Testing for SARS-CoV-2: lessons learned and current use cases.

SUMMARYThe emergence and worldwide dissemination of SARS-CoV-2 required both urgent development of new diagnostic tests and expansion of diagnostic testing capacity on an unprecedented scale. The rapid evolution of technologies that allowed testing to move out of traditional laboratories and into point-of-care testing centers and the home transformed the diagnostic landscape. Four years later, with the end of the formal public health emergency but continued global circulation of the virus, it is important to take a fresh look at available SARS-CoV-2 testing technologies and consider how they should be used going forward. This review considers current use case scenarios for SARS-CoV-2 antigen, nucleic acid amplification, and immunologic tests, incorporating the latest evidence for analytical/clinical performance characteristics and advantages/limitations for each test type to inform current debates about how tests should or should not be used.

Cellulitis and bacteremia caused by the fish pathogen,Streptococcus iniae, in an immunocompromised patient: Case report and mini-review of zoonotic disease, lab identification, and antimicrobial susceptibility.

Streptococcus iniae is a fish pathogen that can also infect mammals including dolphins and humans. Its prevalence in farmed fish, particularly tilapia, provides potential for zoonotic infections, as documented by multiple case reports. Systematic clinical data beyond cellulitis for S. iniae infection in humans, including antimicrobial susceptibility data, are unfortunately rare. Here, we present a case of cellulitis progressing to bacteremia caused by Streptococcus iniae in a functionally immunocompromised patient based on CDK4/CDK6 inhibitor and endocrine therapy, and we discuss risk factors, identification, and antimicrobial susceptibility of this rare pathogen.

Evaluation of the Abbott Alinity m STI assay for diagnosis of the primary causes of sexually transmitted infections in the United States.

Objectives: The sexually transmitted infections, Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG), have similar risk factors and symptoms, supporting use of a quadruplex test as an efficient diagnostic modality.We assessed the clinical and analytical performance of the Abbott Alinity m STI assay to detect these pathogens. Design and methods: Urine and genital swabs from 142 patient samples were tested from an adult outpatient population in the Northeast United States. The positive and negative percent agreement for CT, NG, and TV were determined by comparison with the Hologic Panther Aptima assay. The analytical sensitivity was determined through serial dilution of standards for CT, NG, TV, and MG in negative urine and swab matrix. Results: The positive and negative percent agreement of the Alinity m assay in comparison with the Hologic Panther Aptima assay were, respectively: CT [100.0% (90.6-100.0%) and 99.1% (94.8-100.0%)], NG [100.0% (89.6-100.0%) and 99.1% (94.9-100.0%)]; and TV [96.3% (81.7-99.8%) and 99.1% (95.2-100.0%)]. The limits of detection in urine and swab matrix were, respectively: CT ≤ 5, ≤1; NG ≤ 5, ≤5; TV ≤ 0.5, ≤0.5; and MG ≤ 500, ≤250 genome copies/mL. Conclusions: The Alinity m assay demonstrated excellent performance characteristics and identifies CT, NG, and TV accurately compared with a well-established comparator.

Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

A Versatile Nanoluciferase Reporter Reveals Structural Properties Associated with a Highly Efficient, N-Terminal Legionella pneumophila Type IV Secretion Translocation Signal.

