key: cord-0871253-8kbzwk9d
authors: Velu, Priya; Craney, Arryn; Ruggiero, Phyllis; Sipley, John; Cong, Lin; Hissong, Erika M.; Loda, Massimo; Westblade, Lars F.; Cushing, Melissa; Rennert, Hanna
title: Rapid implementation of SARS-CoV-2 emergency use authorization RT-PCR testing and experience at an academic medical institution
date: 2020-12-05
journal: J Mol Diagn
DOI: 10.1016/j.jmoldx.2020.10.019
sha: abbbdb7423244ebe4c4aa84ed94b7a92ed36927b
doc_id: 871253
cord_uid: 8kbzwk9d

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 (NYPH) system following the emergency use authorization (EUA) issued by the US Food and Drug Administration. Validation was performed on nasopharyngeal and sputum specimens (n=174) using newly designed dual-target rRT-PCR (altona RealStar SARS-CoV-2 Reagent) for detecting SARS-CoV-2 in upper respiratory (URT) and lower respiratory tract (LRT) specimens. Accuracy testing demonstrated excellent assay agreement between expected and observed values and comparable diagnostic performance to reference tests. 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 URT specimens 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. Here 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.

The novel coronavirus SARS-CoV-2 is a member of the Betacoronavirus genera in the subfamily Coronavirinae, which are known to cause respiratory illness and gastroenteritis in humans 1, 2 . An outbreak of respiratory disease caused by SARS-CoV-2, first detected in Wuhan, China in December 2019, rapidly spread to other countries, including the United States 3, 4 . New York City in particular became an epicenter of the pandemic 5 . Given the devastating impact on the healthcare system and the need for accurate and quick diagnosis of SARS-CoV-2 infection the Food and Drug Administration (FDA) has established a rapid pathway for using laboratorydeveloped tests (LDTs) that was outlined in a guidance document published on The scale of demand for diagnostic testing and the shortage of supplies led to the need for high throughput testing that could be readily implemented in a variety of laboratories.

Here we describe the validation and implementation of an EUA test in respiratory including nasopharyngeal (NP) and sputum specimens using research use only (RUO)

RealStar SARS-CoV-2 Reagent (Altona Diagnostics). We also detail workflow considerations and results using the test from the early days of the COVID-19 outbreak in New York City (March 11, 2020, through March 31, 2020) for upper tract respiratory (URT) specimens and after the peak number of cases (April 17, 2020 to May 15, 2020) for lower respiratory (LRT) specimens. 

The FDA EUA guidance specified four distinct performance characteristics consisting of limit of detection (LOD), inclusivity (analytical sensitivity), cross-reactivity (analytical specificity), and clinical evaluation. For the LOD studies, six 10-fold serial dilutions (1x10 1 to 10 7 ) were performed with three replicates at each concentration by spiking J o u r n a l P r e -p r o o f WRCEVA RNA (6x10 5 pfu/µL stock WRCEVA RNA reference material; ~6x10 7 genomic copies/µL) into NP and sputum RNA eluates obtained from pooled-negative patient NP or sputum specimens 6 . LOD was confirmed with 20 additional replicates for each type of sample (sputum and NP). For the accuracy studies, a total of 104 positive (54 NP and 60 sputum) and 70 negative (40 NP swabs and 30 sputum) specimens were used.

Positive specimens were either real patient specimens tested by orthogonal methods, or contrived specimens that were created by spiking WRCEVA RNA into pooled negative NP or sputum specimens RNA eluates, as described above. Twenty of the contrived clinical specimens were spiked at a concentration of 1x-2x LOD, with the remainder of samples spanning the assay testing range. FDA guidance defined the acceptance criteria for test performance as 95% agreement at 1x-2x LOD, and 100% agreement at all other concentrations and negative specimens 6 . Inclusivity and cross-reactivity studies were performed by Altona Diagnostics. Additional cross-reactivity studies were performed using 10 NP samples that tested positive by the RP2 panel for other coronaviruses defined as high priority pathogens from the same genetic family by the FDA.

Data analyses, including statistics and plot generation, were performed using R programming language v 3.6.0 7 . LOD was determined through a probit regression model using the glm function following CLSI EP17A2E Guidance with Application to Quantitative Molecular Measurement Procedures 8 .

Dilution series studies on pooled negative NP specimens spiked with WRCEVA RNA reference material, with three replicates across a viral range of 1 gene copy/reaction to 1,000,000 gene copies/reaction (1 to 6 log 10 ), demonstrated an accurate and linear response across five logs of detection for NP and four logs of detection for sputum ( Table 1 and Figure 1 ). Probit analysis was applied to the NP data after an additional J o u r n a l P r e -p r o o f five replicates of testing were performed at 0.8, 0.6, 0.5, 0.4, and 0.2 gene copies/reaction, and narrowed the LOD to 2.7 gene copies/reaction at 95% detection rate (Figure 2) . A similar LOD series and probit analysis was performed on sputum at 80, 60, 50, 40, and 20 gene copies/reaction, and showed a lower sensitivity with a LOD of 23.0 gene copies/reaction at 95% detection rate. All additional 20/20 NP and 23/23 sputum replicates tested at respective LODs resulted as positive.

