Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses.

BACKGROUND: Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers prohibit the widespread adoption of this technology in clinical laboratories. We validate an end-to-end mNGS assay for detection of respiratory viruses. Our assay is optimized to reduce turnaround time, lower cost-per-sample, increase throughput, and deploy secure and actionable bioinformatic results. METHODS: We validated our assay using residual nasopharyngeal swab specimens from Vancouver General Hospital (n = 359), RT-PCR-positive, or negative for Influenza, SARS-CoV-2, and RSV. We quantified sample stability, assay precision, the effect of background nucleic acid levels, and analytical limits of detection. Diagnostic performance metrics were estimated. RESULTS: We report that our mNGS assay is highly precise, semi-quantitative, with analytical limits of detection ranging from 103-104 copies/mL. Our assay is highly specific (100%) and sensitive (61.9% Overall: 86.8%; RT-PCR Ct < 30). Multiplexing capabilities enable processing of up to 55-specimens simultaneously on an Oxford Nanopore GridION device, with results reported within 12-hours. CONCLUSIONS: This study outlines the diagnostic performance and feasibility of mNGS for respiratory viral diagnostics, infection control, and public health surveillance. We addressed translational barriers to widespread mNGS adoption.

Retrospective validation of MetaSystems' deep-learning-based digital microscopy platform with assistance compared to manual fluorescence microscopy for detection of mycobacteria.

This study aimed to validate Metasystems' automated acid-fast bacilli (AFB) smear microscopy scanning and deep-learning-based image analysis module (Neon Metafer) with assistance on respiratory and pleural samples, compared to conventional manual fluorescence microscopy (MM). Analytical parameters were assessed first, followed by a retrospective validation study. In all, 320 archived auramine-O-stained slides selected non-consecutively [85 originally reported as AFB-smear-positive, 235 AFB-smear-negative slides; with an overall mycobacterial culture positivity rate of 24.1% (77/320)] underwent whole-slide imaging and were analyzed by the Metafer Neon AFB Module (version 4.3.130) using a predetermined probability threshold (PT) for AFB detection of 96%. Digital slides were then examined by a trained reviewer blinded to previous AFB smear and culture results, for the final interpretation of assisted digital microscopy (a-DM). Paired results from both microscopic methods were compared to mycobacterial culture. A scanning failure rate of 10.6% (34/320) was observed, leaving 286 slides for analysis. After discrepant analysis, concordance, positive and negative agreements were 95.5% (95%CI, 92.4%-97.6%), 96.2% (95%CI, 89.2%-99.2%), and 95.2% (95%CI, 91.3%-97.7%), respectively. Using mycobacterial culture as reference standard, a-DM and MM had comparable sensitivities: 90.7% (95%CI, 81.7%-96.2%) versus 92.0% (95%CI, 83.4%-97.0%) (P-value = 1.00); while their specificities differed 91.9% (95%CI, 87.4%-95.2%) versus 95.7% (95%CI, 92.1%-98.0%), respectively (P-value = 0.03). Using a PT of 96%, MetaSystems' platform shows acceptable performance. With a national laboratory staff shortage and a local low mycobacterial infection rate, this instrument when combined with culture, can reliably triage-negative AFB-smear respiratory slides and identify positive slides requiring manual confirmation and semi-quantification. IMPORTANCE: This manuscript presents a full validation of MetaSystems' automated acid-fast bacilli (AFB) smear microscopy scanning and deep-learning-based image analysis module using a probability threshold of 96% including accuracy, precision studies, and evaluation of limit of AFB detection on respiratory samples when the technology is used with assistance. This study is complementary to the conversation started by Tomasello et al. on the use of image analysis artificial intelligence software in routine mycobacterial diagnostic activities within the context of high-throughput laboratories with low incidence of tuberculosis.

Clostridioides difficile PCR Tcdb Cycle Threshold predicts toxin EIA positivity but not severity of infection.

BACKGROUND: Diagnosis of Clostridioides difficile Infection (CDI) entails compatible clinical presentation and laboratory findings. We evaluated real-time polymerase chain reaction (qPCR) cycle threshold (CT) as a predictor for disease severity and TcdB enzyme immunoassay (EIA) results. METHODS: Inpatients or emergency department patients who tested positive for tcdB gene by PCR were evaluated. Patients' stools underwent testing for GDH and TcdA/B by EIA. Medical health records were reviewed for demographic, clinical presentation, laboratory, treatment and outcome data. Severity of CDI was calculated using various severity score indexes. RESULTS: The median CT of cases was 32.05 ± 5.45. The optimal cut-off for predicting toxin EIA positivity and severe CDI based on chart review was 32.6 and 29.8, respectively, with the area under the receiver operator characteristics curve (AUC) of 0.74 and 0.60 respectively. CONCLUSION: CT value was an acceptable predictor for EIA toxin but less so for clinical severity. Our study potentially supports a diagnostic algorithm including CT value to reduce the number of EIA toxin assays performed.

