COVID-19 infection and transmission includes complex sequence diversity.

SARS-CoV-2 whole genome sequencing has played an important role in documenting the emergence of polymorphisms in the viral genome and its continuing evolution during the COVID-19 pandemic. Here we present data from over 360 patients to characterize the complex sequence diversity of individual infections identified during multiple variant surges (e.g., Alpha and Delta). Across our survey, we observed significantly increasing SARS-CoV-2 sequence diversity during the pandemic and frequent occurrence of multiple biallelic sequence polymorphisms in all infections. This sequence polymorphism shows that SARS-CoV-2 infections are heterogeneous mixtures. Convention for reporting microbial pathogens guides investigators to report a majority consensus sequence. In our study, we found that this approach would under-report sequence variation in all samples tested. As we find that this sequence heterogeneity is efficiently transmitted from donors to recipients, our findings illustrate that infection complexity must be monitored and reported more completely to understand SARS-CoV-2 infection and transmission dynamics. Many of the nucleotide changes that would not be reported in a majority consensus sequence have now been observed as lineage defining SNPs in Omicron BA.1 and/or BA.2 variants. This suggests that minority alleles in earlier SARS-CoV-2 infections may play an important role in the continuing evolution of new variants of concern.

CLOVER: A Phase 3 Randomized Trial Investigating the Efficacy and Safety of a Detoxified Toxin A/B Vaccine in Adults 50 Years and Older at Increased Risk of Clostridioides difficile Infection.

BACKGROUND: Clostridioides difficile infection (CDI) causes substantial mortality and healthcare burden. We assessed the detoxified toxin-A/B PF-06425090 vaccine for primary CDI prevention. METHODS: This phase 3 observer-blinded study randomized (1:1) ≥50-year-olds at increased CDI risk (N=17,535) to receive 3 PF-06425090 or placebo doses (0,1,6-months). Primary endpoints were first CDI episode (≥3 unformed stools within 24 hours; central laboratory-confirmed toxin A/B positive) ≥14 days post-dose 3 (PD3; first primary) and post-dose 2 (PD2; second primary). CDI duration, need for CDI-related medical attention (secondary endpoints), and antibiotic use (post hoc analysis) PD3 were evaluated. Tolerability/safety was assessed. RESULTS: The primary endpoint was not met (17 PF-06425090 and 25 placebo recipients had first CDI episode ≥14 days PD3 [vaccine efficacy (VE)=31.0% (96.4%CI: -38.7%-66.6%)]; 24 PF-06425090 and 34 placebo recipients had first CDI episode ≥14 days PD2 [VE=28.6% (-28.4%-61.0%)]). Median CDI duration was lower with PF-06425090 (1 day) versus placebo (4 days; 2-sided nominal P=0.02). Of participants with first CDI episode, 0 PF-06425090 and 11 placebo recipients sought CDI-related medical attention (post hoc analysis estimated VE=100% [95%CI: 59.6%-100.0%]) and 0 PF-06425090 and 10 placebo recipients required antibiotic treatment (VE=100% [54.8%-100.0%]). Local reactions were more frequent in PF-06425090 recipients and systemic events were generally similar between groups; most were mild-to-moderate. AE rates were similar between groups. CONCLUSIONS: Three PF-06425090 doses were safe and well-tolerated. Although the primary endpoint was not met, PF-06425090 reduced symptom duration, CDI requiring medical attention, and CDI-directed antibiotic treatment, highlighting its potential to reduce CDI-associated healthcare burden. NCT03090191.

Pulsed dosing and extended daily dosing of oral vancomycin do not facilitate clearance of Clostridioides difficile colonization in mice.

Vancomycin taper and pulse regimens are commonly used to treat recurrent Clostridioides difficile infections, but the mechanism by which these regimens might reduce recurrences is unclear. Here, we used a mouse model to test the hypothesis that pulse dosing of vancomycin after a 10-day treatment course enhances clearance of C. difficile from the intestinal tract. Mice with C. difficile colonization received 10 days of once-daily oral vancomycin followed by 20 days of treatment with saline (controls), daily vancomycin, or pulse dosing of vancomycin every 2 or 3 days. Stool samples were collected to measure the concentration of C. difficile during and after treatment, vancomycin concentrations, and growth of vegetative C. difficile during every 3 days dosing. Pulse dosing of vancomycin was not effective in maintaining suppression of C. difficile (P > 0.05 in comparison to saline controls); growth of vegetative C. difficile occurred between pulse doses when vancomycin decreased to undetectable levels. Daily dosing of vancomycin suppressed C. difficile during treatment, but recurrent colonization occurred after treatment in more than 75% of mice, and by post-treatment day 14, there was no significant difference among the control, pulse dosing, and daily dosing groups (P > 0.05). These findings demonstrate that pulse dosing of vancomycin every 2 or 3 days does not facilitate the clearance of C. difficile spores in mice. Studies are needed to examine the impact of vancomycin taper and pulsed regimens in patients.

