Validation of saline, PBS and a locally produced VTM at varying storage conditions to detect the SARS-CoV-2 virus by qRT-PCR

Coronavirus Disease-2019 tests require a Nasopharyngeal (NP) and/or Oropharyngeal (OP) specimen from the upper airway, from which virus RNA is extracted and detected through quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR). The viability of the virus is maintained after collection by storing the NP/OP swabs in Viral Transport Media (VTM). We evaluated the performance of four transport media: locally manufactured (“REVITAL”) Viral Transport Media (RVTM), Standard Universal Transport Media (SUTM), PBS and 0.9% (w/v) NaCl (normal saline). We used laboratory cultured virus to evaluate: i) viral recovery and maintaining integrity at different time periods and temperatures; ii) stability in yielding detectable RNA consistently for all time points and conditions; and iii) their overall accuracy. Four vials of SARS-CoV-2 cultured virus (2 high and 2 low concentration samples) and 1 negative control sample were prepared for each media type (SUTM, RVTM, PBS and normal saline) and stored at the following temperatures, -80°C, 4°C, room temperature (25°C) and 37°C for 7 days. Viral Ribonucleic acid (RNA) extractions and qRT-PCR were done on the following days after inoculation with the cultured virus, days 1, 2, 3, 4 and 7 to assess virus stability and viral recovery. CT values fell over time at room temperature, but normal saline, PBS, RVTM and SUTM all showed comparable performance in maintaining virus integrity and stability allowing for the detection of SARS-CoV-2 viral RNA. Overall, this study demonstrated that normal saline, PBS and the locally manufactured VTM can be used for COVID-19 sample collection and testing, thus expanding the range of SARS-CoV-2 viral collection media.

Kenya, like several countries during the COVID-19 pandemic, suffered from disruptions in the 80 supply of reagents required for diagnosis. It was therefore imperative to seek alternatives that 81 would cushion diagnostic centres from unforeseen shortages. Consequently, the present study 82 evaluated the performance of four transport media, i.e., a locally manufactured (i.e. "REVITAL") States). We first generated a10-fold dilution series (viral dilutions 1 to 10, S1 Table)   control) were prepared for each media type and stored at the following temperatures, -80°C, 4°C, 104 room temperature (25°C) and 37°C for 7 days (Fig 1), generating a total of 80 vials. Viral 105 Ribonucleic acid (RNA) extractions and qRT-PCR were done on the following days after 106 inoculation with the cultured virus, days 1, 2, 3, 4 and 7 to assess virus stability and viral recovery. QIAamp Mini Spin Columns. The spin columns were spun at 6,000 x g for 1 minute, the elute was 128 discarded and 500μl of buffer AW1 was added followed by centrifugation at 6,000 x g for 1 minute.
The flowthrough was discarded and 500μl of buffer AW2 was added followed by centrifugation 130 at 16,000 x g for 3 minutes. The elute was discarded and the spin columns allowed to air dry for 5 131 minutes before elution with 60μl buffer AVE.  presentations were produced in R version 3.6.1(13). First, we note that there were 128 negative samples defined as "undetermined" that were replaced with an arbitrary high Ct for negative (i.e., 152 37) to uncover the bias that might occur due to their exclusion during statistical analysis. Temporal

Comparisons between the 4 media types 187
For all temperature levels, media types and days the Ct values were consistently highest in the low 188 concentration samples (between Cts 25 and 28) and they were significantly higher than those for 189 high concentration samples (between Ct 18 and 23) (Tables 1 and 2, Fig 2). The Ct values varied 190 over time in the four temperature levels examined (Table 2). Ct values generally increased over 191 time (day) in the media stored at -80ºC and 4ºC, decreased and remained the same at room 192 temperature and 37ºC, respectively (Fig 2). The multiple linear regression analysis demonstrated 193 there were no statistically significant differences in Ct values across the media types (    qRT-PCR test is reached and taking into consideration the variability between qRT-PCR kits.

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There were some contaminated negative control samples (Ct<36) in <2% of the samples examined.

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The contamination may have occurred during extraction or the compilation of qRT-PCR 259 reagents(18) due to amplicon carry-over given the high number of SARS-CoV-2 amplicons being 260 generated in the lab. It may also be a reflection of the high signal-to-noise ratio at high Ct values. 261 We had mitigated against contamination by incorporating proper disinfection, and use of dedicated 262 working spaces ensuring minimal viral contamination (18). Samples paired with positive negative 263 controls were regarded as unreliable and were removed from further analyses.

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These results based on laboratory cultured samples stored at a range of temperatures suggest that 265 samples swabbed directly from patients could be transported in the different media types examined 266 in this study, given the comparable ability to detect the viral RNA. However, we did not test 267 whether the different media types support viable culturable virus.

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The cultured virus we used may be at higher concentration than samples taken in the field, and 269 storage conditions during transport may be more variable. However, we did not observe evidence 270 of any differences in RNA levels based on the PCR results by VTM, including when virus was 271 stored at room temperature.

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Overall, this study demonstrated that normal saline, PBS and the locally manufactured VTM can 273 be used for COVID-19 sample collection and testing, thus expanding the range of SARS-CoV-2 274 viral collection media. 276 We thank Prof George Warimwe's group from KEMRI-Wellcome Trust Research Programme for 277 the cultured SARS-CoV-2 virus aliquot they kindly provided for use in this study. We thank the 278 KEMRI Director for permission to publish this work.