Many Gram-negative pathogens rely on type IV secretion systems (T4SS) for infection. One limitation has been the lack of ideal reporters to identify T4SS translocated effectors and study T4SS function. Most reporter systems make use of fusions to reporter proteins, in particular, ß-lactamase (TEM) and calmodulin-dependent adenylate cyclase (CYA), that allow detection of translocated enzymatic activity inside host cells. However, both systems require costly reagents and use complex, multistep procedures for loading host cells with substrate (TEM) or for analysis (CYA). Therefore, we have developed and characterized a novel reporter system using nanoluciferase (NLuc) fusions to address these limitations. Serendipitously, we discovered that Nluc itself is efficiently translocated by Legionella pneumophila T4SS in an IcmSW chaperone-dependent manner via an N-terminal translocation signal. Extensive mutagenesis in the NLuc N terminus suggested the importance of an α-helical domain spanning D5 to V9, as mutations predicted to disrupt this structure, with one exception, were translocation defective. Notably, NLuc was capable of translocating several proteins examined when fused to the N or C terminus, while maintaining robust luciferase activity. In particular, it delivered the split GFP11 fragment into J774 macrophages transfected with GFPopt, thereby resulting in in vivo assembly of superfolder green fluorescent protein (GFP). This provided a bifunctional assay in which translocation could be assayed by fluorescence microplate, confocal microscopy, and/or luciferase assays. We further identified an optimal NLuc substrate which allowed a robust, inexpensive, one-step, high-throughput screening assay to identify T4SS translocation substrates and inhibitors. Taken together, these results indicate that NLuc provides both new insight into and also tools for studying T4SS biology. IMPORTANCE Type IV secretion systems (T4SS) are used by Gram-negative pathogens to coopt host cell function. However, the translocation signals recognized by T4SS are not fully explained by primary amino acid sequence, suggesting yet-to-be-defined contributions of secondary and tertiary structure. Here, we unexpectedly identified nanoluciferase (NLuc) as an efficient IcmSW-dependent translocated T4SS substrate, and we provide extensive mutagenesis data suggesting that the first N-terminal, alpha-helix domain is a critical translocation recognition motif. Notably, most existing reporter systems for studying translocated proteins make use of fusions to reporters to permit detection of translocated enzymatic activity inside the host cell. However, existing systems require extremely costly substrates, complex technical procedures to isolate eukaryotic cytoplasm for analysis, and/or are insensitive. Importantly, we found that NLuc provides a powerful, cost-effective new tool to address these limitations and facilitate high-throughput exploration of secretion system biology.

RND Pump-Mediated Efflux of Amotosalen, a Compound Used in Pathogen Inactivation Technology to Enhance Safety of Blood Transfusion Products, May Compromise Its Gram-Negative Anti-Bacterial Activity.

Pathogen inactivation is a strategy to improve the safety of transfusion products. The only pathogen reduction technology for blood products currently approved in the US utilizes a psoralen compound, called amotosalen, in combination with UVA light to inactivate bacteria, viruses, and protozoa. Psoralens have structural similarity to bacterial multidrug efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through genetic, biophysical, and molecular modeling analysis. The main efflux systems in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa are tripartite resistance-nodulation-cell division (RND) systems, which span the inner and outer membranes of Gram-negative pathogens, and expel antibiotics from the bacterial cytoplasm into the extracellular space. We provide evidence that amotosalen is an efflux substrate for the E. coli AcrAB, Acinetobacter baumannii AdeABC, and P. aeruginosa MexXY RND efflux pumps. Furthermore, we show that the MICs for contemporary Gram-negative bacterial isolates for these species and others in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to identify possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens. IMPORTANCE Pathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by major efflux pumps in E. coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the MICs for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to identify possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

Sars-Cov-2 antigen tests predict infectivity based on viral culture: comparison of antigen, PCR viral load, and viral culture testing on a large sample cohort.

