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In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin–angiotensin–aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

An epidemic caused by an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in December 2019 has since rapidly spread internationally, requiring urgent response from the clinical diagnostics community. We present a detailed overview of the clinical validation and implementation of the first laboratory-developed real-time RT-PCR test offered in the NewYork-Presbyterian Hospital system following the Emergency Use Authorization issued by the US Food and Drug Administration. Validation was performed on nasopharyngeal and sputum specimens (n = 174) using newly designed dual-target real-time RT-PCR (altona RealStar SARS-CoV-2 Reagent) for detecting SARS-CoV-2 in upper respiratory tract and lower respiratory tract specimens. Accuracy testing demonstrated excellent assay agreement between expected and observed values and comparable diagnostic performance to reference tests. The limit of detection was 2.7 and 23.0 gene copies per reaction for nasopharyngeal and sputum specimens, respectively. Retrospective analysis of 1694 upper respiratory tract specimens from 1571 patients revealed increased positivity in older patients and males compared with females, and an increasing positivity rate from approximately 20% at the start of testing to 50% at the end of testing 3 weeks later. Herein, we demonstrate that the assay accurately and sensitively identifies SARS-CoV-2 in multiple specimen types in the clinical setting and summarize clinical data from early in the epidemic in New York City.

An epidemic caused by an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in December 2019 has since rapidly spread internationally, requiring urgent response from the clinical diagnostics community. We present a detailed overview of the clinical validation and implementation of the first laboratory-developed real-time reverse-transcription-PCR (rRT-PCR) test offered in the NewYork-Presbyterian Hospital system following the emergency use authority (EUA) guidance issued by the US Food and Drug Administration. Validation was performed on nasopharyngeal and sputum specimens (n=124) using newly designed dual-target rRT-PCR (altona RealStar® SARS-CoV-2 Reagent) for detecting of SARS-CoV-2 in upper respiratory and lower respiratory tract specimens, including bronchoalveolar lavage and tracheal aspirates. Accuracy testing demonstrated excellent assay agreement between expected and observed values. The limit of detection (LOD) was 2.7 and 23.0 gene copies/reaction for nasopharyngeal and sputum specimens, respectively. Retrospective analysis of 1,694 tests from 1,571 patients revealed increased positivity in older patients and males compared to females, and an increasing positivity rate from approximately 20% at the start of testing to 50% at the end of testing three weeks later. Our findings demonstrate that the assay accurately and sensitively identifies SARS-CoV-2 in multiple specimen types in the clinical setting and summarizes clinical data from early in the epidemic in New York City.

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin–angiotensin–aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies., Here, using clinical samples and autopsy tissues, the authors combine fast-colorimetric test (LAMP) for SARS-CoV-2 infection and large-scale shotgun metatranscriptomics for host, viral, and microbial profiling and provide a map of the viral genetic features of the New York City outbreak and associate specific host responses and gene expression perturbations with SARS-CoV-2 infection.

Objectives Viral load testing for BK virus (BKV) has become the standard of care for diagnosing BKV infection and monitoring therapy in kidney transplant patients. However, there are currently no US Food and Drug Administration–approved assays and no standardization among available tests. Methods This study evaluated the performance of the analyte-specific reagent (ASR) BKV primers r-gene and probe r-gene reagents (bioMerieux, Marcy l’Etoile, France) soon to become available on the US market for accuracy, linearity, precision, analytical sensitivity, specificity, and correlation with the Qiagen (Germantown, MD) BKV ASR test using commercial material and patient plasma samples. Results The assay was linear from 204 to 3.92 million (2.31–6.6 log10) DNA copies/mL (coefficient of determination: R 2 =0.999). A dilution series demonstrated limits of detection and quantitation of 2.14 log10 and 2.30 log10 copies/mL (95% hit rate detection), respectively. Interrun precision was highly reproducible, with coefficients of variance ranging from 2.2% to 6.0%. A comparison of 34 matched samples showed a good agreement ( R 2 = 0.87) between the bioMerieux BKV laboratory test and the Qiagen BKV ASR assay results, with an average negative bias (−0.28 log10 copies/mL). Conclusions The laboratory-developed test with bioMerieux BKV reagents is a reliable and sensitive assay for BKV DNA quantitation compared with the Qiagen ASR test.

