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.

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.

Analytical Validation of HEARTBiT: A Blood-Based Multiplex Gene Expression Profiling Assay for Exclusionary Diagnosis of Acute Cellular Rejection in Heart Transplant Patients.

BACKGROUND: HEARTBiT is a whole blood-based gene profiling assay using the nucleic acid counting NanoString technology for the exclusionary diagnosis of acute cellular rejection in heart transplant patients. The HEARTBiT score measures the risk of acute cellular rejection in the first year following heart transplant, distinguishing patients with stable grafts from those at risk for acute cellular rejection. Here, we provide the analytical performance characteristics of the HEARTBiT assay and the results on pilot clinical validation. METHODS: We used purified RNA collected from PAXgene blood samples to evaluate the characteristics of a 12-gene panel HEARTBiT assay, for its linearity range, quantitative bias, precision, and reproducibility. These parameters were estimated either from serial dilutions of individual samples or from repeated runs on pooled samples. RESULTS: We found that all 12 genes showed linear behavior within the recommended assay input range of 125 ng to 500 ng of purified RNA, with most genes showing 3% or lower quantitative bias and around 5% coefficient of variation. Total variation resulting from unique operators, reagent lots, and runs was less than 0.02 units standard deviation (SD). The performance of the analytically validated assay (AUC = 0.75) was equivalent to what we observed in the signature development dataset. CONCLUSION: The analytical performance of the assay within the specification input range demonstrated reliable quantification of the HEARTBiT score within 0.02 SD units, measured on a 0 to 1 unit scale. This assay may therefore be of high utility in clinical validation of HEARTBiT in future biomarker observational trials.

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.

Transitioning cytomegalovirus viral load testing from a laboratory developed test to the cobas® CMV quantitative nucleic acid assay.

Commutability between human cytomegalovirus (CMV) viral load assays (VLA) is poor, despite the development of a WHO CMV International Standard (CMV IS). We evaluated a new CMV VLA, cobas® CMV, as compared to our current laboratory developed CMV VLA (LDT), for clinical use. Both the LDT and cobas® CMV were run in parallel for 109 patient samples. In addition, 104 replicates, over 8 dilutions, of the CMV IS were tested. Conversion factors and correlation between the two assays were calculated. The correlation coefficient between the LDT and cobas® CMV was 0.91 for patient samples. The Bland-Altman graph displayed a systematic bias of +0.31 log10 for the cobas® CMV as compared to the LDT. The bias was greater for lower CMV viral loads. This increase in CMV viral loads was not seen with testing of the CMV IS dilutions by both the LDT and cobas® CMV. CMV VLA inter-assay commutability continues to be an issue when switching CMV testing platforms and requires communication between the laboratory and clinicians during the transition period to prevent misinterpretation of results.

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.

Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia.

BACKGROUND: Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP. METHODS: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ-]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP. RESULTS: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%. CONCLUSION: PJ real-time PCR improved detection of PJ in immunocompromised patients.

HISTORIQUE: Le Pneumocystis jirovecii (PJ), un champignon pathogène, provoque une grave pneumonie à Pneumocystis interstitielle (PPC) chez les patients immunodéprimés. Les chercheurs ont validé un test de réaction en chaîne par polymérase (PCR) en temps réel pour détecter le PJ et ainsi améliorer le diagnostic de PPC. MÉTHODOLOGIE: Les chercheurs ont d'abord vérifié 40 prélèvements de liquide bronchoalvéolaire (LBA) entreposés (20 cas positifs connus au PJ [PJ+] et 20 cas négatifs connus au PJ [PJ−]) au moyen du test moléculaire. Ils ont ensuite analysé 92 prélèvements séquentiels de LBA au moyen d'un test par immunofluorescence (IFA), puis d'un test de PCR en temps réel du PJ. Ils ont résolu les résultats divergents au moyen d'un nouveau test par PCR en temps réel des prélèvements de LBA axée sur une autre cible. Ils ont comparé le résultat du test de PCR en temps réel du PJ à la référence absolue (l'IFA) et à une référence modifiée, dans laquelle un véritable cas positif désignait un prélèvement positif par deux méthodes sur trois chez un patient atteint d'une PPC présumée. RÉSULTATS: Quatre-vingt-dix prélèvements de LBA (68 %) sur 132 provenaient de patients immunodéprimés. Treize prélèvements de LBA (14 %) sur 92 étaient PJ+ d'après l'IFA. Dans la présente étude, 40 prélèvements de LBA étaient PJ+, y compris tous les prélèvements positifs à l'IFA (n=13), tous les prélèvements de LBA PJ+ aiguillés (n=20) et sept autres prélèvements de LBA négatifs à l'IFA, mais positifs selon la référence modifiée. Par rapport à l'IFA, la PCR en temps réel du PJ avait une sensibilité, une spécificité et des valeurs prédictives positive et négative de 100 %, 91 %, 65 % et 100 %, respectivement. Par rapport à la référence modifiée, la PCR en temps réel du PJ avait une sensibilité, une spécificité et des valeurs prédictives positive et négative de 100 %. CONCLUSION: La PCR en temps réel du PJ en améliore la détection chez les patients immunodéprimés.

Testing the limits of multiplex respiratory virus assays for SARS-CoV-2 at high cycle threshold values: Comparative performance of cobas 6800/8800 SARS-CoV-2 & Influenza A/B, Xpert Xpress SARS-CoV-2/Flu/RSV, and cobas Liat SARS-CoV-2 & Influenza A/B.

