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Published in final edited form as: J Breath Res. 2021 Dec 20;16(1):10.1088/1752-7163/ac3f24. doi: 10.1088/1752-7163/ac3f24

Inactivation of SARS-CoV-2 in clinical exhaled breath condensate samples for metabolomic analysis

Shuang Hu 1, Mitchell M McCartney 2,3,4, Juan Arredondo 1, Sumathi Sankaran-Walters 1, Eva Borras 2,3, Richart W Harper 3,4,5, Michael Schivo 3,4,5, Cristina E Davis 2,3,4, Nicholas J Kenyon 3,4,5, Satya Dandekar 1,*
PMCID: PMC9809239  NIHMSID: NIHMS1857782  PMID: 34852327

Abstract

Exhaled breath condensate (EBC) is routinely collected and analyzed in breath research. Because it contains aerosol droplets, EBC samples from SARS-CoV-2 infected individuals harbor the virus and pose the threat of infectious exposure. We report for the first time a safe and consistent method to fully inactivate SARS-CoV-2 in EBC samples and make EBC samples safe for processing and analysis. EBC samples containing infectious SARS-CoV-2 were treated with several concentrations of acetonitrile. The most commonly used 10% acetonitrile treatment for EBC processing failed to completely inactivate the virus in samples and viable virus was detected by the assay of SARS-CoV-2 infection of Vero E6 cells in a biosafety level 3 laboratory. Treatment with either 50% or 90% acetonitrile was effective to completely inactivate the virus, resulting in safe, non-infectious EBC samples that can be used for metabolomic analysis. Our study provides SARS-CoV-2 inactivation protocol for the collection and processing of EBC samples in the clinical setting and for advancing to metabolic assessments in health and disease.

Keywords: exhaled breath condensate (EBC), SARS-CoV-2, COVID-19, metabolomics analysis, infectious material

1. Introduction

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is cause of the devastating pandemic, known as coronavirus disease 2019 (COVID-19), that has resulted in massive loss of human lives and economy globally [1, 2]. The lung is major target organ of SARS-CoV-2 and the respiratory tract presents as the primary site of infection. The virus attaches to lung epithelial cells through binding with angiotensin converting enzyme 2 [3], in synergy with the host factors, TMPRSS [4] and Cathepsin L [5]. Both TMPRSS and Cathepsin L cleave and activate the SARS-CoV-2 spike protein, and promote viral entry and pathogenicity [4, 5]. Susceptible cell targets of the virus are also located in several tissues including liver and gastrointestinal tract and the virus has been detected in these tissues [6–8]. The virus is readily detected in nasal swabs, saliva and exhaled breath condensate (EBC) samples [9, 10]. These samples are useful to identify virally-induced changes in the lung microenvironment and are valuable for the viral diagnosis and for monitoring progression of viral infection as well as host response to therapy.

EBC samples are collected through non-invasive methodology and are valuable in the evaluation of respiratory infections and non-infectious diseases [11]. EBC contains a combination of water vapor and breath aerosols, and is a readily available biospecimen that can be collected from healthy as well as clinically symptomatic individuals. Because its chemical contents include endogenous metabolites and exogenous compounds, biomarkers in EBC correlate with health status and reveal disruption of lung functions [12]. Researchers have investigated the utility of EBC in monitoring of the clinical outcomes during asthma [13, 14], allergies [15], drug use [16], inflammation [17], oxidative stress [18], occupational exposure and public health [19]. The use of EBC analytes has not been well explored in evaluating host response to SARS-CoV-2 infection and for monitoring disease outcomes. Several investigators have detected the presence of the SARS-CoV-2 virus nucleic acids in EBC samples [9, 20]. The EBC samples from COVID-19 patients are being collected by researchers for further analysis and for research investigations. It is of concern that the presence of infectious SARS-CoV-2 in EBC samples may pose threat of infection to researchers and clinical staff who are performing collection and processing of the samples [9]. There is an urgent need to develop safe methods for the collection and processing of EBC samples for biomarker evaluation.

Prior to the COVID-19 pandemic, EBC was considered a relatively safe biospecimen to collect, and use in several clinical and translational studies [19]. Since SARS-CoV-2 virus spreads primarily through fine exhaled aerosol droplets, it has raised serious concerns on how to safely collect, process and analyze breath samples. Because EBC contains aerosol droplets, there must be safety considerations for the subject and operator during breath sample collection. These are accomplished through standardized methods to collect infectious material including the use of personal protective equipment, biological safety cabinets and BSL-2 laboratories. EBC samples are routinely transported from point-of-sample collection sites to other laboratory spaces or facilities for chemical analysis by mass spectrometry, which may not always be equipped to manage samples containing pathogens such as SARS-CoV-2. It is well recognized that many SARS-CoV-2 infected individuals remain clinically asymptomatic but their EBC may still harbor infectious viral particles. Processing of EBC samples routinely incorporates the use of organic solvents, such as acetonitrile [13–16, 18], and it is assumed that the solvent is sufficient in concentration and time to inactivate infectious agents. Currently, EBC samples are collected in 10% acetonitrile prior to metabolic analysis [18], but the efficacy of 10% acetonitrile in inactivating SARS-CoV-2 virus has not been reported. Therefore, there is an urgent need to determine whether standard EBC sample preparation procedures inactivate infectious virus for safety considerations.