Natural History of Clostridioides difficile Colonization and Infection Following New Acquisition of Carriage in Healthcare Settings: A Prospective Cohort Study.

BACKGROUND: Limited information is available on the natural history of Clostridioides difficile colonization and infection in patients with new acquisition of C. difficile in healthcare settings. METHODS: In 3 hospitals and affiliated long-term care facilities, we collected serial perirectal cultures from patients with no diarrhea on enrollment to identify new acquisition of toxigenic C. difficile carriage and determined the duration and burden of carriage. Asymptomatic carriage was defined as transient if only 1 culture was positive, with negative cultures before and after, or persistent if 2 or more cultures were positive. Clearance of carriage was defined as 2 consecutive negative perirectal cultures. RESULTS: Of 1432 patients with negative initial cultures and at least 1 follow-up culture, 39 (2.7%) developed C. difficile infection (CDI) without prior detection of carriage and 142 (9.9%) acquired asymptomatic carriage, with 19 (13.4%) subsequently diagnosed with CDI. Of 82 patients analyzed for persistence of carriage, 50 (61.0%) had transient carriage and 32 (39.0%) had persistent carriage, with an estimated median of 77 days to clearance of colonization (range, 14-133 days). Most persistent carriers had a relatively high burden of carriage and maintained the same ribotype over time, whereas most transient carriers had a low burden of carriage detected only using broth enrichment cultures. CONCLUSIONS: In 3 healthcare facilities, 9.9% of patients acquired asymptomatic carriage of toxigenic C. difficile, and 13.4% were subsequently diagnosed with CDI. Most carriers had transient rather than persistent carriage and most patients developing CDI did not have prior detection of carriage.

A MUC5B Gene Polymorphism, rs35705950-T, Confers Protective Effects Against COVID-19 Hospitalization but Not Severe Disease or Mortality.

Rationale: A common MUC5B gene polymorphism, rs35705950-T, is associated with idiopathic pulmonary fibrosis (IPF), but its role in severe acute respiratory syndrome coronavirus 2 infection and disease severity is unclear. Objectives: To assess whether rs35705950-T confers differential risk for clinical outcomes associated with coronavirus disease (COVID-19) infection among participants in the Million Veteran Program (MVP). Methods: The MUC5B rs35705950-T allele was directly genotyped among MVP participants; clinical events and comorbidities were extracted from the electronic health records. Associations between the incidence or severity of COVID-19 and rs35705950-T were analyzed within each ancestry group in the MVP followed by transancestry meta-analysis. Replication and joint meta-analysis were conducted using summary statistics from the COVID-19 Host Genetics Initiative (HGI). Sensitivity analyses with adjustment for additional covariates (body mass index, Charlson comorbidity index, smoking, asbestosis, rheumatoid arthritis with interstitial lung disease, and IPF) and associations with post-COVID-19 pneumonia were performed in MVP subjects. Measurements and Main Results: The rs35705950-T allele was associated with fewer COVID-19 hospitalizations in transancestry meta-analyses within the MVP (Ncases = 4,325; Ncontrols = 507,640; OR = 0.89 [0.82-0.97]; P = 6.86 × 10-3) and joint meta-analyses with the HGI (Ncases = 13,320; Ncontrols = 1,508,841; OR, 0.90 [0.86-0.95]; P = 8.99 × 10-5). The rs35705950-T allele was not associated with reduced COVID-19 positivity in transancestry meta-analysis within the MVP (Ncases = 19,168/Ncontrols = 492,854; OR, 0.98 [0.95-1.01]; P = 0.06) but was nominally significant (P < 0.05) in the joint meta-analysis with the HGI (Ncases = 44,820; Ncontrols = 1,775,827; OR, 0.97 [0.95-1.00]; P = 0.03). Associations were not observed with severe outcomes or mortality. Among individuals of European ancestry in the MVP, rs35705950-T was associated with fewer post-COVID-19 pneumonia events (OR, 0.82 [0.72-0.93]; P = 0.001). Conclusions: The MUC5B variant rs35705950-T may confer protection in COVID-19 hospitalizations.

