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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.

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 hours after initial specimen collection, informing appropriate transport time and conditions.

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.

In 2015, the Clinical and Laboratory Standards Institute (CLSI) updated its breakpoints for penicillin susceptibility in Corynebacterium species from

Summary During the 2017/18 influenza season, the authors' virology laboratory implemented the cobas® Influenza A/B & RSV (Roche Molecular Diagnostics, Pleasanton, CA, USA) for influenza outbreak management in two scenarios: initial outbreak investigation or at outbreak conclusion to avoid prolonged measures. Twenty-seven investigations were conducted, including declaration of 11 influenza A/B outbreaks. Thirty percent of investigations would have missed the standard batched daily laboratory-developed respiratory polymerase chain reaction (PCR), and delayed outbreak confirmation until the following day. The average reduction in turnaround time for influenza A/B testing was 10.2 h. A rapid molecular PCR in specific outbreak scenarios improved timely management of influenza outbreaks.

Quantitative viral load assays have transformed our understanding of – and ability to manage − viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time reverse transcriptase-polymerase chain reaction (RT-PCR), yield semi-quantitative results only. Reverse transcriptase droplet digital PCR (RT-ddPCR), a technology that partitions each reaction into 20,000 nanolitre-sized droplets prior to amplification, offers an attractive platform for SARS-CoV-2 RNA quantification. We evaluated eight primer/probe sets originally developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests for use in RT-ddPCR, and identified three (Charité-Berlin E-Sarbeco and Pasteur Institute IP2 and IP4) as the most efficient, precise and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. Analytical efficiency of the E-Sarbeco primer/probe set, for example, was ~83%, while assay precision, as measured by the coefficient of variation, was ~2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. We further calibrated RT-ddPCR-derived SARS-CoV-2 E gene copy numbers against cycle threshold (Ct) values from a commercial real-time RT-PCR diagnostic platform. The resulting log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from Ct values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.

Using a real-time RT-PCR-based algorithm to detect SARS-CoV-2 variants of concern, we rapidly identified 77 variants (57-B.1.1.7, 7-B.1.351, and 13-B.1.1.28/P.1). This protocol enabled our laboratory to screen all SARS-CoV-2 positive samples for variants, and identified a cluster of B.1.1.28/P.1 cases, a variant not previously known to circulate in British Columbia.

Highlights • There is increasing global demand for enhanced SARS-CoV-2 testing capacity. • Shortages of swabs and transport media necessitate use of alternate specimen types. • Saliva has been described as an acceptable specimen type for SARS-CoV-2 detection. • Saliva heterogeneity and viscosity can challenge laboratory processing and workflow. • Feasibility of automated, high-volume processing of saliva is demonstrated., With surging global demand for SARS-CoV-2 testing capacity, laboratories seek automated, high-throughput molecular solutions, particularly for specimens not requiring specialized collection devices or viral transport media. Saliva specimens submitted from patients under investigation for COVID-19 from March to July 2020 were processed in the laboratory with sterile phosphate-buffered saline in a 1:2 dilution and tested using manual extraction and a commercial assay for detection of the SARS-CoV-2 E gene (LightMix®) in comparison to the Roche cobas® SARS-CoV-2 Test on the cobas® 6800 instrument. 34.4% (22/64) of saliva samples were positive for SARS-CoV-2. Positive and negative concordance between the LightMix® and cobas® assays were 100%. The overall invalid rate for saliva on the cobas® 6800 (1/128, 0.78%) was similar to the baseline invalid rate observed for nasopharyngeal swabs/viral transport media. Saliva is a feasible specimen type for SARS-CoV-2 testing on the cobas® 6800 platform, with potential to improve turnaround time and enhance testing capacity.

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.

In response to the COVID-19 pandemic, commercial molecular assays for SARS-CoV-2 testing have been rapidly developed and broadly deployed in laboratories worldwide. Although these assays have been reported to correlate well, we sought to compare the Xpert® Xpress SARS-CoV-2 to the cobas® SARS-CoV-2 or the Lightmix® Modular SARS and Wuhan CoV E-gene assay for nasopharyngeal (NP) swabs with low levels of SARS-CoV-2 RNA. Thirty-seven NP swabs were studied, including 10 samples with a moderate cycle threshold (Ct) between 30-33.9, and 22 with Ct≥34, and 5 negative for SARS-CoV-2. Overall concordance on initial comparison was 86.5 % (32/37), which was 100 % concordance for samples with Ct values ranging between 30-33.9. Discordance amongst samples showing a Ct ≥34 was 22.7 % (5/22). Endpoint value analysis on the Xpress SARS-CoV-2 within the discordant samples noted two with an endpoint value >5, which were detected by the cobas® or Lightmix®. Testing of SARS-CoV-2 on the three commercial assays was comparable for NP swabs with moderate Ct values, while high Ct values were less concordant. Importantly, analysis of Xpert® endpoint values improved interpretation of discrepant results.

