4. MALDI-MS Investigations Targeting SARS-CoV-2

In this section, we report the MALDI-MS-based investigations which aimed to detect SARS-CoV-2 infection with the use of “proteotyping” ^[52], “biotyping” ^[47][48], “genotyping”^[49][50][51], and biomolecular host profiling^{[53][54][55][56][57]} approaches. Interestingly, two of these investigations used both biotyping and biomolecular host profiling strategies^[47][48]. All these investigations were extensively reviewed in a very recently published paper ^[65].

4.1. Proteotyping Approach for SARS-CoV-2 Detection by MALDI-MS

Dollman and colleagues applied a proteotyping strategy for the detection of peptides unique to the SARS-CoV-2 by mass alone; they used High Resolution MALDI-FT-ICR Mass Spectrometry^[52]. In particular, nasopharyngeal (NP) swabs from infected patients were cultured in Vero E6 cells or directly prepared before the MALDI-MS analysis. The use of a virus culture provided a more reliable approach to overcome potential false negatives deriving from specimens with low viral loads. The authors performed the MALDI-FT-ICR-MS analyses on the . In the case of cell-cultured SARS-CoV-2 samples with higher virus titers in comparison to other samples, twenty of the most prominent signals were confidently matched to fragments of the N, M, and S proteins with a high mass accuracy (<3 ppm) (Table 1). Detected peptides covered a large range of identified N- and C-terminal, fusion peptide receptor, and RNA-binding domains.

The acquired mass spectra from the non-cell-cultured NP samples showed peptide ions with reduced intensity and lower signal-to-noise ratio (S/N) as compared to those from experiments using cell-cultured samples; in this case, additional matrix cluster ions were also observed. However, despite the lower virus titers, the tryptic peptides of M, N, and S proteins were detected, including a new peptide at m/z 843.4255. In particular, six peptides were always detected in ten different NP samples, and five represent reliable signature peptides for SARS-CoV-2 proteotyping. In fact, due to the shared clinical symptoms presented by the SARS-CoV-2 with other circulating influenza viruses, the authors, by means of a specifically constructed algorithm and database, showed that these peptides were detected in all SARS-CoV-2 strains and exhibited a difference in mass greater than 3 ppm in comparison to those fragments detected in more than 95% of the cases from all influenza virus proteins of the circulating strains.

Table 2 reports the limit of detection (LOD) of the various analytical assays (including LOD related to conventional RT-PCR ^[66]) calculated based on the information from each of the authors, in order to make the comparison more straightforward for the readers. To determine the LOD of this MS platform, serial dilution experiments on the cell-cultured sample were performed, and the most abundant viral peptide signal at m/z 1635.8238 was monitored by MS. The peak was detected down to 0.75 ng, matching 7.5 × 10⁵ copies (Table 2).

This represents an excellent result, considering that a recent study reported maximum estimated levels of virus collected by NP swabs varying from 10⁶ to 10⁹ copies 

Interestingly, the authors stressed that this copy number is comparable with the 10⁵10⁶ copies of virus necessary for high-quality PCR sequencing; however, as they additionally claimed, it is also higher than the 10³–10⁴ copies usually required for virus detection by PCR (Table 2). As underlined by the authors, the detection limits of 10⁵ copies could be significantly improved when operating in an automated selected ion monitoring (SIM) strategy for the detection of only selected peptides for a proteotyping assay (Table 2).

Table 2. Diagnostic performances and estimated costs of the different tests used for SARS-CoV-2 detection.

4.2. Biotyping Approach for SARS-CoV-2 Detection by MALDI-MS

With the aim of carefully and systematically analyzing all the challenging issues of an MS-based diagnostic process, Iles and colleagues developed a clinical MALDI-TOF MS assay for SARS-CoV-2 detection in a saliva or gargle solution ^[47]. The authors performed several experiments in order to optimize the MALDI-TOF detection of viral protein and its proteolytic peptide detection. They finally developed a clinical diagnostic protocol based on (1) the collection of gargle/saliva samples; (2) the rapid processing of the gargle samples by filtration, acetone precipitation with subsequent pellet resuspension, and protein solubilization; (3) MALDI TOF analysis; (4) output data processing by an appropriate software (see Table 1).

Interestingly, through the clinical information extrapolated by MALDI-TOF analysis, the authors demonstrated not only the possibility to detect S protein fragments, but also other peaks matching with Immunoglobulin light chains indicative of an oral upper respiratory immune response, and elevated levels of gargle/saliva IgA heavy chain peak indicative of a viral immune response.

