After COVID-19 declared as a global pandemic, the numbers of suspected cases were increased day today and that needs to maximize laboratory testing capacity by high throughput automated and point of care testing instruments for SARS-CoV-2 detection [
6,
9]. For these types of platforms sample inactivation is an important procedure to protect health care workers from the exposure of SARS-CoV-2 infection [
9]. However, some studies showed that sample inactivation by using heat can leads to RNA degradation and false-negative results [
15,
16]. Therefore, this study was evaluated the effect of heat inactivation at 56 °C for 30 min for SARS-CoV-2 detection. In this study, seven samples (3.7%) had discordant results between those heat-inactivated and without heat-inactivated matched samples. Out of the total discordant samples, 71.4% of samples were double gene negative during the heat-inactivated procedure; but either double or single gene-positive in those without the heat-inactivated procedure. Those discordant samples having with single or double gene-positive were had more than 35 Ct value. A similar study performed in Beijing, China, showed that, the impact of heat inactivation on samples those had high Ct values (33.37–36.89) were high [
16]. This indicates heat inactivation may have a negative impact on decreasing viral load of those samples having Ct values greater than 35. Early identification and detection, early prevention, and control are the currently available method to curbing the rapid spread of COVID-19 infection. However, it may have a negative impact on these measures. Although heat inactivation makes safe for health care workers, false-negative individuals in the community may be transmitted to a wide range of people. On the other hand, the positivity and negativity of the original sample and the heat-inactivated sample result in this study was showed that had no statistically significant difference after a chi-square test (p > 0.05). Similar results were obtained from Renmin Hospital of Wuhan University, China [
17], Zhujiang Hospital, China [
18], and a study conducted in USA on sputum sample [
9] has no significant difference between the non-inactivated and inactivated sample at 56 °C for 30 min for the qualitative detection of SARS-CoV-2. On the contrary, a study conducted in Beijing, China [
16] and Yongchuan District Center for Disease Control and Prevention of Chongqing, China [
19] were indicated that the qualitative detection of SARS-CoV-2 was significantly impacted with heat inactivation at 56 °C for 30 min. This difference might be due to variation in sample size between experiments, type of detection/extraction reagents, sample type and viral strain difference by itself. However, it is difficult to know the impact of this result on the transmission of SARS-CoV-2, as the virus is new, with lots of unknown characteristics and spreading as fast as a forest fire. In this study, the effect of heat inactivation at 56 °C for 30 min on the Ct value of matched samples was analyzed, and the average Ct value difference between non-inactivated and heat-inactivated at 56 °C for 30 min were 0.04 and 0.38 for ORF1a/b gene and N gene respectively. It means that after heat inactivation the ORF1a/b gene and N gene Ct was increased by 0.04 and 0.38 averagely on each sample respectively. Based on paired T-test analysis, the ORF1a/b gene Ct value increment after heat inactivation was not statistically significant (t = 0.28; p > 0.05), whereas the N gene Ct values between inactivated and the non-inactivated group were statistically significant (t = 2.64; p = 0.01). In addition to this, the mean Ct value difference of heat-treated and untreated group sample greater than 30 Ct of ORF1a/b and N gene were 1.26 and 1.00 respectively. These average Ct value increment while heat inactivation was statistically significant in both N and ORF1a/b gene (p < 0.05). Similar studies reported by Pan et al. [
16] and Chen et al. [
15] both were from China and indicated that heat inactivation prior to extraction can significantly reduce the number of RNAcopies (increased Ct values compared to the original sample). This may result in false-negative during heat inactivation procedure and favour viral transmission. However, the studies conducted in Charite University, Berlin, Germany [
13], Renmin Hospital of Wuhan University, Huazhong China [
17], Republic of South Korea [
20] and Zhejiang University School of Medicine, China [
21] revealed that Ct values of heat-inactivated at 56 °C for 30 min group and the non-inactivated group had no statistically significant difference. These difference might be due to sample size, type of sample or and type of strain difference circulating in the community which need additional investigations.
In this experimental study, the correlation between inactivated and non-inactivated samples was also examined. As a result, Pearson correlation analysis was performed and the analysis showed that, the ORF1a/b and N gene Ct values of inactivated sample and non-inactivated sample had excellent correlation (r = 0.978; p < 0.001); and (r = 0.969; p < 0.001) respectively (Figs.
2 and
3). The result reported from the First People’s Hospital of Zhaoqing, Zhao Qing City, China, the Ct value of ORF1a/b gene and N gene were perfectly correlated between inactivated at 56 °C for 30 min and non-inactivated samples [
15].
Our study has some limitations; first, we have used only Oro-pharyngeal swabs, so we cannot conclude for another type of samples; like sputum, saliva, blood and stool samples. Second, we didn’t perform SARS-CoV-2 viral quantification to show the exact viral copy difference between inactivated and non-inactivated sample results. And third, we did not perform viral infectivity analysis because of lack of infrastructure.