Verification, Analytical Sensitivity, Cost-effectiveness, and Comparison of 4 Candida auris Screening Methods.

In this verification study, we compare and contrast the performance characteristics of chromogenic agar culture, direct polymerase chain reaction (PCR), and broth enrichment followed by culture or PCR for the detection of Candida auris colonization. We find that culture and PCR both offer excellent performance, with broth enrichment offering little performance advantage given its cost.

Genomic and transcriptomic characterization of delta SARS-CoV-2 infection in free-ranging white-tailed deer (Odocoileus virginianus).

White-tailed deer (WTD) are susceptible to SARS-CoV-2 and represent an important species for surveillance. Samples from WTD (n = 258) collected in November 2021 from Québec, Canada were analyzed for SARS-CoV-2 RNA. We employed viral genomics and host transcriptomics to further characterize infection and investigate host response. We detected Delta SARS-CoV-2 (B.1.617.2) in WTD from the Estrie region; sequences clustered with human sequences from October 2021 from Vermont, USA, which borders this region. Mutations in the S-gene and a deletion in ORF8 were detected. Host expression patterns in SARS-CoV-2 infected WTD were associated with the innate immune response, including signaling pathways related to anti-viral, pro- and anti-inflammatory signaling, and host damage. We found limited correlation between genes associated with innate immune response from human and WTD nasal samples, suggesting differences in responses to SARS-CoV-2 infection. Our findings provide preliminary insights into host response to SARS-CoV-2 infection in naturally infected WTD.

Use of whole genome sequencing to identify low-frequency mutations in SARS-CoV-2 patients treated with remdesivir.

BACKGROUND: Remdesivir (RDV) has been shown to reduce hospitalization and mortality in COVID-19 patients. Resistance mutations caused by RDV are rare and have been predominantly reported in patients who are on prolonged therapy and immunocompromised. We investigate the effects of RDV treatment on intra-host SARS-CoV-2 diversity and low-frequency mutations in moderately ill hospitalized COVID-19 patients and compare them to patients without RDV treatment. METHODS: From March 2020 to April 2022, sequential collections of nasopharyngeal and mid-turbinate swabs were obtained from 14 patients with and 30 patients without RDV treatment. Demographic and clinical data on all patients were reviewed. A total of 109 samples were sequenced and mutation analyses were performed. RESULTS: Previously reported drug resistant mutations in nsp12 were not identified during short courses of RDV therapy. In genes encoding and surrounding the replication complex (nsp6-nsp14), low-frequency minority variants were detected in 7/14 (50%) and 18/30 (60%) patients with and without RDV treatment, respectively. We did not detect significant differences in within-host diversity and positive selection between the RDV-treated and untreated groups. CONCLUSIONS: Minimal intra-host variability and stochastic low-frequency variants detected in moderately ill patients suggests little selective pressure in patients receiving short courses of RDV. The barrier to RDV resistance is high in patients with moderate disease. Patients undergoing short regimens of RDV therapy should continue to be monitored.

Lachnoanaerobaculum orale bacteremia in a patient with acute myeloid leukemia and stomatitis: An emerging pathogen.

Background: Lachnoanaerobaculum orale is a newly described, obligately anaerobic gram-positive bacillus. The first report of invasive disease caused by L. orale was described in a patient with acute lymphocytic leukemia following systematic chemotherapy. Here we describe another case of L. orale bacteremia in a patient with a hematologic malignancy following chemotherapy-induced neutropenia. Methods: We present a case of a 46-year-old woman with a recent diagnosis of AML who presented to Sunnybrook Health Sciences Center with febrile neutropenia following induction chemotherapy with daunorubicin-cytarabine (3 +7 regimen) with Gemtuzumab and Ozogamycin. Despite being on intravenous pipercillin-tazobactam she remained febrile. Following our clinical assessment and investigations, potential sources of infection included a swollen digit and severe mucositis. Results: One blood culture from admission grew Lachnoanaerobaculum orale in the anaerobic bottle, identified by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF). The isolate also underwent whole-genome sequencing using methods that have been described previously. Results demonstrated the organism was susceptible to cefoxitin, clindamycin, meropenem, metronidazole, penicillin, and piperacillin-tazobactam. We concluded that the source of this patient's bloodstream infection to be chemotherapy-induced stomatitis. Conclusion: With the increasing use of intensive immunosuppressive regimens and hematopoietic stem cell transplantation for patients with hematologic malignancies, there has been an increase in the incidence and detection of bloodstream infections due to anaerobic organisms. This is only the second case report of L. orale bacteremia, highlighting its emerging role as an opportunistic pathogen in immunocompromised patients.

Divergent SARS-CoV-2 variant emerges in white-tailed deer with deer-to-human transmission.

