key: cord-0984767-1omc7l17 authors: Wong, River Chun-Wai; Wong, Ann Han; Ho, Yolanda Iok-Ieng; Siu, Gilman Kit-Hang; Lee, Lam-Kwong; Leung, Eddie Chi-Man; Lai, Raymond Wai-Man title: Application of digital PCR to determine the reliability of Xpert Xpress SARS-CoV-2 assay with envelope (E) gene negative & nucleocapsid (N2) gene positive results date: 2022-05-20 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2022.115726 sha: 7baabeaf40e4ae3f01a8f51e4759f85f191fdb38 doc_id: 984767 cord_uid: 1omc7l17 This study used digital polymerase chain reaction (dPCR) to determine whether envelope (E) gene-negative and nucleocapsid (N2) gene-positive (E-N+) results obtained with the Cepheid Xpert Xpress SARS-CoV-2 assay are reliable. Using droplet digital PCR results as a reference, 18 out of 22 E-N+ samples with a low viral load (81.8%) were identified as true positives. In Cepheid Xpert Xpress SARS-CoV-2 (Xpert Xpress) assay, the dual targets of the envelope (E) and nucleocapsid (N2) genes are amplified. The result is interpreted as positive if either both genes or N2 gene alone is detected [1] . E-N+ results account for 3.2% of the positive SARS-CoV-2 samples tested in our center and similar rate has been reported by Khoshchehreh et al. [2] . Falasca et al. showed that centrifugation to concentrate the Xpert Xpress E-N+ samples by 10-fold, followed by a repeated test, gave a negative result [3] . It is, therefore, recommended that E-N+ results should be interpreted with caution as they may be false positives [3] . Droplet digital PCR (ddPCR) has superior analytical sensitivity, especially for the detection of SARS-CoV-2 in samples with a low viral load [4] [5] [6] [7] [8] [9] [10] . There are two types of digital PCR (dPCR) platforms: droplet-based (e.g., Bio-Rad QX200 ddPCR system), which is well-established, and nanoplate-based (e.g., Qiagen QIAcuity One system), which is new to the market. This study aimed to use dPCR to determine whether E-N+ results obtained with the Xpert Xpress assay are reliable. Twenty-two respiratory samples collected from 19 patients with a previous E-N+ result (Ct values ranging from 37.7 to 44.8) from the Xpert Xpress assay were included in the study. The samples were stored at -80°C for 3 months before retrieval for dPCR. 200 μL of samples was used for RNA extraction and eluted in 50 μL as previously described [11] . To assist in the determination of infection status, testing for IgG antibodies against SARS-CoV-2 nucleocapsid protein (anti-NP IgG) was performed on serum samples, if available, using the Alinity i qualitative assay (Abbott, Sligo, Ireland). Samples were analyzed according to the manufacturer's recommendations. The limit of detection (LoD) claimed is 250 copies/mL. The ddPCR test on the QX200 system (Bio-Rad Laboratories, CA, USA) was performed according to the manufacturer's recommendations. All samples were tested in triplicate. The results were analyzed by using QX Manager, standard edition. Samples were interpreted as positive if ≥ 1 droplets were detected for either one (N1 or N2) or both targets and confirmed by comparing with the positive control. The LoD claimed for the Bio-Rad SARS-CoV-2 ddPCR kit is 150 copies/mL. The Qiagen SARS-CoV-2 N1+N2 assay (Qiagen, Hilden, Germany) was performed using the Nanoplate 26k 24-Well protocol according to the manufacturer's recommendations. All samples were tested in triplicate, and results were analyzed by QIAcuity One system. The LoD claimed is 250 copies/mL. Using QX200 ddPCR assay as a reference method, 18 of the 22 samples (81.8%), with droplet concentrations ranging from 61 to 1,872 copies/mL, were confirmed as positive, while 15 samples (68.2%) were detected with the QIAcuity One assay (Table 1) . It showed that E-N+ results generated with the Xpert Xpress assay were mostly true positives. The positive percentage agreement between the two dPCR platforms was 77.78% (Table 2) . For the 18 true positive samples confirmed by ddPCR, the Ct values of the N2 gene ranged from 37.7 to 44.4, and 16 of the Ct values were greater than or equal to 39. Of the four samples with no positive droplet detected by ddPCR, sample 6 tested positive using the QIAcuity One assay, with a concentration of 50 copies/mL. This was below the LoD of the QX200 ddPCR assay. For the three true negative samples (false positives with the Xpert Xpress assay) that were negative by both dPCR assays, the Ct values of the N2 gene ranged from 40.7 to 43.7. Discordant