Determination of the cycle threshold value of the Xpert Xpress SARS-CoV-2/Flu/RSV test that corresponds to the presence of infectious SARS-CoV-2 in anterior nasal swabs

ABSTRACT Despite having high analytical sensitivities and specificities, qualitative SARS-CoV-2 nucleic acid amplification tests (NAATs) cannot distinguish infectious from non-infectious virus in clinical samples. In this study, we determined the highest cycle threshold (Ct) value of the SARS-CoV-2 targets in the Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4plex) test that corresponded to the presence of detectable infectious SARS-CoV-2 in anterior nasal swab samples. A total of 111 individuals with nasopharyngeal swab specimens that were initially tested by the Xpert Xpress SARS-CoV-2 test were enrolled. A healthcare worker subsequently collected anterior nasal swabs from all SARS-CoV-2-positive individuals, and those specimens were tested by the Xpert 4plex test, viral culture, and laboratory-developed assays for SARS-CoV-2 replication intermediates. SARS-CoV-2 Ct values from the Xpert 4plex test were correlated with data from culture and replication intermediate testing to determine the Xpert 4plex assay Ct value that corresponded to the presence of infectious virus. Ninety-eight of the 111 (88.3%) individuals initially tested positive by the Xpert Xpress SARS-CoV-2 test. An anterior nasal swab specimen collected from positive individuals a median of 2 days later (range, 0–9 days) tested positive for SARS-CoV-2 by the Xpert 4plex test in 39.8% (39/98) of cases. Of these samples, 13 (33.3%) were considered to contain infectious virus based on the presence of cultivable virus and replication intermediates, and the highest Ct value observed for the Xpert 4plex test in these instances was 26.3. Specimens that yielded Ct values of ≤26.3 when tested by the Xpert 4plex test had a likelihood of containing infectious SARS-CoV-2; however, no infectious virus was detected in specimens with higher Ct values. IMPORTANCE Understanding the correlation between real-time PCR test results and the presence of infectious SARS-CoV-2 may be useful for informing patient management and workforce return-to-work or -duty. Further studies in different patient populations are needed to correlate Ct values or other biomarkers of viral replication along with the presence of infectious virus in clinical samples.

capable of identifying patients who are no longer at high risk of transmitting SARS-CoV-2 are therefore of great interest.The ability to isolate virus in culture is considered the reference method for transmissibility (2), though SARS-CoV-2 culture is not routinely performed in clinical laboratories due to long turnaround times, and the requirements for biosafety level-3 (BSL-3) containment and highly trained staff.As an alternative to virus culture, several biomarkers have been proposed as correlates of active viral replication (3).These include nucleocapsid antigen as well as SARS-CoV-2 genome replication intermediates such as minus-strand and subgenomic RNA (4)(5)(6).Perhaps most commonly, cycle threshold (Ct) values from real-time reverse-transcription qualitative PCR (RT-qPCR) are compared to virus isolation in culture and utilized to help predict the risk of transmission (7)(8)(9)(10).
A substantial challenge in setting Ct value cutoffs for active replication is that these values are not necessarily commutable between test systems and cannot be used a priori across different RT-qPCR tests (11,12).The Xpert Xpress CoV-2/Flu/RSV (Xpert 4plex) real-time reverse-transcription PCR test (Cepheid; Sunnyvale, CA) is widely used globally and has not yet been evaluated with virus culture as a reference (13).In this study, we determined the highest Xpert 4plex SARS-CoV-2 Ct value that corresponds to the presence of infectious SARS-CoV-2 in clinical nasal swab samples.Infectious SARS-CoV-2 was defined by the presence of cultivable virus and/or detectable replication intermedi ates in RT-qPCR-positive specimens.

Primary samples and initial testing
One hundred and eleven nasopharyngeal (NP) swabs were collected between 17 November 2020 and 26 January 2022 from study subjects who included active-duty male soldiers and affiliates of the United States Department of Defense aged 18-61 years.These samples were collected using flocked swabs and placed in Universal Transport Medium (UTM; Copan Italia s.p.a.; Brescia, Italy).The UTM was tested using the Xpert Xpress SARS-CoV-2 test (Cepheid), an RT-qPCR targeting the SARS-CoV-2 envelope and nucleocapsid genes (14).

Secondary samples and testing
Anterior nasal swabs (Puritan; Guilford, ME) were subsequently collected from consen ted individuals who tested positive by initial testing and placed in UTM (n = 98).The samples were tested using the Xpert 4plex test, an RT-PCR that detects influenza A, influenza B, respiratory syncytial virus (RSV), and SARS-CoV-2.The SARS-CoV-2 compo nent targets the nucleocapsid and envelope genes and produces a single Ct value if either or both gene targets are detected (12).Post-market in silico surveillance data shows consistent coverage of SARS-CoV-2 gene targets across all variants circulating during the study period (unpublished data, communicated by M. Loeffelholz, Cepheid).Secondary specimens were shipped to testing laboratories on dry ice and were stored in the laboratories at −80°C until the time of SARS-CoV-2 culture and replication intermedi ate testing at the Indiana University School of Medicine and Stanford University School of Medicine, respectively.