We developed and optimized protocol that will effectively inactivate SARS-CoV-2 in EBC samples and make them safe and accessible for laboratory analysis. We hypothesize that infectious SARS-CoV-2 can be irreversibly inactivated by the acetonitrile treatment and loss of viral infectivity can be detected using viral infectivity cell culture assay. We evaluated the efficacy of acetonitrile at 10%, 50% and 90% for inactivating the infectious SARS-CoV-2 by measuring its infectivity in Vero E6 cells culture assay. We found that 50% and 90% of acetonitrile fully inactivated the virus, but not with 10% acetonitrile treatment. Our data demonstrate an effective protocol to fully inactivate infectious SARS-CoV-2 in clinical EBC samples to enable further processing for biomarker and metabolomic analyses. This procedure can be advanced to the clinical setting and will be useful for clinical and translational researchers worldwide to perform safe sample collection and processing.

2. Methods

2.1. SARS-CoV-2 virus strain and propagation

The mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) virus harbors mNeonGreen (mNG) fluorescent gene that emits green signal under UV light after infection (received from Dr Scott Weaver, the UTMB World Reference Center for Emerging Viruses and Arboviruses) [21]. The mNG reporter gene was stably inserted into ORF7 of the SARS-CoV-2 viral genome [21]. This engineered virus allows longitudinal monitoring of virally infected cells since cells supporting active viral replication can be visualized under microscope with green fluorescence of mNG. Infectivity, cytopathicity and replication kinetics of the mNG SARS-CoV-2 are comparable to the unmodified wildtype SARS-CoV-2 [21].

High titer mNG SARS-CoV-2 viral stock was developed by infecting and expanding the virus in Vero E6 cells as previously described [21]. The Vero E6 cells were grown in T75 tissue culture flasks (ThermoFisher Scientific) in Dulbecco’s modified Eagle’s medium (DMEM) glutamix (Gibco) with 10% fetal bovine serum (FBS; Gibco). The viral stock was maintained at the titer of 1.53 × 107 plaque forming units ml−1 and was used for the viral infection studies [22, 23]. The viral titers were measured using the viral plaque assay, which was used to measure the number of infectious viral particles capable of forming plaques in Vero E6 cells [24]. The viral cell culture studies were performed in a biosafety level 3 (BSL-3) laboratory. The laboratory personnel were required to wear powered air purifying respirators (Breathe Easy, 3 M) with Tyvek suits, aprons, booties and double gloves.

2.2. SARS-CoV-2 infection of vero E6 cells and detection by microscopy

We determined the susceptibility of the Vero E6 cells to SARS-CoV-2 infection. The Vero E6 cells (2 × 105 cell per well) were dispensed in 6-well plate and were infected with SARS-CoV-2 at different viral titers (1.53, 153 and 15 300 PFU) in BSL3 laboratory. The cells were infected with different concentrations of the virus (153 PFU and 15 300 PFU) and monitored for the presence of the viral infection. Cells without viral exposure served as negative controls for the measurement of cell growth and viability. We previously reported that cells infected with 1 PFU SARS-CoV-2 has detectable cytopathic effects and high mNG expression during the course of five days cell culture [25].

The Vero E6 cells with productive SARS-CoV-2 infection were visualized by the green fluorescence and detected using Primovert iLED (Zeiss 491207– 0005-000) microscope at 20X and Zeiss Labscope (v3.2). Five different regions in the culture well were randomly selected and imaged under the same settings of bright field and florescent channel. Active viral infection is detected by the presence of green florescent cells, while total absence of green florescent cells is an indication of the lack of viral infection.

2.3. Acetonitrile treatment for viral inactivation

To determine whether acetonitrile inactivated infectious SARS-CoV-2 particles, the mNG SARS-CoV-2 at different titers (1 PFU, 153 PFU or 15 300 PFU) was incubated in acetonitrile at three different concentrations (10%, 50% and 90%) and used for testing its infectivity in Vero E6 cells. In addition, EBC samples containing SARS-CoV-2 were also evaluated for the viral inactivation following the treatment with acetonitrile. The EBC samples were spiked with 153 or 15 300 PFU and incubated with 50% or 90% acetonitrile (MilliporeSigma AX0156-1, LC-MS grade) for 15 min at room temperature. The treated samples were tested for viral infectivity by using the Vero E6 cell assay. Samples were added to the final volume of 2.3 ml DMEM media in the cell cultures and cells were maintained for five days. Cells positive for viral infection were identified by visualizing green florescence under the microscope. Acetonitrile concentrations from the treated samples were diluted to 4.3% and 7.8% acetonitrile in the media.

