Diagnosing COVID-19: diagnostic importance of detecting E gene of the SARS-CoV-2 genome

More than 632 million confirmed cases of COVID-19 have been reported by WHO thus far, including more than 6.5 million deaths [1]. Various molecular tests for SARS-CoV-2 RNA detection have been developed since the onset of the pandemic primarily tests based on the real-time reverse transcription polymerase chain reaction (rRT-PCR), which exhibit high diagnostic sensitivity and reliability [2].

Nasopharyngeal swab collection is recommended for rRT-PCR testing, as it provides a higher viral load compared with oropharyngeal swabs [3]. Specificity, sensitivity and included target regions of viral RNA vary between different tests.

The virus genome encodes 27 proteins and currently, rRT-PCR-based tests enable the detection of several genes specific for SARS-CoV-2: N, E, S, Orf1a/b gene region or a part of it; RdRp gene [4,5]. N protein, composed of 419 amino acids, is a part of viral replication machinery – it contains five domains and it is assumed that all five bind RNA and perform viral genome packaging [6,7]. One study found that the N protein also assists in integrating the S protein into the envelope [8]. The N gene has lower mutation rates than the S gene; however, studies have found N gene deletions of the omicron variant that negatively impact the PCR diagnostics of COVID-19 [9]. E protein, the smallest of all (75 amino acids), plays a role in virion assembly. Also, the transmembrane domain of the E protein forms viral ion channels in the membrane that are significant for viral infection and disease development [10,11]. It is expressed in a small amount in the host cell, mainly within the rough endoplasmic reticulum and Golgi complex [12]. E gene is highly conserved and has very low mutability – only two mutations that cause amino acid substitutions in the E protein were detected within 797 SARS-CoV-2 genome sequences, which makes it a suitable target in PCR diagnostics [10,13]. S protein recognises and binds to ACE2 receptors in human cells in the lungs, heart, kidneys and intestines [14]. ORF1a/b region contains genes that encode 16 nonstructural proteins (NSPs), that participate in replicase–transcriptase complex: NSP1 – interferes with the function of the host’s innate immune system, structural characterisation of NSP2 and NSP6 suggests that they play roles in host cell invasion, NSP3, NSP4, NSP7, NSP8, NSP9 and NSP13 are involved in the process of viral replication, NSP5 (protease) is a regulatory protein, NSP12 is a primary RdRp protein, NSP10 and NSP16 encode 2′O-methyltransferases, NSP13 is a helicase, NSP14 and NSP 15 are nucleases and function of the NSP11 is still unknown [15–17]. The RdRp protein (932 amino acids in length) is a key enzyme involved in viral genome replication and in the transcription of sgmRNAs that encode viral RNA and are translated into virion proteins [14,18]. Its structure comprises five domains: thumb, fingers, palm and Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domains responsible for RNA replication and the interface domain that binds accessory factor NSP8. NiRAN domain expresses a kinase-like activity; therefore, many studies have focused on kinase inhibiting drugs as a potential treatment for COVID-19 [19]. More than 300 mutations in the RdRp gene have been discovered so far and some could interfere with the PCR diagnostics of COVID-19 [20]. M gene (encodes the membrane [matrix] protein) and accessory proteins are rarely used as targets for detecting SARS-CoV-2 RNA. M protein plays a role in shaping virus particles and accessory proteins: orf3a, orf6, orf7a, orf7b, orf8 and orf10 assist in viral replication, assembly and infection processes [21,22]. SARS-CoV-2 proteins also affect host translation and innate immune mechanisms – for example, E protein has the potential to alter host gene expression. It interacts with intracellular vesicular transport, while the N protein acts as an interferon inhibitor and affects RNA processing and signal transduction pathway in the cell [21,22]. When testing for the presence of any of these genes in borderline cases, there is a possibility of obtaining a false-positive or a false-negative result. The only way to prevent the occurrence of false positives or false negatives completely is to perform viral RNA sequencing for each sample. Still, it would be less cost-effective, very time-consuming and inefficient. This study aimed to establish whether it is justified to analyse the E gene of the SARS-CoV-2 genome together with the N and RdRp genes, which increases the cost of diagnostic testing compared with the analysis of one or two genes, and to determine which of the analysed target regions (N, RdRp or E) exhibits higher sensitivity for SARS-CoV-2 detection.

Materials & methods

The prospective study included 155 patients, either in an inpatient or in ambulant care at the tertiary healthcare facility at the Clinical Center of Vojvodina between 29 March and 4 April 2021, who reported symptoms of respiratory infection.