Houston, We Have a Problem: Reports of Clostridioides difficile Isolates With Reduced Vancomycin Susceptibility.

During the past 4 decades, oral vancomycin has been a mainstay of Clostridioides difficile infection (CDI) therapy with no reports of treatment failure due to emergence of vancomycin resistance. However, C. difficile isolates with high-level phenotypic resistance to vancomycin have recently been reported in 3 distinct geographic regions. There is an urgent need for surveillance to determine if strains with reduced vancomycin susceptibility are circulating in other areas. In a Cleveland-area hospital, screening of 176 CDI stool specimens yielded no C. difficile isolates with reduced vancomycin susceptibility and highlighted the potential for false-positive results due to contamination with vancomycin-resistant enterococci. Additional studies are needed to clarify whether reduced vancomycin susceptibility is an emerging problem that will alter clinical practice. Clinicians should alert their health department if they observe a substantial increase in the frequency of vancomycin treatment failure in patients diagnosed with CDI with no alternative explanation for diarrhea.

Airflow Patterns in Double-Occupancy Patient Rooms May Contribute to Roommate-to-Roommate Transmission of Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Hospitalized patients are at risk to acquire severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from roommates with unrecognized coronavirus disease 2019 (COVID-19). We hypothesized that airflow patterns might contribute to SARS-CoV-2 transmission in double-occupancy patient rooms. METHODS: A device emitting condensed moisture was used to identify airflow patterns in double-occupancy patient rooms. Simulations were conducted to assess transfer of fluorescent microspheres, 5% sodium chloride aerosol, and aerosolized bacteriophage MS2 between patient beds 3 meters apart and to assess the effectiveness of privacy curtains and portable air cleaners in reducing transfer. RESULTS: Air flowed from inlet vents in the center of the room to an outlet vent near the door, resulting in air currents flowing toward the bed adjacent to the outlet vent. Fluorescent microspheres (212-250-µm diameter), 5% sodium chloride aerosol, and aerosolized bacteriophage MS2 released from the inner bed were carried on air currents toward the bed adjacent to the outlet vent. Closing curtains between the patient beds reduced transfer of each of the particles. Operation of a portable air cleaner reduced aerosol transfer to the bed adjacent to the outlet vent but did not offer a benefit over closing the curtains alone, and in some situations, resulted in an increase in aerosol exposure. CONCLUSIONS: Airflow patterns in double-occupancy patient rooms may contribute to risk for transmission of SARS-CoV-2 between roommates. Keeping curtains closed between beds may be beneficial in reducing risk.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Patient Transport Van.

We report 2 episodes of potential SARS-CoV-2 transmission from infected van drivers to passengers despite masking and physical distancing. Whole-genome sequencing confirmed relatedness of driver and passenger SARS-CoV-2. With the heater operating, fluorescent microspheres were transported by airflow >3 meters from the front to the back of the van.

Small phenolic and indolic gut-dependent molecules in the primate central nervous system: levels vs. bioactivity.

INTRODUCTION: A rapidly growing body of data documents associations between disease of the brain and small molecules generated by gut-microbiota (GMB). While such metabolites can affect brain function through a variety of mechanisms, the most direct action would be on the central nervous system (CNS) itself. OBJECTIVE: Identify indolic and phenolic GMB-dependent small molecules that reach bioactive concentrations in primate CNS. METHODS: We conducted a PubMed search for metabolomic studies of the primate CNS [brain tissue or cerebrospinal fluid (CSF)] and then selected for phenolic or indolic metabolites that (i) had been quantified, (ii) were GMB-dependent. For each chemical we then conducted a search for studies of bioactivity conducted in vitro in human cells of any kind or in CNS cells from the mouse or rat. RESULTS: 36 metabolites of interests were identified in primate CNS through targeted metabolomics. Quantification was available for 31/36 and in vitro bioactivity for 23/36. The reported CNS range for 8 metabolites 2-(3-hydroxyphenyl)acetic acid, 2-(4-hydroxyphenyl)acetic acid, 3-(3-hydroxyphenyl)propanoic acid, (E)-3-(3,4-dihydroxyphenyl)prop-2-enoic acid [caffeic acid], 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2-acetamido-3-(1H-indol-3-yl)propanoic acid [N-acetyltryptophan], 1H-indol-3-yl hydrogen sulfate [indoxyl-3-sulfate] overlapped with a bioactive concentration. However, the number and quality of relevant studies of CNS neurochemistry as well as of bioactivity were highly limited. Structural isomers, multiple metabolites and potential confounders were inadequately considered. CONCLUSION: The potential direct bioactivity of GMB-derived indolic and phenolic molecules on primate CNS remains largely unknown. The field requires additional strategies to identify and prioritize screening of the most promising small molecules that enter the CNS.

