Introduction

As of February 2023, there have been 753,651,712 reported COVID-19 (coronavirus disease of 2019) infections since the WHO announced the global COVID-19 pandemic. The current pandemic has resulted in 6,813,845 deaths worldwide [1]. These figures may be underestimated [2]. Coronaviruses are members of the family Coronaviridae. They are single-stranded, nonsegmented, enveloped, positive-sense RNA viruses. The name comes from their crown-like shape, seen on electron microscopy, with spike protein projections around their surface [3]. This group of viruses can infect both humans and animals, resulting in a spectrum of clinical syndromes, including, most often, acute respiratory illness [4]. In addition to SARS-CoV-2, six recognized coronaviruses can currently infect humans. HCoV-229E and HCoVNL63 belong to the Alphacoronavirus genus. HCoV-HKU1, HCoV-OC43, MERS-CoV and SARS-CoV belong to the Betacoronavirus genus [3]. Some strains are zoonotic and associated with severe outcomes [5].

The early identification and isolation of infectious patients played an important role in preventing the spread and propagation of this pandemic. Microbiology laboratories are playing a crucial role in diagnosing COVID-19 cases. Various methodologies and diagnostic platforms are available, including nucleic acid amplification tests, serology and antigen detection [6, 7]. Over the past couple of years, there have been tremendous efforts to improve the detection and diagnostic testing for COVID-19. We recognize the importance of increasing laboratory capacity to better support our community. This necessitates overcoming limitations posed by reagent costs, supply chain obstructions, and instrument availability.

The current “gold standard” for COVID-19 diagnosis is real-time reverse transcriptase polymerase chain reaction (RT-PCR). This method is of the highest sensitivity but requires specialized equipment and expensive reagents [8]. Here we assess the performance of the MassARRAY System (Agena Bioscience, San Diego, CA, USA). This assay combines RT-PCR with high-throughput mass spectrometry (MS) processing compared to the RT-PCR assay used in our laboratory. MassARRAY detects RT-PCR products using matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF) mass spectrometry. MALDI/TOF has been widely used in clinical microbiology to identify bacterial isolates from clinical specimens [9]. It generates protein spectra that serves as the finger print for each organism. It has been recently used to characterize fungi and viruses [10]. Below is a summary of our assessment.

Methods

MassARRAY SARS-CoV-2 panel

Residual de-identified RNA extracts previously tested for SARS-CoV-2 were used for this study. The MassARRAY SARS-CoV-2 Panel is a multiplexed assay that targets five SARS-CoV-2 specific regions in the nucleocapsid (N) gene, ORF1 gene, and ORF1ab gene along with a control assay for RNase P (MS2) (Agena Bioscience, Inc. San Diego, CA, USA). The assay was performed following the manufacturer's recommendations. In brief, 3 µl of RNA extract was used for a one-step reverse transcription and PCR amplification of the target regions. The product then transferred to the chip and loaded into the analyzer. Laser is applied for the desorption and ionization step. The molecules are separated and accelerate through the tube towards the detector based on their mass. A spectrum is generated and the data is then analyzed by the software (Fig. 1). Generation of spectra eliminate the need of fluorescent detection. Assay interpretation is summarized in Table 2.

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MassARRAY system

Citation: European Journal of Microbiology and Immunology 13, 1; 10.1556/1886.2023.00013

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Table 2.

MassARRAY SARS-CoV-2 panel interpretation

N1	N2	N3	ORF1	ORF1 ab	MS2	QC Status	Report
Any 2 or more +					+	Pass	SARS-CoV-2 detected
Any 1 +					+	Pass	SARS-CoV-2 inconclusive
None +					+	Pass	SARS-CoV-2 not detected
Any 2 or more +					–	Warning	SARS-CoV-2 detected
Any 1 +					–	Warning	SARS-CoV-2 inconclusive
None +					–	Warning	Invalid

(Agena Bioscience, Inc. San Diego, CA, USA).

Results

186 patient specimens were analyzed using the MassARRAY SARS-CoV-2 Panel. Nucleic acids were extracted for all specimens and tested according to the standard of care in our laboratory. Saved eluates were run on the MassARRAY SARS-CoV-2 Panel. As seen in Fig. 2, 135 specimens were in complete categorical agreement. Twenty-five specimens with ct values >35 resulted as detected with the MassARRAY panel. These specimens were not considered as discordance as they were detected by the standard PCR assay at higher ct values. Nineteen specimens showed discordant results. Seven specimens were excluded (invalid), 4 of these invalid results on the MassARRAY panel were detected by the standard PCR assay at ct values 30–35. The invalid results were due to the failure of internal control with no positive target on the MassARRAY. Unfortunately, there was no eluate/specimen or enough volume left for retesting with the third assay. All those 4 specimens also showed failure of the standard PCR internal control, but the E gene was detected at the following ct values: 30.2, 30.4, 32.5, 34.7. This may be due to some level of inhibition present in these specimens. Stratified results are summarized in Table 3, and calculated performance characteristics are tabulated in Table 4.

