key: cord-0855825-5n617kpv
authors: Yilmaz Gulec, Elif; Cesur, Nevra P.; Yesilyurt Fazlioğlu, Gonca; Kazezoğlu, Cemal
title: Effect of different storage conditions on COVID‐19 RT‐PCR results
date: 2021-07-28
journal: J Med Virol
DOI: 10.1002/jmv.27204
sha: 1a5c2bfc2b0450a5925bcbe1a32aa07d38d9bad7
doc_id: 855825
cord_uid: 5n617kpv

Reliable and rapid detection of severe acute respiratory syndrome coronavirus 2 in laboratory setting is critical to control the pandemic. We aimed to an evaluated polymerase chain reaction (PCR) efficiency of nasopharyngeal swabs stored in viral transport medium (VTM) in different temperatures. Ninety swabs taken into VTM were analyzed at the first hour, then divided into two groups with similar numbers of positive and negative samples. Positive samples of each group were also subgrouped according to Fam CT values as low CT (<25), medium CT (25–32), and high CT (32–38) groups. One group was stored at 4°C, while the other was stored at room temperature, PCR analyses were repeated every 24 h for 5 days and on Day 12. There was a total of 30 positive samples (12 low CT, 11 medium CT, and 7 high CT). The CT values of both groups remained unchanged in first 3 days while the CT values of the room temperature group increased after the third day. All of the positive samples remained positive in both groups for the first 5 days. On the 12th day, the total number of positives decreased to 8 in the room temperature group and 11 in the 4°C groups. All the low CT samples remained positive in both groups. In conclusion, it is safe to store positive samples in room temperature for up to 5 days. Only samples with high viral loads remain positive for 12 days, regardless of whether stored at room temperature or 4°C. Negative samples don't turn to invalid if stored in VTM.

In COVID-19 (coronavirus disease 2019), people get infected usually through the airway with respiratory droplets, although infections via contaminated surfaces and close contacts were also shown. Following infection, an incubation period of SARS-CoV-2 is on average 5-6 days up to 14 days. 3 Viral load and the contagiousness of the disease are very high during incubation and the first days of the disease. The symptoms show up mostly with fever, fatigue, myalgia, back pain, cough, loss of taste and smell, and diarrhea. In severe cases, it deteriorates to pneumonia and respiratory failure. 4 On the other hand, asymptomatic cases have been reported with positive polymerase chain reaction (PCR) results depending on their viral loads. Estimates of the proportion of asymptomatic cases range from 8% to 80%. 5 In the clinical practice of COVID-19 fast and reliable diagnosis of the infected people gain importance to limit the spread of the infection. The gold standard of the diagnosis is a positive real-time reverse transcriptase-polymerase chain reaction test (RT-PCR). 6 Various techniques and materials have been developed such as various sampling methods, transport media, test kits, PCR conditions, and storage conditions to avoid false test results. 7 Although the number of molecular virology laboratories increases day by day, the number of infected people is very high and increases in different periods that this number may exceed the capacity of the laboratories. Thus, the need for storing the samples for long periods before RT-PCR could arise. The recommended storage condition for nasopharyngeal swabs is +4°C during transport and it is recommended that RT-PCR be performed as soon as possible. It is stated that the sample can be stored at 4°C or between 2°C and 8°C for up to 4 or 5 days if the swab is in viral transport medium (VTM). 6 Bio-Speedy SARS CoV-2 Double Gene RT-qPCR Kit (version 1) kit was used for PCR; according to kit protocol, 5 µl patient samples with VTM were added to 15 µl ready kit mixture to achieve 20 µl PCR mixture in total. Thermal cycle parameters of RT-PCR amplification were as follows: 52°C for 5 min for reverse transcription, 95°C for 10 s for holding, then 40 cycles of 95°C for 1 s, and 55°C for 30 s for denaturation, annealing, and extension, respectively. 

Statistical analysis was performed using Microsoft Excel version 2019. The correlation coefficient was calculated by t-test; p value was chosen as 0.05. When p value is made meaningful by using descriptive statistics, no significant difference was detected between CT values according to the p < 0.05 results of CT values. (Table 1) . We grouped the positives according to CT values as low (CTs < 25, nr: 12), medium (CTs between 25 and 32, nr: 11), and high (CTs between 32 and 38, nr: 7) ( Table 1) . CT values did not change significantly in the first 3 days in any of the groups, CT values started to increase in samples stored at room temperature on the fourth day ( Table 1) trol and prevention centers) recommends that samples be stored at 2-8°C for up to 4 days, while WHO recommends that they can be stored at 4°C for a maximum of 5 days. 6, 8 VTMs are used to preserve viruses during transport and storage before PCR or virus cultures. 9, 10 They are designed to preserve the virus being dealt with, while preventing contamination with other agents such as bacteria or fungi. In different articles, it was recommended to use dry swabs taken into saline solutions like phosphate-buffered saline or Tris EDTA buffer in the absence or depletion of VTM in pandemics like nowadays. 11, 12 While VTMs are used for the preservation of the viruses they can also be used for virus inactivation and extraction of the viral nucleic acid for PCR.

Our COVID-19 diagnostic center is one of the most high-capacity centers in Turkey. As around the world, at the beginning of the pandemic, the supply of standard VTMs was not sufficient for high demand, and swab samples were transferred to our center as a dry swab or saline solution. In those days, we observed that samples differences between the CT values of positive samples, even when stored in saline, were not more than two amplification cycles and did not turn negative even on the 14th day. 13 In the study of Rogers et al., 13 they observed a maximum increase of two amplification cycles in samples stored at room temperature even on Day 14. In a study with respiratory viruses, the authors detected and isolated viral nucleic acid from a dry nasopharyngeal swab even after 2 weeks. 16 The maximum duration of research for SARS-CoV-2 in the literature was 14 days. 13 In Table 1 , we observed significant differences between the results of the same day or consecutive days for a few samples, although the same expert operators performed the PCR procedures, we think this was due to manual errors.

We chose to analyze samples on two different platforms to evaluate the efficiency of the platforms we use in our routine laboratory. Platforms did not differ significantly over the course of the study.

In conclusion, it is best to store samples in VTM to get the most reliable results and to avoid test and sample repeats due to invalid results. Even under the best conditions where swab samples are taken into VTMs and stored after nucleic acid extraction, samples can only be stored at room temperature for up to 3 days without a reduction in viral load. Positive samples can be stored for at least 5 days at both room temperature and 4°C without loss of positivity.

Samples with high viral loads with CTs below 25 can be stored at both room temperature and 4°C without loss of positivity for up to 12 days, however, samples with lower viral loads with CTs above 25 cannot be stored for 12 days. For samples with CT values above 32, we recommend repeating the sample to avoid misinterpretation of cases.

With this study, we tried to create a guide on under which conditions we can obtain more reliable results in routine laboratory studies of SARS-CoV-2.

The present work was supported by Bioeksen AR-GE teknolojileri Ltd Şti. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Special thanks to Arzu Tanrıverdi and Ayhan Demir for their valuable contribution to this study. We would like to thank all COVID-19

Diagnostic Center staff for their devoted efforts during the entire pandemic period.

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Effect of different storage conditions on COVID-19 RT-PCR results

The authors declare that there are no conflict of interests.

Elif Yilmaz Gulec: Developed the protocol, abstracted and analyzed data, wrote the manuscript, and is the guarantor. Nevra P. Cesur:Contributed to the development of the protocol, methodology, review, and editing. Gonca Yesilyurt Fazlioglu: Contributed to the development of the protocol, methodology, review, and editing.Cemal Kazezoglu: contributed to the development of protocol and methodology.