Evaluation of the Aptima BV and CV/TV assays compared to conventional laboratory based testing methods for the diagnosis of vaginitis.

PURPOSE: Vaginitis is caused by bacterial vaginosis (BV), Candida vaginitis (CV) and Trichomonas vaginalis (TV). This retrospective study evaluates the performance of the Aptima CV/TV, and BV assays on the automated Panther system. METHODS: Two hundred forty-two multitest swabs were tested on the CV/TV assay and 422 on the BV assay. Positive and negative percent agreement (PPA, NPA) of the Candida glabrata (CG), Candida species group (CSG), TV and BV targets were calculated using a modified gold standard, with review of Gram smear and the usage of the Allplex Vaginitis Screening Assay to resolve discrepancies. RESULTS: The PPA and NPA were respectively 98.4% and 95.9% for BV, 100% and 95.4% for CSG, 100% and 99% for CG, and 100% and 100% for TV, and when compared to consensus results. CONCLUSION: The CV/TV and BV assays surpassed the acceptance criteria threshold of 95%, and proved to be an excellent alternative to conventional testing.

Quick versus Quantitative: Evaluation of Two Commercial Real-Time PCR Assays for the Detection of Pneumocystis jirovecii from Bronchoalveolar Lavage Fluids.

Two commercial real-time PCR assays for the detection of Pneumocystis jirovecii were compared, the quantitative RealStar P. jirovecii assay and the qualitative DiaSorin P. jirovecii assay, the latter of which can be used without nucleic acid extraction. Archived bronchoalveolar lavage (BAL) specimens (n = 66), previously tested by molecular methods, were tested by both assays, and the results were compared to the respective original result. The RealStar P. jirovecii assay demonstrated good positive percent agreement (PPA) (90% [95% confidence interval (CI), 72 to 97%]; 27/30) and negative percent agreement (NPA) (100% [95% CI, 88 to 100%]; 36/36) with the reference method. The DiaSorin P. jirovecii assay concordantly detected P. jirovecii in 19 of 24 positive BAL samples (PPA = 73% [95% CI, 52 to 88%]). All negative BAL samples gave concordant results (NPA = 100% [95% CI, 87 to 100%]; 34/34). Discordant results occurred mostly in samples with low fungal loads. In conclusion, the RealStar assay demonstrated good concordance with reference results, and the DiaSorin P. jirovecii assay performed well for negative BAL and positive BAL samples with P. jirovecii concentrations of greater than 260 copies/mL. IMPORTANCE Pneumonia, caused by the opportunistic fungus Pneumocystis jirovecii, poses a significant risk for immunocompromised individuals. Laboratory testing for P. jirovecii is progressively shifting toward the use of molecular tests such as real-time PCR; however, this is often performed at reference laboratories. Many frontline laboratories are looking into improving their service and reducing turnaround times for obtaining P. jirovecii results by bringing molecular P. jirovecii testing in-house. We evaluated and compared two commercial real-time PCR assays with different workflows for the detection of P. jirovecii from bronchoalveolar lavage specimens. The RealStar P. jirovecii assay requires nucleic acid extraction and provides a quantification of fungal load for positive samples. The DiaSorin P. jirovecii assay offers a simple workflow without nucleic extraction from patient samples and qualitative results. Results from this study provide valuable information on performance and workflow considerations for laboratories that wish to implement P. jirovecii molecular testing.

Abbott ID NOW™ COVID-19 assay: do not discard the swab.

We compared the performance of ID NOW™ COVID-19 assay nasal swabs with RT-PCR of nasopharyngeal swabs for SARS-CoV-2 in an outbreak setting, determining whether addition of RT-PCR of residual nasal swabs (rNS) (post ID NOW™ elution) would increase overall analytic sensitivity. Devices were placed at 2 long term and 1 acute care sites and 51 participants were recruited. Prospective paired nasopharyngeal and nasal samples were collected for RT-PCR and ID NOW™.  ID NOW™ had a positive and negative categorical agreement of 86% and 93% compared to RT-PCR of nasopharyngeal swabs. Sensitivity and specificity of the ID NOW™ was 86% and 100%, positive and negative predictive value was 100% and 95% (COVID-19 positivity rate: 8%). Addition of rNS RT-PCR increased the positive and negative categorical agreement to 93% and 97%. Based on these results, we propose an alternative workflow which includes complementary testing of rNS on a secondary assay.