2.4. Measurement of viral RNA load using real-time PCR assay

Samples of Vero E6 cell culture supernatant (200 μl) were collected at five days post-infection and mixed with three volumes of TRIzol LS (Thermo Fisher Scientific). Viral RNA extraction was performed using Direct-zol™ RNA Miniprep kit (ZYMO RESEARCH cat. #R2050). The RNA samples were suspended in 30 μl water and used (10 μl) to generate cDNA by using the SuperScriptIII with random hexamer primers (ThermoFisher kit; cat. #18080051). SARS-CoV-2 nucleocapsid (N1) specific primers were used in the real time PCR assay (40 cycles) that included N1 gene segment (wtN_F4, 5′-GTTTGGTGGACCCTCAGATT-3′; wtN_R4, 5′-GGTGAACCAAGACGCAGTAT-3′; wtN_P4, 5′-/56 FAM/TAACCAGAA/ZEN/TGGAGAACGCAGTGGG/3IABkFQ/-3′) and viral RNA copy numbers were determined [26].

2.5. Measurement of cell viability of vero E6 cells

To determine whether acetonitrile had cytotoxic effects on the Vero E6 cells in culture, we utilized MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenly tetrazolium bromide) colorimetric assay (Sigma-Aldrich, cat. #CT02) for assessing cell metabolic activity and cell viability. In this assay, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-dependent cellular oxidoreductase enzymes, indicators of cell viability and metabolism, are capable of reducing the tetrazolium dye MTT to its insoluble formazan, which has a purple color [27]. Acetonitrile associated cytotoxicity on Vero E6 cells was evaluated by adding mock viral infection samples treated with acetonitrile to the cell cultures. Cells (2 × 104) were placed on 96-well plate in DMEM glutamix with 10% FBS for two days. For mock infections, acetonitrile (50% and 90%) dilutions were prepared in in a volume of 20 μl and further diluted to 30 μl using DMEM media supplemented with 10% FBS. This was added to Vero E6 cells for 1 h of mock infection. Addition of DMEM media to the cells resulted in the final concentration of acetonitrile to 4.3% and 7.8%. On day 1 and day 4, cells were monitored at 1 and 4 days post-mock infection under EVOS™ FL Auto 2 Imaging System (Fisher Scientific). MTT reagent was added to cells and the absorbance was measured at 650 nm in Spectrophotometer Plate Reader (BIORAD xMARK™). Cell survival rate was interpreted as the percentage relative to untreated control.

2.6. Collection and preparation of EBC samples

The EBC samples were obtained from participants at UC Davis who did not have documented COVID-19 symptoms. The samples were collected using a custom EBC sampler previously detailed for several clinical studies [13, 28, 29]. The user exhales into the sampler using normal, tidal breathing for 15–20 min through a disposable valved mouthpiece without a nose clip. Exhaled breath passes through a trap that passively separates saliva and larger contaminants from exhaled aerosols. The aerosols then pass through the interior of a hollow glass tube that is surrounded by dry ice. The glass surface achieves temperatures that condensate exhaled breath aerosols in the tube. After the user is finished breathing, the EBC is retrieved from the glass tube, and stored at −80 °C. The EBC samples were collected from men (n = 5) and women (n = 4) participants. Their age ranged from 20 to 34 years. These participants were healthy and did not have any clinical symptoms at the time of the sample collection. The EBC sample collection was performed under the human subjects protocol reviewed and approved by the UC Davis IRB, protocol #1636182.

To assess the effects of acetonitrile incubation on EBC metabolite content, a liquid chromatography-quadrupole time of flight-mass spectrometry (LC-qTOF-MS) assay was performed. Samples from several subjects were pooled. From this pool, 150 μl of EBC was used per sample. Twelve EBC samples were prepared: three replicates within four treatment groups: 90%, 50% and 0% acetonitrile incubation, and an additional untreated control group that was processed by previously reported protocols [14, 16, 28, 29].

The pooled EBC samples (150 μl) from three healthy participants were treated with 2.7, 1.5 and 0 ml of acetonitrile for the 90%, 50% and 0% acetonitrile groups, respectively. Deionized water was added to a final volume of 3 ml. Acetonitrile-treated samples were incubated for 15 min at room temperature and acetonitrile was then removed by drying with ultra-high purity nitrogen. The EBC samples without acetonitrile incubation served as untreated controls. All samples were lyophilized until complete dryness, then reconstituted with mobile phase (95:5 water:acetonitrile). In addition to EBC samples, matrix blank samples were prepared for each treatment group (no EBC used during preparation).