OBJECTIVE: To define the relationship of SARS-CoV-2 antigen, viral load determined by RT-qPCR, and viral culture detection. Presumptively, viral culture can provide a surrogate measure for infectivity of sampled individuals and thereby inform how and where to most appropriately deploy antigen and nucleic acid amplification-based diagnostic testing modalities. METHODS: We compared the antigen testing results from three lateral flow and one microfluidics assay to viral culture detection and viral load determination performed in parallel in up to 189 nasopharyngeal swab samples positive for SARS-CoV-2. Sample viral loads, determined by RT-qPCR, were distributed across the range of viral load values observed in our testing population. RESULTS: Antigen tests were predictive of viral culture positivity, with the LumiraDx microfluidics method showing enhanced sensitivity (90%; 95% CI 83-94%) compared with the BD Veritor (74%, 95% CI 65-81%), CareStart (74%, 95% CI 65-81%) and Oscar Corona (74%, 95% CI 65-82%) lateral flow antigen tests. Antigen and viral culture positivity were also highly correlated with sample viral load, with areas under the receiver operator characteristic curves of 0.94 to 0.97 and 0.92, respectively. A viral load threshold of 100 000 copies/mL was 95% sensitive (95% CI, 90-98%) and 72% specific (95% CI, 60-81%) for predicting viral culture positivity. Adjusting for sample dilution inherent in our study design, sensitivities of antigen tests were ≥95% for detection of viral culture positive samples with viral loads >106 genome copies/mL, although specificity of antigen testing was imperfect. DISCUSSION: Antigen testing results and viral culture were correlated. For culture positive samples, the sensitivity of antigen tests was high at high viral loads that are likely associated with significant infectivity. Therefore, our data provides support for use of antigen testing in ruling out infectivity at the time of sampling.

Rapid pathogen identification and phenotypic antimicrobial susceptibility directly from urine specimens.

Implementing effective antimicrobial therapy close to the onset of infection lowers morbidity and mortality and attenuates the spread of antimicrobial resistance. Current antimicrobial susceptibility testing (AST) methods, however, require several days to determine optimal therapies. We present technology and an automated platform that identify (ID) Urinary Tract Infection pathogens in 45 min and provide phenotypic AST results in less than 5 h from urine specimens without colony isolation. The ID and AST tests count cells fluorescently labeled with specific rRNA probes using non-magnified digital imaging. The ID test detected five pathogens at ≤ 7,000 CFU/mL and had a linear range of ~ 4 orders of magnitude. For contrived specimens, AST tests gave 93.1% categorical agreement with 1.3% Very Major Errors (VME), 0.3% Major Errors (ME), and 6.3% minor Errors (mE) compared to the broth microdilution (BMD) reference method. For clinical specimens, the ID test had 98.6% agreement and the AST test had 92.3% categorical agreement with 4.2% mE, 3.4% ME and 4.0% VME compared to BMD. Data presented demonstrates that direct-from-specimen AST tests can accurately determine antimicrobial susceptibility/resistance for each pathogen in a specimen containing two pathogens. The method is robust to urine matrix effects and off-target commensal and contaminating bacteria.

Proceedings of the Clinical Microbiology Open 2018 and 2019 - a Discussion about Emerging Trends, Challenges, and the Future of Clinical Microbiology.

Clinical Microbiology Open (CMO), a meeting supported by the American Society for Microbiology's Clinical and Public Health Microbiology Committee (CPHMC) and Corporate Council, provides a unique interactive platform for leaders from diagnostic microbiology laboratories, industry, and federal agencies to discuss the current and future state of the clinical microbiology laboratory. The purpose is to leverage the group's diverse views and expertise to address critical challenges, and discuss potential collaborative opportunities for diagnostic microbiology, through the utilization of varied resources. The first and second CMO meetings were held in 2018 and 2019, respectively. Discussions were focused on the diagnostic potential of innovative technologies and laboratory diagnostic stewardship, including expansion of next-generation sequencing into clinical diagnostics, improvement and advancement of molecular diagnostics, emerging diagnostics, including rapid antimicrobial susceptibility and point of care testing (POCT), harnessing big data through artificial intelligence, and staffing in the clinical microbiology laboratory. Shortly after CMO 2019, the coronavirus disease 2019 (COVID-19) pandemic further highlighted the need for the diagnostic microbiology community to work together to utilize and expand on resources to respond to the pandemic. The issues, challenges, and potential collaborative efforts discussed during the past two CMO meetings proved critical in addressing the COVID-19 response by diagnostic laboratories, industry partners, and federal organizations. Planning for a third CMO (CMO 2022) is underway and will transition from a discussion-based meeting to an action-based meeting. The primary focus will be to reflect on the lessons learned from the COVID-19 pandemic and better prepare for future pandemics.

