When rare becomes common: N2 gene-positive, E gene-negative SARS-CoV-2 PCR results between 2021 and 2022.

Nucleocapsid gene-positive, envelope gene-negative (N2+/E-) SARS-CoV-2 PCR results obtained with the Cepheid Xpert Xpress SARS-CoV-2 assay are an infrequent phenomenon. We assessed the validity of the N2+/E- cases with an indirect approach by analyzing their occurrence in relation to overall positive PCR rates and absolute number of PCR tests (24,909 samples, collected June 2021 to July 2022). Additionally, 3022 samples were analyzed with the Xpert Xpress CoV-2-plus assay in August/September 2022. The incidence of monthly N2+/E- cases closely followed the overall frequency of positive tests (p < 0.001), while there was no correlation with the monthly number of PCR test. The observed distribution of N2+/E- cases implicates, that they are not merely artefacts, but rather represent samples with a very low viral load. This phenomenon will persist with the Xpert Xpress SARS-CoV-2 plus assay, which also produced more than 10% results where only one target gene replicated with a very high Ct value.

Clinical Evaluation of BioFire COVID-19 Test, BioFire Respiratory Panel 2.1, and Cepheid Xpert Xpress SARS-CoV-2 Assays for Sample-to-Answer Detection of SARS-CoV-2.

Background: Due to the extreme infectivity of SARS-CoV-2, sample-to-answer SARS-CoV-2 reverse transcription (RT) polymerase chain reaction (PCR) assays are urgently needed in order to facilitate infectious disease surveillance and control. The purpose of this study was to evaluate three sample-to-answer SARS-CoV-2 RT-PCR assays­BioFire COVID-19 Test, BioFire RP 2.1, and Cepheid Xpert Xpress SARS-CoV-2­using clinical samples. Methods: A total of 77 leftover nasopharyngeal swab (NP) swabs (36 positives and 41 negatives) confirmed by reference SARS-CoV-2 RT real-time (q) PCR assay were collected. The clinical sample concordance, as specified by their respective emergency use authorizations (EUAs), in comparison to the reference SARS-CoV-2 RT-qPCR assay, was assessed. Results: The results showed that all three sample-to-answer SARS-CoV-2 RT-PCR assays provided perfectly concordant results consistent with the reference SARS-CoV-2 RT-qPCR assay. The BioFire COVID-19 Test exhibited the best turnaround time (TAT) compared to the other assays, regardless of the test results, using one-way analysis of variance followed by Scheffe's post hoc test (p < 0.001). The Xpert Xpress SARS-CoV-2 showed a shorter average TAT (mean ± standard deviation, 49.9 ± 3.1 min) in the positive samples compared to that (55.7 ± 2.5 min) of the negative samples. Conclusions: Our evaluation demonstrates that the BioFire COVID-19 Test, BioFire RP 2.1, and Cepheid Xpert Xpress SARS-CoV-2 assays compare favorably to the reference SARS-CoV-2 RT-qPCR assay, along with a 100% concordance in assay results for clinical samples and an acceptable analytical performance at their guaranteed limits of detection. The addition of a widely used simultaneous sample-to-answer SARS-CoV-2 RT-PCR assay will contribute to the number of medical laboratories able to test for COVID-19.

Application of digital PCR to determine the reliability of Xpert Xpress SARS-CoV-2 assay with envelope (E) gene negative and nucleocapsid (N2) gene positive results.

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 of 22 E-N+ samples with a low viral load (81.8%) were identified as true positives.

Tracheal Aspirate and Bronchoalveolar Lavage as Potential Specimen Types for COVID-19 Testing Using the Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV.