Samples were analyzed on an Agilent 1290 series HPLC system with an Agilent 6530 qTOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Injection volume was 20 μl with samples held at 5 °C. An InfinityLab Poroshell 120 EC-C18 column was used for chromatography (3.0 mm × 50 mm × 2.7 μm, Agilent Technologies). The column compartment was set to 35 °C with a flow rate of 600 μl min−1. A solvent gradient was used with mobile phase A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid). Samples were injected once in electrospray ionization (ESI) positive and once in negative mode, capturing mass ranges of 60–1000 and 100–970 m z−1 respectively, acquired at a scan rate of 2 Hz.

2.7. Quantification and statistical analysis

Data represent the mean ± SEM, calculated using all data points from at least three independent experiments. Statistical significance was determined using nonparametric Mann–Whitney U test for samples with only two groups. All analysis was performed using Graphpad Prism version (9.2.0).

LC-qTOF-MS data were deconvoluted and aligned using Profinder B.08 (Agilent Technologies). Data were cleaned by removing signal from matrix blanks corresponding to each sample treatment. An ANOVA was performed with a p value ⩽0.05 to compare the number of detected chemical features.

3. Results

3.1. Incomplete inactivation of SARS-CoV-2 with 10% acetonitrile treatment

A commonly used protocol for EBC sample processing involves the collection of EBC samples in 10% acetonitrile and lyophilization of these samples prior to transporting for metabolic analysis (figure 1). However, the efficacy of acetonitrile in inactivating infectious reagents in the EBC has not been investigated. We sought to investigate whether acetonitrile treatment inactivates viral infectivity in the EBC samples since EBC samples are dispensed in acetonitrile during the analysis. To detect the effects of acetonitrile on the infectivity of SARS-CoV-2, we utilized Vero E6 cell cultures to infect with SARS-CoV-2. A single plaque forming unit (PFU) of SARS-CoV-2 is sufficient to infect Vero E6 cells, disseminate infection to majority of cells in the culture and cause cytopathicity. We have previously reported that 1 PFU of SARS-CoV-2 induced cytopathic effects and mNG expression were detected in Vero E6 cells during the five days time course [25]. In the current study, our goal was to examine whether 10% acetonitrile treatment is effective in inactivating 1 PFU of the virus which can be detected by the lack of mNG-expressing cells in the Vero E6 culture assay. We assessed the viral infectivity of 1 PFU of SARS-CoV-2 previously treated with 10% acetonitrile in the Vero E6 cells. The SARS-CoV-2 infected cells were readily detected (as green fluorescent cells positive for SARS-CoV-2, figure 2(B)) showing that 10% acetonitrile failed to inactive even one PFU of SARS-CoV-2.

Figure 1.

Figure 1.

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Procedure of exhaled breath condensate (EBC) collection and sample preparation.

Figure 2.

Figure 2.

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Detection of SARS-CoV-2 infected cells. The cells were visualized by microscopy at 20X. (A) Uninfected cells (under bright field), and (B) SARS-CoV-2 infected cells (seen as green under UV light) were detected. Vero E6 cells were infected with 1 PFU of SARS-CoV-2 virus that had prior 10% acetonitrile treatment.

3.2. Complete inactivation of SARS-CoV-2 in acetonitrile at higher concentrations

We sought to determine the effects of higher levels of acetonitrile on inactivating SARS-CoV-2 infectivity. The virus (at 153 PFU or 15 300 PFU concentrations) was suspended in acetonitrile (10%, 50% and 90%) for 15 min. Following this incubation time, the viral sample was added to Vero E6 cells, resulting in the dilution of acetonitrile in the culture media. The Vero E6 cells were cultured for five days and examined for the virally infected cells and for the cytopathic effects of the viral infection were measured following microscopic examination. The Vero E6 cells infected with 153 PFU (figure 3(A)) or 15 300 PFU (figure 3(E)) of SARS-CoV-2 showed a high level of the viral infection as evidenced by the presence of increased numbers of mNG-positive green fluorescent cells. Cytopathic effects of the viral infection were evident by the detachment and loss of adherent Vero E6 cells (figures 3(A) and (E)). In contrast, 10% acetonitrile treated viral samples had a substantial loss in their infectivity which was evident by decreased number of mNG-positive cells compared to untreated controls (figures 3(B) and (F)). The data show that 10% acetonitrile treatment reduced the viral infectivity but failed to completely inactivate it. In contrast, treatment of the virus with 50% (figures 3(C) and (G)) and 90% acetonitrile (figures 3(D) and (H)) resulted in complete inactivation of the viral infectivity and this was demonstrated by the complete absence of mNG-positive Vero E6 cells as compared to untreated controls. This correlated with the maintenance of cell viability of Ver E6 cells which was comparable to untreated controls.

Figure 3.

Figure 3.

Figure 3.

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Loss of SARS-CoV-2 infectivity following acetonitrile treatment. The virus (153 or 15 300 PFU) was dispensed in 10%, 50% or 90% acetonitrile and the viral infectivity was measured in Vero E6 cells. Virally infected (green) and uninfected cells were microscopically visualized at 20×. Cells were infected with (A) 153 PFU of SARS-CoV-2 and with the same dose of virus after treatment with (B) 10%, (C) 50% and (D) 90% of acetonitrile. Cells were infected with (E) 15 300 PFU of SARS-CoV-2 and with the same dose of virus after treatment with (F) 10%, (G) 50% and (H) 90% of acetonitrile.

