate the association between ACE-I or ARB use with outcomes. RESULTS: Use of ACE-I or ARB medications was not associated with increased risk of hospitalization, intensive care unit admission, or death. Compared to patients with charted past medical history, there was a lower risk of hospitalization for patients on ACE-I (odds ratio (OR) 0.43; 95% confidence interval (CI) 0.19-0.97; P = 0.0426) and ARB (OR 0.39; 95% CI 0.17-0.90; P = 0.0270). Compared to patients with hypertension not on ACE-I or ARB, patients on ARB medications had a lower risk of hospitalization (OR 0.09; 95% CI 0.01-0.88; P = 0.0381). CONCLUSIONS: These findings suggest that the use of ACE-I and ARB is not associated with adverse outcomes and may be associated with improved outcomes in COVID-19, which is immediately relevant to care of the many patients on these medications.

Clinical characteristics associated with COVID-19 severity in California.

Given the rapidly progressing coronavirus disease 2019 (COVID-19) pandemic, this report on a US cohort of 54 COVID-19 patients from Stanford Hospital and data regarding risk factors for severe disease obtained at initial clinical presentation is highly important and immediately clinically relevant. We identified low presenting oxygen saturation as predictive of severe disease outcomes, such as diagnosis of pneumonia, acute respiratory distress syndrome, and admission to the intensive care unit, and also replicated data from China suggesting an association between hypertension and disease severity. Clinicians will benefit by tools to rapidly risk stratify patients at presentation by likelihood of progression to severe disease.

Detection, survival and infectious potential of Mycobacterium tuberculosis in the environment: a review of the evidence and epidemiological implications.

Much remains unknown about Mycobacterium tuberculosis transmission. Seminal experimental studies from the 1950s demonstrated that airborne expulsion of droplet nuclei from an infectious tuberculosis (TB) patient is the primary route of transmission. However, these findings did not rule out other routes of M. tuberculosis transmission. We reviewed historical scientific evidence from the late 19th/early 20th century and contemporary studies investigating the presence, persistence and infectiousness of environmental M. tuberculosis We found both experimental and epidemiological evidence supporting the presence and viability of M. tuberculosis in multiple natural and built environments for months to years, presumably following contamination by a human source. Furthermore, several studies confirm M. tuberculosis viability and virulence in the environment using guinea pig and mouse models. Most of this evidence was historical; however, several recent studies have reported consistent findings of M. tuberculosis detection and viability in the environment using modern methods. Whether M. tuberculosis in environments represents an infectious threat to humans requires further investigation; this may represent an untapped source of data with which to further understand M. tuberculosis transmission. We discuss potential opportunities for harnessing these data to generate new insights into TB transmission in congregate settings.

Metformin Use Reverses the Increased Mortality Associated With Diabetes Mellitus During Tuberculosis Treatment.

Background: The global type 2 diabetes mellitus (DM) epidemic threatens progress made in reducing tuberculosis (TB)-related mortality worldwide. Previous clinical studies have not fully evaluated potential confounding variables in addressing the impact of DM on TB treatment outcomes. The antidiabetic agent metformin regulates autophagy and may play a role as a host-directed therapeutic adjuvant to antitubercular treatment. Methods: We conducted a retrospective cohort study comprising patients aged ≥13 years undergoing treatment for culture-confirmed, drug-susceptible pulmonary TB. We assessed the effect of DM on mortality during TB treatment and 2-month TB sputum-culture conversion. We also evaluated the effect of metformin use on survival during TB treatment. Results: Among 2416 patients undergoing TB treatment, after adjusting for age, sex, chronic kidney disease, cancer, hepatitis C, tobacco use, cavitary disease, and treatment adherence, patients with DM had 1.91 times higher odds (95% confidence interval [CI], 1.51-2.40) of death during TB treatment than patients without DM, and 1.72 (95% CI, 1.25-2.38) times higher odds of remaining culture-positive at 2 months. Metformin use in patients with DM was significantly associated with decreased mortality during TB treatment (hazard ratio, 0.56 [95% CI, .39-.82]), and metformin users had similar mortality as patients without DM. Conclusions: This study suggests that despite multiple potential confounding variables, DM poses an increased risk of adverse TB treatment outcomes. There was a significant association between metformin use and decreased mortality during TB treatment, suggesting a potential role for this agent as adjunctive, host-directed therapy.

Comparison of Digital Chest Radiography to Purified Protein Derivative for Screening of Tuberculosis in Newly Admitted Inmates.

This study compares purified protein derivative (PPD) screening to digital chest radiography (CXR) screening for tuberculosis (TB) in newly admitted inmates in the San Diego County Jail system. The study period lasted from 2002 to 2014, during which 45 cases of active TB were detected, a rate of 69.2 cases per 100,000 person-years. Compared to PPD, CXR reduces the median number of days active TB cases were in the general population from 44.4 to 5.2 days and the number of exposures from 1,222 to 138 persons. These results confirm that CXR remains a more effective method for preventing exposure to active TB in jail facilities.

Strategies for folding of affinity tagged proteins using GroEL and osmolytes.

Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.

The role of specific beta-gamma subunit interactions in oxyanion stimulation of the MgATP hydrolysis of a hybrid photosynthetic F1-ATPase.

Pairs of cysteine residues were introduced into the twisted N- and C-terminal helices of the gamma subunit of the chloroplast F1-ATPase to test, via disulfide cross-linking, potential inter-helical movements involved in catalysis of ATP hydrolysis. The extent of disulfide cross-linking was determined by estimating the amount of free sulfhydryl available for labeling with fluoresceinyl maleimide before and after cross-linking. Significant disulfide formation (50-75%) was observed between cysteines introduced at positions 30 and 31 in the N-terminal helix and 276 and 278 in the C-terminal helix. Cross-linking had no apparent effect on catalysis, therefore eliminating the involvement of large-scale inter-helical movements within this region of the gamma subunit in cooperative ATP hydrolysis. However, the presence of the two cysteines together in the gammaV31C/A276C double mutant, irrespective of whether or not they were cross-linked together, lowered the MgATPase activity by more than 70% and completely eliminated the well-known activating effect of the oxyanion sulfite. The CaATPase activity was unaffected. Similar but less pronounced effects were seen with the gammaK30C/A276C double mutant. The results indicate that residues at or near positions 31 and 276 within the twisted helical pair of the gamma subunit are required to overcome Mg2+ inhibition of ATP hydrolysis. These residues are likely to be involved in forming a point of contact between the gamma and beta subunits that is responsible for this effect.

C-Terminal mutations in the chloroplast ATP synthase gamma subunit impair ATP synthesis and stimulate ATP hydrolysis.

Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. The catch is considered to play a critical role in the binding change mechanism whereby binding of ATP to one catalytic site releases the catch and induces a partial rotation of the gamma subunit. This role is supported by the observation that mutation of the equivalent arginine and glutamine residues in the Escherichia coli F1 gamma subunit drastically reduced all ATP-dependent catalytic activities of the enzyme [Greene, M. D., and Frasch, W. D. (2003) J. Biol. Chem. 278, 5194-5198]. In this study, we show that simultaneous substitution of the equivalent residues in the chloroplast F1 gamma subunit, arginine 304 and glutamine 305, with alanine decreased the level of proton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATP hydrolysis activities increased by more than 3-fold as a result of these mutations; however, the sulfite-stimulated activity decreased by more than 60%. The Mg2+-dependent, but not the Ca2+-dependent, ATPase activity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicating selective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catch residues are required for efficient proton coupling and for activation of multisite catalysis when MgATP is the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore, for rotation of the gamma subunit.