Nasopharyngeal swab specimens (NPS) were collected from all participants in 3 ml collection tubes pre-filled with the viral transport medium (SANLI Medical Technology Development Co., Liuyang, Hunan, China) and stored and transported at 4°C before analysis. A laboratory request form containing, inter alia, clinical data were received for each patient. All samples were tested within 12–24 h. Before testing, samples were subjected to heat inactivation at 56°C for 35 min to reduce the risk of SARS-CoV-2 transmission. Two tests were performed on all NPS specimens by the Virology Laboratory in the Clinical Center of Vojvodina: the GeneFinder™ and the ARGENE^® test. Both GeneFinder and ARGENE are commercially available rRT- PCR assays for the qualitative detection of SARS-CoV-2. GeneFinder is manufactured for triple-target (N, RdRp and E gene) testing and ARGENE for dual-target (N and RdRp gene) testing.

For the GeneFinder and the ARGENE analysis, RNA was extracted via magnetic beads based isolation method using Viral DNA and RNA Extraction Kit (Xi’an Tianlong Science and Technology Co., Ltd., Xi’an City, China), following the manufacturer’s instructions. 200 μl of each sample was added to a pre-filled plate and placed into GeneRotex 96 full-automatic nucleic acid extractor system manufactured by the same company. PCR amplification and detection of target genes for both tests were performed on Gentier 96e real-time quantitative PCR system (Xi’an Tianlong Science and Technology Co., Ltd., Xi’an City, China).

For testing with GeneFinder COVID-19 Plus RealAmp Kit, 5 μl of each RNA sample was added to a 15 μl reaction mix (10 μl of COVID-19 PLUS Reaction mixture + 5 μl of COVID-19 PLUS Probe mixture). About 5 μl of positive and negative control were added to the reaction mix in separate wells, for every run on an instrument. Reaction conditions for RT- PCR assay were set according to the kit protocol: reverse transcription for 20 min at 50°C, pre denaturation for 5 min at 95°C and 45 cycles of PCR (denaturation for 15 s at 95°C and annealing/elongation for 60 s at 58°C). Ct values (threshold cycle – a number of a PCR cycle at which fluorescence is significantly increased over the baseline signal) using FAM dye labeled probe for RdRp gene, HEX dye labeled probe for N gene, Texas Red-dye labeled probe for E gene and Cy5- dye labeled probe for internal control (IC) were acquired. Amplification curve with Ct values not higher than 40 in the FAM, Texas Red or HEX channel indicated a positive result, according to GeneFinder manufacturer’s protocol. IC should have been amplified in all samples, with Ct values not higher than 35 in the Cy5 channel, otherwise, a result was considered invalid. Positive control has produced a positive result, and negative control has produced a negative result in each channel and every run. If amplification was detected in the Texas Red channel only, the sample was retested, and if any of the target genes were detected – a positive result was confirmed. If none of the targets was detected, the result would be reported as inconclusive [23].

For testing with the ARGENE SARS-COV-2 R-GENE^® Real-Time Detection Kit, 10 μl of IC1 was added to 200 μl of each sample before RNA extraction. About 10 μl of RNA sample extracted with IC1 was added to a 15 μl reaction mix (14.85 μl of amplification reaction premix + 0.15 μl of diluted reverse transcriptase [1:10]). About 10 μl of positive and negative control samples were included in each assay in separate wells. RT-PCR assay conditions were set according to the protocol: reverse transcription for 5 min at 50°C, Taq Polymerase activation and predenaturation for 15 min at 95°C and 45 cycles of PCR (denaturation for 10 s at 95°C, annealing for 40 s at 60°C and elongation for 25 s at 72°C). Ct values using FAM-dye-labeled probe for N gene, Cy5-dye-labeled probe for RdRp gene and HEX-dye-labeled probe for IC were obtained during amplification. Gentier 96e system software analyzed PCR amplification curves and scored the samples as positive or negative. Any Ct value was considered a positive result according to the ARGENE manufacturer’s protocol.

Statistical analysis

Data were analyzed using the IBM^® SPSS Statistics for Windows software (version 20.0, IBM SPSS Inc., NY, USA). The statistical significance level was set at p < 0.05.

