Penicillin-binding protein redundancy in Bacillus subtilis enables growth during alkaline shock.

Penicillin-binding proteins (PBPs) play critical roles in cell wall construction, cell shape maintenance, and bacterial replication. Bacteria maintain a diversity of PBPs, indicating that despite their apparent functional redundancy, there is differentiation across the PBP family. Apparently-redundant proteins can be important for enabling an organism to cope with environmental stressors. In this study, we evaluated the consequence of environmental pH on PBP enzymatic activity in Bacillus subtilis. Our data show that a subset of PBPs in B. subtilis change activity levels during alkaline shock and that one PBP isoform is rapidly modified to generate a smaller protein (i.e., PBP1a to PBP1b). Our results indicate that a subset of the PBPs are favored for growth under alkaline conditions, while others are readily dispensable. Indeed, we found that this phenomenon could also be observed in Streptococcus pneumoniae, implying that it may be generalizable across additional bacterial species and further emphasizing the evolutionary benefit of maintaining many, seemingly-redundant periplasmic enzymes.IMPORTANCEMicrobes adapt to ever-changing environments and thrive over a vast range of conditions. While bacterial genomes are relatively small, significant portions encode for "redundant" functions. Apparent redundancy is especially pervasive in bacterial proteins that reside outside of the inner membrane. While conditions within the cytoplasm are carefully controlled, those of the periplasmic space are largely determined by the cell's exterior environment. As a result, proteins within this environmentally exposed region must be capable of functioning under a vast array of conditions, and/or there must be several similar proteins that have evolved to function under a variety of conditions. This study examines the activity of a class of enzymes that is essential in cell wall construction to determine if individual proteins might be adapted for activity under particular growth conditions. Our results indicate that a subset of these proteins are preferred for growth under alkaline conditions, while others are readily dispensable.

Severe Acute Respiratory Syndrome Coronavirus 2 Viremia Is Associated With Coronavirus Disease 2019 Severity and Predicts Clinical Outcomes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA) is detected in the bloodstream of some patients with coronavirus disease 2019 (COVID-19), but it is not clear whether this RNAemia reflects viremia (ie, virus particles) and how it relates to host immune responses and outcomes. METHODS: SARS-CoV-2 vRNA was quantified in plasma samples from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-intensive care unit [ICU]), and 23 ICU patients. vRNA levels were compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in plasma. RESULTS: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6%, and 11.1% of ICU, non-ICU, and outpatients, respectively. Virions were detected in plasma pellets using electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (P < .0001); for inpatients, plasma vRNA levels were strongly associated with higher World Health Organization (WHO) score at admission (P = .01), maximum WHO score (P = .002), and discharge disposition (P = .004). A plasma vRNA level >6000 copies/mL was strongly associated with mortality (hazard ratio, 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (P < .01) but not with plasma neutralizing antibody titers (P = .8). CONCLUSIONS: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia correlate strongly with disease severity, patient outcome, and specific inflammatory biomarkers but not with neutralizing antibody titers.

Considerations regarding Interpretation of Positive SARS-CoV-2 Molecular Results with Late Cycle Threshold Values.

COVID-19 disease lies on a spectrum, ranging from completely asymptomatic to mild disease to severe and critical disease. Studies have shown that prolonged shedding or sporadic detection of SARS-CoV-2 RNA can occur long after symptom resolution. Adding to these clinical complexities is the demand for testing for SARS-CoV-2 at all stages of diseases, frequently driven by screening of asymptomatic persons, something that traditionally has not been performed for other viral respiratory diseases. This can lead to positive results from nucleic acid amplification tests (NAATs), such as RT-PCR, with late cycle threshold (CT) values near the test's limit of detection. In this commentary, we review unique attributes of COVID-19 and causes of NAAT late CT values. We provide interpretation considerations as well as strategies to aid in test interpretation.