Xpert Xpress SARS-CoV-2/Flu/RSV is a rapid diagnostic test currently approved for the detection of SARS-CoV-2 using upper respiratory tract specimens. This study attempts to assess the performance of this assay using upper and lower respiratory tract specimens by comparing its results to the lab-developed PCR test. We assessed the performance of GeneXpert for the detection of SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus for upper respiratory tract specimens. In addition, the SARS-CoV-2 detection was evaluated for lower respiratory tract specimens (bronchoalveolar lavage and tracheal aspirate). Precision and reproducibility of the test were also assessed using samples with varying cycle threshold values. Xpert Xpress SARS-CoV-2/Flu/RSV shows 100% positive and negative agreements for all four targets when tested using upper respiratory tract specimens. For lower respiratory tract specimens, tracheal aspirate and bronchoalveolar lavage samples respectively show 96% and 100% positive percent agreement for SARS-Cov-2 target only. No positive flu/RSV samples were included for lower respiratory tract specimens. Both samples show 100% negative percent agreement. The precision and reproducibility assay also showed 100% correspondence. Xpert Xpress SARS-CoV-2/Flu/RSV can be potentially used for SARS-Cov-2 detection in lower respiratory tract specimens. Performance passed our study acceptance criteria and shows promising implications as a point of care detection assay. IMPORTANCE Cepheid's Xpert Xpress SARS-CoV-2/Flu/RSV provides a means of rapid diagnosis that can help in hospital bed management and patient flow. It is also important for each microbiology lab to increase its capacity and most importantly have a different platform to overcome the anticipated reagent shortage at times of peak community transmission. There is limited evidence on using it for lower respiratory tract specimens. Here we present our validation for upper respiratory tract specimens as well as a potential use for lower respiratory specimens (BAL and TA), and we discuss some of the applications we have been using in our organization.

Presumptive positive with the Cepheid Xpert Xpress SARS-CoV-2 assay due to N mutations in the Delta variant.

The Cepheid Xpert® Xpress SARS-CoV-2 assay is 1 of the several real-time reverse transcription polymerase chain reaction (RT-PCR) assays that received Emergency Use Authorization from the United States Food and Drug Administration (FDA) for detection of SARS-CoV-2. Here we report 4 SARS-CoV-2 samples that were reported as presumptive positives on the Cepheid platform while reported as positives on alternative RT-PCR platforms. Whole genome sequencing indicated that the samples were Delta variants and had point mutations in the N gene which potentially interfered with SARS-CoV-2 detection. Two types of point mutations were found in these samples in the US CDC 2019-nCoV Real time PCR N2 Probe region: C29203T and C29200T. C29203T is a novel point mutation, and C29200T has not been previously reported in the Delta variants. This underlines the fact that mutations in the real-time RT-PCR assay target region could hinder accurate detection of SARS-CoV-2.

Prevalence of a Single-Nucleotide Variant of SARS-CoV-2 in Korea and Its Impact on the Diagnostic Sensitivity of the Xpert Xpress SARS-CoV-2 Assay.

The sensitivity of molecular diagnostics could be affected by nucleotide variants in pathogen genes, and the sites affected by such variants should be monitored. We report a single-nucleotide variant (SNV) in the nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., G29179T, which impairs the diagnostic sensitivity of the Xpert Xpress SARS-CoV-2 assay (Cepheid, Sunnyvale, CA, USA). We observed significant differences between the threshold cycle (Ct) values for envelope (E) and N genes and confirmed the SNV as the cause of the differences using Sanger sequencing. This SNV, G29179T, is the most prevalent in Korea and is associated with the B.1.497 virus lineage, which is dominant in Korea. Clinical laboratories should be aware of the various SNVs in the SARS-CoV-2 genome and consider their potential effects on the diagnosis of coronavirus disease 2019.

Comparative evaluation of the Thermo fisher TaqPath™ COVID-19 combo kit with the Cepheid Xpert® Xpress SARS-CoV-2 assay for detecting SARS-CoV-2 in nasopharyngeal specimens.