Wildlife reservoirs of broad-host-range viruses have the potential to enable evolution of viral variants that can emerge to infect humans. In North America, there is phylogenomic evidence of continual transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (Odocoileus virginianus) through unknown means, but no evidence of transmission from deer to humans. We carried out an observational surveillance study in Ontario, Canada during November and December 2021 (n = 300 deer) and identified a highly divergent lineage of SARS-CoV-2 in white-tailed deer (B.1.641). This lineage is one of the most divergent SARS-CoV-2 lineages identified so far, with 76 mutations (including 37 previously associated with non-human mammalian hosts). From a set of five complete and two partial deer-derived viral genomes we applied phylogenomic, recombination, selection and mutation spectrum analyses, which provided evidence for evolution and transmission in deer and a shared ancestry with mink-derived virus. Our analysis also revealed an epidemiologically linked human infection. Taken together, our findings provide evidence for sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, Beta, Delta, and Omicron variants.

Background: SARS-CoV-2 Omicron variant of concern (VOC) has evolved multiple mutations within the spike protein, raising concerns of increased antibody evasion. In this study, we assessed the neutralization potential of COVID-19 convalescent sera and sera from vaccinated individuals against ancestral SARS-CoV-2 and VOCs. Methods: The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2 and Beta, Delta, and Omicron VOCs was assessed using a micro-neutralization assay. Findings: Convalescent sera from unvaccinated individuals infected by the ancestral virus demonstrated reduced neutralization against Beta and Omicron VOCs. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of the Omicron variant relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Infection alone, either with ancestral SARS-CoV-2 or the Delta variant, was not sufficient to induce high neutralizing antibody titers against Omicron. Conclusions: In summary, we demonstrate that convalescent and vaccinated sera display varying levels of SARS-CoV-2 VOC neutralization. Data from this study will inform booster vaccination strategies against SARS-CoV-2 VOCs. Funding: This research was funded by the Canadian Institutes of Health Research (CIHR). VIDO receives operational funding from the Government of Saskatchewan through Innovation Saskatchewan and the Ministry of Agriculture and from the Canada Foundation for Innovation through the Major Science Initiatives for its CL3 facility.

Surface and Air Contamination With Severe Acute Respiratory Syndrome Coronavirus 2 From Hospitalized Coronavirus Disease 2019 Patients in Toronto, Canada, March-May 2020.

BACKGROUND: We determined the burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in air and on surfaces in rooms of patients hospitalized with coronavirus disease 2019 (COVID-19) and investigated patient characteristics associated with SARS-CoV-2 environmental contamination. METHODS: Nasopharyngeal swabs, surface, and air samples were collected from the rooms of 78 inpatients with COVID-19 at 6 acute care hospitals in Toronto from March to May 2020. Samples were tested for SARS-CoV-2 ribonucleic acid (RNA), cultured to determine potential infectivity, and whole viral genomes were sequenced. Association between patient factors and detection of SARS-CoV-2 RNA in surface samples were investigated. RESULTS: Severe acute respiratory syndrome coronavirus 2 RNA was detected from surfaces (125 of 474 samples; 42 of 78 patients) and air (3 of 146 samples; 3 of 45 patients); 17% (6 of 36) of surface samples from 3 patients yielded viable virus. Viral sequences from nasopharyngeal and surface samples clustered by patient. Multivariable analysis indicated hypoxia at admission, polymerase chain reaction-positive nasopharyngeal swab (cycle threshold of ≤30) on or after surface sampling date, higher Charlson comorbidity score, and shorter time from onset of illness to sampling date were significantly associated with detection of SARS-CoV-2 RNA in surface samples. CONCLUSIONS: The infrequent recovery of infectious SARS-CoV-2 virus from the environment suggests that the risk to healthcare workers from air and near-patient surfaces in acute care hospital wards is likely limited.

Whole genome sequence of Corynebacterium kroppenstedtii isolated from a case of recurrent granulomatous mastitis.

We describe the case of a 33-year-old woman with recurrent granulomatous mastitis associated with Corynebacterium kroppenstedtii. This organism has been increasingly associated with granulomatous mastitis, specifically the cystic neutrophilic histopathologic variant, although currently there is a paucity both of reported cases and genomic sequence data. We highlight the challenges in the diagnosis and treatment of this entity, in particular focusing on the various methods of microbiologic identification, including MALDI-TOF, 16 s rRNA PCR and whole-genome sequencing.

A Comparison of Whole Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random Hexamers, and Bait Capture.

Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.

Isolation, Sequence, Infectivity, and Replication Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2.

Since its emergence in Wuhan, China, in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected ≈6 million persons worldwide. As SARS-CoV-2 spreads across the planet, we explored the range of human cells that can be infected by this virus. We isolated SARS-CoV-2 from 2 infected patients in Toronto, Canada; determined the genomic sequences; and identified single-nucleotide changes in representative populations of our virus stocks. We also tested a wide range of human immune cells for productive infection with SARS-CoV-2. We confirm that human primary peripheral blood mononuclear cells are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single-nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype by using in vitro and in vivo infection models.