Alterations in the nasopharyngeal microbiome associated with SARS-CoV-2 infection status and disease severity.

OBJECTIVES: The COVID-19 pandemic and ensuing public health emergency has emphasized the need to study SARS-CoV-2 pathogenesis. The human microbiome has been shown to regulate the host immune system and may influence host susceptibility to viral infection, as well as disease severity. Several studies have assessed whether compositional alterations in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection. However, the results of these studies were varied, and many did not account for disease severity. This study aims to examine whether compositional differences in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection status and disease severity. METHODS: We performed Nanopore full-length 16S rRNA sequencing on 194 nasopharyngeal swab specimens from hospitalized and community-dwelling SARS-CoV-2-infected and uninfected individuals. Sequence data analysis was performed using the BugSeq 16S analysis pipeline. RESULTS: We found significant beta (PERMANOVA p < 0.05), but not alpha (Kruskal-Wallis p > 0.05) diversity differences in the nasopharyngeal microbiota among our study groups. We identified several differentially abundant taxa associated with SARS-CoV-2 infection status and disease severity using ALDEx2. Finally, we observed a trend towards higher abundance of Enterobacteriaceae in specimens from hospitalized SARS-CoV-2-infected patients. CONCLUSIONS: This study identified several alterations in the nasopharyngeal microbiome associated with SARS-CoV-2 infection status and disease severity. Understanding the role of the microbiome in infection susceptibility and severity may open new avenues of research for disease prevention and treatment.

Automated 16S Sequencing Using an R-Based Analysis Module for Bacterial Identification.

Sanger sequencing of the 16S rRNA gene is routinely used for the identification of bacterial isolates. However, this method is still performed mostly in more-specialized reference laboratories, and traditional protocols can be labor intensive. In this study, 99 clinical bacterial isolates were used to validate a fast, simplified, and largely automated protocol for 16S sequencing. The workflow combines real-time PCR of the first 500 bp of the bacterial 16S rRNA gene and amplicon sequencing on an automated, cartridge-based sequence analyzer. Sequence analysis, NCBI BLAST search, and result interpretation were performed using an automated R-based script. The automated workflow and R analysis described here produced results equal to those of manual sequence analysis. Of the 96 sequences with adequate quality, 90 were concordantly identified to the genus (n = 62) or species level (n = 28) compared with routine laboratory identification of the organism. One organism identification was discordant, and 5 resulted in an inconclusive identification. For sequences that gave a valid result, the overall accuracy of identification to at least the genus level was 98.9%. This simplified sequencing protocol provides a standardized approach to clinical 16S sequencing, analysis, and quality control that would be suited to frontline clinical microbiology laboratories with minimal experience. IMPORTANCE Sanger sequencing of the 16S rRNA gene is widely used as a diagnostic tool for bacterial identification, especially in cases where routine diagnostic methods fail to provide an identification, for organisms that are difficult to culture, or from specimens where cultures remain negative. Our simplified protocol is tailored toward use in frontline laboratories with little to no experience with sequencing. It provides a highly automated workflow that can deliver fast results with little hands-on time. Implementing 16S sequencing in-house saves additional time that is otherwise required to send out isolates/specimens for identification to reference laboratories. This makes results available much faster to physicians who can in turn initiate or adjust patient treatment accordingly.

BugSplit enables genome-resolved metagenomics through highly accurate taxonomic binning of metagenomic assemblies.

A large gap remains between sequencing a microbial community and characterizing all of the organisms inside of it. Here we develop a novel method to taxonomically bin metagenomic assemblies through alignment of contigs against a reference database. We show that this workflow, BugSplit, bins metagenome-assembled contigs to species with a 33% absolute improvement in F1-score when compared to alternative tools. We perform nanopore mNGS on patients with COVID-19, and using a reference database predating COVID-19, demonstrate that BugSplit's taxonomic binning enables sensitive and specific detection of a novel coronavirus not possible with other approaches. When applied to nanopore mNGS data from cases of Klebsiella pneumoniae and Neisseria gonorrhoeae infection, BugSplit's taxonomic binning accurately separates pathogen sequences from those of the host and microbiota, and unlocks the possibility of sequence typing, in silico serotyping, and antimicrobial resistance prediction of each organism within a sample. BugSplit is available at https://bugseq.com/academic .

Nanopore metagenomic sequencing for detection and characterization of SARS-CoV-2 in clinical samples.