The Impact of Clinical Decision Support Alerts on Clostridioides difficile Testing: A Systematic Review.

BACKGROUND: Several studies have investigated the utility of electronic decision support alerts in diagnostic stewardship for Clostridioides difficile infection (CDI). However, it is unclear if alerts are effective in reducing inappropriate CDI testing and/or CDI rates. The aim of this systematic review was to determine if alerts related to CDI diagnostic stewardship are effective at reducing inappropriate CDI testing volume and CDI rates among hospitalized adult patients. METHODS: We searched Ovid Medline and 5 other databases for original studies evaluating the association between alerts for CDI diagnosis and CDI testing volume and/or CDI rate. Two investigators independently extracted data on study characteristics, study design, alert triggers, cointerventions, and study outcomes. RESULTS: Eleven studies met criteria for inclusion. Studies varied significantly in alert triggers and in study outcomes. Six of 11 studies demonstrated a statistically significant decrease in CDI testing volume, 6 of 6 studies evaluating appropriateness of CDI testing found a significant reduction in the proportion of inappropriate testing, and 4 of 7 studies measuring CDI rate demonstrated a significant decrease in the CDI rate in the postintervention vs preintervention period. The magnitude of the increase in appropriate CDI testing varied, with some studies reporting an increase with minimal clinical significance. CONCLUSIONS: The use of electronic alerts for diagnostic stewardship for C. difficile was associated with reductions in CDI testing, the proportion of inappropriate CDI testing, and rates of CDI in most studies. However, broader concerns related to alerts remain understudied, including unintended adverse consequences and alert fatigue.

Environmental Contamination of Contact Precaution and Non-Contact Precaution Patient Rooms in Six Acute Care Facilities.

BACKGROUND: Environmental contamination is an important source of hospital multidrug-resistant organism (MDRO) transmission. Factors such as patient MDRO contact precautions (CP) status, patient proximity to surfaces, and unit type likely influence MDRO contamination and bacterial bioburden levels on patient room surfaces. Identifying factors associated with environmental contamination in patient rooms and on shared unit surfaces could help identify important environmental MDRO transmission routes. METHODS: Surfaces were sampled from MDRO CP and non-CP rooms, nursing stations, and mobile equipment in acute care, intensive care, and transplant units within 6 acute care hospitals using a convenience sampling approach blinded to cleaning events. Precaution rooms had patients with clinical or surveillance tests positive for methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, carbapenem-resistant Enterobacteriaceae or Acinetobacter within the previous 6 months, or Clostridioides difficile toxin within the past 30 days. Rooms not meeting this definition were considered non-CP rooms. Samples were cultured for the above MDROs and total bioburden. RESULTS: Overall, an estimated 13% of rooms were contaminated with at least 1 MDRO. MDROs were detected more frequently in CP rooms (32% of 209 room-sample events) than non-CP rooms (12% of 234 room-sample events). Surface bioburden did not differ significantly between CP and non-CP rooms or MDRO-positive and MDRO-negative rooms. CONCLUSIONS: CP room surfaces are contaminated more frequently than non-CP room surfaces; however, contamination of non-CP room surfaces is not uncommon and may be an important reservoir for ongoing MDRO transmission. MDRO contamination of non-CP rooms may indicate asymptomatic patient MDRO carriage, inadequate terminal cleaning, or cross-contamination of room surfaces via healthcare personnel hands.

Unique Clindamycin-Resistant Clostridioides difficile Strain Related to Fluoroquinolone-Resistant Epidemic BI/RT027 Strain.

During a surveillance study of patients in a long-term care facility and the affiliated acute care hospital in the United States, we identified a Clostridioides difficile strain related to the epidemic PCR ribotype (RT) 027 strain associated with hospital outbreaks of severe disease. Fifteen patients were infected with this strain, characterized as restriction endonuclease analysis group DQ and RT591. Like RT027, DQ/RT591 contained genes for toxin B and binary toxin CDT and a tcdC gene of identical sequence. Whole-genome sequencing and multilocus sequence typing showed that DQ/RT591 is a member of the same multilocus sequence typing clade 2 as RT027 but in a separate cluster. DQ/RT591 produced a similar cytopathic effect as RT027 but showed delayed toxin production in vitro. DQ/RT591 was susceptible to moxifloxacin but highly resistant to clindamycin. Continued surveillance is warranted for this clindamycin-resistant strain that is related to the fluoroquinolone-resistant epidemic RT027 strain.

