The Limit of Detection Matters: The Case for Benchmarking Severe Acute Respiratory Syndrome Coronavirus 2 Testing.

BACKGROUND: Resolving the coronavirus disease 2019 (COVID-19) pandemic requires diagnostic testing to determine which individuals are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard is to perform reverse-transcription polymerase chain reaction (PCR) on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of approximately 100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss infected patients. However, the relative clinical sensitivity of these assays remains unknown. METHODS: Here we model the clinical sensitivities of assays based on their LoD. Cycle threshold (Ct) values were obtained from 4700 first-time positive patients using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization test. We derived viral loads from Ct based on PCR principles and empiric analysis. A sliding scale relationship for predicting clinical sensitivity was developed from analysis of viral load distribution relative to assay LoD. RESULTS: Ct values were reliably repeatable over short time testing windows, providing support for use as a tool to estimate viral load. Viral load was found to be relatively evenly distributed across log10 bins of incremental viral load. Based on these data, each 10-fold increase in LoD is expected to lower assay sensitivity by approximately 13%. CONCLUSIONS: The assay LoD meaningfully impacts clinical performance of SARS-CoV-2 tests. The highest LoDs on the market will miss a majority of infected patients. Assays should therefore be benchmarked against a universal standard to allow cross-comparison of SARS-CoV-2 detection methods.

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.

Visualizing the effects of lactate dehydrogenase (LDH) inhibition and LDH-A genetic ablation in breast and lung cancer with hyperpolarized pyruvate NMR.

In many tumors, cancer cells take up large quantities of glucose and metabolize it into lactate, even in the presence of sufficient oxygen to support oxidative metabolism. It has been hypothesized that this malignant metabolic phenotype supports cancer growth and metastasis, and that reversal of this so-called "Warburg effect" may selectively harm cancer cells. Conversion of glucose to lactate can be reduced by ablation or inhibition of lactate dehydrogenase (LDH), the enzyme responsible for conversion of pyruvate to lactate at the endpoint of glycolysis. Recently developed inhibitors of LDH provide new opportunities to investigate the role of this metabolic pathway in cancer. Here we show that magnetic resonance spectroscopic imaging of hyperpolarized pyruvate and its metabolites in models of breast and lung cancer reveal that inhibition of LDH was readily visualized through reduction in label exchange between pyruvate and lactate, while genetic ablation of the LDH-A isoform alone had smaller effects. During the acute phase of LDH inhibition in breast cancer, no discernible bicarbonate signal was observed and small signals from alanine were unchanged.

Open Development and Clinical Validation of Multiple 3D-Printed Nasopharyngeal Collection Swabs: Rapid Resolution of a Critical COVID-19 Testing Bottleneck.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a severe international shortage of the nasopharyngeal swabs that are required for collection of optimal specimens, creating a critical bottleneck blocking clinical laboratories' ability to perform high-sensitivity virological testing for SARS-CoV-2. To address this crisis, we designed and executed an innovative, cooperative, rapid-response translational-research program that brought together health care workers, manufacturers, and scientists to emergently develop and clinically validate new swabs for immediate mass production by 3D printing. We performed a multistep preclinical evaluation of 160 swab designs and 48 materials from 24 companies, laboratories, and individuals, and we shared results and other feedback via a public data repository (http://github.com/rarnaout/Covidswab/). We validated four prototypes through an institutional review board (IRB)-approved clinical trial that involved 276 outpatient volunteers who presented to our hospital's drive-through testing center with symptoms suspicious for COVID-19. Each participant was swabbed with a reference swab (the control) and a prototype, and SARS-CoV-2 reverse transcriptase PCR (RT-PCR) results were compared. All prototypes displayed excellent concordance with the control (κ = 0.85 to 0.89). Cycle threshold (CT ) values were not significantly different between each prototype and the control, supporting the new swabs' noninferiority (Mann-Whitney U [MWU] test, P > 0.05). Study staff preferred one of the prototypes over the others and preferred the control swab overall. The total time elapsed between identification of the problem and validation of the first prototype was 22 days. Contact information for ordering can be found at http://printedswabs.org Our experience holds lessons for the rapid development, validation, and deployment of new technology for this pandemic and beyond.

PFKFB3-mediated Pro-glycolytic Shift in Hepatocellular Carcinoma Proliferation.