Great ape health watch: Enhancing surveillance for emerging infectious diseases in great apes.

Infectious diseases have the potential to extirpate populations of great apes. As the interface between humans and great apes expands, zoonoses pose an increasingly severe threat to already endangered great ape populations. Despite recognition of the threat posed by human pathogens to great apes, health monitoring is only conducted for a small fraction of the world's wild great apes (and mostly those that are habituated) meaning that outbreaks of disease often go unrecognized and therefore unmitigated. This lack of surveillance (even in sites where capacity to conduct surveillance is present) is the most significant limiting factor in our ability to quickly detect and respond to emerging infectious diseases in great apes when they first appear. Accordingly, we must create a surveillance system that links disease outbreaks in humans and great apes in time and space, and enables veterinarians, clinicians, conservation managers, national decision makers, and the global health community to respond quickly to these events. Here, we review existing great ape health surveillance programs in African range habitats to identify successes, gaps, and challenges. We use these findings to argue that standardization of surveillance across sites and geographic scales, that monitors primate health in real-time and generates early warnings of disease outbreaks, is an efficient, low-cost step to conserve great ape populations. Such a surveillance program, which we call "Great Ape Health Watch" would lead to long-term improvements in outbreak preparedness, prevention, detection, and response, while generating valuable data for epidemiological research and sustainable conservation planning. Standardized monitoring of great apes would also make it easier to integrate with human surveillance activities. This approach would empower local stakeholders to link wildlife and human health, allowing for near real-time, bidirectional surveillance at the great ape-human interface.

Performance of SARS-CoV-2 antigen testing in symptomatic and asymptomatic adults: a single-center evaluation.

BACKGROUND: Antigen testing offers rapid and inexpensive testing for SARS-CoV-2 but concerns regarding performance, especially sensitivity, remain. Limited data exists for use of antigen testing in asymptomatic patients; thus, performance and reliability of antigen testing remains unclear. METHODS: 148 symptomatic and 144 asymptomatic adults were included. A nasal swab was collected for testing by Quidel Sofia SARS IFA (Sofia) as point of care. A nasopharyngeal swab was also collected and transported to the laboratory for testing by Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV RT-PCR (Cepheid). RESULTS: Overall, Sofia had good agreement with Cepheid (> 95%) in adults, however was less sensitive. Sofia had a sensitivity of 87.8% and 33.3% for symptomatic and asymptomatic patients, respectively. Among symptomatic patients, testing > 5 days post symptom onset resulted in lower sensitivity (82%) when compared with testing within 5 days of symptom onset (90%). Of the four Sofia false-negative results in the asymptomatic cohort, 50% went on to develop COVID-19 disease within 5 days of testing. Specificity in both symptomatic and asymptomatic cohorts was 100%. CONCLUSIONS: Sofia has acceptable performance in symptomatic adults when tested < 5 days of symptom onset. Caution should be taken when testing patients with ≥ 5 days of symptoms. The combination of low prevalence and reduced sensitivity results in relatively poor performance of in asymptomatic patients. NAAT-based diagnostic assays should be considered in when antigen testing is unreliable, particularly in symptomatic patients with > 5 days of symptom onset and asymptomatic patients.

Understanding, Verifying, and Implementing Emergency Use Authorization Molecular Diagnostics for the Detection of SARS-CoV-2 RNA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has brought a new wave of challenges to health care, particularly in the area of rapid diagnostic test development and implementation. The diagnosis of acute coronavirus disease 2019 (COVID-19) is critically dependent on the detection of SARS-CoV-2 RNA from clinical specimens (e.g., nasopharyngeal swabs). While laboratory-developed testing for SARS-CoV-2 is an essential component of diagnostic testing for this virus, the majority of clinical microbiology laboratories are dependent on commercially available SARS-CoV-2 molecular assays. In contrast to assays approved or cleared by the U.S. Food and Drug Administration (FDA) for in vitro diagnostic use, assays for the detection of SARS-CoV-2 nucleic acids have emergency use authorization (EUA) from the FDA. Outside of highly specialized academic and commercial laboratory settings, clinical microbiology laboratories are likely unfamiliar with the EUA classification, and thus, assay verification can be daunting. Further compounding anxiety for laboratories are major issues with the supply chain that are dramatically affecting the availability of test reagents and requiring laboratories to implement multiple commercial EUA tests. Here, we describe guidance for the verification of assays with EUA for the detection of SARS-CoV-2 nucleic acid from clinical specimens.

