WGS of a cluster of MDR Shigella sonnei utilizing Oxford Nanopore R10.4.1 long-read sequencing.

OBJECTIVES: To utilize long-read nanopore sequencing (R10.4.1 flowcells) for WGS of a cluster of MDR Shigella sonnei, specifically characterizing genetic predictors of antimicrobial resistance (AMR). METHODS: WGS was performed on S. sonnei isolates identified from stool and blood between September 2021 and October 2022. Bacterial DNA from clinical isolates was extracted on the MagNA Pure 24 and sequenced on the GridION utilizing R10.4.1 flowcells. Phenotypic antimicrobial susceptibility testing was interpreted based on CLSI breakpoints. Sequencing data were processed with BugSeq, and AMR was assessed with BugSplit and ResFinder. RESULTS: Fifty-six isolates were sequenced, including 53 related to the cluster of cases. All cluster isolates were identified as S. sonnei by sequencing, with global genotype 3.6.1.1.2 (CipR.MSM5), MLST 152 and PopPUNK cluster 3. Core genome MLST (cgMLST, examining 2513 loci) and reference-based MLST (refMLST, examining 4091 loci) both confirmed the clonality of the isolates. Cluster isolates were resistant to ampicillin (blaTEM-1), trimethoprim/sulfamethoxazole (dfA1, dfrA17; sul1, sul2), azithromycin (ermB, mphA) and ciprofloxacin (gyrA S83L, gyrA D87G, parC S80I). No genomic predictors of resistance to carbapenems were identified. CONCLUSIONS: WGS with R10.4.1 enabled rapid sequencing and identification of an MDR S. sonnei community cluster. Genetic predictors of AMR were concordant with phenotypic antimicrobial susceptibility testing.

Hepatitis B virus genotype surveillance in Canadian blood donors and a referred patient population, 2016-2021.

BACKGROUND AND OBJECTIVES: Hepatitis B virus (HBV) genotypes (A-H) have a distinct geographic distribution and are highly associated with the country of birth. Canada has experienced increased immigration over the past decade, primarily from regions where HBV is endemic. This study investigated the proportions and trends of HBV genotypes within blood donor and clinical populations of Canada over the period 2016-2021. MATERIALS AND METHODS: Study samples involved two cohorts: (1) Canadian blood donors (n = 246) deferred from donation due to HBV test positivity and (2) chronic HBV patients from across Canada (clinically referred population, n = 3539). Plasma or serum was extracted, and the surface antigen and/or polymerase-coding region was amplified and sequenced to determine genotype by phylogenetic analysis. RESULTS: Six (A-E, G) and eight (A-H) HBV genotypes were detected among deferred blood donors and the clinically referred population, respectively. Differences in HBV genotype proportions between the two cohorts were observed across Canada. Males comprised most of the referred population among genotypes A-E (p < 0.0001), except for genotypes B and C. The median age was younger among blood donors (36 years [range 17-72]) compared with the referred population (41 years [range 0-99]). Distinct trends of increasing (E, referred; B, blood donor) and decreasing genotype prevalence were observed over the study period. CONCLUSION: HBV genotypes in Canada are highly diverse, suggesting a large immigrant population. Observed trends in genotype prevalence and proportional differences among cohorts imply shifts among the HBV-infected population of Canada, which warrants continued surveillance.

People With Human Immunodeficiency Virus Receiving Suppressive Antiretroviral Therapy Show Typical Antibody Durability After Dual Coronavirus Disease 2019 Vaccination and Strong Third Dose Responses.

BACKGROUND: Longer-term humoral responses to 2-dose coronavirus disease 2019 (COVID-19) vaccines remain incompletely characterized in people living with human immunodeficiency virus (HIV) (PLWH), as do initial responses to a third dose. METHODS: We measured antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, angiotensin-converting enzyme 2 (ACE2) displacement, and viral neutralization against wild-type and Omicron strains up to 6 months after 2-dose vaccination, and 1 month after the third dose, in 99 PLWH receiving suppressive antiretroviral therapy and 152 controls. RESULTS: Although humoral responses naturally decline after 2-dose vaccination, we found no evidence of lower antibody concentrations or faster rates of antibody decline in PLWH compared with controls after accounting for sociodemographic, health, and vaccine-related factors. We also found no evidence of poorer viral neutralization in PLWH after 2 doses, nor evidence that a low nadir CD4+ T-cell count compromised responses. Post-third-dose humoral responses substantially exceeded post-second-dose levels, though Omicron-specific responses were consistently weaker than responses against wild-type virus. Nevertheless, post-third-dose responses in PLWH were comparable to or higher than controls. An mRNA-1273 third dose was the strongest consistent correlate of higher post-third-dose responses. CONCLUSION: PLWH receiving suppressive antiretroviral therapy mount strong antibody responses after 2- and 3-dose COVID-19 vaccination. Results underscore the immune benefits of third doses in light of Omicron.