This study evaluated the performance of the Altona Diagnostics RealStar® Adenovirus Research Use Only (RUO) real-time PCR reagents for HAdV quantitation in plasma samples from immunodeficient patients. The assay was linear from 2.30-9.17 log10 copies/mL (coefficient of determination; R2=0.998) with limits of detection and quantification of 2.19 log10 and 2.30 log10 copies/mL (>95% positivity rate), respectively. Assay precision was highly reproducible with coefficients of variance ranging from 0% to 4.7%. A comparison of 66 matched samples showed good agreement (R2=0.845) between the Altona and the reference laboratory assay, with an average negative bias (-0.24 log10 copies/mL). Genotyping analysis demonstrated that HAdV species B and C accounted for 77% of the positive samples. A significant (≥0.9 log10) difference in quantitation between both tests was found for three HAdV types (HAdV types A12, B14 and F41). In conclusion, the Altona RealStar® test is a reliable and sensitive assay for HAdV DNA quantitation.

Background Viral load testing for BK Virus (BKV) has become the standard of care for the diagnosis of infection and monitoring of therapy of kidney transplant patients infected with BKV. However, there are currently no FDA-approved BKV quantification assays and no standardization among available tests. Objective and study design This study evaluated the performance of the Artus BK Virus RG PCR (RUO) assay (QIAGEN) for accuracy, linearity, precision, analytical sensitivity, specificity, and correlation with a referral laboratory test in patient samples. Results Linear regression analysis of the quantitative results demonstrated a linear range of quantification from 192 to 194 million (2.28 to 8.29 log 10 ) DNA copies/mL and a coefficient of determination ( R 2 ) of 0.994. A dilution series demonstrated a limit of detection and a limit of quantification of 2.00 log 10 , and 2.30 log 10 copies/mL (>95% positivity rate), respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.2% to 7.0%. A comparison of 34 matched samples showed a good agreement ( R 2 = 0.983) between the Artus BK test and the referral laboratory results, with an average positive bias (0.39 log 10 copies/mL). Genotyping analysis using large-T antigen sequences demonstrated that 90% of the positive samples were BKV type I, and that there was no significant difference in quantification between the referral laboratory and Artus BK Virus tests. Conclusions The Artus BK Virus RG PCR test is a reliable and sensitive assay for BKV DNA quantification as compared to the referral laboratory test.

OBJECTIVE We sought to develop a score to predict sustained virological response (SVR) in racially diverse HIV/hepatitis C virus (HCV)-coinfected and HCV-monoinfected pegylated interferon/ribavirin-treated patients. METHODS We retrospectively evaluated 374 patients (259 monoinfected and 115 coinfected) treated at a single tertiary care center. The IL28B rs12979860 single nucleotide polymorphism genotyping was performed in 335 patients, and plasma CXCL10 levels were measured by enzyme-linked immunosorbent assay in 171 patients. RESULTS Of the 374 patients, 64.9% were white, 17.2% were African American, 76.5% were HCV genotype 1 infected, and 49.3% had advanced fibrosis. Sustained virological response was achieved by 151 (40.4%) patients, 106 (40.9%) patients monoinfected, and 45 (39.1%) patients coinfected. Patients with IL28B C/C genotype were significantly more likely to achieve an SVR compared with non-C/C genotype patients, but only if they were infected with HCV genotypes 1/4 (59.1% vs 21.1%, P < 0.0001). No significant differences existed in IL28B predictive capacity between coinfected and monoinfected patients. Pretreatment CXCL10 levels were significantly higher in nonresponders, both monoinfected and coinfected, compared with SVR patients (P = 0.0018). Coinfected patients had higher CXCL10 levels compared with monoinfected patients (P = 0.03). The combination of IL28B genotype, pretreatment CXCL10 and HCV RNA levels, and HCV genotype had the best ability to predict treatment response in both patient groups (area under the receiver operating characteristic curve = 0.85). Among all patients, a cutoff score of -0.94 or more had a sensitivity of 0.93 and specificity of 0.59. In coinfected patients, a score of -0.55 or more had sensitivity of 0.81 and specificity of 0.80. CONCLUSIONS IL28B genotype, pretreatment CXCL10, and HCV RNA levels have very good capacity to predict pegylated interferon/ribavirin-treatment outcome in both HIV/HCV coinfected and HCV monoinfected patients.

