Assessment of different experimental setups to determine viral filtration efficiency of face masks

Abstract

As a result of the pandemic COVID -19 many new materials and masks came on the market. To determine their suitability, several standards specify which properties to test, including bacterial filtration efficiency (BFE), while none describe how to determine viral filtration efficiency (VFE), a property that is particularly important in times of pandemic. Therefore, we focused our research on evaluating the suitability and efficiency of different systems for determining VFE. Here, we evaluated the VFE of 6 mask types (e.g., a surgical mask, a respirator, material for mask production and cloth masks) with different filtration efficiencies in four experimental setups and compared the results with BFE results. The study included 17 BFE and 22 VFE experiments with 73 and 81 mask samples tested, respectively. We have shown that the masks tested had high VFE (>99% for surgical masks and respirators, ≥98% for a material and 87-97% for cloth masks) and that all experimental setups provided highly reproducible and reliable VFE results (coefficient of variation < 6%). Therefore, the VFE tests described in this study can be integrated into existing standards for mask testing.

Introduction

COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has drastically changed our daily lives. Even though more than two years have passed since the pandemic began, there are still some questions that have not been fully answered. One of them is the exact mode of SARS-CoV-2 transmission¹. It is known that close proximity to an infected person presents the highest risk of contracting the virus through inhalation or direct deposition of infected droplets on mucous membranes (direct transmission), but it is not entirely clear if the larger droplets or the smaller droplets/aerosols are mainly responsible for virus transmission in such cases². The importance of airborne transmission with aerosols over longer distances (> 2 m; indirect transmission) has also become clear, especially in closed, crowded spaces, with inadequate ventilation^2,3. Indeed, a growing body of evidence points to the importance of such transmission⁴, which is also supported by findings on other respiratory pathogens⁵.  Furthermore,  it is known that SARS-CoV-2 can remain infectious on different surfaces⁶; however, this type of indirect transmission has been shown to have the least impact on the spread of SARS-CoV-2². This ambiguity regarding the most important modes of transmission is not specific only to SARS-CoV-2, but also to other respiratory pathogens⁵. 

Larger droplets are generally considered to be larger and heavier particles, which quickly fall on the ground before they evaporate completely.  Therefore, they are only present in the close proximity of the infected person (up to 2 m)⁷. Aerosols, on the other hand, are smaller and lighter droplets that remain airborne for longer periods of time, even hours⁷, and can travel farther than 2 m⁸. The cut-off between larger droplets and aerosols is often considered to be 5 μm (even by major health agencies including the World Health Organization and the Centers for Disease Control and Prevention^9,10) but some studies also indicate otherwise (i.e., the cut-off should be set higher), as discussed in Wang et al., 2021⁷. The spread of aerosols and consequently their contents (like viruses) in the air depends on environmental factors such as relative humidity and temperature⁷. Aerosols are particularly important for the spread of viruses, not only because they can linger in the air and cause infections over longer distances, but also because they usually contain more viruses and can invade the lower respiratory tract⁷. It has been shown that the viral load of SARS-CoV-2 is higher and that it persists longer in the lower respiratory tract than in the upper respiratory tract^11,12. 

Regardless of which particles are the most important in transmission of SARS-CoV-2, the virus can be transmitted with droplets of different sizes produced by symptomatic or asymptomatic individuals when coughing, sneezing, singing or even just talking and breathing⁷. Therefore, the transmission of pathogens must be stopped at the source, i.e., at the mouth and the use of masks, and especially their proper use, is of crucial importance. 

