BACKGROUND:Rapid detection of syphilis infection enables timely antibiotic treatment to prevent long-term complications and further transmission. We implemented a treponemal rapid lateral flow immunochromatographic test that was performed by trained clinical laboratory personnel on a 24/7 basis in an urban county hospital. METHODS:From March 2023 to February 2024, 461 rapid syphilis tests (RSTs) were performed on serum from 449 patients. Specimens were also tested by a conventional reverse algorithm beginning with an enzyme immunoassay, followed by a rapid plasma reagin test, and when necessary, an additional treponemal test to resolve discordant results. RESULTS:Most patients (349, 77.7%) initially presented to the emergency department. The most common patient risk factors for syphilis were substance use disorder (51.9%) and unstable housing (42.8%). Compared with the reverse algorithm, rapid syphilis test (RST) sensitivity was 52.2%, specificity 98.2%, positive predictive value 83.7%, and negative predictive value 92.1%. After excluding 45 patients who had a documented prior history of syphilis, RST sensitivity was 83.3%, specificity 98.2%, positive predictive value 74.1%, and negative predictive value 99.0%. Nearly all of the 33 patients testing negative by the RST but positive by enzyme immunoassay were more likely to have been treated for a previous infection: 27 had a documented history of treatment, and 30 had undetectable or low rapid plasma reagin titers (≤1:2). The median RST turnaround time was 1.6 hours compared with 197.8 hours for reverse algorithm screening. CONCLUSIONS:RST is a useful option for high-risk patients in urban settings. Other testing methods are preferable for previously treated syphilis, and conventional follow-up testing is required to confirm results.

BACKGROUND:Candidozyma auris (C. auris) causes prolonged asymptomatic colonization and widespread silent healthcare transmission. An unknown proportion of patients who are colonized will progress to invasive candidiasis. Although culture has been the traditional method of colonization detection, PCR-based assays are now increasingly used for diagnosis and for evaluation of healthcare outbreaks. METHODS:During a six-month, multi-hospital unit C. auris outbreak in a 500-bed urban public hospital, screening transitioned from chromogenic culture to open-access low-throughput PCR and house-wide high-throughput PCR, mandated via electronic admission order sets. Infection prevention and control interventions included electronic health record contact tracing, "Contact Plus" precautions, double-bleach cleaning, dual UV-C disinfection, and ATP environmental monitoring. Whole genome sequencing evaluated clonality. RESULTS:Admission screening was performed for all newly admitted patients and weekly surveillance testing for all inpatients during the outbreak, with testing scaled from 1,181 cultures to 1,539 low-throughput PCRs and 8,918 high-throughput PCRs, reducing median turnaround times from 82.1 to 37.6 and 20.3 hours, respectively. Twenty-nine colonized patients were identified, with 50% of secondary cases linked to bed spaces previously occupied by colonized individuals. Sequencing confirmed a highly clonal Clade I cluster. Transmission was halted only after introducing universal PCR screening alongside enhanced infection prevention and control interventions. Notably, no patients experienced symptomatic infection with C. auris or required antifungal therapy. CONCLUSIONS:High-throughput, rapid PCR screening combined with rigorous infection prevention and control practices can successfully contain healthcare-associated C. auris outbreaks. Transitioning laboratory-developed tests to open-access, high-throughput platforms significantly enhances hospital outbreak response capacity.