The convergent total synthesis and antibacterial profile of the natural product streptothricin F.

A convergent, diversity-enabling total synthesis of the natural product streptothricin F has been achieved. Herein, we describe the potent antimicrobial activity of streptothricin F and highlight the importance of a total synthesis that allows for the installation of practical divergent steps for medicinal chemistry exploits. Key features of our synthesis include a Burgess reagent-mediated 1,2-anti-diamine installation, diastereoselective azidation of a lactam enolate, and a mercury(ii) chloride-mediated desulfurization-guanidination. The development of this chemistry enables the synthesis and structure-activity studies of streptothricin F analogs.

Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms. METHODS: To overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years. RESULTS: Our CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment. CONCLUSIONS: Collectively, the framework we developed provides a clearer, high-resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

Transcriptomics Reveals How Minocycline-Colistin Synergy Overcomes Antibiotic Resistance in Multidrug-Resistant Klebsiella pneumoniae.

Multidrug-resistant Gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using transcriptome sequencing (RNA-Seq), we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline.

Verification of the Abbott Alinity m Resp-4-Plex assay for detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus.

COVID-19 symptomology may overlap with other circulating respiratory viruses that may also cause severe disease and for which there are specific and potentially life-saving treatments. The Abbott Alinity m Resp-4-Plex assay is a multiplex PCR assay that simultaneously detects and differentiates infection with SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV). We characterized its accuracy, precision, and analytical sensitivity. All were found to be robust for measures examined. In the context of sample-to-answer, near random access automation on the Alinity m platform, we believe that the Resp-4-Plex assay offers significant utility in addressing the current needs of the SARS-CoV-2 pandemic and future needs during anticipated endemic circulation of SARS-CoV-2 with other respiratory viruses.

Saliva is Comparable to Nasopharyngeal Swabs for Molecular Detection of SARS-CoV-2.

The continued need for molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the potential for self-collected saliva as an alternative to nasopharyngeal (NP) swabs for sample acquisition led us to compare saliva to NP swabs in an outpatient setting without restrictions to avoid food, drink, smoking, or tooth-brushing. A total of 385 pairs of NP and saliva specimens were obtained, the majority from individuals presenting for initial evaluation, and were tested on two high-sensitivity reverse transcriptase PCR (RT-PCR) platforms, the Abbott m2000 and Abbott Alinity m (both with limits of detection [LoD] of 100 copies of viral RNA/ml). Concordance between saliva and NP swabs was excellent overall (Cohen's κ = 0.93) for both initial and follow-up testing, for both platforms, and for specimens treated with guanidinium transport medium as preservative as well as for untreated saliva (κ = 0.88 to 0.95). Viral loads were on average 16× higher in NP specimens than saliva specimens, suggesting that only the relatively small fraction of outpatients (∼8% in this study) who present with very low viral loads (<1,600 copies/ml from NP swabs) would be missed by testing saliva instead of NP swabs when using sensitive testing platforms. Special attention was necessary to ensure leak-resistant specimen collection and transport. The advantages of self-collection of saliva, without behavioral restrictions, will likely outweigh a minor potential decrease in clinical sensitivity in individuals less likely to pose an infectious risk to others for many real-world scenarios, especially for initial testing. IMPORTANCE In general, the most accurate COVID-19 testing is hands-on and uncomfortable, requiring trained staff and a "brain-tickling" nasopharyngeal swab. Saliva would be much easier on both fronts, since patients could collect it themselves, and it is after all just spit. However, despite much interest, it remains unclear how well saliva performs in real-world settings when just using it in place of an NP swab without elaborate or cumbersome restrictions about not eating/drinking before testing, etc. Also, almost all studies of COVID-19 testing, whether of NP swabs, saliva, or otherwise, have been restricted to reporting results in the abstruse units of "CT values," which only mean something in the context of a specific assay and testing platform. Here, we compared saliva versus NP swabs in a real-world setting without restriction and report all results in natural units-the amount of virus being shed-showing that saliva is essentially just as good as NP swabs.