These features reveal the diagnostic utility of MALDI-TOF MS as a powerful and economically ideal solution, with its ease of sampling and speed of analysis. However, other studies and validation tests on saliva/gargle spiked with cultured virus, or a direct comparison of the MALDI-TOF MS analysis with an RT-PCR detection of COVID-19 in clinical samples is required.

Starting from the results obtained by the study of Iles and coworkers, Chivte and colleagues analyzed gargle samples with MALDI-TOF MS to assess the presence of SARS-CoV-2 infection and compared the resulting spectra with the corresponding results of an RT-qPCR from NP swabs ^[48]. In particular, they analyzed 60 gargle samples (see Table 1) from volunteer student athletes, including 30 PCR positive and 30 PCR negative samples. Spectral clear differences were observed in the m/z range from 20,000 to 200,000. This extended m/z range allowed them to analyze the area under the curve (AUC) of putative viral and host proteins in order to assess the presence of SARS-CoV-2 infection. The receiver operating characteristic (ROC) analysis identified five peaks which showed highly sensitive and specific discrimination between COVID-19 positive and negative samples, including peaks which probably corresponded to the S1 and S2 fragments of the SARS-CoV-2 S protein, a potential biomarker near m/z 112,000, and peaks putatively assigned as human immunoglobulins or human α-amylase (Tables 1 and 3). For these biomarkers, an elevated concordance was achieved (90%) with the RT-PCR results, demonstrating that this methodology is a promising tool for a rapid and inexpensive COVID-19 assay. 

Table 3. Sample size,protein patterns, and their expression trend in COVID-19 vs non-COVID-19 samples in Biomolecular Host Profiling studies.

⁽¹⁾ Area under the receiver operating characteristic (ROC) curve; ⁽²⁾ Receiver operating characteristic; ⁽³⁾ Machine learning; ⁽⁴⁾ Support vector machine with a radial kernel; ⁽⁵⁾ Principal component analysis; ⁽⁶⁾ DNN: deep neural network; ⁽⁷⁾ GBM: XGBoost gradient boosting machine; ⁽⁸⁾ PPA: positive percent agreement; ⁽⁹⁾ NPA: negative percent agreement; ⁽¹⁰⁾ Logistic regression.

4.3. Genotyping Approach for SARS-CoV-2 Detection by MALDI-MS

Wandernoth and colleagues evaluated the ability of MALDI-TOF MS to detect nucleic acid from SARS-CoV-2 in biological samples and compared the results to those of rRT-PCR ^[49]. In particular, they analyzed oral and NP swab samples from 22 patients who tested positive and 22 patients who tested negative by RT-PCR for SARS-CoV-2 infection (see Table 1). They used the new commercially available MS-based assay ^® (Agena Bioscience®, San Diego, CA, USA), a genotyping panel for the detection of SARS-CoV-2 developed by Agena Bioscience, which received the CE-IVD mark in Europe for the detection of nucleic acid from SARS-CoV-2 in respiratory specimens. This platform provides a robust route for the detection of the SARS-CoV-2 virus in human samples. The results of the rRT-PCR and MALDI-TOF MS analyses were comparable in all samples. The LOD was estimated at 10 genome copies (Table 2). Very recently, Rybicka and colleagues ^[50] also tested the new commercially available MS-based assay MassARRAY^® SARS-CoV-2 Panel (Agena Bioscience) and compared it to the RT-PCR diagnostic test. Oral and NP swabs from 168 suspected COVID-19 patients with symptoms of respiratory infection were simultaneously processed with both assays (Table 1). Among the 168 analyzed samples, different results were obtained for 10.12% (17 samples). Using the MassARRAY^® SARS-CoV-2 Panel, the authors were able to detect SARS-CoV-2 in low viral load specimens and with very few microliters of viral RNA. They pointed out that the MassARRAY^® System provides automation, minimal hands-on time, and onboard data analysis, delivering easy-to-interpret data with a simple and fast workflow.

Also Hernandez and colleagues compared the already mentioned RT-PCR/MALDI-TOF MS-based assay (AGENA MASS ARRAY) with the cobas^® 6800/8800 SARS-CoV2 real-time RT-PCR (Roche, Basel, Switzerland) for conventional real-time RT-PCR ^[51]. They collected saliva samples from 60 patients, which were previously subjected to molecular testing for SARS-CoV-2 in NP or anterior nare specimens. 