The most commonly used masks to protect against SARS-CoV-2 include respirators, surgical or medical masks, and cloth masks¹³. Respirators are masks that fit closely to the face of the wearer and therefore provide the best protection for and from the wearer. They are multi-layered (often four-layered) and made of non-woven fibrous materials such as polypropylene. This allows for high filtration efficiency (FE), resulting in great protection. They may be classified differently depending on the region of the world, i.e., in Europe they are classified into different filtering facepieces (FFP1, FFP2, and FFP3), while in the United States letters (N, R, P) and numbers are used to describe a type of respirator (e.g., N95)^13,14. Surgical masks are also multi-layered (often three-layered) and are made of the similar materials as respirators, often polypropylene. Based on their bacterial filtration efficiency (BFE) (among other technical parameters) they can be classified into Type I or II, with the BFE of ≥95 or ≥98, respectively¹⁵. Due to their high filtration performance, they also provide a high level of protection. However, they are not as fitted as respirators, so the user and its environment are not as well protected as with respirators because the gaps between the mask and the user can serve as a pathway for infection^13,14. Both respirators and surgical masks are usually intended for single use and undergo rigorous testing before they reach the market, whereas cloths masks do not¹³. There are many advantages to using cloth masks, especially from an environmental standpoint, as they can be reused. They can be made of different materials such as cotton or silk, with a different number of layers. Their filtration efficiency can vary drastically depending on the characteristics of the mask, such as the type of material, the number of layers, the thread count, and the fit of the mask¹⁶. Although most cloth masks do not provide as high level of protection for and from the wearer compared to surgical masks and respirators, they always provide some level of protection¹⁴. 

To ensure the quality of masks and determine their efficacy, it is important to test various parameters, including FE. In Europe, the standard for surgical masks EN 14683:2019+AC:2019¹⁵ specifies the properties of surgical masks that must be tested. This includes bacterial filtration efficiency (BFE), using Staphylococcus aureus. However, there is no standardized method for testing of viral filtration efficiency (VFE). Nevertheless, some laboratories are already performing such tests¹⁷. In terms of research, only few studies are available on determining VFE of different masks^18–20, reporting limited amount of experimental data. Therefore, there is a need for a study that evaluates the suitability and efficiency of different systems for determining the VFE. We developed four different experimental setups for VFE testing based on the standard EN 14683:2019+AC:2019 using bacteriophage MS2 and determined their applicability on six types of masks (e.g., a surgical mask, a respirator, materials for mask production and cloth masks) made from various materials. In comparison, we determined the BFE of these masks in two different experimental setups. This allowed us to assess the performance of a non-standardized (VFE) method in relation to a standardized (BFE) method that has been used for years. Furthermore, we evaluated suitability of bacteriophage MS2 as a model virus for different experimental setups of the VFE¹⁹. Thus, this study included numerous experiments, namely 17 BFE and 22 VFE experiments with 73 and 81 mask samples, respectively. It was shown that the results of all experimental setups used for VFE determination were repeatable and reliable. Therefore, the developed system for VFE testing could be implemented in the existing standards for mask testing, if needed. In addition, the results obtained may serve as a good starting point for other research groups working in the important area of personal protection and prevention of pathogen transmission.

Materials And Methods

2.1 Types of masks tested

We tested 6 types of masks or materials for masks (henceforth referred to as mask samples) that were either purchased or homemade and thus not officially on the market (Figure 1). Two of the mask samples were produced in accordance with standards, i.e., a three-layer polypropylene surgical mask classified as Type II (EN 14683:2019+AC:2019¹⁵) (A) and a five-layer respirator classified as FFP2 (EN 149:2001+A1:2009²¹) made of nonwoven, meltblown and cotton fabric (E). A three-layer polypropylene material for mask production (B) was provided by a local mask manufacturer. A homemade reusable two-layer cotton mask (C) and a reusable two-layer mask with an outer layer of cotton and polyester and an inner layer of polypropylene (D), were provided by small private mask producers. A reusable two-layer cotton mask (F) was purchased in a local pharmacy shop. Each mask was conditioned in a chamber with 250 g of KCl and 0.5 L of dH₂0 for a minimum of 4 h at 21 °C ± 5 °C and relative humidity of 85 %RH ± 5 %RH prior to testing.

2.2 Experimental setups and performance of FEs

A system constructed for determination of BFE in accordance with EN 14683:2019+AC:2019 standard was used for all experiments (Table 1, Figure 2) differing mainly by the type of sampler, airflow, and, in the case of the experimental setup VI, also the pump. Two experimental setups were used to determine the BFE. In the first, bacteria were collected directly on the plates using a 6-stage Andersen sampler, while in the second they were collected in a liquid medium in the impinger type 1 (Table 1). To determine the VFE, four experimental setups were used. Viruses were either collected with Andersen sampler, or with type 1 or type 2 impinger (Table 1). The main difference between the impingers was their size, radius of the top of the impinger, complexity of the hooks and manufacturing as the impinger type 1 was homemade, larger, with narrower top of the impinger and simpler hooks, while the impinger type II was standardized and purchased from the SKC BioLite. The impinger type 2 was also used in combination with a different vacuum pump (Table 1).