SARS-CoV-2 culture titration
To roughly correlate Xpert 4plex test SARS-CoV-2 Ct values and the quantity of detect able SARS-CoV-2 plaque forming units (PFUs) in a sample, SARS-CoV-2 isolate 2019-nCoV/USA/WA1/2020 (BEI Resources, Manassas, VA), was first cultured in Vero E6 cells and titered by plaque assay (15).Stocks were adjusted to a theoretical titer of 1.0 × 10 6 PFU/mL, diluted 10-fold to a final dilution of 10 −8 PFU/mL, and tested in triplicate using the Xpert 4plex test.Aliquots of each dilution were back-titered in duplicate by plaque assay to determine their observable titers; reported results represent the average of both assays.

SARS-CoV-2 culture of clinical swab samples
Aliquots of nasal swab samples were cultured on monolayers of Vero E6 cells in six-well tissue culture plates, as previously described (16).Cell cultures were tested on days 0 and 8 of incubation using a laboratory-developed RT-qPCR to determine the presence of actively replicating SARS-CoV-2: a decrease of at least 4 Ct values during culture incubation indicated viral replication.Infected cultures also displayed cytopathic effects typical of SARS-CoV-2 (rounding and detachment of cells from the substrate).

Replication intermediate testing
Aliquots of nasal swab samples were tested for SARS-CoV-2 replication intermediates, including minus-strand species and subgenomic RNA, which are only detectable during coronavirus replication.Strand-specific testing involved a two-step RT-qPCR with separate reactions for the plus and minus strands, as previously described (4).In the first step, strand-specific primers were used to create cDNA: a reverse envelope (E) gene primer generated cDNA to plus strand; separately, a forward E gene primer generated cDNA to the minus strand.In the second step, the cDNAs were amplified by real-time PCR using E gene primers and probes.In addition, these specimens were tested for subgenomic RNA with a one-step RT-qPCR using the leader sequence forward primer, E gene reverse primer, and E gene probe, as modified from Wolfel et al. (17,18).

RESULTS
Of the 111 primary NP swabs submitted for testing, 98 (88.3%) tested positive for SARS-CoV-2 using the Xpert Xpress SARS-CoV-2 test.A second specimen (anterior nasal swab) was collected from SARS-CoV-2-positive individuals a median of 2 days later (range, 0-9 days).39.8% (39/98) of these anterior nasal swabs tested positive by the Xpert 4plex test.Of these positive samples, 33.3% (13/39) had a Ct value of ≤26.3, contained cultivable virus, and had detectable replication intermediates, indicating the presence of infectious SARS-CoV-2.To roughly correlate Xpert 4plex test SARS-CoV-2 Ct values and the quantity of detectable SARS-CoV-2 PFU in a sample, a titered stock of infectious SARS-CoV-2 was diluted and tested in triplicate using the 4plex test.The lowest titer of the diluted stock that yielded culture-detectable virus was the 1 × 10 1 PFU/mL dilution, which had a mean Ct value of 29 (range, 28.9-29.0;Table 1).Figure 1 compares Ct values for culture-positive and culture-negative samples.Table 2 presents data from the comparison of Cepheid Ct value, minus-strand RNA RT-qPCR, and subgenomic RNA RT-qPCR, with virus culture as reference.The Xpert 4plex Ct value of the cultivatable specimen that had detectable sgRNA but non-detectable minus-strand RNA was 26.3 cycles.