Background: Multiplex real-time RT-PCR assays for respiratory pathogens are valuable tools to optimize laboratory workflow and turnaround time. At a time when resurgence of influenza and respiratory syncytial virus (RSV) cases have been widely observed along with continued transmission of SARS-CoV-2, timely identification of all circulating respiratory viruses is crucial. This study evaluates the detection of low viral loads of SARS-CoV-2 by four multiplex molecular assays: Roche cobas 6800/8800 SARS-CoV-2 & Influenza A/B Test, Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV, cobas Liat SARS-CoV-2 & Influenza A/B, and a laboratory-developed test (LDT). Methods: Retrospective upper respiratory tract specimens positive for various respiratory viruses at a range of cycle threshold (Ct) values (18-40) were tested by four multiplex assays. Positive and negative percent agreement (PPA and NPA) with validated RT-PCR assays were calculated. Results: A total of 82 samples were assessed, with discordant results observed in a portion of the samples (10/82, 12.2%) where Ct values were >33. The majority of the discordant results (6/10, 60%) were false negatives. Overall, PPA was 100% (58/58) for cobas 6800, 97.4% (38/39) for GeneXpert, 100% (17/17) for Liat, and 90.5% (57/63) for the LDT. PPA for the LDT increased to 92.1% after manual review of amplification curves. Conclusions: Commercial multiplex respiratory virus assays have good performance for samples with medium to high viral loads (Ct values <33). Laboratories should consider appropriate test result review and confirmation protocols to optimize sensitivity, and may consider reporting samples with additional interpretive comments when low viral loads are detected.

Historique: Les dosages multiplex par RT-PCR en temps réel (amplification en chaîne par polymérase avec transcription inverse en temps réel) des agents pathogènes respiratoires sont des outils précieux pour optimiser le flux de travail et le temps de traitement en laboratoire. Alors qu'on observe une résurgence générale des cas d'influenza et du virus respiratoire syncytial (VRS) et une transmission continue du SRAS-CoV-2, il est crucial de détecter rapidement tous les virus respiratoires en circulation. Dans la présente étude, les chercheurs ont évalué la détection des faibles charges virales du SRAS-CoV-2 à l'aide de quatre dosages moléculaires multiplex : le test cobas 6800/8800 SRAS-CoV-2 et influenza A/B de Roche, le test Xpress SRAS-CoV-2/influenza/VRS de Cepheid Xpert, le test cobas SRAS-CoV-2 et influenza A/B de Liat et un test créé par le laboratoire (TCL). Méthodologie: Les chercheurs ont procédé au dépistage rétrospectif d'échantillons ayant obtenu un résultat positif à divers virus respiratoires à une série de valeurs de cycle seuil (Ct) (18­40) à l'aide de quatre dosages multiplex. Ils ont calculé le pourcentage de concordance positif (PCP) et négatif (PCN) avec les dosages par RT-PCR validés. Résultats: Au total, les chercheurs ont évalué 82 échantillons et observé des résultats discordants dans une partie des échantillons (dix sur 82, 12,2 %), pour lesquels les valeurs Ct étaient supérieures à 33. La majorité de ces résultats discordants (six sur dix, 60 %) étaient faussement négatifs. Dans l'ensemble, le PCP atteignait 100 % (58 sur 58) selon le test cobas 6800, 97,4 % (38 sur 39) selon le test GeneXpert, 100 % (17 sur 17) selon le test Liat et 90,5 % (57 sur 63) selon le TCL. Le PCP du TCL est passé à 92,1 % après l'examen manuel des courbes d'amplification. Conclusions: Les dosages multiplex commerciaux des virus respiratoires donnent un bon rendement pour les échantillons contenant une charge virale modérée à élevée (valeurs Ct inférieures à 33). Les laboratoires devraient envisager de procéder à une analyse des résultats du dépistage et à des protocoles de confirmation appropriés pour en optimiser la sensibilité et pourraient également envisager d'ajouter des commentaires interprétatifs aux résultats des échantillons lorsque la charge virale décelée est faible.

Stability of BK polyomavirus DNA in urine over time and analytical evaluation of an automated BKV quantitative nucleic acid test.

Introduction. BK polyomavirus (BKPyV) quantitative testing is an important screening tool post-transplantation, although interpretation can be challenging due to lack of standardization, assay heterogeneity and variability of BKPyV DNA over time (in urine).Methods. Remnant clinical EDTA plasma and urine samples were tested by the cobas BKV test and a validated laboratory-developed test (LDT). Accuracy [positive and negative percent agreement (PPA and NPA), Pearson's correlation, Bland-Altman analysis] and reproducibility were evaluated. To assess BKPyV DNA stability in urine, prospective urine samples were maintained at two different storage temperatures and tested in triplicate over 7 days.Results. Overall PPA was 95.6 % (43/45) and NPA was 94.4 % (170/180). For plasma, Pearson's correlation (0.950) and Bland-Altman analysis (0.113±0.22 log10 IU ml-1) showed high agreement. For neat urine, Pearson's correlation (0.842) and Bland-Altman analysis (0.326±0.80 log10 IU ml-1) showed somewhat higher variability. Reproducibility was high for the cobas BKV versus the LDT. BKPyV DNA levels in neat urine remained relatively stable over 7 days at both storage temperatures, although outlier results were intermittently detected.Conclusion. The cobas BKV test showed high agreement and reproducibility compared to the reference LDT. BKPyV viral load testing in urine has known limitations, but neat urine can be processed by the cobas BKV.