








key: cord-265740-wjdeps3h
authors: Radbel, Jared; Jagpal, Sugeet; Roy, Jason; Brooks, Andrew; Tischfield, Jay; Sheldon, Michael; Bixby, Christian; Witt, Dana; Gennaro, Maria Laura; Horton, Daniel B.; Barrett, Emily S.; Carson, Jeffrey L.; Panettieri, Reynold A.; Blaser, Martin J.
title: Detection of SARS-CoV-2 is comparable in clinical samples preserved in saline or viral transport media
date: 2020-05-13
journal: J Mol Diagn
DOI: 10.1016/j.jmoldx.2020.04.209
sha: 
doc_id: 265740
cord_uid: wjdeps3h

As the COVID-19 pandemic sweeps across the world, the availability of viral transport media (VTM) has become severely limited, contributing to delays in diagnosis and rationing of diagnostic testing. Given that SARS-CoV-2 viral RNA has demonstrated stability, we posited that phosphate buffered saline (PBS) may be a viable transport medium, as an alternative to VTM), for clinical qPCR testing. We assessed the intra- and inter-individual reliability of SARS-CoV-2 qPCR in clinical endotracheal secretion samples transported in VTM or PBS, evaluating the stability of the RT-qPCR signal for three viral targets (N gene, ORF1ab, and S gene) when samples were stored in these media at room temperature for up to 18 hours. We report that using PBS as a transport medium has high intra-and inter-individual reliability, maintains viral stability, and is comparable to VTM in the detection of the three SARS-CoV-2 genes through 18 hours of storage. Our study establishes PBS as a clinically useful medium that can be readily deployed for transporting and short-term preservation of specimens containing SARS-CoV-2. Use of PBS as a transport medium has the potential to increase testing capacity for SARS-CoV-2, aiding more widespread screening and early diagnosis of COVID-19.

In December 2019, a novel coronavirus was recognized as causing a cluster of pneumonia cases in Wuhan, China (1) . The infectious agent, an RNA virus, was termed acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and was identified as the cause of COVID-19, a clinical syndrome manifested by an influenza-like illness that can progress to acute lung injury or acute respiratory distress syndrome (ARDS) with substantial mortality (2) . COVID-19 has affected more than 3.1 million people causing more than 220,000 deaths worldwide (https://coronavirus.jhu.edu/map.html, last accessed April 29, 2020).

SARS-CoV-2 detection using standard testing of upper airway secretions requires a nasopharyngeal (NP) or oropharyngeal (OP) swab that is transported to a clinical laboratory using viral transport media (VTM) (https://www.fda.gov/medical-devices/emergency-situations-medical-devices/faqs-diagnostic-testingsars-cov-2#offeringtests, last accessed April 29 2020). Recently, OP and saliva testing using VTM also were shown to be comparable to NP swabs for detection of the virus (3, 4) . As the COVID-19 pandemic has swept across the world, availability of VTM has become severely limited, impairing local and regional capacity for diagnosis. Since SARS-CoV2 has capped RNA with a 5′ GTP resembling host RNA and that the virus sgRNA manifests remarkable stability (5), we posited that qPCR detection of SARS-CoV-2 in specimens preserved in phosphate buffered saline (PBS), which is readily available, would be comparable to those in VTM. Here, we report that sample preservation in PBS or VTM are comparably effective for the preservation of SARS-CoV-2 in endotracheal secretions.

Source materials. For transport media, we followed procedures outlined in standard references (5) (6) (7) (8) .

We used phosphate buffered saline (PBS), a water-based salt solution containing disodium hydrogen phosphate, sodium chloride, potassium chloride and potassium dihydrogen phosphate, pH 7.2 (Sigma Aldrich, Saint Louis MO). Viral transport media (VTM) was derived according to the Centers for Disease Control and Prevention (CDC) Coronavirus outbreak response (https://www.cdc.gov/coronavirus/2019ncov/downloads/Viral-Transport-Medium.pdf, last accessed April 29, 2020). In brief, a solution with Hanks Balanced Salt Solution (HBSS), heat-inactivated fetal bovine serum (final concentration 2%), gentamicin 100µg /mL and amphotericin B 0.5 µg /mL was prepared and aliquoted into 2 ml screw top vials (6) . Tubes then were stored at 4°C until use.