BACKGROUND & AIMS: Metabolic reprogramming, in particular, glycolytic regulation, supports abnormal survival and growth of hepatocellular carcinoma (HCC) and could serve as a therapeutic target. In this study, we sought to identify glycolytic regulators in HCC that could be inhibited to prevent tumor progression and could also be monitored in vivo, with the goal of providing a theragnostic alternative to existing therapies. METHODS: An orthotopic HCC rat model was used. Tumors were stimulated into a high-proliferation state by use of off-target liver ablation and were compared with lower-proliferating controls. We measured in vivo metabolic alteration in tumors before and after stimulation, and between stimulated tumors and control tumors using hyperpolarized 13C magnetic resonance imaging (MRI) (h13C MRI). We compared metabolic alterations detected by h13C MRI to metabolite levels from ex vivo mass spectrometry, mRNA levels of key glycolytic regulators, and histopathology. RESULTS: Glycolytic lactate flux increased within HCC tumors 3 days after tumor stimulation, correlating positively with tumor proliferation as measured with Ki67. This was associated with a shift towards aerobic glycolysis and downregulation of the pentose phosphate pathway detected by mass spectrometry. MRI-measured lactate flux was most closely coupled with PFKFB3 expression and was suppressed with direct inhibition using PFK15. CONCLUSIONS: Inhibition of PFKFB3 prevents glycolytic-mediated HCC proliferation, trackable by in vivo h13C MRI.

Hyperpolarized [1-13C]Pyruvate Magnetic Resonance Spectroscopic Imaging for Evaluation of Early Response to Tyrosine Kinase Inhibition Therapy in Gastric Cancer.

PURPOSE: To evaluate the use of hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging (HP-13C MRSI) for quantitative measurement of early changes in glycolytic metabolism and its ability to predict response to pan-tyrosine kinase inhibitor (Pan-TKI) therapy in gastric cancer (GCa). PROCEDURES: Pan-TKI afatinib-sensitive NCI-N87 and resistant SNU16 human GCa cells were assessed for GLUT1, hexokinase-II (HKII), lactate dehydrogenase (LDHA), phosphorylated AKT (pAKT), and phosphorylated MAPK (pMAPK) at 0-72 h of treatment with 0.1 µM afatinib. Subcutaneous NCI-N87 tumor-bearing nude mice underwent [18F]FDG PET/MRI and HP-13C MRSI at baseline and 4 days after treatment with afatinib 10 mg/kg/day or vehicle (n = 10/group). Changes in PET and HP-13C MRSI metabolic parameters were compared between the two groups. Imaging findings were correlated with tumor growth and histopathology over 3 weeks of treatment. RESULTS: In vitro analysis showed a continuous decrease in LDHA, pAKT, and pMAPK in NCI-N87 compared to SNU16 cells within 72 h of treatment with afatinib, without a significant change in GLUT1 and HKII in either cell type. [18F]FDG PET of NCI-N87 tumors showed no significant change in PET measures at baseline and day 4 of treatment in either treatment group (SUVmean day 4/day 0: 2.7 ± 0.42/2.34 ± 0.38, p = 0.57 in the treated group vs. 1.73 ± 0.66/2.24 ± 0.43, p = 0.4 in the control group). HP-13C MRSI demonstrated significantly decreased lactate-to-pyruvate ratio (L/P) in treated tumors (L/P day 4/day 0: 0.83 ± 0.30/1.10 ± 0.20, p = 0.012 vs. 0.94 ± 0.20/0.98 ± 0.30, p = 0.75, in the treated vs. control group, respectively). Response to afatinib was confirmed with decreased tumor size over 3 weeks (11.10 ± 16.50 vs. 293.00 ± 79.30 mm3, p < 0.001, treated group vs. control group, respectively) and histopathologic evaluation. CONCLUSIONS: HP-13C MRSI is a more representative biomarker of early metabolic changes in response to pan-TKI in GCa than [18F]FDG PET and could be used for early prediction of response to targeted therapies.

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.

SARS-CoV2 Testing: The Limit of Detection Matters.

Resolving the COVID-19 pandemic requires diagnostic testing to determine which individuals are infected and which are not. The current gold standard is to perform RT-PCR on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of ~100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss more infected patients, resulting in more false negatives. However, the false-negative rate for a given LoD remains unknown. Here we address this question using over 27,500 test results for patients from across our healthcare network tested using the Abbott RealTime SARS-CoV-2 EUA. These results suggest that each 10-fold increase in LoD is expected to increase the false negative rate by 13%, missing an additional one in eight infected patients. The highest LoDs on the market will miss a majority of infected patients, with false negative rates as high as 70%. These results suggest that choice of assay has meaningful clinical and epidemiological consequences. The limit of detection matters.

Nasal-Swab Testing Misses Patients with Low SARS-CoV-2 Viral Loads.

