key: cord-0767236-30d4wtxr
authors: Hernandez, Matthew M.; Banu, Radhika; Shrestha, Paras; Patel, Armi; Chen, Feng; Cao, Liyong; Fabre, Shelcie; Tan, Jessica; Lopez, Heidi; Chiu, Numthip; Shifrin, Biana; Zapolskaya, Inessa; Flores, Vanessa; Lee, Pui Yiu; Castañeda, Sergio; Ramírez, Juan David; Jhang, Jeffrey; Osorio, Giuliana; Gitman, Melissa R.; Nowak, Michael D.; Reich, David L.; Cordon‐Cardo, Carlos; Sordillo, Emilia Mia; Paniz‐Mondolfi, Alberto E.
title: RT‐PCR/MALDI‐TOF mass spectrometry‐based detection of SARS‐CoV‐2 in saliva specimens
date: 2021-05-19
journal: J Med Virol
DOI: 10.1002/jmv.27069
sha: 4835be48468c14c9590443725259c941ea22450c
doc_id: 767236
cord_uid: 30d4wtxr

As severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infections continue, there is a substantial need for cost‐effective and large‐scale testing that utilizes specimens that can be readily collected from both symptomatic and asymptomatic individuals in various community settings. Although multiple diagnostic methods utilize nasopharyngeal specimens, saliva specimens represent an attractive alternative as they can rapidly and safely be collected from different populations. While saliva has been described as an acceptable clinical matrix for the detection of SARS‐CoV‐2, evaluations of analytic performance across platforms for this specimen type are limited. Here, we used a novel sensitive RT‐PCR/MALDI‐TOF mass spectrometry‐based assay (Agena MassARRAY®) to detect SARS‐CoV‐2 in saliva specimens. The platform demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of the platform and determined the limit of detection of the assay to be 1562.5 copies/ml. Furthermore, across the five individual target components of this assay, there was a range in analytic sensitivities for each target with the N2 target being the most sensitive. Overall, this system also demonstrated comparable performance when compared to the detection of SARS‐CoV‐2 RNA in saliva by the cobas® 6800/8800 SARS‐CoV‐2 real‐time RT‐PCR Test (Roche). Together, we demonstrate that saliva represents an appropriate matrix for SARS‐CoV‐2 detection on the novel Agena system as well as on a conventional real‐time RT‐PCR assay. We conclude that the MassARRAY® system is a sensitive and reliable platform for SARS‐CoV‐2 detection in saliva, offering scalable throughput in a large variety of clinical laboratory settings.

Accurate and rapid testing is vital to informing the response to the coronavirus disease 2019 (COVID- 19) pandemic. Since its inception, nucleic acid amplification testing (NAAT) for SARS-CoV-2 RNA in nasopharyngeal (NP) specimens has been the mainstay in diagnosis.

Collection of such specimens requires trained healthcare professionals who need materials, such as swabs and viral transport medium (VTM), which may not be available in all settings. [1] [2] [3] Saliva has garnered attention as an alternative specimen type given its minimal discomfort and its ability to be self-collected. As of Indeed, recent reviews of studies demonstrated that saliva NAAT diagnostic performance is comparable to that of NP specimens. 5, 6 While studies have compared detection of SARS-CoV-2 across matched NP and saliva specimens, there is large variability in specimen collection, processing methods, and testing platforms. [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] Moreover, studies that assess the analytical performance of detection in saliva across platforms are lacking.

Since the identification of SARS-CoV-2, large-scale testing has been difficult to achieve given various hurdles, including instrument availability and supply chain limitations. [15] [16] [17] [18] Recently, a novel multiplex reverse transcription (RT-PCR)/MALDI-TOF assay from Agena Bioscience has received EUA for SARS-CoV-2 detection in upper respiratory specimens. 19 The MassARRAY ® SARS-CoV-2 Panel and MassARRAY ® System have the potential to increase diagnostic capacity and complement standard NAAT technologies. This is particularly promising for utilizing saliva in large community-based testing efforts.