Navigating Clinical Utilization of Direct-from-Specimen Metagenomic Pathogen Detection: Clinical Applications, Limitations, and Testing Recommendations.

BACKGROUND: Metagenomic next generation sequencing (mNGS) is becoming increasingly available for pathogen detection directly from clinical specimens. These tests use target-independent, shotgun sequencing to detect potentially unlimited organisms. The promise of this methodology to aid infection diagnosis is demonstrated through early case reports and clinical studies. However, the optimal role of mNGS in clinical microbiology remains uncertain. CONTENT: We reviewed studies reporting clinical use of mNGS for pathogen detection from various specimen types, including cerebrospinal fluid, plasma, lower respiratory specimens, and others. Published clinical study data were critically evaluated and summarized to identify promising clinical indications for mNGS-based testing, to assess the clinical impact of mNGS for each indication, and to recognize test limitations. Based on these clinical studies, early testing recommendations are made to guide clinical utilization of mNGS for pathogen detection. Finally, current barriers to routine clinical laboratory implementation of mNGS tests are highlighted. SUMMARY: The promise of direct-from-specimen mNGS to enable challenging infection diagnoses has been demonstrated through early clinical studies of patients with meningitis or encephalitis, invasive fungal infections, community acquired pneumonia, and other clinical indications. However, the proportion of patient cases with positive clinical impact due to mNGS testing is low in published studies and the cost of testing is high, emphasizing the importance of improving our understanding of 'when to test' and for which patients mNGS testing is appropriate.

Outbreak of Adenovirus in a Neonatal Intensive Care Unit: Critical Importance of Equipment Cleaning During Inpatient Ophthalmologic Examinations.

PURPOSE: Outbreaks of adenovirus in neonatal intensive care units (NICUs) can lead to widespread transmission and serious adverse outcomes. We describe the investigation, response, and successful containment of an adenovirus outbreak in a NICU associated with contaminated handheld ophthalmologic equipment used during retinopathy of prematurity (ROP) screening. DESIGN: Epidemiologic outbreak investigation. PARTICIPANTS: A total of 23 hospitalized neonates, as well as NICU staff and parents of affected infants. MAIN OUTCOME MEASURES: Routine surveillance identified an adenovirus outbreak in a level IV NICU in August 2016. Epidemiologic investigation followed, including chart review, staff interviews, and observations. Cases were defined as hospital-acquired adenovirus identified from any clinical specimen (NICU patient or employee) or compatible illness in a family member. Real-time polymerase chain reaction (PCR) and partial- and whole-genome sequencing assays were used for testing of clinical and environmental specimens. RESULTS: We identified 23 primary neonatal cases and 9 secondary cases (6 employees and 3 parents). All neonatal case-patients had respiratory symptoms. Of these, 5 developed pneumonia and 12 required increased respiratory support. Less than half (48%) had ocular symptoms. All neonatal case-patients (100%) had undergone a recent ophthalmologic examination, and 54% of neonates undergoing examinations developed adenovirus infection. All affected employees and parents had direct contact with infected neonates. Observations revealed inconsistent disinfection of bedside ophthalmologic equipment and limited glove use. Sampling of 2 handheld lenses and 2 indirect ophthalmoscopes revealed adenovirus serotype 3 DNA on each device. Sequence analysis of 16 neonatal cases, 2 employees, and 2 lenses showed that cases and equipment shared 100% identity across the entire adenovirus genome. Infection control interventions included strict hand hygiene, including glove use; isolation precautions; enhanced cleaning of lenses and ophthalmoscopes between all examinations; and staff furlough. We identified no cases of secondary transmission among neonates. CONCLUSIONS: Adenovirus outbreaks can result from use of contaminated ophthalmologic equipment. Even equipment that does not directly contact patients can facilitate indirect transmission. Patient-to-patient transmission can be prevented with strict infection control measures and equipment cleaning. Ophthalmologists performing inpatient examinations should take measures to avoid adenoviral spread from contaminated handheld equipment.

CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests.

Effective evaluations of antimicrobial susceptibility tests (ASTs) require robust study design. The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing has recognized that many published studies reporting the performance of commercial ASTs (cASTs) suffer from major design and/or analysis flaws, rendering the results difficult or impossible to interpret. This minireview outlines the current consensus of the Methods Development and Standardization Working Group of the CLSI Subcommittee on Antimicrobial Susceptibility Testing regarding best practices for systematic evaluation of the performance of an AST, including the analysis and presentation of essential data intended for publication.

Lower Respiratory Multiplex Panels for the Detection of Bacterial and Viral Infections.

Development of commercial multiplex panels for the detection and diagnosis of lower respiratory tract infections is rapidly progressing, and FDA-cleared assays are currently available. This review provides a comprehensive overview of the current or soon-to-be available commercial assays, focusing on their analytical performance, advantages, and challenges and the potential impact on patient outcomes when laboratories deploy the assays.

Bacteremia caused by the photosynthetic environmental bacterium Rhodopseudomonas.

We report a case of persistent Rhodopseudomonas bacteremia in a patient two months after an allogeneic bone marrow transplant for acute myeloid leukemia. The bacteremia persisted until IV catheter removal. To our knowledge, this is the first report of Rhodopseudomonas causing infection in humans.

The VieB auxiliary protein negatively regulates the VieSA signal transduction system in Vibrio cholerae.

BACKGROUND: Vibrio cholerae is a facultative pathogen that lives in the aquatic environment and the human host. The ability of V. cholerae to monitor environmental changes as it transitions between these diverse environments is vital to its pathogenic lifestyle. One way V. cholerae senses changing external stimuli is through the three-component signal transduction system, VieSAB, which is encoded by the vieSAB operon. The VieSAB system plays a role in the inverse regulation of biofilm and virulence genes by controlling the concentration of the secondary messenger, cyclic-di-GMP. While the sensor kinase, VieS, and the response regulator, VieA, behave similar to typical two-component phosphorelay systems, the role of the auxiliary protein, VieB, is unclear. RESULTS: Here we show that VieB binds to VieS and inhibits its autophosphorylation and phosphotransfer activity thus preventing phosphorylation of VieA. Additionally, we show that phosphorylation of the highly conserved Asp residue in the receiver domain of VieB regulates the inhibitory activity of VieB. CONCLUSION: Taken together, these data point to an inhibitory role of VieB on the VieSA phosphorelay, allowing for additional control over the signal output. Insight into the function and regulatory mechanism of the VieSAB system improves our understanding of how V. cholerae controls gene expression as it transitions between the aquatic environment and human host.

Penicillin-binding protein redundancy in Bacillus subtilis enables growth during alkaline shock.

Penicillin-binding proteins (PBPs) play critical roles in cell wall construction, cell shape, and bacterial replication. Bacteria maintain a diversity of PBPs, indicating that despite their apparent functional redundancy, there is differentiation across the PBP family. Seemingly redundant proteins can be important for enabling an organism to cope with environmental stressors. We sought to evaluate the consequence of environmental pH on PBP enzymatic activity in Bacillus subtilis. Our data show that a subset of B. subtilis PBPs change activity levels during alkaline shock and that one PBP isoform is rapidly modified to generate a smaller protein (i.e., PBP1a to PBP1b). Our results indicate that a subset of the PBPs are preferred for growth under alkaline conditions, while others are readily dispensable. Indeed, we found that this phenomenon could also be observed in Streptococcus pneumoniae, implying that it may be generalizable across additional bacterial species and further emphasizing the evolutionary benefit of maintaining many, seemingly redundant periplasmic enzymes.