First, 13 μL of a liquid sample containing either a bacterium (Staphylococcus aureus, ATCC 6538) or a virus (bacteriophage MS2, ATCC 15597-B1) (Figure 2a) was aerosolized (Figure 2b), producing droplets and aerosols of different sizes, with an average value of 3.1 ± 0.3 μm (Supplementary Tables S1-S6). The generated droplets and aerosols were mixed with ambient air at a flow rate of 28.3 L/min (setups I and III), 31.2 L/min (setups II and IV), 10.3 L/min (setup V), or ~6.1 L/min (setup VI). After the 1-minute aerosolization, air flowed through the system for another minute (for BFE, and VFE in Andersen sampler) or two minutes (for other setups of VFE) at the same flow rate as just described. The airflow carried droplets and aerosols through the glass chamber with standardized dimensions (Figure 2c), where they reached the two-piece component in which the mask was tightly clamped, with the inside of the mask facing upward (Figure 2d). In the case of the positive controls (PCs), there was no mask in the two-piece component. Droplets and aerosols were then collected in either a 6-stage Andersen sampler or a type 1 or 2 impinger (Figure 2e). In each experiment, the first PC was performed first, then 3-5 mask subsamples were tested, followed by the second PC. After each experiment, a negative control (NC; air flowing for 2 or 3 minutes without bacteria or viruses) was performed. The pressure was always maintained at 0.35 bar. At the end, the system was first cleaned by the aerosolization of 70 % ethanol or 4.9 % hydrogen peroxide and then Milli-Q water, and the equipment was washed, autoclaved and/or sterilized with the UV light.

Most of the experiments were performed with mask samples A and B. This allowed us to directly compare the results of different experimental setups, and to determine the reliability and reproducibility of the VFE tests. Other masks were included in the experiments to investigate whether the developed VFE tests are suitable for evaluating the FE of masks of different quality, to determine whether MS2 is a suitable virus for VFE tests performed in different experimental setups, and to obtain a more comprehensive overview of the FE of different masks.

2.2.1 BFE

2.2.1.1 BFE with 6-stage Andersen sampler (experimental setup I)

In experimental setup I, in which a 6-stage Andersen sampler was used, 11 experiments were performed and a total of 45 mask subsamples were tested (Table 1, Supplementary Table S1).  BFE was determined according to the standard EN 14683:2019+AC:2019¹⁵, described in Košir et al., 2022²², with small modifications, including a wider range of average bacterial concentration in positive controls that was considered to be appropriate, i.e., 1.28 x 10³ – 3.07 x 10³ colony forming units (CFU)/test.  For each PC, mask sample or NC, bacteria were collected on 6 plates in 6-stage Andersen sampler. Each stage had 400 openings of different diameters, with the largest at the top (first stage) and the smallest at the bottom (sixth stage) to mimic the flow of inhaled particles in the human respiratory system (the diameters of the openings on the same stage were the same)²². Plates were prepared from 40 g/L tryptic soy agar (TSA) (Fluka). After the experiments, plates were incubated overnight at 37 °C, the colonies were counted and the CFU was determined taking into account the positive hole correction^22,23. The final BFE was calculated as described in the section Calculation of BFE and VFE. The mean particle size was calculated as described in EN 14683:2019+AC:2019¹⁵ and Košir et al., 2022²².

2.2.1.2 BFE with impinger type 1 (experimental setup II)

In the experimental setup II, where the Andersen sampler was replaced by the impinger type 1, 6 experiments were conducted and 28 mask subsamples were tested (Table 1, Supplementary Table S2). The impinger contained 30 mL of peptone water prepared from 10 g/L Bacto peptone (DB) and 5 g/L NaCl (Merck). A new glass cup i.e., lower sampling part of the impinger was used for every subsample (e.g., PC, mask subsample or NC). At the end of each experiment, 100 μL of undiluted or diluted peptone water from each subsample was spread on two TSA plates. Plates were incubated overnight at 37 °C and the bacterial colonies were counted. Bacterial concentrations (CFU/mL) were determined considering bacterial dilutions and plating volumes. The final BFE was calculated as described in the section Calculation of BFE and VFE. 