Results

A total of 155 NPS samples were tested by two assays (GeneFinder and ARGENE), 109 of which were positive (70.32%). Samples were obtained from adults aged 17–91 years, with a male–female ratio of 42.6–57.4%, respectively. According to GeneFinder manufacturer’s protocol, a specimen was considered positive for SARS-CoV-2 if a sigmoidal amplification curve in the FAM, Texas Red or JOE/VIC channel was detected, with Ct values ≤40. According to the ARGENE manufacturer’s protocol, any Ct value detected in the FAM or Cy5 channel was considered a positive result. There was only one sample with a Ct value higher than 40 when tested with ARGENE, but the amplification curve shape was not sigmoidal – so we retested it and the result was negative. GeneFinder assay exhibited 1.36-fold higher sensitivity, as more positive cases were detected with GeneFinder (109) than with ARGENE (80). It detects the E gene in addition to the N and RdRp genes, which are also detected by the ARGENE assay. Clinical data of each patient was analyzed and it has been observed that three cases with symptoms consistent with an active COVID-19 infection tested positive with GeneFinder, but negative with ARGENE, which makes GeneFinder the preferred assay for detection of SARS-CoV-2. We performed further testing of the GeneFinder assay for a difference between acquired Ct values for each of the three target genes to improve the interpretation of obtained results and reduce the occurrence of false positives or false negatives in the future.

A Kruskal–Wallis H test showed a statistically significant difference in Ct value between the analyzed genes in the group of positives, p < 0.001. Dunn–Bonferroni post hoc test was carried out to adjust the significance for each pairwise test and determine which particular gene Ct values significantly differ from the other. There was a significant difference between Ct values obtained for the E and N gene, and N and RdRp gene (p = 0.12, p < 0.001 and p < 0.001 – for comparison of the E and RdRp gene, E and N gene and N and RdRp gene, respectively) (Figure 1).

We divided the positives into three subgroups based on the number of targets detected. All three genes were detected in 50 samples (45.87%). There was no significant difference in Ct value between N, E and RdRp genes in that group (p = 0.42) with a median Ct value of 29.30 (95% CI: 27.18–30.20 and standard deviation [SD]) of 4.50 for RdRp gene; a median Ct value of 28.39 (95% CI: 27.21–29.14) and SD of 3.91 for the N gene and a median Ct value of 28.71 (95% CI: 26.61–29.60) and SD of 5.21 for E gene (Figure 2).

In 21 samples (19.27%) N and RdRp genes were detected, with a median Ct value of 36.57 (95% CI: 43.82–38.23) and SD of 5.96 for the RdRp gene and median Ct value of 34.03 (95% CI: 32.71–35.02) and SD of 5.58 for the N gene. Higher Ct values were recorded for the RdRp gene and the Mann–Whitney U test confirmed that the difference was significant (p = 0.019) (Figure 3).

Only the N gene was detected in 38 samples (34.86%), with a median Ct value of 37.24 (95% CI: 36.90–37.91), ranging from 33.46 to 39.69 with an SD of 1.48. A Kruskal–Wallis H test was carried out with Dunn–Bonferroni post hoc pairwise comparison to analyze if there was a statistically significant difference in Ct value of N gene between groups positive for all three genes, positive for N and RdRp genes and positive for N gene only. The results showed that the N gene Ct value was highest in the group of samples where no other target genes were detected and lowest in the group positive for all three targets (p = 0.001, p < 0.001 and p = 0.002, for comparison of positives for all three genes and N and RdRp gene, positives for all three genes and the N gene only and positives for N and RdRp gene and the N gene only, respectively) (Figure 4).

Discussion

Serology testing, rapid antigen testing, analysis of clinical features and radiological characteristics (the chest x-ray and CT scan results) are used to diagnose individuals with suspected COVID-19 [24,25]. However, rRT-PCR is considered the gold standard for diagnostic detection of COVID-19; although, it has been reported that sometimes problems occur with false-positive and false-negative samples. In the past two and a half years, many rRT-PCR-based tests have been developed according to protocols approved by WHO and came into wide use (Charité Berlin, CDC China, Institut Pasteur Paris, National Institute of Health, Thailand and US CDC protocol) [26]. As mentioned, a variation in sensitivity has been noticed between assays used by institutions around the world targeting different genes of the SARS-CoV-2 genome. There are several opinions on which of the targets are crucial for diagnosis [26].