results for the two dPCR assays were observed in sample no. 3, 4, 6, 8 and 9. They had viral loads below or close to the LoD. We verified the analytical sensitivity of both dPCR assays using purified intact SARS-CoV-2 viral particles (NATtrol™ SARS-Related Coronavirus 2; ZeptoMetrix, NY, USA) and determined that the LoDs were 100 copies/mL. Further testing of five external quality assurance samples showed 100% concordance with the intended qualitative results, with a similar viral load detected by both platforms (Table 3 ). This provided further evidence that discrepancies occur mainly in samples with viral loads close to the LoD. There was no positive droplet being detected by QX200 ddPCR on two samples (no. 6 & 15) and three samples (no. 3, 8 &15) were not detected by QIAcuity One assay, while these patients were seropositive that indicates a prior COVID-19 infection. Sample no. 15 & 16 were collected from the same patient on the same day and discrepancies in dPCR results was probably associated with sample quality as no. 15 was a saliva sample that self-collected by patient while no. 16 was a nasopharyngeal sample that collected by healthcare workers. These patients were likely have resolving infections with prolonged viral RNA shedding in their respiratory samples. As Hong Kong has adopted a Zero-COVID strategy, clinically stable patients will be monitored for the presence of SARS-CoV-2 before discharge. Our findings suggested majority of E-N+ results obtained with the Xpert Xpress assay were true positives, which is discrepant to the results reported by Falasca et al. [3] . It may due to different approaches used for the confirmation of such weakly positive results. In our laboratory, any sample with a Ct value ≥ 35 is confirmed using another RT-PCR platform, and a positive result is only reported if SARS-CoV-2 RNA is detected by both platforms. Otherwise, the result is reported as indeterminate. Clinicians are strongly advised to collect another sample (preferably a lower respiratory tract specimen) from patients with indeterminate results and manage them as presumptive positive while recommending repeat sampling. Testing for the presence of Anti-NP IgG could also be considered to investigate the possible false positive RT-PCR result. Stoichiometric variability may also have contributed to the discrepancies between the two dPCR assays. The input volume of RNA was different, with 5.5 μL used for each of the 22 μL reactions in the QX200 ddPCR assay and 10 μL used for each of the 40 μL reactions in the QIAcuity One assay. Such a difference in the volume of template added may affect the amount of viral RNA available for the RT-PCR reaction. In conclusion, our study showed that most of the E-N+ Xpert Xpress results were true positives. There are several limitations of this study. First, the sample size was small. Second, dPCR was performed retrospectively and not a "head-to-head" comparison with the Xpert Xpress assay, which may have introduced bias into the analysis. Third, RNA may be degraded after prolonged storage or multiple freeze/thaw processes. Further prospective studies are therefore recommended. This research did not receive a specific grant from a funding agency in the public, commercial or not-for-profit sectors. The authors declare that we have no conflicts of interest. Evaluation on testing of deep throat saliva and lower respiratory tract specimens with Xpert Xpress SARS-CoV-2 assay A needle in the haystack? Assessing the significance of envelope (E) gene-negative, nucleocapsid (N2) gene-positive SARS-CoV-2 detection by the Cepheid Xpert Xpress SARS-COV-2 assay Detection of SARS-COV N2 Gene: Very low amounts of viral RNA or false positive? Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge. medRxiv ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR Analytical and Clinical Performance of Droplet Digital PCR in the Detection and Quantification of SARS-CoV-2 Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification Rapid adaptation and continuous performance evaluation of SARS-CoV-2 envelope gene (E-gene) real-time RT-PCR assays to support the hospital surge in test demand The authors would like to thank all of the technical staff in the Department of Microbiology, Prince of Wales Hospital for their herculean efforts and contributions to SARS-CoV-2 testing during the COVID-19 pandemic.We would also like to thank Qiagen for providing the QIAcuity One system and reagents used in this study.