OBJECTIVES: The COVID-19 pandemic has underscored the need for rapid novel diagnostic strategies. Metagenomic Next-Generation Sequencing (mNGS) may allow for the detection of pathogens that can be missed in targeted assays. The goal of this study was to assess the performance of nanopore-based Sequence-Independent Single Primer Amplification (SISPA) for the detection and characterization of SARS-CoV-2. METHODS: We performed mNGS on clinical samples and designed a diagnostic classifier that corrects for barcode crosstalk between specimens. Phylogenetic analysis was performed on genome assemblies. RESULTS: Our assay yielded 100% specificity overall and 95.2% sensitivity for specimens with a RT-PCR cycle threshold value less than 30. We assembled 10 complete, and one near-complete genomes from 20 specimens that were classified as positive by mNGS. Phylogenetic analysis revealed that 10/11 specimens from British Columbia had a closest relative to another British Columbian specimen. We found 100% concordance between phylogenetic lineage assignment and Variant of Concern (VOC) PCR results. Our assay was able to distinguish between the Alpha and Gamma variants, which was not possible with the current standard VOC PCR being used in British Columbia. CONCLUSIONS: This study supports future work examining the broader feasibility of nanopore mNGS as a diagnostic strategy for the detection and characterization of viral pathogens.

Evaluation of the BioFire® COVID-19 test and Respiratory Panel 2.1 for rapid identification of SARS-CoV-2 in nasopharyngeal swab samples.

The BioFire® COVID-19 Test and Respiratory Panel 2.1 (RP2.1) are rapid, fully automated assays for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs. In the case of the RP2.1, an additional 21 viral and bacterial pathogens can be detected. Both tests have received emergency use authorization from the U.S. Food & Drug Administration and Interim Order authorization from Health Canada for use in clinical laboratories. We evaluated the performance characteristics of these tests in comparison to a laboratory-developed real-time PCR assay targeting the viral RNA-dependent RNA polymerase and E genes. A total of 78 tests were performed using the BioFire COVID-19 Test, including 30 clinical specimens and 48 tests in a limit of detection study; 57 tests were performed using the RP2.1 for evaluation of SARS-CoV-2 detection, including 30 clinical specimens and 27 tests for limit of detection. Results showed 100% concordance between the BioFire assays and the laboratory-developed test for all clinical samples tested, and acceptable performance of both BioFire assays at their stated limits of detection. Conclusively, the BioFire COVID-19 Test and RP2.1 are highly sensitive assays that can be effectively used in the clinical laboratory for rapid SARS-CoV-2 testing.

Approach to Assessment of New Swabs and Viral Transport Media for SARS-CoV-2 Testing.

In light of the present pandemic of novel coronavirus disease 2019 (COVID-19) and the unprecedented high demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing worldwide, there are shortages of established specimen collection devices for respiratory viral testing for diagnostic microbiology laboratories. This creates the need to validate unverified collection devices from manufacturers that may not be a registered supplier for medical devices. As clinical laboratories do not routinely perform quality control of established collection devices, there is a need to have a systematic, robust approach to the assessment of substitute unregistered collection swabs and viral transport media (VTM). A discussion of the aspects requiring consideration when determining the suitability and implementation of new collection devices is presented. These specific assessment criteria include an inspection of device integrity, determination of swab and VTM sterility and in vitro performance, VTM stability, and examination of the clinical performance of the device. This method was used in a front-line medical microbiology laboratory on swabs and VTM from an unregistered manufacturer, with suboptimal results that precluded implementation. As the pandemic continues, it will be important for diagnostic laboratories to adopt a flexible and streamlined approach to maintaining adequate supply chains for testing reagents and materials.

FilmArray respiratory panel assay: An effective method for detecting viral and atypical bacterial pathogens in bronchoscopy specimens.

The BioFire FilmArray Respiratory Panel (FA RP) is a rapid multiplexed molecular assay approved for detection of viral and atypical bacterial pathogens in nasopharyngeal specimens. This study aimed to evaluate the performance of the BioFire FilmArray Respiratory Panel v1.7 on bronchoscopy specimens. We tested 133 bronchial specimens (87 archived and 46 prospectively collected) with the FA RP and compared the results to the Luminex NxTAG Respiratory Pathogen Panel (NxTAG RPP). After discordant analysis, 123 specimens gave concordant results using the FA RP and the NxTAG RPP for an overall agreement of 93.9% (kappa = 0.88 [95% CI 0.80-0.96]), a positive percent agreement of 93.7% (95% CI 83.7-97.7) and a negative percent agreement of 94.1% (95% CI 84.9-98.1). In conclusion, the BioFire FilmArray RP performed reliably to detect a broad range of respiratory pathogens in bronchoscopy specimens.