Here, we report the first evaluation, to date, of this platform ("Agena") to detect SARS-CoV-2 RNA in saliva and compare it to the performance of the cobas ® 6800/8800 SARS-CoV-2 real-time RT-PCR Test ("Roche") for the same specimen type. Furthermore, we also compared the analytic sensitivity of each platform and its component targets. 

Saliva specimens were collected from 60 deidentified patients who underwent molecular testing for SARS-CoV-2 in NP or anterior nares (AN) specimens collected in the previous 48 h. Saliva was collected in sterile containers (35 2070; Corning) and volumes ranged from 0.5 to 1.5 ml. Upon receipt in MSHS CML, 1 ml of VTM (R99; Hardy Diagnostics) was added to each. Mixtures were vortexed for 30 s, and 1 ml of each was incubated at 55°C for 15 min before SARS-CoV-2 testing. Extension products (analytes) were desalted, transferred to a SpectroCHIP ® Array (silicon chip with prespotted matrix crystal), and loaded into the MassARRAY ® Analyzer (a MALDI-TOF mass spectrometer). The analyte/matrix cocrystals were irradiated by a laser inducing desorption and ionization, and positively charged molecules accelerated into a flight tube toward a detector. Separation occurred by time-of-flight, which is proportional to molecular mass. After data processing, a spectral fingerprint was generated for each analyte that characterizes the mass/charge ratio and relative intensity of the molecules. Data acquired by the MassARRAY ® Analyzer was processed with the MassARRAY ® Typer software and SARS-CoV-2 Report software. The assay detects five viral targets: three in the nucleocapsid (N) gene (N1, N2, N3) and two in the ORF1ab gene (ORF1, Orf1ab). If the IC was detected, results were interpreted as positive if ≥2 targets were detected or negative if <2 targets were detected. If no IC and no targets were detected, the result was invalid and required rerunning of the specimen.

For the cobas ® 6800/8800 SARS-CoV-2 real-time RT-PCR Test (09175431190; Roche), processed saliva specimens were run as previously described for NP specimens. 20 Briefly, the assay utilizes two targets to detect SARS-CoV-2 RNA: the SARS-CoV-2-specific ORF1ab gene (T1) and the pan-Sarbecovirus envelope E gene (T2). A result was positive for SARS-CoV-2 if both T1 and T2 were detected, or if T1 was detected alone. A result was presumptive positive if T2 was detected alone. A result was negative if neither T1 nor T2 was detected.

The LoD was determined across both platforms using known concentrations of a SARS-CoV-2 standard spiked into saliva clinical matrix.

Briefly, an in-house SARS-CoV-2 standard was generated by of each dilution were determined by extrapolation from standard curves generated across T1 and T2 targets ( Figure S1 ) for each dilution.

The stock concentration was calculated as the average of the extrapolated concentrations at each dilution. Aliquots of stock were stored at −80°C to prevent multiple freeze-thaw cycles.

To simulate the collection of saliva for testing, saliva from healthy donors was combined one-to-one with VTM and spiked with the SARS-CoV-2 standard. Dilutions of the spiked specimens were generated over a range of 3125.0-97.7 copies/ml (cp/ml) and 12 500-195.3 cp/ml for testing on the Roche and Agena platforms, respectively. For each platform, ten replicates of each dilution were generated, and 10 replicates of saliva-VTM spiked with the SARS-CoV-2-negative NP diluent to serve as negative controls. Spiked saliva-VTM specimens were run as described above. SARS-CoV-2 was not detected in any of the negative controls, and all results were valid across both platforms.

For each platform, the LoD of each assay and component targets were determined. The experimental LoD represents the lowest concentration with ≥95% detection. The probit LoD was determined by 95% detection based on a probit regression model. Figure S1 ).