Experimental Protocols: Respiratory secretions from 16 confirmed COVID-19 positive subjects were collected over a four-day period from an intensive care unit at Robert Wood Johnson University Hospital in New Brunswick NJ, according to a protocol approved by the Rutgers IRB (Protocol # Pro2020000800).

All subjects were mechanically ventilated for acute hypoxemic respiratory failure due to confirmed COVID-19 pneumonia. Specimens were collected into a sterile container via closed circuit, in-line catheter suction of respiratory secretions from the endotracheal tube (ET), as part of routine clinical care. Swabs were then dropped into vials containing PBS or VTM and transported to the RUCDR laboratory for analysis. To test intra-and inter-patient variation in efficacy of detecting SARS-CoV2 from ET-derived samples, three experimental procedures were performed. Eight samples from two subjects (four from each subject) were harvested at the same time and transported in either VTM or PBS. Samples were processed immediately (at 0 hours) or after 2 hours at room temperature (RT). Real time-qPCR (RT-qPCR) was performed on 1 sample from each transport medium at each incubation time (0 or 2 hours) and Ct values for the SARS-CoV-2 nucleocapsid (N), open reading frame 1ab ORF1ab, and spike protein (S) genes were compared; bacteriophage MS2 (MS2) spiked into the samples was used as a positive control. In a parallel experiment, the stability of detection of SARS-CoV2 sgRNA by RT-qPCR in samples transported in PBS and VTM also was examined after remaining at room temperature for time points ranging from 0 to 18 hours. These experiments mimicked field conditions, in which specimens remain in transport at RT for periods up to 18 hours. Twenty samples from each of two subjects were kept for 0, 2, 4, 6 h or overnight at RT in VTM or PBS to mimic these real-world transport condition. Ct values were again compared for the RT-qPCRs for the three viral genes described above across the indicated time points. To examine inter-subject variance, we examined samples from an additional 12 patients whose ET secretions were transported in either PBS or VTM; RT-qPCR was performed immediately on arrival in the lab for these 24 samples, and Ct values were again compared between those transported in VTM or PBS. Statistical analysis. Pearson correlation was used to quantify association between repeated samples within subject, between transport media, and between the three genes. Linear mixed effects models were used to address several of our research questions. These models were fitted separately for each of the 3 genes. The outcomes were the Ct values, while the predictors included the transport media (VTM versus PBS) and hours in storage (0, 2h, 4h, 6h, or 18h). The models included a random intercept for each subject, to account for repeated observations (within-subject correlation). We used lme4 and ggplot2 packages in R for the linear mixed effects models and plots, respectively (https://www.Rproject.org) (9) .

Analysis of repeated samples from the same subject. From two unique subjects, we obtained paired identical swab specimens that were transported in PBS and VTM. A total of 39 samples were analyzed, representing 10 for each subject in PBS and 9 in VTM, and the variation in Ct plotted (Figure 1) . As Next, we used a linear mixed effects model (see Methods) to compare the sensitivity of the two transport media in the timed samples, from 0 h to overnight (18h) storage. For all three genes, there were no significant differences, and in each case, the VTM values trended higher than for PBS. We next evaluated if across individual samples, the values obtained in testing one gene correlated with the results for the other genes. Additionally, we tested whether the choice of transport medium made a difference. In total, for the 13 samples transported in VTM, the pair-wise correlations between the three genes ranged from 0.947 to 0.956. For the 14 samples transported in PBS, the correlations ranged from 0.963 to 0.991. Thus, the results for the three genes were highly correlated independent of transport medium type.