Triple-target testing is more expensive and takes more time than dual-target testing. Since, we often analyzed 700 samples per day in the virology laboratory for the past two and a half years, it would be beneficial for us to use the assay that analyses two instead of three genes if the two assays had equal analytical performances. Therefore, we tested the same 155 samples with both assays and compared the results. We found the assay that detects N, E and RdRp genes to be more sensitive than the one that only detects N and RdRp genes.

The CDC recommended that positive specimens should have a Ct value ≤35; however, European Commission states: “An exact value needs to be defined locally in a laboratory and may be specific to the assay used.” [27,28]. The vast majority of samples in our study were taken from hospitalized patients, many of whom were in a serious or even critical condition – patients with acute leukemia, patients waiting for a heart, kidney or liver transplant, etc. Infection with SARS-CoV-2 would generally put them and patients in the same wards, at higher risk for a fatal outcome, so we did not lower the cut-off Ct value recommended by the manufacturer.

In most positive cases (45.87%), all three genes were detected; in 19.27% cases, N and RdRp genes were detected and in 34.86% cases, only the N gene was detected. Therefore, diagnostic based on the N gene detection shows high sensitivity, while the E gene detection provides high specificity, in other words, a higher proportion of true negatives. Our study showed a significant difference between Ct values obtained for E and N gene and the N and RdRp gene. Considering that in some samples, only the N gene was detected with a high Ct value, this was the expected result. There was no significant difference in Ct value between N, E and RdRp genes in the group positive for all three targets. Significantly higher Ct values were recorded for the RdRp gene in a group that tested positive for both N and RdRp genes but negative for the E gene. Some other studies also reported higher Ct values of RdRp compared with the N gene. In contrast, others showed the opposite, suggesting that the efficiency of target gene amplification varies between assays and changes with PCR conditions [26,29]. The N gene Ct value was lowest in the group positive for all three targets, indicating that those patients were in the acute phase of infection.

Taking an rRT-PCR test after the incubation phase (5–7 days after exposure is recommended, but sometimes testing extends to 14 days [30], which could lead to a false-negative result. Only the N gene was detected in 38 samples that tested positive; with a median Ct value of 37.24, which is significantly higher than the median Ct value obtained for positives for the N and RdRp genes and higher than that obtained for positives for all three genes. Those samples were taken between 2 and 5 weeks after the onset of symptoms from patients ready to be discharged from the hospital. Such results require interpretation considering clinical observation. Serology testing can be done; if no SARS-CoV-2 antibodies are present, it suggests a negative result [31]. However, the presence of COVID-19 IgG antibodies cannot be used to diagnose current infection because individuals will test positive for a while after recovery [28].

Samples that tested positive for N and RdRp genes but not for the E gene were also tested at least 2 weeks after symptoms appeared, and showed low viral load (Ct>36 and 34, respectively). The median Ct value was significantly higher than that obtained for positives for all three targets. Those samples were taken from patients in whom symptoms appeared and who had a previous positive result more than a week ago; therefore, there is a great possibility that detected fragments represent amplification of residual viral RNA [32]. Some of the patients from this group were tested again in 2 days and the results returned negative.

Our findings show that analysis of three target genes allows the differentiation of infectious patients from those that need to be retested and whose clinical features should be evaluated.

Since SARS-CoV-2 is an RNA virus that mutates at a higher rate than DNA viruses, new variants are constantly discovered [13]. Diagnostic tests are designed to target sequences with lower mutability. Nevertheless, mutations in the N gene and the RdRp gene have been identified that could cause diagnostic errors, lower test sensitivity and increase the risk of false-negative results [9,20]. Since the E gene demonstrates high stability, it might be used to develop new mRNA vaccines to provide an immune response against infections caused by new SARS-CoV-2 strains [4]. Also, E and M gene sequences are highly conserved among known coronaviruses. They have a lower probability of mutation occurrence than the S gene, making the E gene a proper diagnostic target that provides a high degree of specificity [13,33]. As new SARS-CoV-2 variants constantly emerge, complicating COVID-19 testing, it is necessary to apply diagnostic tests targeting less mutable genes to identify individuals infected with current and future variants.

Conclusion

This data suggests that analysis of more than two target genes of viral RNA increases testing accuracy. With the detection of only one gene at Ct ≥37, there is a potential for a false-positive result. Inclusion of the E gene target in testing increases specificity, and in the case of detection of N and RdRp genes, without the E gene, retesting is advised. Since the E gene sequence shows high stability, its incorporation in diagnostic testing reduces the probability that infections with new variants will be undetected. Providing sufficient clinical information is also essential for interpreting obtained results.