The standard was spiked into saliva collected from healthy donors and ten replicates of serial dilutions were run side-by-side on each platform. On the Agena platform, the experimental LoD was 1562.5 cp/ml (Table 2) , which is lower than the LoD reported by manufacturers for NP specimens (2500 cp/ml). 19 Across five Agena targets, the most sensitive was the N2 target (1562.5 cp/ml) followed by the N1 target (3125 cp/ml) ( Table 2 and Figure 2a) . The least sensitive was the Orf1ab target whose LoD could not be determined from the range of concentrations tested. This reflected a gradient in sensitivity across the individual Agena targets.

On the Roche platform, the experimental LoD was lower than that of Agena (390.6 cp/ml). The Ct values for these saliva specimens is lower (228.6 cp/ml). However, the CI for this value is broad (151.4-3.7 × 10 10 ) given that the concentration at which no specimens were detected was not determined in our study.

Saliva represents an attractive specimen type for SARS-CoV-2 testing given its limited invasiveness, ability to be self-collected, and its reduced need for limited supplies. These characteristics make this specimen type invaluable as it has the potential for scalable and cost-effective SARS-CoV-2 screening and detection efforts across diverse groups of populations (e.g., young, old; symptomatic, asymptomatic). A number of groups have demonstrated that saliva is an acceptable specimen type when compared to other upper respiratory specimens. 5, 8, 9, [12] [13] [14] However, the analytical performance of this specimen type has yet to be evaluated across the multitude of platforms utilized. Here, we demonstrate the utility of saliva as a diagnostic specimen on the Agena platform with a high agreement with a real-time RT-PCR diagnostic platform (Roche). Furthermore, saliva specimens collected within 2 days are equivocally sensitive and specific across both methods when compared to matched upper respiratory specimens.

It is important to note that these platforms tested differ by their b LoD determined by probit analysis. "NA" reflects the inability to perform probit analyses due to lack of sufficient probit points. c 95% fiduciary confidence interval.

diagnostic platforms. Most studies have not evaluated the LoD across platforms in a standardized method for saliva specimens. 6 In addition, the analytic sensitivity of component targets is not systematically evaluated, reported, or compared across platforms. 6, 7, [24] [25] [26] Our study demonstrates a slightly lower sensitivity in the Agena platform when compared to that of the Roche for saliva specimens ( Figure 2 ). Moreover, we find that there are differences in analytic sensitivity across Agena targets with the N2 as the most sensitive target (Figure 2 and Table 2 ), which is also seen on the Roche platform (e.g., T2) ( Figure 2 ). These metrics are indispensable as they can inform how diagnostics address new circulating viral variants whose mutations may interfere with molecular methods.

Our study does have limitations in that our saliva collection methods did not occur at one time point but rather randomly within 2 days of initial NP/AN collection. While the utility of standardized collection methods (e.g., early morning) remains to be further assessed, this is not a variable we controlled. In addition, we utilized a pooled positive NP specimen to serve as our analyte to assess sensitivity. As a result, analytic sensitivities are based on a potentially heterogenous mixture of viral variants.

To minimize the heterogeneity of our pooled positive NP specimen, we pooled specimens isolated from 2 consecutive days to ensure a sampling of viruses from the predominant clade at the given time.

Overall, we demonstrate that the novel Agena RT-PCR/MALDI-TOF mass spectrometry-based system is capable of detecting SARS-CoV-2 RNA in saliva specimens. This platform is unique, has the potential for scalable throughput design, and is comparable in performance to the more ubiquitous real-time RT-PCR technology.

Given the continued spread and rise of new SARS-CoV-2 variants, there is a critical need to understand the analytic capabilities of these technologies. This is especially necessary for large-scale screening efforts where saliva has the potential to be further exploited for its utility. This understanding of assay and target sensitivity is essential to informing both effective detection efforts and broader public health measures to ultimately quell the COVID-19

pandemic.

We thank the members of the MSHS CML for providing any assistance when needed throughout this study. We also would like to thank the patients and healthy donors for providing specimens to complete this study.

The authors declare that there are no conflict of interests. 

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No new data were generated in this study; thus, sharing of data does not apply to this study.