Older Adults Mount Less Durable Humoral Responses to Two Doses of COVID-19 mRNA Vaccine but Strong Initial Responses to a Third Dose.

BACKGROUND: Third coronavirus disease 2019 (COVID-19) vaccine doses are broadly recommended, but immunogenicity data remain limited, particularly in older adults. METHODS: We measured circulating antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, ACE2 displacement, and virus neutralization against ancestral and omicron (BA.1) strains from prevaccine up to 1 month following the third dose, in 151 adults aged 24-98 years who received COVID-19 mRNA vaccines. RESULTS: Following 2 vaccine doses, humoral immunity was weaker, less functional, and less durable in older adults, where a higher number of chronic health conditions was a key correlate of weaker responses and poorer durability. One month after the third dose, antibody concentrations and function exceeded post-second-dose levels, and responses in older adults were comparable in magnitude to those in younger adults at this time. Humoral responses against omicron were universally weaker than against the ancestral strain after both the second and third doses. Nevertheless, after 3 doses, anti-omicron responses in older adults reached equivalence to those in younger adults. One month after 3 vaccine doses, the number of chronic health conditions, but not age, was the strongest consistent correlate of weaker humoral responses. CONCLUSIONS: Results underscore the immune benefits of third COVID-19 vaccine doses, particularly in older adults.

Clinical and Infection Prevention Applications of Severe Acute Respiratory Syndrome Coronavirus 2 Genotyping: An Infectious Diseases Society of America/American Society for Microbiology Consensus Review Document.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into a world of maturing pathogen genomics, with >2 million genomes sequenced at this writing. The rise of more transmissible variants of concern that affect vaccine and therapeutic effectiveness has led to widespread interest in SARS-CoV-2 evolution. Clinicians are also eager to take advantage of the information provided by SARS-CoV-2 genotyping beyond surveillance purposes. Here, we review the potential role of SARS-CoV-2 genotyping in clinical care. The review covers clinical use cases for SARS-CoV-2 genotyping, methods of SARS-CoV-2 genotyping, assay validation and regulatory requirements, clinical reporting for laboratories, and emerging issues in clinical SARS-CoV-2 sequencing. While clinical uses of SARS-CoV-2 genotyping are currently limited, rapid technological change along with a growing ability to interpret variants in real time foretell a growing role for SARS-CoV-2 genotyping in clinical care as continuing data emerge on vaccine and therapeutic efficacy.

Clinical and Infection Prevention Applications of Severe Acute Respiratory Syndrome Coronavirus 2 Genotyping: an Infectious Diseases Society of America/American Society for Microbiology Consensus Review Document.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into a world of maturing pathogen genomics, with more than 2 million genomes sequenced at the time of writing. The rise of more transmissible variants of concern that impact vaccine and therapeutic effectiveness has led to widespread interest in SARS-CoV-2 evolution. Clinicians are also eager to take advantage of the information provided by SARS-CoV-2 genotyping beyond surveillance purposes. Here, we review the potential role of SARS-CoV-2 genotyping in clinical care. The review covers clinical use cases for SARS-CoV-2 genotyping, methods of SARS-CoV-2 genotyping, assay validation and regulatory requirements, and clinical reporting for laboratories, as well as emerging issues in clinical SARS-CoV-2 sequencing. While clinical uses of SARS-CoV-2 genotyping are currently limited, rapid technological change along with a growing ability to interpret variants in real time foretells a growing role for SARS-CoV-2 genotyping in clinical care as continuing data emerge on vaccine and therapeutic efficacy.

Rapid Detection of SARS-CoV-2 Variants of Concern, Including B.1.1.28/P.1, British Columbia, Canada.

To screen all severe acute respiratory syndrome coronavirus 2-positive samples in Vancouver, British Columbia, Canada, and determine whether they represented variants of concern, we implemented a real-time reverse transcription PCR-based algorithm. We rapidly identified 77 samples with variants: 57 with B.1.1.7, 7 with B.1.351, and an epidemiologic cluster of 13 with B.1.1.28/P.1.

Repeat virological and serological profiles in hospitalized patients initially tested by nasopharyngeal RT-PCR for SARS-CoV-2.