The 

Clinical evaluation studies resulted in the detection of SARS-CoV-2 in all specimens contrived by spiking WRCEVA RNA reference material (n=20) into pooled SARS-CoV-2 negative NP VTM or sputum eluates and all four positive patient samples tested by NYC-DOH ( Table 2 and Supplemental Table S1 ). Figure   S1 ). Similar clinical evaluation studies were performed for sputum specimens considered the most challenging sample type by the FDA, also with 100% concordance ( Table 2 and Supplemental Table S2 ). All archived sputum and NP specimens that tested negative on the RP2 panel, also tested negative on the SARS-CoV-2 rRT-PCR assay ( (Figure 3) . Ct values were not significantly different for the E gene, S gene, and IC targets between positive BAL and tracheal aspirate samples (Supplemental Figure S1 ). The mean number of LRT samples received per day was 7

(range 1-25), which was significantly lower compared to the number of URT specimens tested (mean 85; range . Given the small sample size, additional statistics on clinical cohort characteristics were not calculated for LRT specimens.

Given the increased rate of SARS-CoV-2 infection and the lack of any commercially available tests, the FDA opened a pathway on February 29, 2020 that allowed laboratories to implement laboratory-developed tests to meet this diagnostic need. we were able to complete the validation process within a week, followed by a successful go-live testing day. Comparable evaluation studies given the regulatory requirements can take months to achieve.

We tested a total of 1,694 URT (40% positive) and 141 LRT (25% positive) specimens from 1,571 and 115 patients, respectively. The lower number of LRT compared to URT specimens reflects hospital policy that restricted LRT testing to intubated patients that needed clearance of isolation (two negative NP swabs plus one negative LRT specimen) or to patients with high suspicion for COVID-19 with repeat negative testing by RT-PCR (two negative NP swabs).

In the cohort of patients tested over three weeks by our assay, positive results were seen more frequently in older males compared to younger and female patients, which has been supported by several studies 15, 16 . Post-menopausal women have been reported to have a greater risk of hospitalization compared to non-menopausal women, an effect that has been attributed to the potential protective effect of estrogen 17 . In this study, older women were more likely to test positive for SARS-CoV-2 compared to younger female patients. However, the difference in detection rate between older and younger women was diminished after removing obstetrics patients screened universally regardless of symptoms, highlighting the importance of restricting comparisons of positivity rates to groups of patients subjected to similar selection criteria and warranting the importance of carefully designed studies.

Among the obstetrics patients, only 7% tested positive for SARS-CoV-2, which is similar to the prevalence (13.5%) obtained for women admitted at delivery at other NYPH campuses 18 . We did not see any differences in the number of positive tests by race, but this was early in the epidemic in New York City. The ED likely had more positive tests since patients tend to be more acutely symptomatic there compared to ambulatory clinics. The percentage of positive tests increased steadily and settled at around 50% three weeks into the epidemic, with later testing on other platforms showing daily positivity rates as high as 75-80% as the epidemic reached its peak in specific boroughs (unpublished data). In this study, 13 patients tested positive after initial negative results in the ED, suggesting they had sufficient symptoms to warrant inpatient admission despite negative testing. This conversion may be due to increased viral burden on subsequent days post-infection, or due to better specimen sampling 19 . eluate and (B) sputum specimen eluate. Six ten-fold dilutions were performed starting at 1,000,000 gene copies/reaction and ending at 1 gene copy/reaction. The apparent LOD was between 1 and 10 gene copies/reaction for NP specimens and between 10 and 100 gene copies/reaction for sputum specimens. Table 3 . Summary table of orthogonal testing of specimens on different specimen types and different platforms. Thirty authentic patient NP swab specimens were orthogonally tested using FDA EUA SARS-CoV-2 RT-PCR assays on the Roche cobas 6800 or Cepheid GeneXpert platforms. The reported Ct values are from targets on the S gene/E gene (Altona); Target 1 SARS-CoV-2 specific/Target 2 pan-Sarbecovirus (cobas 6800); and E gene/N2 gene (Cepheid). Additionally, four NP specimens were tested by NYS-DOH EUA assay. Twenty sputum specimens were orthogonally tested at ARUP reference laboratory (Hologic SARS-CoV-2 assay on Panther System). Eighteen of twenty specimens were contrived positive samples obtained by spiking a range of concentrations (1:10 to 1:12,800) from a real patient lower respiratory tract sample confirmed to have SARS-CoV-2 infection by a SARS-CoV-2 EUA assay into pooled leftover negative sputum samples. Two of the samples (#49-50) were authentic patient sputum samples. 

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