ABSTRACT Molecular syndromic panels such as the BioFire FilmArray Gastrointestinal Panel (BF-GIP) have been widely adopted for gastrointestinal illness diagnosis due to their fast turnaround times and broad pathogen coverage. Recently, the BF-GIP demonstrated increased rates of norovirus false-positive detections, prompting a Class II recall of more than two million tests in February 2024 and resulting in an estimated burden of more than a quarter-million false-positive norovirus results reported over the last 5 years. We examined the prevalence of BF-GIP norovirus false positives across four hospitals from December 2024 to June 2025. Among 185 BF-GIP norovirus-positive results confirmed with the BD MAX Enteric Viral Panel, the false discovery rate ranged from 31% to 74% across sites, with the highest rate seen at a specialized cancer care hospital. Deep sequencing of BF-GIP pouches ( n = 42) confirmed the Noro-1 assay as the primary source of off-target amplification, identifying 78 off-target species—predominantly commensal stool bacteria—compared to only two species for the Noro-2 assay. Off-target species amplified by the Noro-1 assay were recovered from both false-positive and true-negative pouches, suggesting no single species accounted for the false-positive results. Partial primer complementarity at off-target loci and amplicon Tm values within the acceptable range support mispriming of gut microbiota as the underlying cause. False-positive pouches exhibited significantly higher crossing point values than true-positive pouches for both assays (Noro-1: 26.6 vs 11.1, P = 0.013; Noro-2: 30.0 vs 13.1, P < 0.001), consistent with low-level off-target amplification. These findings highlight the high false discovery rate of the Noro-1 assay, identify bacterial species involved in mispriming, and demonstrate the need to redesign this assay to ensure reliable testing and improved patient care. IMPORTANCE Syndromic molecular panels have revolutionized gastrointestinal diagnostics. However, recent data have suggested significant norovirus false-positive results associated with the BioFire FilmArray Gastrointestinal Panel. Here, we investigate a major diagnostic failure associated with the 2024 Class II recall of this assay, revealing norovirus false discovery rates as high as 74% in certain clinical settings. By deep sequencing amplicons from FilmArray pouches, we identified widespread cross-reactivity of the Noro-1 assay with stool microbiome nucleic acid. Off-target Noro-1 amplicons were detected from 78 bacterial species across 42 clinical pouches. For 16 species with the highest read counts per pouch, amplicons mapped to discrete genomic loci with partial primer overlap, consistent with mispriming. The identification of these discrete loci, combined with the repeatedly high false discovery rate reported across multiple studies, creates a clinical imperative to redesign this assay. Our work also highlights the ongoing need for rigorous post-market surveillance and the utility of deep sequencing in troubleshooting diagnostic assay failures.

Abstract Molecular syndromic panels such as the BioFire FilmArray Gastrointestinal Panel (BF-GIP) have been widely adopted for gastrointestinal illness diagnosis due to their fast turnaround times and broad pathogen coverage. Recently, the BF-GIP demonstrated increased rates of norovirus false-positive detections, prompting a Class II recall of more than two million tests in February 2024. We examined the prevalence of BF-GIP norovirus false positives across four hospitals from December 2024 to June 2025. Among 185 BF-GIP norovirus-positive results confirmed with the BD MAX Enteric Viral Panel, the false discovery rate ranged from 31 to 74% across sites, with the highest rate seen at a specialized cancer care hospital. Deep sequencing of BF-GIP pouches (n=42) confirmed the Noro-1 assay as the primary source of off-target amplification, identifying 78 off-target species – predominantly commensal stool bacteria – compared to only two species for the Noro-2 assay. Off-target species amplified by the Noro-1 assay were recovered from both false-positive and true-negative pouches, suggesting no single species accounted for the false-positive results. Partial primer complementarity at off-target loci and amplicon Tm values within the acceptable range support mispriming of gut microbiota as the underlying cause. False-positive pouches exhibited significantly higher Cp values than true positives for both assays (Noro-1: 26.6 vs. 11.1, p=0.013; Noro-2: 30.0 vs. 13.1, p<0.001), consistent with low-level off-target amplification. These findings highlight the high false discovery rate of the Noro-1 assay, identify bacterial species involved in mispriming, and demonstrate the need to redesign this assay to ensure reliable testing and improved patient care. Importance Syndromic molecular panels have revolutionized gastrointestinal diagnostics. However, recent data have suggested significant norovirus false-positive results associated with the BioFire FilmArray Gastrointestinal Panel. Here, we investigate a major diagnostic failure associated with the 2024 Class II recall of this assay, revealing norovirus false discovery rates as high as 74% in certain clinical settings. By deep sequencing amplicons from FilmArray pouches, we identified widespread cross-reactivity of the Noro-1 assay with stool microbiome nucleic acid. Off-target Noro-1 amplicons were detected from 78 bacterial species across 42 clinical pouches. For 16 species with the highest read counts per pouch, amplicons mapped to discrete genomic loci with partial primer overlap, consistent with mispriming. The identification of these discrete loci, combined with the repeatedly high false discovery rate reported across multiple studies, creates a clinical imperative to redesign this assay. Our work also highlights the ongoing need for rigorous post-market surveillance and the utility of deep sequencing in troubleshooting diagnostic assay failures.