PURPOSE: With over 50 SARS-CoV-2 gene amplification assays that have been EUA cleared with minimal experimental validation performed, it is likely that not all of these assays are comparable in their ability to detect SARS-CoV-2 in clinical specimens. Thermo Fisher Scientific is a relatively new company in the molecular diagnostics field and the purpose of this study was to compare the performance of the Thermo Fisher TaqPath™ Combo Kit with an established test, the Cepheid Xpert® Xpress SARS-CoV-2 assay, for its ability to detect SARS-CoV-2 in nasopharyngeal specimens. METHODS: A total of 300 randomly selected nasopharyngeal specimens were evaluated and tested by the TaqPath and GeneXpert assays. Discordant test specimens were arbitrated by performing an alternative PCR assay and Sanger sequencing. RESULTS: The TaqPath assay had a 96.7 and 99.6% positive and negative agreement respectively when compared to the Xpert Xpress test. However, after test arbitration, the three discordant specimens were arbitrated in favor of the TaqPath assay producing a positive and negative percent agreement of 100% for the TaqPath Combo Kit while the Xpress SARS-CoV-2 assay had a positive and negative percent agreement of 98.3 and 99.2% respectively. CONCLUSIONS: The TaqPath Combo Kit is a high complexity assay that compares favorably with the Xpert Xpress test and can be reliably used for the detection of SARS-CoV-2 in nasopharyngeal specimens.

Evaluation of automated molecular tests for the detection of SARS-CoV-2 in pooled nasopharyngeal and saliva specimens.

BACKGROUND: Pooling of samples for SARS-CoV-2 testing in low-prevalence settings has been used as an effective strategy to expand testing capacity and mitigate challenges with the shortage of supplies. We evaluated two automated molecular test systems for the detection of SARS-CoV-2 RNA in pooled specimens. METHODS: Pooled nasopharyngeal and saliva specimens were tested by Qiagen QIAstat-Dx Respiratory SARS-CoV-2 Panel (QIAstat) or Cepheid Xpert Xpress SARS-CoV-2 (Xpert), and the results were compared to that of standard RT-qPCR tests without pooling. RESULTS: In nasopharyngeal specimens, the sensitivity/specificity of the pool testing approach, with 5 and 10 specimens per pool, were 77%/100% (n = 105) and 74.1%/100% (n = 260) by QIAstat, and 97.1%/100% (n = 250) and 100%/99.5% (n = 200) by Xpert, respectively. Pool testing of saliva (10 specimens per pool; n = 150) by Xpert resulted in 87.5% sensitivity and 99.3% specificity compared to individual tests. Pool size of 5 or 10 specimens did not significantly affect the difference of RT-qPCR cycle threshold (CT ) from standard testing. RT-qPCR CT values obtained with pool testing by both QIAstat and Xpert were positively correlated with that of individual testing (Pearson's correlation coefficient r = 0.85 to 0.99, p < 0.05). However, the CT values from Xpert were significantly stronger (p < 0.01, paired t test) than that of QIAstat in a subset of SARS-CoV-2 positive specimens, with mean differences of -4.3 ± 2.43 and -4.6 ± 2 for individual and pooled tests, respectively. CONCLUSION: Our results suggest that Xpert SARS-CoV-2 can be utilized for pooled sample testing for COVID-19 screening in low-prevalence settings providing significant cost savings and improving throughput without affecting test quality.

Copan eNAT Transport System To Address Challenges in COVID-19 Diagnostics in Regions with Limited Testing Access.