Interestingly, SARS-CoV-2 detection in saliva samples by the AGENA system showed high sensitivity and specificity (Table 1) when compared to RT-PCR results from NP or anterior nare specimens. Analogue sensitivity and specificity were obtained by cobas^® 6800/8800 SARS-CoV-2 real-time RT-PCR (Roche).

These data demonstrate that saliva constitutes an appropriate matrix for SARS-CoV-2 detection on the novel Agena system, with a comparable performance to the more ubiquitous real-time RT-PCR technology. 

4.4. Biomolecular Host Profiling Approach for SARS-CoV-2 Detection by MALDI-MS

In this section, we report on MALDI-MS studies performed on a variety of biological specimens in which host diagnostic biomarker patterns had been extrapolated rather than direct SARS-CoV-2 detection ^{[53][54][55][56][57]}. The investigations by Iles and colleagues ^[47] and by Chivte and colleagues ^[48], which assessed the presence of both viral and host proteins, were already reported in the previous section (see Table 1 and Table 3). Many of these investigations used oral or respiratory samples ^{[53][54][55][56]},which are the commonly accepted specimens for SARS-CoV-2 detection; interestingly, one such study was conducted using serum samples to decipher the molecular changes induced by the systemic effects after infection ^[57]. In these investigations, MALDI-TOF readouts were processed by machine learning (ML) techniques (Table 1) to extrapolate novel or unknown diagnostic information from a mass spectra dataset. Additionally, the robustness and clinical significance of the ML models employed were assessed.

Nachtigall et al. described a method of detecting SARS-CoV-2 in NP swabs using the MALDI-TOF MS and ML analyses ^[53]. They proposed this approach starting from the rationale that several clinical laboratories use MALDI-MS as a high-throughput, cost-effective, and robust technology for the conventional diagnosis of pathogen infections.

They analyzed a total of 362 NP swab samples, 211 RT-PCR positives and 151 RT-PCR negatives, from subjects coming from three different countries (Argentina, Chile, and Peru) (Table 1).

The authors tested different feature selection methods and ML approaches to determine the top performing analysis strategies. Feature selection methods can be used in data pre-processing to obtain efficient data reduction, a useful step in order to identify accurate data models [96]. Through a comparison of the results obtained from RT–PCR and MALDI-TOF coupled with ML, the authors found a concordance rate that was acceptable as a clinical diagnostic approach (>80%), confirming that the MALDI-MS and ML analyses represent promising alternatives as fast screening assays for SARS-CoV-2.

Tran and colleagues described as proof-of-concept a novel, automated, ML-enhanced MALDI-TOF MS approach for analyzing nasal swabs from patients with suspected COVID-19 ^[54].

The authors evaluated the performance of the MALDI-TOF MS method using SARS-CoV-2 RNA PCR positive and negative samples in order to determine the accuracy, the positive percent agreement (PPA), and the negative percent agreement (NPA) of the MALDI-TOF MS method as compared to the PCR method. They collected 226 nasal swab samples that were analyzed both by RT-PCR and by MALDI-TOF MS. Normalized mass spectra were subjected to ML analysis by the Auto-ML MILO (Machine Intelligence Learning Optimizer) platform ^[67]. The MILO as ML platform consists in a series of algorithms, scalers, scorers, and feature selectors/transformers, which are used to generate models that are then statistically assessed to ultimately identify the best performing model for a specific purpose. The authors described their ML-enhanced MALDI-TOF approach as an attempt to address both throughput and speed limitations observed in molecular platforms; interestingly, the authors demonstrated the ability of both PCA and ML models to classify MALDI-TOF mass spectra for discriminating positive and negative COVID-19 cases.

The high-throughput performance of this platform was also demonstrated by the short time required for the analysis (total turnaround time was less than 1 h, with the potential of performing up to 1104 analyses per day, per instrument) as compared to commercial RT-PCR platforms, which need, for example, batch testing for optimal reagent use.

Another investigation that described the analysis of NP samples by MALDI-TOF MS coupled with ML was performed by Deulofeu and colleagues ^[55]. In particular, they used different ML approaches for the analysis of the MALDI-TOF mass spectra from 237 NP samples in order to identify a signature characterizing negative and positive samples for SARS-CoV-2 infection (Table 1). These samples were analyzed by both RT-PCR and MALDI-TOF MS. The obtained data demonstrated the utility of the developed method for the detection of SARS-CoV-2-positive samples and the simplicity, safety, rapidity, and cheapness of MALDI-TOF MS as a diagnostic test. The authors estimated the cost of MALDI-TOF MS to be 25% more than the cost of the RT-PCR analyses. They also stated that the time required to receive results is around 6 h for a conventional RT-PCR, whereas the analyses of the NP samples by MALDI-TOF MS take less than one-third of this (Table 1).