Background Quantitative HCV RNA testing is considered standard of care for monitoring during treatment of patients infected with HCV. The COBAS ® AmpliPrep/COBAS ® TaqMan ® HCV Test fully automates specimen processing and reaction assembly for HCV viral load testing using reverse transcription and real-time PCR amplification. Objectives The performance of the COBAS ® AmpliPrep/COBAS ® TaqMan ® HCV Test was evaluated in a multi-center study. Study design Typical plasma based specimens were tested for accuracy, analytic range of measurement, reproducibility and genotype specific quantitation. Results Linear regression analysis of the quantitative results demonstrated a linear range of detection from 50 to 5 million (1.7–6.7 log 10 ) IU/mL and a coefficient of determination ( R 2 ) of 0.9948. The precision of the assay was highly reproducible within and between runs and among laboratories with coefficients of variance (CV) ranging from 6.7% to 40.0% across the seven laboratories. A representative sample for each of the six major HCV genotypes demonstrated reproducible quantitation between the seven laboratories . Conclusions The COBAS ® AmpliPrep/COBAS ® TaqMan ® HCV Test is a reliable and sensitive assay for HCV RNA quantitation.

Summary Bacteriophage T7 infection has been studied in Escherichia coli strains showing both increased and decreased ribosome fidelity and in the presence of streptomycin, which stimulates translational misreading, in an effort to determine effects on the apparent programmed translational frameshift that occurs during synthesis of the gene 10 capsid protein. Quantitation of the protein bands from SDS-PAGE failed to detect any significant effects on the amounts of the shifted 10B protein relative to the in-frame 10A protein under all fidelity conditions tested. However, any changes in fidelity conditions led to inhibition of phage morphogenesis in single-step growth experiments, which could not be accounted for by reduced amounts of phage protein synthesis, nor, at least in the case of decreased accuracy, by reduced amounts of phage DNA synthesis. Reduction in phage DNA synthesis did appear to account for a substantial proportion of the reduction in phage yield seen under conditions of increased accuracy. Similar effects of varying ribosomal fidelity on growth were also seen with phage T3, and to a lesser extent with phage T4. The absence of change in the high-frequency T7 gene 10 frameshift differs from earlier reports that ribosomal fidelity affects low-frequency frameshift errors.

We have tested the effect of increased ribosomal fidelity on a modified version of the programmed release factor 2 (RF2) translational frameshift. In the constructs tested, the original UGA codon at the site of the shift was replaced by either of two sense codons, UGG (tryptophan), which allows a frameshift of approximately 13%, or CUG (leucine), which allows a frameshift of only approximately 2%. We confirmed the results of Curran and Yarus [Curran, J. F. & Yarus, M. (1989) J. Mol. Biol. 209, 65-77] in a wild-type ribosomal host, including a reduction of the UGG shift following induction of tRNA(Trp) from a plasmid copy of the tRNA gene. But to our surprise, in a hyperaccurate streptomycin pseudo-dependent host, the UGG frameshift increased to more than 50%. When we added a tRNA(Trp) plasmid to these cells, induction of the tRNA(Trp) gene reduced the shift back to approximately 7%. Messenger RNA levels did not vary greatly under these different induced conditions. Other increased accuracy alleles also showed increased frameshifting with UGG at the frameshift site. All increased accuracy alleles led to slower translation rates, and there appeared to be a proportionality between the extent of reduction of synthesis for the in-frame reporter and the extent of UGG frameshift for the out-of-frame reporter. There were little effects of increased accuracy on the lower level CUG frameshift. However, over-production of the cognate tRNA(1Leu) dramatically reduced even this lower level of shift, despite the fact that tRNA(1Leu) is already the most abundant isoacceptor in Escherichia coli. These results can be rationalized by following the hypothesis of Curran and Yarus as follows: with wild-type ribosomes, limited availability of tRNA(Trp) (about 1% of total tRNA) facilitates a pause at the UGG codon (due to the vacant A site), allowing increased opportunity for ribosome realignment. Excess tRNA(Trp) reduces the time the A site is vacant and thus reduces the frameshift. The slower hyperaccurate ribosomes increase the pause time and thus increase the opportunity for shifting, a process again reversed by increasing the in-frame cognate tRNA(Trp). These data provide strong support for a model in which the extent of ribosome pause time at a programmed frameshift site is a major determinant in the efficiency of the frameshift and in which tRNA availability can be a major influence on this process.

In recent years, a number of “programmed” translational frameshifts have been identified, in which ribosomes, to varying extents, shift their reading frame at specific sites while translating a message. This has been found in diverse species including eucaryotic viruses and bacteria, among others (reviewed in Atkins et al., 1990).