Background: Diarrhea is common among patients with cancer, and rapid diagnosis of gastrointestinal (GI) infections using a multiplex stool enteric pathogen panel (EPP) may expedite clinical decision-making and inform infection prevention measures. However, the diagnostic yield of EPPs in patients with cancer with hospital-onset diarrhea is not well defined. At our center, inpatient diarrhea testing guidelines recommend EPP testing primarily in those with community-onset diarrhea. We aimed to assess adherence to these EPP testing guidelines and describe EPP diagnostic yield in hospitalized patients with cancer to identify opportunities for diagnostic stewardship. Methods: We retrospectively reviewed EPP and stand-alone C. difficile (SACD) tests ordered between July 1, 2021 – May 23, 2025 among hospitalized patients (age ≥18 years) with a malignancy diagnosis. Our center recommends EPP testing (BIOFIRE® FILMARRAY® GI Panel) for community-onset diarrhea (on or before hospital day [HD] 3) and SACD testing for hospital-onset diarrhea (after HD 3). EPPs may be ordered after HD 3 for patients with unexplained prolonged or bloody diarrhea. Results: A total of 3338 stool tests (1433 EPP, 1905 SACD) were performed among 2131 patients. Among EPPs, 468 (33%) were ordered after HD 3 whereas 1621 (85%) SACD tests were ordered after HD 3 (Figure 1). The proportion of EPPs ordered after HD 3 was highest among patients with hematologic malignancies (44%) compared to other cancer types and higher among hematopoietic cell transplant (HCT) recipients versus non-HCT recipients (54% vs. 22%; Figure 2). Among EPPs ordered after HD 3, 102/468 (22%) were positive for ?1 pathogen compared to 288/965 (30%) EPPs ordered on HD 1-3. At least 1 non-C. difficile pathogen was detected in 49/468 (10%) EPPs ordered after HD 3 compared to 146/965 (15%) on HD 1-3 (Table 1). Among EPPs with non-C. difficile pathogens detected after HD 3, norovirus was most frequent (26/468; 6%; Table 1). Conclusion: EPP detection of non-C. difficile pathogens among hospitalized patients with cancer was lower in patients with hospital-onset diarrhea compared to those with community-onset diarrhea. Most EPP orders after HD 3 at our center occurred in HCT recipients and patients with hematologic malignancies. Additional studies are needed to determine the clinical significance of positive EPP results beyond HD 3 and understand which patients may benefit most from EPP testing beyond HD 3.

Universal admission screening for Candidozyma auris (C. auris) identified C. auris colonization in 25/22742 (0.1%) of patients. Most patients (20/25, 80%) did not meet public health screening criteria, suggesting known risk factors for C. auris may not sufficiently capture the impacted population. Findings may not be generalizable to higher-incidence settings.