Vaccine hesitancy in pregnant Women: A narrative review.

Pregnant women are often at higher risk for morbidity and mortality due to contracting vaccine-preventable diseases that result in adverse pregnancy outcomes such as spontaneous abortion, preterm births, and congenital fetal defects. For example, health care provider recommendation is correlated with maternal acceptance of influenza vaccination, however, up to 33 % of pregnant women remain unvaccinated irrespective of provider recommendation. Vaccine hesitancy is a multifactorial problem that both the medical and public health systems need to address synergistically. Vaccine education should incorporate balanced perspectives to deliver vaccine education. This narrative review addresses four questions: 1) what are the primary concerns of pregnant women that lead them to be hesitant about receiving vaccinations; 2) to what extent does the source (e.g. provider, friend, family) of vaccine advice and information influence a pregnant person's decision to accept a vaccine; 3) how does the delivery method of vaccine education influence their decision; 4) how can categorizing patients into four distinct groups based on their opinions and behavior regarding vaccines be used to improve provider-patient communication and increase vaccine acceptance. Results from the literature show that the three most common reasons for vaccine hesitancy include: i.) fear of side effects or adverse events; ii.) lack of confidence in vaccine safety; iii.) low perception of being at high risk of infection during pregnancy and/or not having previously received the vaccination when not pregnant. We conclude that vaccine hesitancy is dynamic therefore people do not always hold a static level of vaccine hesitancy. People may move between a continuum of vaccine hesitancy for a multifactorial reasons. A framework, characterized by levels of vaccine hesitancy before and during pregnancy, was constructed to help providers find balance between promoting individual health and public health while providing vaccine education.

Pregnant Women's Concerns Regarding COVID-19 and Their Willingness to Be Vaccinated.

Objectives: We investigated coronavirus disease 2019 (COVID-19) opinions, experiences, and willingness to accept COVID-19 vaccination during pregnancy at two prenatal clinics in early 2021 and early 2022. Materials and Methods: Paper questionnaires were distributed to pregnant women at prenatal care facilities in Virginia and Florida between January and April 2021 and January and April 2022. Questions regarding acceptance and opinions of the influenza vaccine served as a baseline to assess COVID-19 vaccine opinions. Associations between demographic parameters and vaccine opinions and acceptance were examined using Chi-square. A COVID-19 concern score was constructed by principal component analysis with differences between groups assessed by analysis of variance (ANOVA) and analysis of covariance (ANCOVA). Results: Many participants (40.6%) reported that the COVID pandemic had affected their pregnancy. Main themes were problems with social networks, increased stress/anxiety, and being more cautious. In 2021, 19.5% reported they would accept a COVID-19 vaccination during their pregnancy, which increased to 45.8% in 2022. Vaccine hesitancy did not vary by race or between sites, but educational attainment was significant (p < 0.001). Women with a higher concern score were more likely to report they would accept a COVID-19 vaccine. Women who would accept COVID vaccination had a positive opinion regarding the influenza vaccine. Main themes for refusing COVID-19 vaccination were concerns about side effects, lack of research/data, and mistrust of vaccines. Conclusions: The proportion of women willing to accept COVID-19 vaccination increased but remained below 50%. Willingness to accept vaccination during pregnancy was associated with higher education, higher concern about COVID-19, and a positive opinion of the influenza vaccine.

Performance of the Sofia SARS-CoV-2 Rapid Antigen Test in Symptomatic and Asymptomatic Pediatric Patients.