DISCUSSION
In this study, the Ct values obtained from testing anterior nasal swabs on the Xpert Xpress SARS-CoV-2/Flu/RSV multiplex test were compared to viral culture and RNA biomarkers for active replication.Specimens that yielded Ct values of 26.3 and lower had a likelihood of containing infectious SARS-CoV-2, and specimens with Ct values greater than 26.3 did not contain detectable infectious virus.Although 6/19 (32%) specimens with Ct values below 26.3 did not yield cultivable virus, it is possible that viral infectivity in those specimens was compromised immediately following collection (e.g., a delay in freezing) or during cold storage prior to viral culture.Interestingly, analysis of titered SARS-CoV-2 stocks indicated that infectious virus could be detected in samples with Ct values of 29 and lower, suggesting that inhibitors of viral infectivity (e.g., neutralizing antibodies) could be present in the clinical specimens.
Various Ct value cutoffs have been proposed in other studies comparing RT-qPCR methods to viral culture.For example, using the same envelope (E) gene primer/probe sets for RT-qPCR (19), cutoffs of 24 (7), 31 (8), and 34 (20) cycles have been reported.This wide range of cutoffs may be due to differences in the analytical sensitivity of the culture method used for comparison, differences in the performance characteristics of the RT-qPCR assay despite the use of the same genomic target (e.g., different nucleic acid extraction and amplification methods), and differences in patient demographics, underlying diseases, time after onset of illness, and specimen types.
Advantages of this study include the use of a widely used, commercially available, sample-to-answer RT-qPCR method in which all steps of the nucleic acid amplification process are standardized.In addition, the analytical characteristics of the culture method are clearly defined, and a single specimen type (anterior nasal swab) was evaluated.While this design may allow other users of the 4plex to use such a cutoff in clini cal practice, further work will be necessary to determine cutoffs using other upper respiratory specimen types.Note that Cepheid now offers an updated version of this test that contains an additional SARS-CoV-2 target, the RNA-dependent RNA polymerase gene, the Xpert Xpress CoV-2/Flu/RSV plus; additional experiments will be required to confirm Ct value agreement for this newer test.Johnson et al. showed that the Ct values obtained using the Xpert Xpress CoV-2/Flu/RSV plus correlate with those of the test used in this study (21).
The initial samples in this study were collected from military personnel, including individuals reporting to Marine Corps Recruit Depot (MCRD).These samples were tested by NHRC as part of MCRD's safety screening to prevent outbreaks at the facility.The secondary anterior nasal swab samples were collected from consented individuals a median of 2 days later.This arrangement allowed assessment of time-based testing, which revealed that 60.2% of secondary tests were negative for SARS-CoV-2 RNA.Given that a single result at diagnosis cannot predict the trajectory of infection, the incorpora tion of a Ct value cutoff on a second test may be a reasonable approach to ensure that individuals with late-Ct values at diagnosis are not simply very early on in the course of their illness.
This study is limited by the small cohort size, the enrolment of individuals that do not represent the general population (18-to 61-year-old healthy males), and the absence of SARS-CoV-2 variant data.All similarly designed studies evaluating biomarkers for active replication, including Ct value cutoffs and detection of replication intermediates, are also limited by the uncertainty with which detection of SARS-CoV-2 in culture correlates with human-to-human transmission.
In summary, the data described in this study offer insight into the correlation between Ct values of a commercial 4plex assay that detects SARS-CoV-2 and the presence of infectious SARS-CoV-2 in clinical specimens: for the Xpert 4plex assay used for this study, we determined that specimens that yielded SARS-CoV-2 Ct values of ≤26.3 may harbor infectious virus, while infectious virus could not be detected in clinical specimens with higher Ct values.Additional studies in different patient popula tions are warranted to further correlate Ct values and other biomarkers of active viral replication with the presence of infectious virus in clinical specimens.

FIG 1 a
FIG 1 Comparison of Ct values for culture-positive and culture-negative samples.Culture-positive samples had a median Ct value of 22 [interquartile range (IQR) 20.6-24.1],whereas culture-negative samples had a median Ct value of 33.9 (IQR 27.1-38.4).The difference in medians was statistically significant (P value ≤ 0.0001; Mann-Whitney U test).
for funding, the U.S. Marine Corps for assistance with sample collection, and the study volunteers for their participation.The use of Xpert Xpress SARS-CoV-2/Flu/RSV Ct values for developing a therapeutic or patient management strategy for COVID-19 patients is considered off-label.Cepheid does not endorse off-label use of Xpert tests.Laboratories must complete a validation study to utilize the test for this purpose.K.V.B. was employed by Indiana University Health at the time of study performance, and M.C. was an employee of the Indiana University School of Medicine Department of Microbiology and Immunology at the time of study performance.Conceptualization, Funding acquisition, Investigation, Methodology, Project administra tion, Resources, Supervision, Writing -review and editing | Benjamin A. Pinsky, Con ceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Writing -original draft, Writing -review and editing | Michael J. Loeffelholz, Concep tualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing -original draft, Writing -review and editing ETHICS APPROVAL See the funding table on p. 6. C.A.M., C.S., and C.B. are military service members or employees of the U.S. Government.This work was prepared as part of their official duties.Title 17, U.S.C. §105 provides that copyright protection under this title is not available for any work of the U.S. Government.Title 17, U.S.C. §101 defines a U.S. Government work as work prepared by a military service member or employee of the U.S. Government as part of that person' s official duties.Report No. 23-103 was supported by the Joint Program Executive Office under work unit no.N2004.The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.

TABLE 2
Comparison of culture results to Ct values and molecular markers of viral replication a sgRNA, subgenomic RNA; PPA, positive percent agreement; NPA, negative percent agreement; CI, confidence interval. a