A sustained outbreak of H5N1 influenza virus among wild fowl and domestic livestock has caused more than 70 zoonotic infections in humans in North America, including two deaths. The United States Centers for Disease Control and Prevention has recommended rapid H5 subtyping for all hospitalized cases with influenza A virus infection to enable prompt initiation of antiviral treatment, as well as infection prevention and implementation of public health measures to control spread. To address these needs, we developed a qualitative multiplex RT-qPCR assay to subtype H5 influenza virus in nasal, nasopharyngeal, and conjunctival specimens with a limit of detection of 250 copies/mL. No cross-reactivity was observed with other common respiratory viruses, including seasonal H3N2 and H1N1 influenza A viruses. We retrospectively subtyped 590 influenza A virus-positive clinical specimens with Ct values less than 31 processed by University of Washington labs between March 2024 and February 2025, including 512 specimens collected during the 2024-2025 influenza season, and detected no H5 positives. After clinical implementation, we performed 150 clinically ordered H5 subtyping tests between February and April 2025 and again detected no positives. This work enhances clinical pandemic preparedness activities and highlights the exceedingly low prevalence of H5N1 influenza virus during the 2024-2025 respiratory season.IMPORTANCEThe spread of H5N1 influenza virus in the United States has led to the culling of almost 200 million birds, infected cow herds across 17 states, and resulted in 70 human infections as of July 2025. Rapid PCR subtyping of H5 influenza virus is critical to inform hospital infection prevention and public health to enable containment of viral transmission. Here, we report the design, validation, and clinical implementation of a qualitative multiplex H5-subtyping RT-qPCR assay for nasopharyngeal, nasal, and conjunctival swab specimens. Additionally, we offer the largest reported study of H5 subtyping of influenza A virus-positive specimens in the United States to date. No H5 infections were detected in 740 samples collected between March 2024 and April 2025 from patients with confirmed influenza A virus infection in a large academic medical system in Seattle, WA.

Objectives:Viridans streptococci (VS) are opportunistic oral commensals and a common cause of bacteraemia in neutropenic patients. In this retrospective single centre cohort study, we investigated the prevalence of ceftriaxone resistance in VS (CRO-R VS) blood isolates between January 2005 and December 2022 from patients treated at a tertiary care hospital. Methods:Blood culture isolates were identified using biochemicals and mass spectrometry. Susceptibility testing was performed by Kirby-Bauer and Epsilometer tests. Demographic data, clinical outcomes and antimicrobial use were assessed through electronic medical record review. Results:Among 791 patients with VS bacteraemia, 31 (4%) had confirmed CRO-R VS bacteraemia over the 18-year period; 20/31 (65%) were patients also treated at the Fred Hutchinson Cancer Center and were the focus of this study. Of these 20 patients, 18 (90%) had a known haematologic malignancy; 14 (70%) had undergone haematopoietic cell transplant (HCT); 18 (90%) were neutropenic at the time of culture. Two (10%) patients died within 30 days of CRO-R VS bacteraemia. All the CRO-R isolates (20/20) were members of the Streptococcus mitis group, 12 were multi-drug resistant; all were susceptible to vancomycin. Most patients received vancomycin once blood cultures were positive for a Gram-positive organism. Conclusions:During the study period, the frequency of VS isolate susceptibility testing increased; however, there was no concomitant increase in the percentage of CRO-R isolates at our facility. These data are important in an era where cefepime monotherapy is often used and reinforces the importance of routine resistance testing among VS bacteraemia.