3.3. Complete inactivation of SARS-CoV-2 in EBC samples with acetonitrile treatment

The EBC samples containing infectious SARS-CoV-2 (153 PFU and 15 300 PFU) were treated with 50% or 90% acetonitrile and evaluated for the viral infectivity by Vero E6 cell assay. While SARS-CoV-2 in untreated EBC samples readily infected Vero E6 cells as detected by a large number of mNG-positive cells (figures 4(B) and (E)), there was complete absence of mNG signal in Vero E6 cells exposed to SARS-CoV-2 from acetonitrile (50% and 90%) treated EBC samples (figures 4(C), (D), (F) and (G)). Our findings suggest that acetonitrile at 50% and 90% completely inactivated SARS-CoV-2 in EBC samples.

Figure 4.

Figure 4.

Figure 4.

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Inactivation of SARS-CoV-2 in EBC samples. The EBC samples containing SARS-CoV-2 (153 and 15 300 PFU) were suspended in acetonitrile and used to infect Ver E6 cells. Cells were evaluated by microscopy under bright field (left), and for the viral infection (seen as green) under UV light (right) at 20×. (A) Uninfected control. Cells were infected with (B) EBC with 153 PFU SARS-CoV-2 and with the virus inactivated with (C) 50% and (D) 90% of acetonitrile. Cells were infected with (E) EBC harboring 15 300 PFU of SARS-CoV-2 and with the virus inactivated with (F) 50% and (G) 90% of acetonitrile.

To determine whether the residual levels of acetonitrile from the treated samples had any effects on Vero E6 cell viability that was independent of viral infection, we performed MTT assay. The concentration of acetonitrile in the EBC samples treated with 50% and 90% acetonitrile for 15 min was diluted to 4.3% and 7.8% respectively in the DMEM media of the cell cultures. Longitudinal monitoring of the cells microscopically revealed that acetonitrile in the samples had transient impact on cells on day 1, causing a minor fraction of cells to detach (supplemental figure 1 (available online at stacks.iop.org/JBR/16/017102/mmedia)). However, the cells proliferated and had cell viability similar to untreated controls by day 4 (supplemental figure 1) and had high cell viability as measured by MTT assay (supplemental figure 2). The Vero E6 cells exposed to mock infection samples (50% and 90% acetonitrile treated) did not show any marked cytotoxic effects of residual acetonitrile. Thus, our findings suggest that the lack of viral infection in Vero E6 cells exposed to SARS-CoV-2 treated with 50% and 90% acetonitrile is due to the effective inactivation of the virus and this was totally independent of any cytotoxic side effects of acetonitrile on the cells.

3.4. Detection of SARS-CoV-2 viral RNA in EBC samples

In SARS-CoV-2 infected cells, active viral infection leads to the release of viral particles from the cells. We utilized a highly sensitive real-time PCR assay to detect and measure the SARS-CoV-2 viral RNA in Vero E6 cell culture supernatants. We envisioned that the spread of viral infection would result in substantial increase in the viral RNA copy numbers in the cell culture supernatants. We measured the viral RNA copy numbers in the viral inoculum and found that 153 PFU corresponded to ~102 viral RNA copies while 15 300 PFU were equivalent to ~104 copies (figure 5). We found high levels of SARS-CoV-2 RNA (108 viral RNA copies per ml) in Vero E6 cell cultures infected with SARS-CoV-2 containing EBC samples (153 and 15 300 PFUs) (figure 5). This demonstrated that a high level of viral replication in cells corresponded to increased viral RNA loads in the supernatants. Acetonitrile treatment resulted in inhibition of viral infection and replication in Vero E6 cells. The real-time PCR assay detected few copies (<82 copies for all the samples and 292 copies for one sample) of viral RNA in cell culture supernatants (figure 5). These copy numbers represented the residual viral inoculum which was biologically inactive due to the loss of viral infectivity.

Figure 5.

Figure 5.

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SARS-CoV-2 RNA levels in the cell culture supernatants. Viral RNA copy numbers were measured using real-time PCR assay. High viral RNA load was detected in the supernatants of SARS-CoV-2 infected cells but not in acetonitrile-inactivated SARS-CoV-2 exposed Vero E6 cells.