Real-time polymerase chain reaction (PCR) for SARS-CoV-2 is the mainstay of COVID-19 diagnosis, yet there are conflicting reports on its diagnostic performance. Wide ranges of false-negative PCR tests have been reported depending on clinical presentation, the timing of testing, specimens tested, testing method, and reference standard used. We aimed to estimate the frequency of discordance between initial nasopharyngeal (NP) PCR and repeat NP sampling PCR and serology in acutely ill patients admitted to the hospital. Panel diagnosis of COVID-19 infection is further utilized in discordance analysis. Included in the study were 160 patients initially tested by NP PCR with repeat NP sampling PCR and/or serology performed. The percent agreement between initial and repeat PCR was 96.7%, while the percent agreement between initial PCR and serology was 98.9%. There were 5 (3.1%) cases with discordance on repeat testing. After discordance analysis, 2 (1.4%) true cases tested negative on initial PCR. Using available diagnostic methods, discordance on repeat NP sampling PCR and/or serology is a rare occurrence.

Practical challenges to the clinical implementation of saliva for SARS-CoV-2 detection.

Due to global shortages of flocked nasopharyngeal swabs and appropriate viral transport media during the COVID-19 pandemic, alternate diagnostic specimens for SARS-CoV-2 detection are sought. The accuracy and feasibility of saliva samples collected and transported without specialized collection devices or media were evaluated. Saliva demonstrated good concordance with paired nasopharyngeal swabs for SARS-CoV-2 detection in 67/74 cases (90.5%), though barriers to saliva collection were observed in long-term care residents and outbreak settings. SARS-CoV-2 RNA was stable in human saliva at room temperature for up to 48 h after initial specimen collection, informing appropriate transport time and conditions.

Suboptimal Biological Sampling as a Probable Cause of False-Negative COVID-19 Diagnostic Test Results.

False-negative severe acute respiratory syndrome coronavirus 2 test results can negatively impact the clinical and public health response to coronavirus disease 2019 (COVID-19). We used droplet digital polymerase chain reaction (ddPCR) to demonstrate that human DNA levels, a stable molecular marker of sampling quality, were significantly lower in samples from 40 confirmed or suspected COVID-19 cases that yielded negative diagnostic test results (ie, suspected false-negative test results) compared with a representative pool of 87 specimens submitted for COVID-19 testing. Our results support suboptimal biological sampling as a contributor to false-negative COVID-19 test results and underscore the importance of proper training and technique in the collection of nasopharyngeal specimens.

Phenotypic and Genotypic Correlates of Penicillin Susceptibility in Nontoxigenic Corynebacterium diphtheriae, British Columbia, Canada, 2015-2018.

In 2015, the Clinical and Laboratory Standards Institute (CLSI) updated its breakpoints for penicillin susceptibility in Corynebacterium species from <1 mg/L to <0.12 mg/L. We assessed the effect of this change on C. diphtheriae susceptibility reported at an inner city, tertiary care center in Vancouver, British Columbia, Canada, during 2015-2018 and performed whole-genome sequencing to investigate phenotypic and genotypic resistance to penicillin. We identified 44/45 isolates that were intermediately susceptible to penicillin by the 2015 breakpoint, despite meeting previous CLSI criteria for susceptibility. Sequencing did not reveal ß-lactam resistance genes. Multilocus sequence typing revealed a notable predominance of sequence type 76. Overall, we saw no evidence of penicillin nonsusceptibility at the phenotypic or genotypic level in C. diphtheriae isolates from our institution. The 2015 CLSI breakpoint change could cause misclassification of penicillin susceptibility in C. diphtheriae isolates, potentially leading to suboptimal antimicrobial treatment selection.

Whole-Genome Sequencing of Corynebacterium diphtheriae Isolates Recovered from an Inner-City Population Demonstrates the Predominance of a Single Molecular Strain.

In some parts of the world, Corynebacterium diphtheriae has reemerged as a pathogen, especially as a cause of infections among impoverished and marginalized populations. We performed whole-genome sequencing (WGS) on all cutaneous C. diphtheriae isolates (n = 56) from Vancouver's inner-city population over a 3-year time period (2015 to 2018). All isolates with complete genome assembly were toxin negative, contained a common set of 22 virulence factors, and shared a highly conserved accessory genome. One of our isolates harbored a novel plasmid conferring macrolide and lincosamide resistance. Fifty-two out of 56 isolates were multilocus sequence type 76, and single nucleotide variants (SNV) and core-genome multilocus sequence typing (cgMLST) analysis demonstrated tight clustering of our isolates relative to all publicly available C. diphtheriae genomes. All sequence type 76 (ST76) study isolates were within a median of 22 SNVs and 13 cgMLST alleles of each other, while NCBI genomes were within a median of 17,436 SNVs and 1,552 cgMLST alleles of each other (both P < 2.2 × 10-16). A single strain of C. diphtheriae appears to be causing cutaneous infections in the low-income population of Vancouver. Further research is needed to elucidate transmission networks in our study population and standardize C. diphtheriae epidemiological typing when whole genomes are sequenced.