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Specimens included in this study and discordant agreement. Indeterminate: Ct 35–39 on the standard assay. Inconclusive: one target positive on the MassARRAY SARS-CoV-2 Panel. Invalid: no positive target and control failure on the MassARRAY SARS-CoV-2 Panel

Citation: European Journal of Microbiology and Immunology 13, 1; 10.1556/1886.2023.00013

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From this primary comparison, the MassARRAY SARS-CoV-2 Panel had an accuracy of 89.39% when compared to our standard of care.

19/186 (10.2%) that results were found to be discordant were assessed by a different assay except one where no eluate or sample was available for repeat testing (Table 5). 14 out of 18 agreed with the MassARRAY, and the performance was recalculated (see Table 6). The overall accuracy was 97.21% after the discordance agreement.

Discussion

While vaccines for COVID-19 have become available, efficient large-scale testing is still essential as the population works toward herd immunity and as virus variants emerge. New testing methods continue to be developed to meet these demands. Our study demonstrates that the MS-based MassARRAY System is an accurate and highly sensitive method for SARS-CoV-2 detection. Following the discordant agreement with an alternative RT-PCR test, the MassARRAY performance was found to have sensitivity, specificity, and accuracy, exceeding 97%, making it a viable diagnostic tool for the laboratory. Previous reports have shown that the MassARRAY system is reliable, with an earlier study of 44 samples finding complete concordance between the MS method and traditional “gold-standard” RT-PCR [12]. Another study of 168 samples [13] concluded MassARRAY to be superior in sensitivity and capable of detecting false negatives reported by RT-PCR. Rybicka et al. found the MassARRAY to be particularly advantageous when detecting patients of low viral load who were likely infected by SARS-CoV-2 and could potentially be infectious to others. It can be noted that laboratories vary in their choice of RT-PCR target genes [12, 13], with different methods targeting combinations of N genes, E gene, and regions of ORF1 and ORF1ab. Discrepancies between RT-PCR results may be attributed in some part to variations in target genes.

Practical benefits of the MassARRAY system include being low-cost to run and requiring no fluorescent reagents. As such, it can be relied upon as an alternative testing method during periods of increased demand or when real-time RT-PCR reagent supply chains, in particular fluorescent reagents, are disrupted. When evaluating different COVID-19 assays, special consideration should be given to the specificity of the test before implementation to limit the number of specimens requiring retesting. This is particularly important when laboratories experience disruption in reagent supply. MassARRAY can achieve a high throughput of approximately 3,000 samples/day [14] and has the added benefit of integrated data analysis software. The system has also been found to accurately detect SARS-CoV-2 from saliva samples [15] which can be collected rapidly and conveniently from large populations.

However, there are some limitations in using the MassARRAY system in the laboratory. The seven-hour run time far exceeds the two-hour run time of our standard real-time RT-PCR assay. Additionally, while the MassARRAY workflow is partially automated, it still requires multiple preparation steps and more hands-on time than our automated laboratory assay. Ultimately, our laboratory was able to increase real-time RT-PCR throughput to 10,000 samples/day, negotiating the need to rely on the MassARRAY system. Nevertheless, this MS-based technology is an accurate means of COVID-19 screening that can supplement the standard testing should the need arise for alternative testing methods. It can be a valuable method for some laboratories when the turn-around time for COVID-19 testing is not critical.

Our study has some limitations. Although it included large and random sampling, it would have benefited from collecting additional clinical information, including symptomatic/asymptomatic status, recent COVID-19 infection, and exposure history, such that MassARRAY performance could be assessed relating to these factors, especially with the discordance results. From our experience at the beginning of this pandemic, indeterminant real-time RT-PCR results often corresponded to asymptomatic, early or resolved infections. Furthermore, suspected patients could have been followed up to determine disease progression. Another limitation of our study is that only one specimen type (nasopharyngeal swab) was evaluated. Lastly, the unavailability of enough specimen for retesting the invalid results, limit further investigation to better understand the reason for those results.

In conclusion, our study demonstrates that the MassARRAY panel can be used as an accurate method for the detection of SARS-CoV-2. It is useful as a supplemental testing or an alternate method when experiencing a supply chain shortage. It can also be used in non-acute hospital laboratories when the turn-around time is not critical.

Contributions

Conceptualization: F AlMutawa, A Cabrera and J Delport; Data curation: F Chen, A Cabrera; Investigation: F Chen; Methodology: F AlMutawa and A Cabrera; Writing-original draft: F AlMutawa and A Cabrera; Writing-review & editing: F AlMutawa, A Cabrera and J Delport.

Funding

This study was not funded.

Data sharing

Available upon request.

Conflict of interest

The authors declare no conflict of interest.