3.5. Evaluation of the metabolite content of EBC

We examined the effects of acetonitrile treatment on the metabolomic contents of EBC using a previously established LC-qTOF-MS assay [14, 16, 29]. Samples were analyzed twice, once in positive and once in negative ESI mode. We determined the number of chemical features detected in each sample treatment, relative to samples prepared using a previously reported protocol that does not include acetonitrile treatment (table 1 and figure 6). These data established the baseline metabolomic content in EBC samples as untreated controls. Four treatment groups for EBC samples were included in the analysis. These included EBC samples treated with 0%, 50% and 90% acetonitrile. In addition, one group of EBC samples was without acetonitrile treatment was processed according to the previous preparation protocol with no incubation at room temperature. This control group was included to test whether incubation of EBC at room temperature for 15 min, (which was not a part of the previous protocol), would alter metabolite content.

Table 1.

Averaged number of chemical features with standard deviation for each EBC preparation protocol, as measured in both LC-qTOF-MS ESI negative and ESI positive modes. All samples were prepared from a stock EBC sample, which was a pool of EBC from multiple subjects. There were n = 3 replicates used for each treatment group. The 0% acetonitrile treatment was included to test whether incubation of EBC at room temperature for 15 min, which was not a part of the previous protocol, would impact metabolite content.

90% Acetonitrile 50% Acetonitrile 00% Acetonitrile Non-incubated samples
ESIneg * 768 ± 31 868 ± 63 1022 ± 47 960 ± 27
ESIpos * 839 ± 81 1112 ± 6 943 ± 32 1119 ± 120
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The asterisk (*) indicates significant difference relative to the previous protocol per an ANOVA. See also figure 6.

Figure 6.

Figure 6.

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Detection of the number of metabolic features in EBC samples. Boxplots show the number of chemical features detectable in EBC samples treated with acetonitrile in comparison to untreated EBC controls. P-values from an ANOVA are shown. The EBC Samples (n = 3 per treatment) were injected once in ESI positive and once in ESI negative LC-qTOF-MS modes.

The metabolomic feature count for acetonitrile-treated samples was compared with untreated controls processed using the previous preparation protocol and the significance of the difference was determined. In both ESI positive and negative modes, samples treated with 90% acetonitrile had significantly fewer features than the untreated samples, with 192 fewer features in ESI negative and 280 fewer in ESI positive modes. The differences observed in the 50% and 0% acetonitrile treatment did not show statistical significance compared to the controls (using previous protocol). The 50% acetonitrile treated samples yielded 92 fewer in ESI negative but only 7 fewer in positive; 0% acetonitrile yielded 62 more in ESI negative but 176 fewer in positive mode. Our data suggest that 50% acetonitrile treatment of EBC samples is sufficient for effective inactivation of the infectious virus and will be suitable for advancing the samples to metabolomic analysis.

4. Discussion

SARS-CoV-2 viral RNA has been detected in clinical EBC samples from patients with COVID-19 [9, 20, 30, 31]. Additionally, many of these patients were shown to exhale a variable levels of viral RNA copy numbers [30]. The spread of SARS-CoV-2 infection through aerosols has been well recognized [32–34]. It is well documented that seasonal human coronaviruses, influenza viruses and rhinoviruses have been detected in exhaled breath of infected individuals [35]. We have established the protocol to completely inactivate SARS-CoV-2 virus infectivity in EBC samples that generates non-infectious samples which are safe for processing in the clinical and research laboratories for metabolomics analysis. In this study, we utilized the SARS-CoV-2 virus labeled with green florescent signals, mNeonGreen, to infect Vero E6 cells and to readily identify infected cells and detect cytopathic effects of the viral infection [21]. The virus was completely inactivated following the treatment with 50% or 90% acetonitrile and the loss of viral infectivity was demonstrated by the Vero E6 cell infection assay. We found that 10% acetonitrile treatment (which is a commonly used laboratory protocol for collecting EBC samples for metabolomics investigations) [18] failed to inactivate the infectious SARS-CoV-2. It was alarming that this treatment did not fully inactivate even one PFU of the virus. We previously reported that 1 PFU of SARS-CoV-2 induced active infection on Vero E6 cells and the effects of the viral infection was well demonstrated by the cytopathic effects and the presence of mNG-expressing cells (>70% of the cells in Vero E6 cell cultures) [25]. In the current study, our goal was to establish a viral inactivation protocol that can effectively inactivate infectious viral loads that are comparable to the viral burden found in clinical samples. This would be relevant for applying to the clinical samples which harbor variable levels of viral loads. Therefore, we selected 153 and 15 300 PFU for viral inactivation protocol with clinical EBC samples. Our findings suggest that clinical EBC samples treated with acetonitrile in the range of 50% to 90% are non-infectious and safe for processing for further analysis in the laboratory setting. The mechanism of SARS-CoV-2 inactivation may be due to the irreversible denaturation of virus structural proteins in acetonitrile and inability of the virus to bind to host cell receptors. It is likely that other enveloped viruses in clinical samples can also be inactivated using acetonitrile treatment.