Are Many Patients Diagnosed With Healthcare-associated Clostridioides difficile Infections Colonized With the Infecting Strain on Admission?

In a cohort of 480 patients admitted to an acute care hospital, 68 (14%) had positive perirectal cultures for toxigenic Clostridioides difficile on admission. Of the 11 patients (2%) diagnosed with healthcare-associated C. difficile infections, 3 (27%) had genetically related admission and infection isolates, based on whole-genome sequencing.

Impact of Antibiotic Treatment on the Burden of Nasal Staphylococcus aureus among Hospitalized Patients.

We examined the impact of systemic antibiotics on the burden of nasal Staphylococcus aureus in hospitalized patients. Of 1,482 patients, 237 (16%) had nasal methicillin-susceptible S. aureus (MSSA) and 92 (6%) had nasal methicillin-resistant S. aureus (MRSA) on admission. Treatment regimens that included agents with inhibitory activity against MRSA or MSSA significantly reduced the burden of carriage, whereas regimens lacking anti-MRSA activity, including fluoroquinolones, promoted MRSA overgrowth.

Tapering Courses of Oral Vancomycin Induce Persistent Disruption of the Microbiota That Provide Colonization Resistance to Clostridium difficile and Vancomycin-Resistant Enterococci in Mice.

Vancomycin taper regimens are commonly used for the treatment of recurrent Clostridium difficile infections. One rationale for tapering and pulsing of the dose at the end of therapy is to reduce the selective pressure of vancomycin on the indigenous intestinal microbiota. Here, we used a mouse model to test the hypothesis that the indigenous microbiota that provide colonization resistance against C. difficile and vancomycin-resistant enterococci (VRE) is repopulated during tapering courses of vancomycin. Mice were treated orally with vancomycin daily for 10 days, vancomycin in a tapering dose for 42 days, fidaxomicin for 10 days, or saline. To assess colonization resistance, subsets of mice were challenged with 104 CFU of C. difficile or VRE at multiple time points during and after completion of treatment. The impact of the treatments on the microbiome was measured by cultures, real-time PCR for selected anaerobic bacteria, and deep sequencing. Vancomycin taper-treated mice developed alterations of the microbiota and disruption of colonization resistance that was persistent 18 days after treatment. In contrast, mice treated with a 10-day course of vancomycin exhibited recovery of the microbiota and of colonization resistance by 15 days after treatment, and fidaxomicin-treated mice maintained intact colonization resistance. These findings demonstrate that alteration of the indigenous microbiota responsible for colonization resistance to C. difficile and VRE persist during and after completion of tapering courses of vancomycin.

Multistep Testing Algorithms for Clostridioides difficile Infection.

A 10-year-old girl with spina bifida and neurogenic bowel hospitalized for abdominal pain, dehydration, and more than 20 episodes of diarrhea per day. A stool sample test result was positive for Clostridioides difficile by polymerase chain reaction and an enzyme immunoassay result for C difficile toxin A and B was negative. How do you interpret these test results?

Intestinal Carriage of Carbapenemase-Producing Organisms: Current Status of Surveillance Methods.

Carbapenemases have become a significant mechanism for broad-spectrum ß-lactam resistance in Enterobacteriaceae and other Gram-negative bacteria such as Pseudomonas and Acinetobacter spp. Intestinal carriage of carbapenemase-producing organisms (CPOs) is an important source of transmission. Isolation of carriers is one strategy that can be used to limit the spread of these bacteria. In this review, we critically examine the clinical performance, advantages, and disadvantages of methods available for the detection of intestinal carriage of CPOs. Culture-based methods (Centers for Disease Control and Prevention [CDC] protocols, chromogenic media, specialized agars, and double-disk synergy tests) for detecting carriage of CPOs are convenient due to their ready availability and low cost, but their limited sensitivity and long turnaround time may not always be optimal for infection control practices. Contemporary nucleic acid amplification techniques (NAATs) such as real-time PCR, hybridization assays, loop-mediated isothermal amplification (LAMP), or a combined culture and NAAT approach may provide fast results and/or added sensitivity and specificity compared with culture-based methods. Infection control practitioners and clinical microbiologists should be aware of the strengths and limitations of available methods to determine the most suitable approach for their medical facility to fit their infection control needs.