Finally, Rocca and colleagues also investigated the potential role of MALDI-TOF MS combined with ML methods in the identification and the discrimination between COVID-19 positive samples and COVID-19 negative ones ^[56]

They used NP swab samples from 311 patients that were analyzed by MALDI-TOF MS analysis without processing (Table 1). From the evaluation of parameters and statistical analysis, six peaks at m/z 3372, 3442, 3465, 3488, 6347, and 10,836 demonstrated a decrease or an absence of COVID-19 positive samples and were considered as potential biomarkers (Table 3).

The preliminary results demonstrated that the method still needs to be improved, and the authors stated that further studies on a larger cohort of patients, evaluating different extraction methods and improving ML algorithms, will be performed. 

Yan et al. utilized a high-throughput serum peptidome profiling method based on MALDI-TOF MS for the detection of COVID-19 ^[57]. They analyzed serum samples from a total of 298 individuals: 146 COVID-19 patients (classified into mild, typical, severe, and critical) and 152 control patients (including 73 non-COVID-19 patients with similar clinical symptoms such as fever/cough, 33 tuberculosis patients, and 46 healthy individuals) (Table 1).

Using ML methods, twenty-five peaks were identified as the distinctive features between COVID-19 patients and control participants, with statistically significant differences (Table 3). 

Starting from the observation that the symptoms of SARS-CoV-2 infected patients are frequently shared by patients affected by other respiratory infections, the authors highlighted that this could lead to the misinterpretation of results; so they emphasized the ability of this platform to discriminate COVID-19 patients from non-COVID-19 subjects with similar clinical symptoms. The authors concluded that these results also demonstrated that MALDI-TOF-based serum profiling could be a powerful tool for screening, routine surveillance, and diagnosis of COVID-19 in populations.

5. Discussion

5.1. Comparison of MALDI-MS vs RT-PCR Techniques for SARS-CoV-2 Detection: Advantages and Limitations

As a resource for new, alternative and/or complementary, rapid COVID-19 diagnostic tests, here we  explored the recent developments and applications of MALDI MS-based technologies for the accurate and unambiguous SARS-CoV-2 clinical diagnosis, which may help to control the COVID-19 pandemic.

MS techniques have rapidly complemented or replaced conventional methods in clinical diagnostic laboratories and have been successfully implemented in the investigation of viruses and their pathogenesis ^[13],^{[15][16][17][18][68][69]}. In particular, MALDI-MS has become an indispensable and versatile method for biochemical and clinical investigations ^[70][71].MALDI-MS represents one such technology, characterized by its ease of use, high-throughput capabilities, and cost effectiveness, thus becoming a routine tool in clinical microbiology laboratories and also proving to be capable of supporting clinical decision-making^[24][44] .

The current gold standard technique for the molecular diagnosis of SARS-CoV-2 infection is the RT-qPCR, which allows for the analysis of thousands of samples in a single day and shows a high testing sensitivity of 95% ^[72] and a low limit of detection of <10 copies/reaction^[73]  under ideal circumstances. Nevertheless, several investigations have reported that clinically evident COVID-19 infections often go undetected by SARS-CoV-2 RT-PCR testing ^[74];the evidence that virus shedding can occur at undetectable levels during the very early and late stages of SARS-CoV-2 infection demonstrates that RT-PCR results should always be interpreted in a wider context. Indeed, the most efficient strategy for the diagnosis of SARS-CoV-2 infection in suspected patients should encompass a combination of SARS-CoV-2 detection by RT-PCR with clinical and epidemiologic observations (symptoms, previous exposure to virus, negative diagnostic tests for other respiratory diseases), whereas additional follow-up testing with RT-PCR should be required from patients with initially negative results and high suspicion of COVID-19.

The time required for conventional PCR can vary from about 4–6 h for sample processing (including sample preparation, RNA extraction, reverse transcription PCR, and readout of amplified DNA products), up to a few days, considering the time needed for the eventual transport of the specimen to the laboratory.

