Investigation of a cluster of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in a hospital administration building.

OBJECTIVE: To investigate a cluster of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in employees working on 1 floor of a hospital administration building. METHODS: Contact tracing was performed to identify potential exposures and all employees were tested for SARS-CoV-2. Whole-genome sequencing was performed to determine the relatedness of SARS-CoV-2 samples from infected personnel and from control cases in the healthcare system with coronavirus disease 2019 (COVID-19) during the same period. Carbon dioxide levels were measured during a workday to assess adequacy of ventilation; readings >800 parts per million (ppm) were considered an indication of suboptimal ventilation. To assess the potential for airborne transmission, DNA-barcoded aerosols were released, and real-time polymerase chain reaction was used to quantify particles recovered from air samples in multiple locations. RESULTS: Between December 22, 2020, and January 8, 2021, 17 coworkers tested positive for SARS-CoV-2, including 13 symptomatic and 4 asymptomatic individuals. Of the 5 cluster SARS-CoV-2 samples sequenced, 3 were genetically related, but these employees denied higher-risk contacts with one another. None of the sequences from the cluster were genetically related to the 17 control sequences of SARS-CoV-2. Carbon dioxide levels increased during a workday but never exceeded 800 ppm. DNA-barcoded aerosol particles were dispersed from the sites of release to locations throughout the floor; 20% of air samples had >1 log10 particles. CONCLUSIONS: In a hospital administration building outbreak, sequencing of SARS-CoV-2 confirmed transmission among coworkers. Transmission occurred despite the absence of higher-risk exposures and in a setting with adequate ventilation based on monitoring of carbon dioxide levels.

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.

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.

Long-term in vitro culture of Plasmodium vivax isolates from Madagascar maintained in Saimiri boliviensis blood.

BACKGROUND: Plasmodium vivax is the most prevalent human malaria parasite and is likely to increase proportionally as malaria control efforts more rapidly impact the prevalence of Plasmodium falciparum. Despite the prominence of P. vivax as a major human pathogen, vivax malaria qualifies as a neglected and under-studied tropical disease. Significant challenges bringing P. vivax into the laboratory, particularly the capacity for long-term propagation of well-characterized strains, have limited the study of this parasite's red blood cell (RBC) invasion mechanism, blood-stage development, gene expression, and genetic manipulation. METHODS AND RESULTS: Patient isolates of P. vivax have been collected and cryopreserved in the rural community of Ampasimpotsy, located in the Tsiroanomandidy Health District of Madagascar. Periodic, monthly overland transport of these cryopreserved isolates to the country's National Malaria Control Programme laboratory in Antananarivo preceded onward sample transfer to laboratories at Case Western Reserve University, USA. There, the P. vivax isolates have been cultured through propagation in the RBCs of Saimiri boliviensis. For the four patient isolates studied to-date, the median time interval between sample collection and in vitro culture has been 454 days (range 166-961 days). The median time in culture, continually documented by light microscopy, has been 159 days; isolate AMP2014.01 was continuously propagated for 233 days. Further studies show that the P. vivax parasites propagated in Saimiri RBCs retain their ability to invade human RBCs, and can be cryopreserved, thawed and successfully returned to productive in vitro culture. CONCLUSIONS/SIGNIFICANCE: Long-term culture of P. vivax is possible in the RBCs of Saimiri boliviensis. These studies provide an alternative to propagation of P. vivax in live animals that are becoming more restricted. In vitro culture of P. vivax in Saimiri RBCs provides an opening to stabilize patient isolates, which would serve as precious resources to apply new strategies for investigating the molecular and cellular biology of this important malaria parasite.

Pharmacologic stimulation of cytochrome P450 46A1 and cerebral cholesterol turnover in mice.

Cytochrome P450 46A1 (CYP46A1) is a brain-specific cholesterol 24-hydroxylase responsible for the majority of cholesterol elimination from the brain. Genetically increased CYP46A1 expression in mice leads to improved cognition and decreases manifestations of Alzheimer disease. We found that four pharmaceuticals (efavirenz (EFV), acetaminophen, mirtazapine, and galantamine) prescribed for indications unrelated to cholesterol maintenance increased CYP46A1 activity in vitro. We then evaluated the anti-HIV medication EFV for the mode of interaction with CYP46A1 and the effect on mice. We propose a model for CYP46A1 activation by EFV and show that EFV enhanced CYP46A1 activity and cerebral cholesterol turnover in animals with no effect on the levels of brain cholesterol. The doses of EFV administered to mice and required for the stimulation of their cerebral cholesterol turnover are a hundred times lower than those prescribed to HIV patients. At such small doses, EFV may be devoid of adverse effects elicited by high drug concentrations. CYP46A1 could be a novel therapeutic target and a tool to further investigate the physiological and medical significance of cerebral cholesterol turnover.

Inhibition and stimulation of activity of purified recombinant CYP11A1 by therapeutic agents.

In vertebrates, the biosynthesis of steroid hormones is initiated by cytochrome P450 CYP11A1 which converts cholesterol to pregnenolone. We investigated whether some of the experimental and FDA-approved therapeutic agents alter the activity of CYP11A1 in the reconstituted system in vitro. We found that under the experimental conditions used and when phospholipids are included, ketoconazole, posaconazole, carbenoxolone, and selegiline inhibit CYP11A1-mediated production of pregnenolone by at least 67%. Conversely, pemirolast, clobenpropit, desogestrel, dexmedetomidine, and tizanidine stimulate the enzyme activity by up to 70%. We then evaluated the identified inhibitors and activators for spectral binding to CYP11A1 and their effect on enzyme activity in the absence of phospholipids. The data obtained provide insight into how different drugs interact with CYP11A1 and demonstrate that P450 association with the lipid bilayer determines, in many cases, a drug's effect on enzyme activity.

In silico and intuitive predictions of CYP46A1 inhibition by marketed drugs with subsequent enzyme crystallization in complex with fluvoxamine.

Cytochrome P450 46A1 (cholesterol 24-hydroxylase) is an important brain enzyme that may be inhibited by structurally distinct pharmaceutical agents both in vitro and in vivo. To identify additional inhibitors of CYP46A1 among U.S. Food and Drug Administration-approved therapeutic agents, we used in silico and intuitive predictions and evaluated some of the predicted binders in the enzyme and spectral binding assays. We tested a total of 298 marketed drugs for the inhibition of CYP46A1-mediated cholesterol hydroxylation in vitro and found that 13 of them reduce CYP46A1 activity by >50%. Of these 13 inhibitors, 7 elicited a spectral response in CYP46A1 with apparent spectral K(d) values in a low micromolar range. One of the identified tight binders, the widely used antidepressant fluvoxamine, was cocrystallized with CYP46A1. The structure of this complex was determined at a 2.5 Å resolution and revealed the details of drug binding to the CYP46A1 active site. The NH(2)-containing arm of the Y-shaped fluvoxamine coordinates the CYP46A1 heme iron, whereas the methoxy-containing arm points away from the heme group and has multiple hydrophobic interactions with aliphatic amino acid residues. The CF(3)-phenyl ring faces the entrance to the substrate access channel and has contacts with the aromatic side chains. The crystal structure suggests that only certain drug conformers can enter the P450 substrate access channel and reach the active site. Once inside the active site, the conformer probably further adjusts its configuration and elicits the movement of the protein side chains.