2.2.2 VFE

2.2.2.1 VFE with Andersen sampler (experimental setup III)

In the experimental setup III, the experiments were performed and the final VFE was calculated in the same way as in the experiments for BFE, experimental setup I. The only difference was the type of plates used. Here, the mixture of 5 ml of melted ‘TSB top agar’ and 100 µL of E. coli in logarithmic phase was poured onto ‘TSB agar’ plates (explained in the section Double-layer plaque assay) and the plates were allowed to harden. Together, 4 experiments were conducted and 12 mask subsamples were tested (Table 1, Supplementary Table S3). 

2.2.2.2 VFE with impingers type 1 and 2 (experimental setups IV, V and VI)

In experimental setup IV, impinger type 1 was used, while in setups V and VI, impinger type 2 was used. In addition, a different vacuum pump was used in setup VI. In the experimental setup IV, 6 experiments with a total of 26 mask subsamples were performed (Supplementary Table S4), in the setup V, there were 9 experiments with 34 mask subsamples (Supplementary Table S5), and in the setup V, there were 3 experiments with 9 mask subsamples (Supplementary Table S6) (Table 1). In the experimental setup IV, viruses were collected in 30 ml of peptone water (it was prepared in the same way as for BFE), while in the setups V and VI they were collected in 10 mL of it. A new glass cup was used for each subsample (e.g., PC, mask subsample or NC). At the end of the experiment, appropriate dilutions of the peptone water were prepared and the virus concentrations were determined using double-layer plaque assay and the final VFE was determined as described in the section Calculation of BFE and VFE.

2.2.2.2.1 Double-layer plaque assay

Three media were used for double-layer plaque assay (DAL), e.g. ‘TSB agar’, ‘TSB top agar’ and ‘liquid TSB’. ‘TSB agar’ prepared from 30 g/L TSB and 15 g/L Bacto agar (BD) was used for agar plates. ‘TSB top agar’ was prepared in the same way except that 7 g of agar was added, and was used as the top layer in this assay. ‘Liquid TSB’ used for cultivation of Escherichia coli CB390²⁴ was prepared from 30 g TSB/L. All media contained 1.93 g/L MgCl₂ × 6H₂O (Duchefa   Biochemie) and 100 mg/L ampicillin (Sigma-Aldrich). 

DAL was performed by adding 0.1 mL of E. coli in logarithmic phase (prepared by inoculating 5 mL of ‘liquid TSB’ with 0.2 mL of ~19 h old bacterial culture followed by 3 h incubation at 37 °C and 230 rpm) and 0.25 mL of undiluted or diluted peptone water with viruses to ~5 mL of melted ‘TSB top agar’ in 15 ml glass tubes. This was then mixed thoroughly and poured onto ‘TSB agar’ plates. Each virus dilution was prepared in duplicates or triplicates. After overnight incubation at 37 °C, the number of plaques was counted and the virus concentrations (plaque forming units, PFU/mL) were calculated considering virus dilutions and plating volumes 

2.3 Calculation of BFE and VFE

The first step in determining BFE or VFE in each experiment was to calculate the average value of the two PCs (the first was performed at the beginning of each experiment, the second after 3-5 mask subsamples). This value was then used to calculate BFE or VFE for each mask subsample according to equation (1): 

Results And Discussion

In the absence of standardized methods and research data for determining the VFE of masks, the present study focused on the evaluation of four different experimental setups that can be used for this purpose. In parallel, BFE was determined on the same mask samples according to the standardized method EN 14683:2019+AC:2019 and its modified version. This allowed a direct comparison and assessment of the standardized and non-standardized method, which further facilitated evaluation of the quality of VFE tests as well as the suitability of MS2 as a model virus for the VFE test in various experimental setups.  

Evaluation of the experimental setups 

Initially, 5 or 6 different experimental setups (including both BFE and VFE) were tested on two mask samples, A and B, both made of three-layer polypropylene, to compare and assess each of the experimental setup. Three to five subsamples of each mask sample were tested in 3-4 independent repetitions, each time giving similar FE results (Supplementary Tables S1-S6), indicating high reproducibility of the developed test systems (coefficient of variation, CV, ≤ 1%) (Table 2).  This was also confirmed by the generation of droplets of similar average diameter of 3.1 μm ± 0.3 μm in the experiments with Andersen sampler along with the maintenance of stable bacterial and viral concentrations in the PCs of the same experimental setup (Supplementary Tables S1-S6).