Most of the reports described here that are related to Biotyping, Proteotyping, and Genotyping approaches are proof-of-principle studies showing the feasibility of viral protein or viral nucleic acid detection by MALDI-MS, which often do not involve stress testing in low viral load samples. Nevertheless, as reported in Table 1 and Table 2, the specificity, sensitivity, and the detection limit of the SARS-CoV-2 virus by MALDI-MS diagnostic methods were high and comparable to reference methods such as PCR.

5.2. Pre-Analytical and Analytical Issues for Molecular Detection of SARS-CoV-2

There are several critical issues and challenges affecting the diagnosis of SARS-CoV-2 infections, including sampling operations, specimen source, and sampling time, which could have an impact on the end result. It is essential to handle bodily fluids according to standardized procedures in order to distinguish reliable molecular biomarkers and to assess the influence of pre-analytical parameters on the final result in order to avoid artifacts. As revealed by the literature reviewed in the previous sections, there is high variability in all of the abovementioned critical technical factors; for example, most studies do not clarify if the tested samples were analyzed by MALDI-MS immediately after collection or if they had been deep-frozen until analysis. This represents an important limitation, considering that several pre-analytical and analytical variables, including storage time and temperature, could alter the analysis of the MS profile of biological specimens, and that several studies demonstrated that the proteins could undergo degradation processes^[37][75][76]. Thus, especially for the biomolecular host profiling studies, it is not possible to exclude the fact that the observed differences in the molecular fingerprints obtained by MALDI-MS could derive from the degradation of the molecular species due to the action of endo-proteases, including those of microbial origin in the nasal environment, or from the inherent biological variability among subjects, also due to genetic factors affecting the phenotype. 

5.3. Detection of SARS-CoV-2 in Different Types of Clinical Specimens

Respiratory sampling is the preferred and widely approved method for SARS-CoV-2 detection by RT-qPCR. The choice of optimal clinical specimens takes into consideration several factors such as ease of accessibility, non-invasive collection procedure, a lower risk to health care professionals during sample collection, and good viral loads for higher possibilities of detection. NP and/or OP swabs are best recommended for the screening or diagnosis of an early infection, when low viral loads are known to occur and false-negative results can derive from differences in analytical sensitivity among methods^[77]. For these reasons, most of the studies reviewed here are based on the use of NP or OP samples.  

An alternative option for collecting upper respiratory tract specimens in order to detect SARS-CoV-2 is self-collected saliva or gargle specimen^[66] . Saliva is a reliable biological fluid that can easily be provided by the patient; it does not require healthcare personnel in a screening program ^[66][78]. In fact, a mass screening program should rely on devices that can be used by non-medical staff to promptly assess whether an individual is positive to COVID-19. Moreover, a gargle sample is obtained from an easy-to-perform procedure and is comfortable for the patient. Reliable and rapid diagnostic techniques based on the use of saliva or gargle specimens might represent a cost-saving procedure considering that their collection does not require the need to develop and maintain specific infrastructure for swab collection as well as the need for dedicated healthcare personnel. 

Serum samples could be used to understand the molecular mechanism for the diagnosis of COVID-19. As a consequence of viral infection, serum may contain mediators produced by the systemic effects or released to the lung, and this could reflect the physiological or pathological state ^[20].

Serum samples, and more generally, serological methods, could play a key role in the epidemiology of COVID-19 and in defining the immune status of asymptomatic patients, but they are not recommended for the screening or diagnosis of early infections ^[79]. However, serology could contribute to the confirmation of the diagnosis of COVID-19 by providing information about the type and the concentration of various immunoglobulins generated after SARS-CoV-2 infection ^[80].

5.4. Asymptomatic Infection and the Spread of New SARS-CoV-2 Variants

Another critical issue is the limited resources employed to facilitate and enable the widespread rapid screening of asymptomatic individuals necessary to control the potential of silent chains of virus transmission. Asymptomatic patients are the carriers of the live, replicating virus and contribute to the unknown viral transmission and spread. In some cases, the methods used for SARS-CoV-2 detection in these subjects can be less sensitive than in the symptomatic ones ^[81]. The here reviewed studies which have considered asymptomatic population demonstrated that the MALDI-TOF MS platform was able to detect SARS-CoV-2 infection in asymptomatic individuals, and with elevated accuracy (see Table 1), which could be of paramount importance for the reopening of businesses, recreational facilities, schools, and all non-hospital settings.

In addition to asymptomatic infection, SARS-CoV-2 variants are another challenge for virus detection^[82][83] . In fact, new variants caused by mutations in the genome of the SARS-CoV-2 are now emerging as the virus spreads throughout the world, and this is of great relevance considering that such mutations may alter various aspects of virus biology such as pathogenicity, infectivity, transmissibility, and/or antigenicity ^[84]]. Thus, alternative and/or complementary methods of rapidly detecting and monitoring the evolution of virus strains are of vital importance.