BackgroundCutibacterium acnes is the bacterium most commonly responsible for shoulder periprosthetic joint infection (PJI) and is often cultured from samples obtained at the time of revision for failed shoulder arthroplasty. We sought to determine whether these bacteria originate from the patient or from exogenous sources. We also sought to identify which C. acnes genetic traits were associated with the development of shoulder PJI.MethodsWe performed bacterial whole-genome sequencing of C. acnes from a single-institution repository of cultures obtained before or during primary and revision shoulder arthroplasty and correlated the molecular epidemiology and genetic content of strains with clinical features of infection.ResultsA total of 341 isolates collected over a four-year period from 88 patients were sequenced. C. acnes cultured from surgical specimens demonstrated significant similarity to the strains colonizing the skin of the same patient (p<0.001). Infrequently, there was evidence of strains shared across unrelated patients, suggesting that exogenous sources of C. acnes culture-positivity were uncommon. Phylotypes IB and II were modestly associated with clinical features of PJI, but all phylotypes appeared inherently capable of causing disease. Chronic shoulder PJI was associated with the absence of common C. acnes genes involved in bacterial quorum-sensing (luxS, tqsA).ConclusionC. acnes strains cultured from deep intraoperative sources during revision shoulder arthroplasty demonstrate strong genetic similarity to the strains colonizing a patient’s skin. Some phylotypes of C. acnes commonly colonizing human skin are modestly more virulent than others, but all phylotypes have a capacity for PJI. C. acnes cultured from cases of PJI commonly demonstrated genetic hallmarks associated with adaptation from acute to chronic phases of infection. This is the strongest evidence to date supporting the role of the patient’s own, cutaneous C. acnes strains in the pathogenesis of shoulder arthroplasty infection. Our findings support the importance of further research focused on perioperative decolonization and management of endogenous bacteria that are likely to be introduced into the arthroplasty wound at the time of skin incision.

BACKGROUND:Nontoxigenic Corynebacterium diphtheriae, often associated with wounds, can rarely cause infective endocarditis (IE). Five patients with C. diphtheriae IE were identified within 12 months at a Seattle-based hospital system. We reviewed prior C. diphtheriae-positive cultures to determine if detections had increased over time and evaluated epidemiologic trends. METHODS:We conducted a formal electronic health record search to identify all patients aged ≥18 years with C. diphtheriae detected in a clinical specimen (ie, wound, blood, sputum) between 1 September 2020 and 1 April 2023. We collected patient demographics, housing status, comorbidities, substance-use history, and level of medical care required at detection. We extracted laboratory data on susceptibilities of C. diphtheriae isolates and on other pathogens detected at the time of C. diphtheriae identification. RESULTS:Between 1 September 2020 and 1 April 2023, 44 patients (median age, 44 years) had a C. diphtheriae-positive clinical culture, with most detections occurring after March 2022. Patients were predominantly male (75%), White (66%), unstably housed (77%), and had a lifetime history of injecting drugs (75%). Most C. diphtheriae-positive cultures were polymicrobial, including wound cultures from 36 (82%) patients and blood cultures from 6 (14%) patients, not mutually exclusive. Thirty-four patients (77%), including all 5 patients with C. diphtheriae IE, required hospital admission for C. diphtheriae or a related condition. Of the 5 patients with IE, 3 died of IE and 1 from COVID-19. CONCLUSIONS:Findings suggest a high-morbidity outbreak disproportionately affecting patients who use substances and are unstably housed.

Whole-genome sequencing (WGS) provides greater resolution than other molecular epidemiology strategies and is emerging as a new gold standard approach for microbial strain typing. The Bruker IR Biotyper is designed as a screening tool to identify bacterial isolates that require WGS to establish accurate relationships, but its performance and utility in nosocomial outbreak investigations have not been thoroughly investigated. Here, we evaluated the IR Biotyper by retrospectively examining isolates tested by WGS during investigations of potential nosocomial transmission events or outbreaks. Ninety-eight clinical isolates from 14 different outbreak investigations were examined: three collections of Acinetobacter baumannii (n = 2, n = 9, n = 5 isolates in each collection), one of Escherichia coli (n = 16), two of Pseudomonas aeruginosa (n = 2 and n = 5), two of Serratia marcescens (n = 9 and n = 7), five of Staphylococcus aureus (n = 8, n = 4, n = 3, n = 3, n = 17), and one of Stenotrophomonas maltophilia (n = 8). Linear regression demonstrated a weak, positive correlation between the number of pairwise genome-wide single-nucleotide polymorphisms (SNPs) and IR Biotyper spectral distance values for Gram-positive (r = 0.43, P ≤ 0.0001), Gram-negative (r = 0.1554, P = 0.0639), and all organisms combined (r = 0.342, P ≤ 0.0001). Overall, the IR Biotyper had a positive predictive value (PPV) of 55.81% for identifying strains that were closely related by genomic identity, but a negative predictive value (NPV) of 86.79% for identifying unrelated isolates. When experimentally adjusted cut-offs were applied to A. baumannii, P. aeruginosa, and E. coli, the PPV was 62% for identifying strains that were closely related and the NPV was 100% for identifying unrelated isolates. Implementation of the IR Biotyper as a screening tool in this cohort would have reduced the number of Gram-negative isolates requiring further WGS analysis by 50% and would reduce the number of S. aureus isolates needing WGS resolution by 48%.