Table 2. Reproducibility of bacterial (BFE) or viral (VFE) filtration efficiency determined with different experimental setups (BFE: setups I and II; VFE: setups III – VI). The coefficient of variation (%) was calculated from all subsamples of each mask sample in the same experimental setup (A – F). 

Interestingly, the results show that the average BFE and VFE are slightly lower when using Andersen sampler than when collecting bacteria and viruses in impingers (Table 3). This could be linked to the initial bacterial and viral concentrations, which were lower in experimental setups with Andersen sampler and up to 3.07 x 10³ CFU or PFU (Supplementary Tables S1-S6). It is very important to use the correct initial concentration in the Andersen sampler, as the plates can become saturated with microorganisms, preventing accurate determination of FE. Hence, the statistical correction, i.e., the positive hole correction²³, is applied to determine CFU and PFU, which anticipates that more than 1 bacteria or virus can pass through each hole of an individual stage (representative plates for BFE and VFE tests in Andersen sampler are shown in Supplementary Figure S1). The determined concentration is thus estimated and can differ from the actual concentration determined by classical growing and counting of CFU and PFU. Since the VFE values obtained with the Andersen sampler are on average lower than the VFE values in the impingers, the experimental setup with the Andersen sampler presents a safer choice, as when it comes to protective equipment, it is better to underestimate the FE and test the “worst case” filtration efficiency than to overestimate it²⁵. Moreover, working with this sampler allows to determine the average droplet size and to work with airflows corresponding to respiration. In addition, a single Andersen sampler is sufficient for all subsamples, unlike impingers, which require the use of a new glass cup for each subsample. In addition, the plates are transferred from the Andersen sampler directly to the incubator without the need for processing as with impingers.

On the other hand, if it is necessary to work with higher initial concentrations of viruses or if the samples require additional processing and testing for other properties, then impingers are a way to go.  However, the type of impinger and pump must be selected based on the desired airflow and considering the practicality of the experimental setup. In our opinion (from the experimental setups that used impingers) the combination in experimental setup V worked the best. A commercial laboratory practice also supports our conclusion, as they use impinger only for determination of VFE with increased challenge, i.e., when the initial viral concentration is higher than 3.3 x 10³ PFU/test, while otherwise use Andersen sampler¹⁷. They, however, do it in combination with phix 174 as a model virus. Only a few other groups have worked on determining VFE in a similar setup. They either worked with MS2¹⁹ or phix 174²⁰, which they sampled using the Andersen sampler. In addition, a completely different experimental setup was also developed for determination of VFE, using the mannequin head with an aerosol source simulator and a SARS-CoV- 2 pseudovirus as a model virus¹⁸.

Table 3. Average bacterial (BFE) and viral filtration efficiency (VFE) 

BFE and VFE values were calculated from all subsamples of the same experimental setup for an individual mask sample; ^aAccording to EN 14683:2019+AC:2019, Type I and II are determined. This is not applicable (NA) to reusable cloth masks. ^bWhen the BFE and VFE values were between 99% and 100%, more decimal places were included to show the exact filtration efficiency of the mask. 

Several other factors are known to affect the FE of masks, including airflow²⁰. Higher airflow decreases FE, likely due to the shorter time available for droplets to diffuse or interact with the electrostatically charged fibers²⁶. In the experimental setups with the Andersen sampler (I and III) and the homemade impinger (II and IV), the airflow velocity was similar (28.3 L/min vs. 31.2 L/min), while much lower velocities were measured with type II impinger (V and VI) (10.3 and ~6.1 L/min, respectively). Since the FE results of the same mask sample were similar, regardless of airflow, they indicate that air velocity is not that crucial in VFE tests. 

In addition to mask samples A and B, four other mask samples were used. This enabled us to evaluate if different experimental setups for VFE testing were suitable for testing of masks of different quality and FE. It also helped in the final determination of whether MS2 is an appropriate viral model for VFE testing. Finally, the inclusion of these mask samples allowed us to evaluate how efficient masks are in general and how much they can mitigate the spread of respiratory viruses when worn properly. As was the case for mask samples A and B, the results obtained for the other masks also indicate the robustness, reliability, and repeatability of the experimental procedures developed, as confirmed by the FE results, generation of droplets of similar average diameter along with the maintenance of stable bacterial and viral concentrations in the positive controls of the same experimental setup (Supplementary Tables S1,S2,S4,S5). In addition, the CVs for mask samples C-F were also low and, as expected, higher for the masks with the lower FE (Table 2). In addition to reliability, the results obtained with the 6 different mask samples also demonstrate that MS2 is a suitable viral model for VFE testing for different experimental setups and that VFE testing can be performed for masks made of different materials and with different FEs.