Community-based health care clinics and hospital outreach services have the potential to expand coronavirus disease 2019 (COVID-19) diagnostics to rural areas. However, reduced specimen stability during extended transport, the absence of a cold chain to centralized laboratories, and biosafety concerns surrounding specimen handling have limited this expansion. In the following study, we evaluated eNAT (Copan Italia, Brescia, Italy) as an alternative transport system to address the biosafety and stability challenges associated with expanding COVID-19 diagnostics to rural and remote regions. In this study, we demonstrated that high-titer severe acute respiratory virus syndrome coronavirus 2 (SARS-CoV-2) lysate placed into eNAT medium cannot be propagated in cell culture, supporting viral inactivation. To account for off-site testing in these settings, we assessed the stability of contrived nasopharyngeal (NP) specimens stored for up to 14 days in various transport media (eNAT, eSwab, viral transport medium [VTM], saline, and phosphate-buffered saline [PBS]) at 4°C, 22 to 25°C, and 35°C. The molecular detection of SARS-CoV-2 was unaffected by sample storage temperature over the 2 weeks when stored in eNAT or PBS (change in cycle threshold, ≤1). In contrast, variable stability was observed across test conditions for other transport media. As eNAT can inactivate SARS-CoV-2, it may support COVID-19 diagnostics at the point of care. Evaluation of compatibility of eNAT with Cepheid Xpert Xpress SARS-CoV-2 assay demonstrated diagnostic accuracy and sensitivity equivalent to those of VTM. Taken together, these findings suggest that the implementation of eNAT as a collection device can expand COVID-19 testing to areas with limited health care access.

Asymptomatic Patient Testing After 10:1 Pooling Using the Xpert Xpress SARS-CoV-2 Assay.

OBJECTIVES: Pool testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) preserves testing resources at the risk of missing specimens through specimen dilution. METHODS: To determine whether SARS-CoV-2 specimens would be missed after 10:1 pooling, we identified 10 specimens with midrange (ie, 25-34 cycles) and 10 with late (ie, >34-45 cycles) crossing threshold (Ct) values and tested these both neat and after 10:1 pooling. Final test results and Ct changes were compared. RESULTS: Overall, 17 of 20 specimens that contained SARS-CoV-2 were detected after 10:1 pooling with the Xpert Xpress SARS-CoV-2 Assay (Cepheid), rendering an 85% positive percentage of agreement. All 10 of 10 specimens with an undiluted Ct in the mid-Ct range were detected after 10:1 pooling, in contrast to 7 of 10 with an undiluted Ct in the late-Ct range. The overall Ct difference between the neat testing and the 10:1 pool was 2.9 cycles for the N2 gene target and 3 cycles for the E gene target. The N2 gene reaction was more sensitive than the E gene reaction, detecting 16 of 20 positive specimens after 10:1 pooling compared with 9 of 20 specimens. CONCLUSIONS: An 85% positive percentage of agreement was achieved, with only specimens with low viral loads being missed following 10:1 pooling. The average impact on both reverse transcription polymerase chain reactions within this assay was about 3 cycles.

Evaluation on testing of deep throat saliva and lower respiratory tract specimens with Xpert Xpress SARS-CoV-2 assay.

BACKGROUND: Xpert® Xpress SARS-CoV-2 assay is only validated on nasopharyngeal specimens for detection of SARS-CoV-2. Other specimen types such as deep throat saliva (DTS), also known as posterior oropharyngeal saliva and lower-respiratorytract specimens (LRT) including sputum, tracheal aspirate and bronchoalveolar lavage are not validated. These non-validated specimen types, however, do have significant diagnostic value. OBJECTIVE: Evaluate the performance of Xpert Xpress SARS-CoV-2 assay for detection of SARS-CoV-2 from DTS and LRT specimens. METHODS: 162 specimens from 158 patients with suspected COVID-19 disease were tested with Xpert Xpress SARS-CoV-2 assay. These included 120 DTS and 42 LRT specimens i.e. 35 sputum, 6 tracheal aspirate and one bronchoalveolar lavage. Results were compared to those by the TIB-Molbiol LightMix® SarbecoV E-gene assay. RESULTS: Xpert Xpress SARS-CoV-2 assay has satisfactory performance when compared with reference method. The positive percent agreement (PPA) of DTS and LRT specimens were 98.86 % & 100 % respectively while the negative percent agreement (NPA) was 100 % for both DTS and LRT specimens. CONCLUSIONS: This study demonstrated with appropriate sample pre-treatment, Xpert Xpress SARS-CoV-2 assay can be used to test on non-validated specimen types including DTS & LRT specimens.