The sensitivity and specificity of SARS-CoV-2 antigen tests have not been widely assessed in children. We evaluated children presenting to outpatient care with Quidel Sofia SARS-CoV-2 antigen test (Sofia-Ag-RDT) compared against Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV reverse transcriptase-polymerase chain reaction test from November 2020 to April 2021. Sofia-Ag-RDT had the highest sensitivity in symptomatic (82%; 95% confidence interval, 68%-91%) children.

Multi-disciplinary Leadership to Mitigate COVID-19 in an Austere West African Military Environment.

INTRODUCTION: The COVID-19 pandemic created challenges for forward-deployed military units to Western Africa. Austere military environments afford multiple avenues to transmit COVID-19 amongst service members. MATERIALS AND METHODS: A COVID-19 outbreak on a military base in Western Africa spanning over 100 days is statistically analyzed using a Pearson's correlation coefficient. Furthermore, a COVID-19 reproductive number (R0) is evaluated to examine the relationship between specific command-directed policies to mitigate COVID-19 transmission. RESULTS: The multidisciplinary partnership of military command, medical, and public health leadership implemented evidence-based and epidemiologically informed COVID-19 preventive base-wide policies, including appropriate isolation/quarantine policies. The R0 for the outbreak was 0.03 and remained <1 for the outbreak duration. This base remained COVID-19 free for multiple weeks after policy implementation. CONCLUSIONS: The implementation of practical mitigating base-wide policies through seamless communication between military command/medical/public health leadership resolved the COVID-19 outbreak while maintaining mission readiness. Weekly COVID-19 testing epidemiological data may be utilized by commanders to direct further decision-making on tightening/loosening base-wide policy restrictions for continued mission-essential operations, e.g., security, food service, or airfield operations.

Factors that shape pregnant women's perceptions regarding the safety of cannabis use during pregnancy.

BACKGROUND: Cannabis use among pregnant women has increased. We surveyed pregnant women in rural Pennsylvania to examine cannabis use and opinions regarding its safety during pregnancy. We examined associations between challenges of pregnancy (e.g., exhaustion, pain, nausea) and cannabis use. METHODS: A cross-sectional survey was administered to a convenience sample of English-speaking pregnant women receiving prenatal care at Geisinger, May-June 2019. Principal component analysis (PCA) was used to construct three scores (overwhelmed/exhausted, happy/optimistic, and health worries) based on 10 questions regarding common experiences during pregnancy (e.g., nausea/vomiting, pain, exhaustion, mood). A score based on four questions regarding cannabis safety during pregnancy was also constructed. RESULTS: From a maximum of 300 surveys distributed, 284 were completed (95%). Most participants were white (87%), married (49%) or living with a partner (38%), and had private health insurance (62%). Most women indicated it was unsafe to use alcohol and tobacco products during pregnancy (> 90%), but that proportion dropped to 82% and 63% regarding recreational cannabis and medical cannabis, respectively. Only women with prior cannabis use (23% of sample) continued to do so during pregnancy: 57% of women reporting daily cannabis use prior to pregnancy continued to use cannabis during pregnancy with 33% reporting daily use. Two thirds of users during pregnancy indicated they were self-medicating for: nausea (90%), anxiety (70%), insomnia (30%), and pain management (30%). Many (56%) of the women who used cannabis during pregnancy believed it is safe. Younger women and women who were overwhelmed/exhausted or less happy/optimistic were more likely to believe cannabis use is safe. Women valued healthcare provider advice more than advice from family and friends. Study strengths include a high response rate. Weaknesses include self-report and that is was a convenience sample; however, the demographics of the sample were similar to past studies. CONCLUSION: Women with a history of cannabis use, especially daily use, are at risk of continuing during pregnancy and should receive counseling. Younger women and women with greater stressors during pregnancy also are at greater risk. Screening for prior use and for stressors may identify patients that would benefit from enhanced counseling.