Molecular assays for fungal pathogen detection directly in patient specimens represent valuable diagnostic tools, with the potential to increase sensitivity and reduce turnaround time. This review highlights existing and emerging molecular methods for the diagnosis of fungal infections, their clinical performance, limitations, and knowledge gaps. Broad-range PCR and metagenomic sequencing detect diverse pathogens but have not replaced traditional methods. Limitations are cost, availability, and limited real-world performance data. Multi-target panels and organism-specific assays can provide increased sensitivity and, frequently, speed. Some single- or multi-target assays are commercially available to many clinical laboratories. Molecular diagnostic methods for fungal pathogens are diverse; complement culture, stains, and biomarkers; and increasingly fulfill case definitions. To maximize yield, assay selection should be guided by suspected organisms and patient-specific risk factors. Collaboration between patient-facing providers and laboratorians/pathologists promotes optimal testing strategies and the deployment of more rapid and specific assays.

Background Shigella spp have been associated with community-wide outbreaks in urban settings. We analysed a sustained shigellosis outbreak in Seattle, WA, USA, to understand its origins and mechanisms of antimicrobial resistance, define ongoing transmission patterns, and optimise strategies for treatment and infection control. Methods We did a retrospective study of all Shigella isolates identified from stool samples at the clinical laboratories at Harborview Medical Center and University of Washington Medical Center (Seattle, WA, USA) from May 1, 2017, to Feb 28, 2022. We characterised isolates by species identification, phenotypic susceptibility testing, and whole-genome sequencing. Demographic characteristics and clinical outcomes of the patients were retrospectively examined. Findings 171 cases of shigellosis were included. 78 (46%) patients were men who have sex with men (MSM), and 88 (52%) were people experiencing homelessness (PEH). Although 84 (51%) isolates were multidrug resistant, 100 (70%) of 143 patients with data on antimicrobial therapy received appropriate empirical therapy. Phylogenomic analysis identified sequential outbreaks of multiple distinct lineages of Shigella flexneri and Shigella sonnei. Discrete clonal lineages (ten in S flexneri and nine in S sonnei) and resistance traits were responsible for infection in different at-risk populations (ie, MSM, PEH), enabling development of effective guidelines for empirical treatment. The most prevalent lineage in Seattle was probably introduced to Washington State via international travel, with subsequent domestic transmission between at-risk groups. Interpretation An outbreak in Seattle was driven by parallel emergence of multidrug-resistant strains involving international transmission networks and domestic transmission between at-risk populations. Genomic analysis elucidated not only outbreak origin, but directed optimal approaches to testing, treatment, and public health response. Rapid diagnostics combined with detailed knowledge of local epidemiology can enable high rates of appropriate empirical therapy even in multidrug-resistant infection.