Decay of the viral signal over time for specimens transported in the two media. We next considered whether there was decay in viral signal over time, and whether it differed according to the transport medium used. From the prior analyses, the coefficient of hours of storage was estimated to be negative in the models of all three genes tested (approximately -0.03 for each gene) and was not significantly different from zero (all p-values >0.5). These data indicate that storage at room temperature for up to 18 hours had little effect on the values detected in the RT-qPCRs for the three SARS-CoV-2 genes tested.

However, these models did not include an interaction term for the transport medium used for storage and time, and there could be differences between the media. To assess this possibility, we performed the same analyses as above, but included interactions between hours of storage and transport medium used. For each of the three genes tested, both the main effect of time and the interaction between time and the transport medium were not statistically significant (all p-values >0.5) and estimates were close to 0. Therefore, we did not detect decay in the viral signal or differential decay by transport medium over the time interval studied.

During pandemics, molecular diagnostics are crucial to obtaining accurate and timely data to influence public health policy decisions in real time (10) . However, mounting demand for testing has caused a depletion of the viral transport media needed to perform SARS-CoV-2 pCR testing (11) . Thus, in the midst of the SARS-CoV-2 pandemic, the FDA has allowed laboratories to consider testing alternative transport media (https://www.fda.gov/medical-devices/emergency-situations-medical-devices/faqsdiagnostic-testing-sars-cov-2, last accessed April 29, 2020). Our experiments using clinical samples demonstrate the efficacy of PBS as a transport medium and its applicability to clinically relevant conditions, such as overnight storage at room temperature. First, we determined that SARS-CoV-2 qPCR detection with PBS as a transport medium has high intra-patient reliability. Next, using PBS for transport, we demonstrated strong inter-patient reliability of SARS-CoV-2 qPCR. We also found strong correlation of Ct values from specimens transported in either PBS or VTM across multiple subjects with unknown viral loads. These results establish PBS as a dependable transport medium for use with clinical samples. Our data are consistent with the recent demonstration that PBS is equivalent to VTM when each medium is spiked with known quantities of SARS-CoV-2 (12) .

With little decay in signal over storage times up to 18h, PBS also has utility for laboratories that test for several SARS-Cov-2 genes that have different specimen processing times. Testing can focus on any or all of the four SARS-Cov-2 structural proteins, including the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins, or on any of their protein domains (13) . That results for all three viral genes tested were strongly correlated across samples from multiple subjects, support the robustness of the entire testing pathway, including transport. Furthermore, delays in getting samples to the testing lab often occur in busy clinical settings (11) . As such, our findings that PBS acts as a stable storage medium with lack of significant viral decay for up to 18 h at room temperature prior to qPCR is advantageous.

Our study is limited in that we used tracheal secretions from mechanically ventilated patients, and we do not know the extent to which our results can be extended to NP, OP swabs and/or saliva testing.

Given the severity of illness in our subjects, they may have had higher viral loads than patients with milder disease in whom increased sensitivity of detection may be needed. However, the stability of the signal, with minimal changes in intensity for 18 hours, indicates the robustness of the methodology. The stability of SARS-CoV-2 in the environment (5) , which contributes to its widespread dissemination, may diminish the need for rapid transport of clinical specimens.

The extent to which clinical laboratories can respond to the COVID-19 pandemic is tied to the ability to develop and deploy proper diagnostic procedures (10) . Early SARS-CoV-2 detection allows prompt treatment of infected patients and rapid implementation of control measures to limit viral transmission (14) . Expanded testing capacity would also facilitate more widespread surveillance and containment of infectious transmission in communities, which could support policies to relax restrictions in work, travel, and social distancing. Our study establishes PBS as a clinically useful transport medium with the potential to increase viral detection capacity, thus improving clinical care and surveillance efforts. 

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Strom, and Helmut Zarbl. We also wish to thank Marten/UPS and dfYoung for in-kind logistics support.Finally, we thank our courageous colleagues serving on the frontlines in the clinic.