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

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.

Introduction The identification of CMV antiviral drug resistance (AVDR) is a critical diagnostic test for immunocompromised patients with CMV infection and a failure of virologic response on optimal antiviral treatment. We developed a next-generation sequencing (NGS) assay for CMV AVDR testing and compared the AVDR mutations identified by NGS to Sanger sequencing. Methods Retrospective review of CMV AVDR testing requests for UL97 and UL54 at our laboratory from 2014 to 2019 was conducted. NGS was performed on the MinION and compared to Sanger sequencing performed at the national reference laboratory. Analysis of the sequences was completed with a novel cloud bioinformatics platform (BugSeq). Results Twenty patient samples previously characterized were included for study on the MinION. NGS captured all of the CMV AVDR mutations identified by Sanger, and identified additional mutations in UL97 and/or UL54 in 8/13 (62%) of the samples. An analysis of the depth of coverage at which we no longer detected minority single nucleotide variants (SNVs) detected in the original data was conducted, estimating a recall of 95% at 1800 fold coverage. Conclusion NGS utilizing MinION technology for the detection of CMV AVDR mutations identified additional minority variants in UL97 and UL54 as compared with Sanger sequencing. Through the application of a bioinformatics pipeline available online, our NGS process eliminates barriers associated with the use of the MinION and NGS in clinical laboratories.

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

Background In clinical settings, multiplex molecular panels are becoming increasingly common for the detection of respiratory pathogens. Little evidence is available to guide appropriate use of respiratory multiplex panels, particularly with respect to the patient populations most likely to benefit from such testing. Methods During the 2018–2019 influenza season, all patients with a nasopharyngeal swab submitted for respiratory virus detection were initially tested on a commercial rapid PCR platform for influenza A/B and respiratory syncytial virus (RSV) (Cepheid GeneXpert, Sunnyvale, CA). Patients with negative swabs were reviewed by a laboratory physician based on pre-defined criteria (Table 1) for additional testing by a laboratory-developed multiplex assay for parainfluenza 1/2/3, adenovirus, and human metapneumovirus (hMPV). Results In total, 1144 nasopharyngeal swabs were tested. 287 (25.1%) were positive on the GeneXpert: influenza A (234, 81.5%), influenza B (13, 4.5%), and RSV (40, 13.9%). Of the patients who tested negative, 234 (27.3%) met criteria for further respiratory virus testing. The most commonly detected viral pathogens on the multiplex assay were hMPV (20/30, 66.7%), parainfluenza 3 (7/30, 23.3%) and adenovirus (3/30, 10%). The yield of the multiplex assay was highest for patients selected for antimicrobial stewardship (AS) criteria (13/56, 23.2%), followed by transplant (2/16, 12.5%), HIV (7/64, 10.9%), cystic fibrosis (2/19, 10.5%), critical care (6/68, 8.8%), and other/upon physician request (0/11, 0%). Of the patients who received multiplex testing for AS criteria and tested positive for a viral pathogen, only 3/13 (23.1%) had antibiotics discontinued by the medical team within 48 hours of the report. Conclusion Additional testing for respiratory viral pathogens had low overall diagnostic yield, and further refinement of the algorithm is needed to better target utilization of respiratory virus testing. The patient population with the highest yield (those who met AS criteria) failed to demonstrate consistent timely discontinuation of unnecessary antibiotics by the medical team. Implementation of respiratory multiplex panels would be strengthened by collaboration with AS teams. Disclosures All authors: No reported disclosures.

e17556 Background: A growing body of evidence supports the use of natural compounds in oncology care. The majority of cancer patients use supplements (supps); however, most rely on questionable sou...

Designing normalized tables is a subject of great difficulty and importance. Unnecessary redundancy may results not only in too much space being used to store the data and poor query performance, but it may also result in inconsistencies as denormalized (unnecessary redundancy) data is much more difficult to keep current. Converting tables from E-R Diagrams (or extended E-R Diagrams or Class Diagrams) is a difficult task for most students. Some students manage this task in their first undergraduate class by simply memorizing the rules and never knowing the reasons behind why the rule was created. Therefore when they get to more advanced classes they had forgotten how to accomplish the conversion because they never understood the reasons behind the rules.