Relevance of reviewing endpoint analysis for negative results on the Xpert Xpress Flu/RSV.

After the implementation of the Xpert Xpress Flu/respiratory syncytial virus (RSV) assay for rapid respiratory molecular testing, we investigated the significance of reported endpoint values for influenza A, influenza B, and RSV). This study prospectively analyzed nasopharyngeal swabs submitted to our virology laboratory in the 2018/19 influenza season. Initial testing was performed on the Xpress Flu/RSV assay. Samples were further tested on a laboratory-developed multiplex polymerase chain reaction (laboratory-developed multiplex respiratory test [LDT]) if the sample was reported as negative by the Xpress Flu/RSV but had an elevated endpoint value ≥5 for any respiratory virus target. There were 1040 negative results on the Xpress Flu/RSV; thirty-one had at least one endpoint value ≥5 [influenza A (25), influenza B (1), RSV (2), influenza A/RSV (1), and influenza A/B/RSV (2)]. Five samples (5/31, 16.1%) were positive on the LDT for influenza A or RSV. In contrast, the positivity rate on the LDT for negative Xpress Flu/RSV samples with endpoint values less than 5 was 0.35% (P < .0001). A threshold for endpoint values could not reliably be established to differentiate a potential influenza A positive result from a negative result on the LDT. Routine evaluation ofendpoint values should be a consideration for laboratories implementing Xpress Flu/RSV, in addition to supplementary respiratory virus testing for clinically relevant situations.

Clinical heterogeneity of patients with stool samples testing PCR+/Tox- from a two-step Clostridium difficile diagnostic algorithm.

The clinical significance of indeterminate (PCR+/Tox-) results for patients tested with a two-step algorithm for Clostridium difficile infection (CDI) is uncertain. We aimed to evaluate the clinical presentation and 8-week outcomes of patients with indeterminate test results. Patients with stool samples testing positive by PCR and negative by toxin A/B immunoassay between February 1, 2017, and April 30, 2018, were assessed by antimicrobial stewardship program (ASP) clinicians and classified as colonized or infected. Retrospective chart review was performed to obtain outcomes occurring within 8 weeks of testing, including recurrent C. difficile diarrhea, subsequent treatment for CDI, follow-up C. difficile testing, all-cause mortality, and CDI-related complications. In total, 110 PCR+/Tox- patients were evaluated. ASP classified 54% of patients as infected and 46% as colonized. Patients assessed and classified as colonized did not have increased adverse outcomes by 8 weeks compared to those assessed as infected, despite not receiving treatment for CDI. We conclude that PCR+/Tox- patients are heterogeneous with respect to clinical presentation. Negative toxin A/B immunoassay in a two-step algorithm should not be interpreted in isolation to distinguish colonization from infection as many PCR+/Tox- results may be clinically significant for CDI.

Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory.

Sanger sequencing or DNA hybridization have been the primary modalities for hepatitis B (HBV) resistance testing and genotyping; however, there are limitations, such as low sensitivity and the inability to detect novel mutations. Next-generation sequencing (NGS) for HBV can overcome these limitations, but there is limited guidance for clinical microbiology laboratories to validate this novel technology. In this study, we describe an approach to implementing deep pyrosequencing for HBV resistance testing and genotyping in a clinical virology laboratory. A nested PCR targeting the pol region of HBV (codons 143 to 281) was developed, and the PCR product was sequenced by the 454 Junior (Roche). Interpretation was performed by ABL TherapyEdge based on European Association for the Study of the Liver (EASL) guidelines. Previously characterized HBV samples by INNO-LiPA (LiPA) were compared to NGS with discordant results arbitrated by Sanger sequencing. Genotyping of 105 distinct samples revealed a concordance of 95.2% (100/105), with Sanger sequencing confirming the NGS result. Resistance testing by NGS was concordant with LiPA in 85% (68/80) of previously characterized samples. Additional mutations were found in 8 samples, which related to the identification of low-level mutant subpopulations present at <10% (6/8). To balance the costs of testing for the validation study, reproducibility of the NGS was investigated through an analysis of sequence variants at loci not associated with resistance in a single patient sample. Our validation approach attempts to balance costs with efficient data acquisition.

Hospital-associated transmission of Brucella melitensis outside the laboratory.

Brucella melitensis was identified in an aspirate obtained from a patient's hip joint during a procedure at a hospital in Canada. We conducted an investigation into possible exposures among hospital workers; 1 worker who assisted with the procedure tested positive for B. melitensis. Aerosol-generating procedures performed outside the laboratory may facilitate transmission of this bacterium.