The EBC samples are easily accessible and provide a source of biomarkers of infectious and non-infectious diseases [19]. For metabolomics investigations, EBC is commonly lyophilized and reconstituted for the analysis of analytes. This process has potential to pose a threat to aerosolize SARS-CoV-2 virus from infected EBC samples [36]. Therefore, processing of clinical EBC samples warrant optimized protocols that can fully inactivate infectious respiratory pathogens. This is more so important due to the potential application of EBC samples in measuring metabolomics signatures and inflammation biomarkers as well as for pharmacokinetics [13–18]. EBC is commonly lyophilized, which is an aerosol generating procedure that could release SARS-CoV-2 virus from EBC into the air. The significance of our study is developing a protocol that would reduce risk of accidental transmission early in sample preparation and certainly before any aerosol generating steps. Our proposed protocols for the virus inactivation using acetonitrile will be of value in processing EBC samples for metabolomics and pharmacokinetics studies.

As a biofluid specimen, EBC contains a wide range of compounds, including analytes that are volatile, nonvolatile, inorganic, organic, redox-relevant, or proteinaceous [36]. It is beyond the scope of this study to examine the effects of acetonitrile treatment on all analytes found in EBC. Instead, we report a method to inactivate SARS-CoV-2 virus in EBC for safer laboratory handling. Further evaluation of potential effects of this viral inactivation procedure on their target compounds will be needed. We performed an LC-qTOF assay to examine acetonitrile treatment effects on the number of metabolites detected. Our data show that incubating EBC at room temperature for 15 min without acetonitrile (0% acetonitrile treatment group) did not impact the number of detected metabolites, in comparison to a previous non-incubated protocol. Treatment of EBC samples with 50% acetonitrile did not significantly alter the number of metabolites measured as compared to the previous protocols, supporting the use of this approach to inactivate the virus in clinical samples. There was a decrease in the number of metabolites detected following the treatment with 90% acetonitrile. Based on these results, EBC treatment with 50% acetonitrile for 15 min is sufficient to inactivate SARS-CoV-2 virus without impacting the number of detectable metabolites.

These protocols are effective in inactivating high titers (>15 000 PFU) of the infectious SARS-CoV-2 in EBC samples. This amount is significantly higher than the levels of viral RNA reported (<105 RNA copies per milliliter) in clinical EBC samples [20, 30, 31]. Therefore, the virus inactivation protocol in our study is applicable for clinical EBC samples and can be used for metabolomics investigations. The implication of this study is in enhancing the capacity of breath researchers for collecting and processing EBC samples in the clinical or research laboratory environment.

5. Conclusion

We have established a safe and reliable strategy to process EBC samples from COVID-19 patients, using an optimized acetonitrile treatment protocol that fully inactivates SARS-CoV-2. This knowledge will benefit clinical and translational researches worldwide, for the exhaled breath analysis in human health and diseases.

Supplementary Material

supplemental

Acknowledgments

This work was partially supported by NIH National Centre for Advancing Translational Sciences (NCATS) through award UL1 TR001860, and 1U18TR003795; NIH awards R37-AI 153025, R01-AI123105, UG3-OD023365, P30ES023513; Department of Medical Microbiology and Immunology COVID funds; University of California CIT-RIS and the Banatao Institute Award 19-0092; the Department of Veterans Affairs Award I01 BX004965; the University of California Tobacco-Related Disease Research Program Award T31IR1614. The contents of this manuscript are solely the responsibility of the authors and do not represent the official views of the funding agencies. We thank the Food Safety and Measurement Facility at UC Davis, especially its Director Dr Larry Lerno, for providing access and maintaining the LC-qTOF.

Footnotes

Supplementary material for this article is available online

Data availability statement

All data that support the findings of this study are included within the article (and any supplementary files).