The urgent need for large-scale diagnostic testing for SARS-CoV-2 has prompted pursuit of sample-collection methods of sufficient sensitivity to replace sampling of the nasopharynx (NP). Among these alternatives is collection of nasal-swab samples, which can be performed by the patient, avoiding the need for healthcare personnel and personal protective equipment. Previous studies have reached opposing conclusions regarding whether nasal sampling is concordant or discordant with NP. To resolve this disagreement, we compared nasal and NP specimens collected by healthcare workers in a cohort consisting of individuals clinically suspected of COVID-19 and outpatients known to be SARS-CoV-2 RT-PCR positive undergoing follow-up. We investigated three different transport conditions, including traditional viral transport media (VTM) and dry swabs, for each of two different nasal-swab collection protocols on a total of 308 study participants, and compared categorical results and Ct values 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 EUA (limit of detection [LoD], 100 copies viral genomic RNA/mL transport medium). We found high concordance (Cohen's kappa >0.8) only for patients with viral loads above 1,000 copies/mL. Those with viral loads below 1,000 copies/mL, the majority in our cohort, exhibited low concordance (Cohen's kappa = 0.49); most of these would have been missed by nasal testing alone. Previous reports of high concordance may have resulted from use of assays with higher LoD (≥1,000 copies/mL). These findings counsel caution in use of nasal testing in healthcare settings and contact-tracing efforts, as opposed to screening of asymptomatic, low-prevalence, low-risk populations. Nasal testing is an adjunct, not a replacement, for NP.

Open Development and Clinical Validation Of Multiple 3D-Printed Sample-Collection Swabs: Rapid Resolution of a Critical COVID-19 Testing Bottleneck.

The SARS-CoV-2 pandemic has caused a severe international shortage of the nasopharyngeal swabs that are required for collection of optimal specimens, creating a critical bottleneck in the way of high-sensitivity virological testing for COVID-19. To address this crisis, we designed and executed an innovative, radically cooperative, rapid-response translational-research program that brought together healthcare workers, manufacturers, and scientists to emergently develop and clinically validate new swabs for immediate mass production by 3D printing. We performed a rigorous multi-step preclinical evaluation on 160 swab designs and 48 materials from 24 companies, laboratories, and individuals, and shared results and other feedback via a public data repository (http://github.com/rarnaout/Covidswab/). We validated four prototypes through an institutional review board (IRB)-approved clinical trial that involved 276 outpatient volunteers who presented to our hospital's drive-through testing center with symptoms suspicious for COVID-19. Each participant was swabbed with a reference swab (the control) and a prototype, and SARS-CoV-2 reverse-transcriptase polymerase chain reaction (RT-PCR) results were compared. All prototypes displayed excellent concordance with the control (κ=0.85-0.89). Cycle-threshold (Ct) values were not significantly different between each prototype and the control, supporting the new swabs' non-inferiority (Mann-Whitney U [MWU] p>0.05). Study staff preferred one of the prototypes over the others and the control swab overall. The total time elapsed between identification of the problem and validation of the first prototype was 22 days. Contact information for ordering can be found at http://printedswabs.org. Our experience holds lessons for the rapid development, validation, and deployment of new technology for this pandemic and beyond.

A microRNA/Runx1/Runx2 network regulates prostate tumor progression from onset to adenocarcinoma in TRAMP mice.

While decades of research have identified molecular pathways inducing and promoting stages of prostate cancer malignancy, studies addressing dynamic changes of cancer-related regulatory factors in a prostate tumor progression model are limited. Using the TRAMP mouse model of human prostate cancer, we address mechanisms of deregulation for the cancer-associated transcription factors, Runx1 and Runx2 by identifying microRNAs with reciprocal expression changes at six time points during 33 weeks of tumorigenesis. We molecularly define transition stages from PIN lesions to hyperplasia/neoplasia and progression to adenocarcinoma by temporal changes in expression of human prostate cancer markers, including the androgen receptor and tumor suppressors, Nkx3.1 and PTEN. Concomitant activation of PTEN, AR, and Runx factors occurs at early stages. At late stages, PTEN and AR are downregulated, while Runx1 and Runx2 remain elevated. Loss of Runx-targeting microRNAs, miR-23b-5p, miR-139-5p, miR-205-5p, miR-221-3p, miR-375-3p, miR-382-5p, and miR-384-5p, contribute to aberrant Runx expression in prostate tumors. Our studies reveal a Runx/miRNA interaction axis centered on PTEN-PI3K-AKT signaling. This regulatory network translates to mechanistic understanding of prostate tumorigenesis that can be developed for diagnosis and directed therapy.