Introduction: Tebipenem is a potential option for the treatment of a range of infections because of its oral dosing coupled with the safety profile of the beta-lactam antimicrobial class.Objectives: To evaluate tebipenem in vitro activity against a challenge set of clinical Enterobacterales collected from outpatient and community settings.Methods: 618 Enterobacterales isolates were submitted by 11 geographically dispersed U.S medical cen-ters that processed cultures from affiliated outpatient centers in 2022. Susceptibility tests for tebipenem and comparator agents were performed by broth microdilution. Extended-spectrum-beta-lactamase (ESBL)-like isolates were identified phenotypically. Multidrug-resistant isolates were non-susceptible to >= 1 agent in >= 3 antimicrobial classes. Genotypic testing (CarbaR) was conducted on select isolates.Results: Isolates (59% Escherichia coli ) were recovered from patients seen predominantly in urol-ogy/nephrology (24%), nursing home/long-term care (21%), and ambulatory/primary care (21%) clinics. Comparator agent susceptibility rates against all isolates were as follows: levofloxacin (67.5%), amox-icillin/clavulanate (73.6%), cefixime (70.4%), cefpodoxime (70%), cephalexin (61.7%), ceftriaxone (74.4%), cefazolin (63.8%), ertapenem (97.6%), meropenem (99.7%), nitrofurantoin (64.9%), and sulfamethoxa-zole/trimethoprim (70.9%). Overall, 90.3% (558/619) of isolates were inhibited at a tebipenem MIC of <= 0.125 mg/L (MIC50/90, 0.016/0.125 mg/L), including 85.7% inhibition of ESBL-phenotype isolates (n = 161; MIC50/90, 0.03/0.25 mg/L), 86.3% of levofloxacin and sulfamethoxazole/trimethoprim co-resistant isolates (n = 95; MIC50/90, 0.016/0.25 mg/L) and 84.3% of multidrug-resistant isolates (n = 172; MIC50/90, 0.03/0.25 mg/L). Carbapenemase genes were observed in 2 ESBL-phenotype isolates with a tebipenem MIC of >= 0.5 mg/L.

AbstractMolecular testing for infectious diseases is generally both very sensitive and specific. Well-designed PCR primers rarely cross-react with other analytes, and specificities seen during test validation are often 100%. However, analytical specificities measured during validation may not reflect real-world performance across the entire testing process. Here, we use the unique environment of SARS-CoV-2 screening among otherwise well individuals to examine the false positivity rate of high throughput so-called “sample-to-answer” nucleic acid amplification testing (NAAT) on three commercial assays: the Hologic Panther Fusion®, Hologic Aptima® transcription mediated amplification (TMA), and Roche cobas® 6800. We used repetitive sampling of the same person as the gold standard to determine test specificity rather than retesting of the same sample. We examined 451 people repetitively sampled over 7 months via nasal swab, comprising 7,242 results. During the study period there were twelve positive tests (0.17%) from 9 people. Eight positive tests (0.11%, five individuals) were considered bona fide true positives based on repeat positives or outside testing and epidemiological data. One positive test had no follow-up testing or metadata and could not be adjudicated. Three positive tests (three individuals) did not repeat as positive on a subsequent collection, nor did the original positive specimen test positive on an orthogonal platform. We consider these three tests false positives and estimate the overall false positive rate of high-throughput automated, sample-to-answer NAAT testing to be approximately 0.041% (3/7242). These data help laboratorians, epidemiologists, and regulators understand specificity and positive predictive value associated with high-throughput NAAT testing.

To maintain their Clinical Laboratory Improvement Amendment (CLIA) accreditation, clinical laboratories are expected to enroll in proficiency testing (PT) to confirm that all parts of the diagnostic testing process perform as expected ([1][1]). In recent years, an increasing number of clinical