References

  • [1].Hu B et al. 2021. Characteristics of SARS-CoV-2 and COVID-19 Nat. Rev. Microbiol. 19 141–54 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Wölfel R et al. 2020 Virological assessment of hospitalized patients with COVID-2019 Nature 581 465–9 [DOI] [PubMed] [Google Scholar]
  • [3].Trypsteen W et al. 2020. On the whereabouts of SARS-CoV-2 in the human body: a systematic review PLoS Pathog.16 e1009037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Mollica V, Rizzo A and Massari F 2020. The pivotal role of TMPRSS2 in coronavirus disease 2019 and prostate cancer Future Oncol. 16 2029–33 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Zhao -M-M et al. 2021. Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development Signal Transduction Targeted Ther. 6 134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Salamanna F et al. 2020. Body localization of ACE-2: on the trail of the keyhole of SARS-CoV-2 Front. Med 7 1–24 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Lamers MM et al. 2020. SARS-CoV-2 productively infects human gut enterocytes Science 369 50–54 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Wang Y et al. 2020. SARS-CoV-2 infection of the liver directly contributes to hepatic impairment in patients with COVID-19 J. Hepatol. 73 807–16 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Ryan DJ et al. 2021. Use of exhaled breath condensate (EBC) in the diagnosis of SARS-COV-2 (COVID-19) Thorax 76 86–88 [DOI] [PubMed] [Google Scholar]
  • [10].Vaz SN et al. 2020. Saliva is a reliable, non-invasive specimen for SARS-CoV-2 detection Braz. J. Infect. Dis. 24 422–7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Urs R et al. 2021. Collecting exhaled breath condensate from non-ventilated preterm-born infants: a modified method Pediatr. Res. 1–3 [DOI] [PubMed] [Google Scholar]
  • [12].Papaioannou AI et al. 2011. Exhaled breath condensate pH as a biomarker of COPD severity in ex-smokers Respir. Res. 12 67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Schmidt AJ et al. 2020. Investigating the relationship between breath aerosol size and exhaled breath condensate (EBC) metabolomic content J. Breath Res. 14 047104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Schmidt AJ et al. 2020. Portable exhaled breath condensate metabolomics for daily monitoring of adolescent asthma J.Breath Res. 14 026001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Fernández-Peralbo MA et al. 2015. Study of exhaled breath condensate sample preparation for metabolomics analysis by LC–MS/MS in high resolution mode Talanta 144 1360–9 [DOI] [PubMed] [Google Scholar]
  • [16].Borras E et al. 2019. Detecting opioid metabolites in exhaled breath condensate (EBC) J. Breath Res. 13 046014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Stiegel MA, Pleil JD, Sobus JR, Morgan MK and Madden MC 2015. Analysis of inflammatory cytokines in human blood, breath condensate, and urine using a multiplex immunoassay platform Biomarkers 20 35–46 [DOI] [PubMed] [Google Scholar]
  • [18].Borras E. et al. Novel LC-MS-TOF method to detect and quantify ascorbic and uric acid simultaneously in different biological matrices. J. Chromatogr. B. 2021;1168:122588. doi: 10.1016/j.jchromb.2021.122588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Pleil JD, Wallace MAG and Madden MC 2018. Exhaled breath aerosol (EBA): the simplest non-invasive medium for public health and occupational exposure biomonitoring J.Breath Res. 12 027110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Sawano M et al. 2021. RT-PCR diagnosis of COVID-19 from exhaled breath condensate: a clinical study J. Breath Res.15 1–12 [DOI] [PubMed] [Google Scholar]
  • [21].Xie X et al. 2020. An Infectious cDNA Clone of SARS-CoV-2 Cell Host Microbe 27 841–848.e3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Case JB et al. 2020. Growth, detection, quantification, and inactivation of SARS-CoV-2 Virology 548 39–48 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Jureka AS, Silvas JA and Basler CF 2020. Propagation, inactivation, and safety testing of SARS-CoV-2 Viruses 12 622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Baer A and Kehn-Hall K 2014. Viral concentration determination through plaque assays: using traditional and novel overlay systems J. Vis. Exp. e52065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Coil DA et al. 2021. SARS-CoV-2 detection and genomic sequencing from hospital surface samples collected at UC Davis PLoS One 16 e0253578 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Corman VM. et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance. 2020;25:2000045. doi: 10.2807/1560-7917.ES.2020.25.3.2000045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Stockert JC et al. 2018. Tetrazolium salts and formazan products in Cell Biology: viability assessment, fluorescence imaging, and labeling perspectives Acta Histochem. 120 159–67 [DOI] [PubMed] [Google Scholar]
  • [28].Zamuruyev KO et al. 2016. Human breath metabolomics using an optimized non-invasive exhaled breath condensate sampler J. Breath Res. 11 016001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Aksenov AA et al. 2017. Analytical methodologies for broad metabolite coverage of exhaled breath condensate J.Chromatogr. B 1061–1062 17–25 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Ma J. et al. Exhaled breath is a significant source of SARS-CoV-2 emission. medRxiv 2020 [Google Scholar]
  • [31].Zhou L. et al. Detection of SARS-CoV-2 in exhaled breath from COVID-19 patients ready for hospital discharge. medRxiv p. 2020:2020.05.31.20115196. [Google Scholar]
  • [32].Tang S et al. 2020. Aerosol transmission of SARS-CoV-2? Evidence, prevention and control Environ. Int. 144 106039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Bazant MZ and Bush JWM 2021. A guideline to limit indoor airborne transmission of COVID-19 Proc. Natl Acad. Sci. 118 e2018995118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Jarvis MC 2020. Aerosol transmission of SARS-CoV-2: physical principles and implications Front. Public Health 8 1–8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Leung NHL et al. 2020. Respiratory virus shedding in exhaled breath and efficacy of face masks Nat. Med. 26 676–80 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Davis MD 2020. Chapter 7—exhaled breath condensate and aerosol Breathborne Biomarkers and the Human Volatilome 2nd edn, ed Beauchamp J, Davis C and Pleil J (Amsterdam: Elsevier; ) pp 109–19 [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplemental
NIHMS1857782-supplement-supplemental.pdf (434.4KB, pdf)

Data Availability Statement

All data that support the findings of this study are included within the article (and any supplementary files).

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