Evaluation of the Synergistic Activity of Antibacterial and Antifungal Drugs against Candida auris Using an Inkjet Printer-Assisted Method.

Candida auris is an emerging multidrug-resistant fungal pathogen that spreads readily in health care settings and has caused numerous hospital outbreaks. Very few treatment options exist for C. auris infections. We evaluated the activity of all two-drug combinations of three antifungal agents (amphotericin B, caspofungin, and voriconazole) and two antibacterial agents (minocycline and rifampin) against a collection of 10 C. auris isolates using an automated, inkjet printer-assisted checkerboard array method. Three antibacterial-antifungal combinations (amphotericin B plus rifampin, amphotericin B plus minocycline, and caspofungin plus minocycline) demonstrated synergistic activity by checkerboard array against ≥90% of strains, with fractional inhibitory concentration index (FICI) values of 0.094 to 0.5. The two amphotericin B-containing combinations were also synergistic using the time-kill synergy testing method, with up to a 4.99-log10 decrease in surviving yeast compared to either agent alone. Our results suggest that combinations of antifungal and antibacterial agents provide a promising avenue for treatment of this multidrug-resistant pathogen.

Pyrazole-Thiazole Core-Containing Analogs Exhibit Adjunctive Activity with Meropenem against Carbapenem-Resistant Enterobacteriaceae (CRE).

Pyrazole-thiazole core-containing compound KP-40 and 20 novel derivatives were designed and synthesized through traditional SAR analysis. These molecules displayed adjunctive activity with meropenem against Gram-negative bacteria evidenced by a range of fractional inhibitory concentration (FIC=0.5-0.25) and minimum adjunctive concentration (MAC=128-32 µM) values. Of this series of molecules, four compounds displayed notable adjunctive potential, with FIC and MAC values of 0.25 and 32 µM, respectively. Moreover, the solubility of these compounds was improved to an acceptable range. Further analysis using our "in house" permeation and efflux multi parameter optimization (PEMPO) algorithm revealed key physicochemical properties that may be critical for the development of active Gram-negative antibacterials. Taking PEMPO scores into consideration prior to executing synthesis of analogs may be a simple, yet rapid and effective strategy that can be used in conjunction with traditional SAR approaches to aid in the design of potent Gram-negative antibacterials.

Nasal Swab Performance by Collection Timing, Procedure, and Method of Transport for Patients with SARS-CoV-2.

The urgent need for large-scale diagnostic testing for SARS-CoV-2 has prompted interest in sample collection methods of sufficient sensitivity to replace nasopharynx (NP) sampling. Nasal swab samples are an attractive alternative; however, previous studies have disagreed over how nasal sampling performs relative to NP sampling. Here, we compared nasal versus NP specimens collected by health care workers in a cohort of individuals clinically suspected of COVID-19 as well as SARS-CoV-2 reverse transcription (RT)-PCR-positive outpatients undergoing follow-up. We compared subjects being seen for initial evaluation versus follow-up, two different nasal swab collection protocols, and three different transport conditions, including traditional viral transport media (VTM) and dry swabs, on 307 total study participants. We compared categorical results and viral loads to those from standard NP swabs collected at the same time from the same patients. All testing was performed by RT-PCR on the Abbott SARS-CoV-2 RealTime emergency use authorization (EUA) (limit of detection [LoD], 100 copies viral genomic RNA/ml transport medium). We found low concordance overall, with Cohen's kappa (κ) of 0.49, with high concordance only for subjects with very high viral loads. We found medium concordance for testing at initial presentation (κ = 0.68) and very low concordance for follow-up testing (κ = 0.27). Finally, we show that previous reports of high concordance may have resulted from measurement using assays with sensitivity of ≥1,000 copies/ml. These findings suggest nasal-swab testing be used for situations in which viral load is expected to be high, as we demonstrate that nasal swab testing is likely to miss patients with low viral loads.