OBJECTIVES:The first coronavirus disease 2019 (COVID-19) case in the United States was reported in Washington State. The pandemic caused drastic disruptions to medical institutions, including medical education. The Department of Laboratory Medicine at the University of Washington responded by rapidly implementing substantial changes to medical student clerkships.METHODS:In real time, we converted one ongoing case- and didactic-based course, LabM 685, to remote learning.RESULTS:Fifteen of 17 scheduled sessions proceeded as planned, including two sessions for student presentations. Two didactics were canceled as the functions of the teleconferencing platform were not sufficient to proceed. One grand rounds speaker canceled due to COVID-19 precautions. Elements of an immersive clinical laboratory clerkship, LabM 680, were repurposed to accommodate 40 medical students per class via remote learning, highlighting clinical laboratory activities that continue throughout the outbreak. A new remote clerkship, MedSci 585C, was developed incorporating distance learning and guided small-group sessions. This coincided with parallel efforts to make resident and fellow service work, conferences, and didactics available remotely to comply with social distancing.CONCLUSIONS:The changes in medical education described reflect the dynamic interplay of current events affecting the world of clinical pathology. Throughout this, technology-while with some limitations-has provided the platform for innovative learning.

More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test. Whole genome sequencing of the specimen uncovered two mutations, T205I and D399N, present in the nucleocapsid protein of the isolate. All six SARS-CoV-2 positive clinical specimens available in our laboratory with a D399N nucleocapsid mutation and CT < 31 were not detected by the Sofia 2 but detected by the Abbott BinaxNOW COVID-19 Ag Card, while clinical specimens with the T205I mutation were detected by both assays. Testing of recombinant SARS-CoV-2 nucleocapsid with these variants demonstrated an approximate 1000-fold loss in sensitivity for the Quidel Sofia SARS Antigen FIA test associated with the D399N mutation, while the BinaxNOW and Quidel Quickvue SARS Antigen tests were unaffected by the mutation. The D399N nucleocapsid mutation has been relatively uncommon to date, appearing in only 0.02% of genomes worldwide at time of writing. Our results demonstrate how routine pathogen genomics can be integrated into the clinical microbiology laboratory to investigate diagnostic edge cases, as well as the importance of profiling antigenic diversity outside of the spike protein for SARS-CoV-2 diagnostics.

Background Carbapenemase-producing, carbapenem-resistant Pseudomonas aeruginosa (CP-CRPA) is a global challenge. However, detection efforts can be laborious because numerous mechanisms produce carbapenem resistance. A minimum inhibitory concentration-based algorithm (imipenem- or meropenem-resistant plus ceftazidime-nonsusceptible plus cefepime-nonsusceptible) was proposed to identify the isolates most likely to harbor a carbapenemase; however, prospective validation in geographies displaying genotypic diversity and varied carbapenemase prevalence is warranted. Methods CRPA isolates were collected during the Enhancing Rational Antimicrobials for P. aeruginosa (ERACE-PA) global surveillance program from 17 sites in 12 countries. Isolates underwent susceptibility testing following local standards to ceftazidime, cefepime, and ceftolozane/tazobactam. Isolates underwent initial phenotypic carbapenemase screening followed by molecular testing if positive. The primary algorithm criteria were applied, and results were compared with phenotypic carbapenemase results to assess the performance of the algorithm. A secondary criterion, the algorithm criterion or imipenem- or meropenem-resistant plus ceftolozane/tazobactam-nonsusceptible, was assessed. Results A total of 807 CRPA were assessed, and 464 isolates met the algorithm criteria described above. Overall, testing was reduced by 43% compared with testing all CRPA. Carbapenemase-positive isolates missed by the algorithm were largely driven by Guiana extended spectrum (GES). Addition of the criterion of imipenem- or meropenem-resistant plus ceftolozane/tazobactam-nonsusceptible decreased the number of CP-CRPA missed by the algorithm (21 vs 40 isolates, respectively), reducing number of isolates tested by 39%. Conclusions Application of the initial algorithm (imipenem- or meropenem-resistant plus ceftazidime-nonsusceptible plus cefepime-nonsusceptible) performed well in a global cohort, with 33% phenotypically carbapenemase-positive isolates. The addition of imipenem- or meropenem-resistant plus ceftolozane/tazobactam-nonsusceptible reduced the number of phenotypically carbapenemase-positive isolates missed and may be useful in areas with a prominence of GES.