Despite some observed differences, the average VFE values of the same mask sample are quite similar regardless of the experimental setup and are comparable to BFE results (Figure 3, Table 3). This is not surprising since the same nebulizer was used in all experiments, producing droplets and aerosols of the same size, with diameters large enough to contain either bacteria (S. aureus has a diameter of 0.5-1 μm) or viruses (MS2 has a diameter of about 27 nm). A similar observation was also made by Rengasamy et al., 2017²⁰, when they compared BFE and VFE values for the same masks. 

We have shown that all experimental setups tested for the determination of VFE can be sufficient and that the decision of which experimental setup to choose depends on several factors, as described above. Therefore, this study can serve as a great foundation for implementing VFE testing into existing standards for mask testing.

Determination of filtration efficiency of masks

As expected, mask sample A, classified as a Type II surgical mask (EN 14683:2019+AC:2019), resulted in BFE and VFE above 99% in all experimental setups (Table 3, Figure 3). A similarly high FE was also obtained for mask sample B, with some observed differences between the experimental setups, which could classify the final product, i.e., surgical masks made from this material as Type I or II. Knowing that the characteristics of the material can vary and depend on several process parameters²⁷, the determined differences in FE could be due to the fact that three separate layers of polypropylene were manually assembled for testing, whereas for the final product,  all three materials are pressed together. Mask sample E also had a very high FE value ≥ 99.9%, which was expected considering that this mask sample was an FFP2 respirator, although a different standard is normally used to determine the FE of respirators. We also tested BFE and VFE of three reusable, cloth mask samples, C, D, and F. Mask samples C and F, both made of two layers of cotton, had FE of >90%. Mask sample D contained polyester and polypropylene in addition to cotton and had lower BFE and VFE of 79 and 87%, respectively. It is known that cotton can ensure high FE, but it depends on the number of layers and thread count, as they assure physical filtration. On the other hand, materials like polyester have  moderate electrostatic discharge, which is better for filtration of smaller aerosols (<300 nm)¹⁶. Since we produced droplets with an average size of 3 µm, we do not know if FE of these masks would be better for smaller particles only and whether mask sample D would be superior in filtering such particles compared to the other two cloth masks.

Similar observations on the FEs of different masks were made by other groups, which found high FEs (either BFE, VFE, or particle filtration efficiency, PFE) in surgical masks and respirators, while cloth masks had different FEs, which in some cases were quite high, above 90%^18–20,28. Caution should be exercised in interpreting these results, because the fit of masks is usually not taken into account when FE is determined, and therefore the protection provided by the masks does not necessarily correspond to the measured FE. For example, the BFE systems described in the standards and the VFE systems based on them usually test the FE of one area of the mask (in our case it was 8 x 8 cm for respirators and 10 x 10 cm for other masks) that is tightly clamped so that the air carrying bacteria/viruses can only pass through the material. In reality, various masks, especially cloth masks, do not always fit tightly against the wearer's face and these openings can serve as a transmission route for various pathogens. Therefore, in addition to FE, another important property of the mask is fit,  because the same mask can protect differently depending on the fit; the better the fit, the better the protection²⁹. Thus, it is not surprising that respirators (which undergo total inward leakage testing) have been found to be most effective in containing the spread of SARS-CoV-2³⁰. 

Face masks, when worn properly, are a very important part of the contingency plan to prevent the spread of respiratory pathogens such as SARS-CoV-2. This has been confirmed by various studies that have shown that surgical masks, respirators, cloth masks, or even just fabrics used to make various textiles¹⁶, have high FEs. The type of mask to choose for protection depends on several factors, such as frequency of contact with infected persons, length of time spent in poorly ventilated enclosed spaces, and the state of the immune system.

Declarations

Declaration in Interest Statement

Declarations of interest: none

Data statement

All data obtained during this study are included in the main text or supplementary information. All raw data can be available upon request.

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Table 1

Table 1 is available in the supplementary files.