Very recently, some studies employed MALDI-MS to study and distinguish strains of the major variants of concern using mass signatures^[83][85]. These studies also demonstrate how such MALDI-MS data can be used to chart viral evolution and to construct mass-based phylogenetic trees, similar to those obtained using conventional gene sequence phylogenetics. The use of MS datasets to track the evolution of SARS-CoV-2 in order to identify such mutations that may limit detection by PCR or those that allow immune escape or reduce the efficacy of existing vaccines and/or therapies have great future potential.

6. Conclusions

Laboratory SARS-CoV-2 testing assays are the primary mitigation measure for the prompt identification and isolation of infected individuals. In fact, the rapid and accurate detection of SARS-CoV-2 infection and the close monitoring of persons in contact with cases are key steps in managing the COVID-19 pandemic, until vaccines can be extensively administered or until antiviral drugs become widely available. In this context, MS-based technologies may represent an alternative resource or may support PCR-based methods as a diagnostic test in the fight against the novel coronavirus. To date, MALDI-MS is routinely used in clinical practice for the rapid, unambiguous, and cost-effective identification of pathogens in biological samples. It is worth noting that several MS-laboratories around the world have been working to deliver guidelines and protocols on sample collection, processing, and data analysis in order to develop rapid and robust methods for the high-throughput screening of SARS-CoV-2 infection. Furthermore, clinical trials using MALDI-MS are currently ongoing for the management of the COVID-19 pandemic. In particular, one of these studies is based on MALDI-TOF MS profiling and combined with ML methods to detect individuals infected with SARS-CoV-2 from saliva samples, while the other one is based on the MALDI-TOF MS analysis of both saliva and blood samples to search for discriminating profiles between COVID-19 and non-COVID-19 individuals. Nevertheless, some limitations still exist for the applications of MALDI-MS technology in routine virus diagnostics. In fact, unlike what happens for bacteria, in the case of biotyping approaches, the major limitations could arise from the relatively low protein content of viruses, the higher molecular weight of viral proteins (>20,000 Da), and a probable carryover of debris from the cell substrate in which the viruses are cultured in vitro.

On the other hand, despite the high costs of the instrument, the high-resolution MALDI MS with proteotyping strategies, based on SARS-CoV-2 signature peptides, has demonstrated excellent performance characteristics especially for the high-accuracy detection of SARS-CoV-2. Considering high accuracy, high throughput, and speed of analysis, the use of more innovative MS-based tools and assays represents an essential and feasible complement to PCR.

MALDI-MS-based genotyping approaches also enable the high-throughput and low-cost detection of SARS-CoV-2. Nevertheless, the level of accuracy of these approaches is not yet comparable to those of proteotyping strategies. In any case, conventional tests such as PCR or immunoassay have become insufficient, also considering the shortages of medium, reagents, collection devices, and consumables that hinder day-to-day laboratory operations and the ability to improve testing capacity. In this scenario, MALDI-MS-based genotyping strategies could represent reliable diagnostic techniques that require alternative consumables. Moreover, recent investigations have demonstrated the ability of these platforms to assess unfolding genomic variation in a timely manner, and have highlighted the potential of diagnostic results to serve as a reliable system for the detection of emergent SARS-CoV-2 variants of concern .

Finally, regarding the proof-of-principle studies, the biomolecular host profiling approaches that assess host response to predict infection are a further intriguing alternative to the current testing methods for the identification of SARS-CoV-2 infected individuals. However, viral load differs among infected individuals, in particular between symptomatic and asymptomatic subjects, which leads to a broad variation in host response. Therefore, these approaches warrant further investigations that would test their threshold for the detection of viral infection and their ability to differentiate types of infectious or non-infectious causes of host modulation, in order to avoid confounding results. Additionally, these strategies also have multiple desirable features for a clinical test during the COVID-19 pandemic, which includes minimal sample preparation, few reagents, and rapid and high-throughput data acquisition.

Arguably, as part of a pandemic preparedness plan, a massive amount of novel scientific knowledge as well as the use of innovative MS-based tools and assays will support the development of diagnostics facilitating a reliable response to emerging infectious diseases.

Certainly, the expanded application of MS-based technologies and related multi-omics strategies could become one of the reference methodologies for other future epidemics/pandemics that may arise.