DESC:DESCRIPTION
[003] A respirator has been constructed and characterised which does following functions ;
- Filters more than 95 % Bacterial Load, tested as per test method ASTM F2101
- Filters more than 95 % of Particulate matters ,tested as per test method ASTM F2299 /F2299M-03(2010)
- Passes Fluid penetration test as per ASTM F2299
- Has excellent Viral filtration properties
- Confirms to flammability testing of class 1
- Is Biocompatible and safe
- Kills bacteria that come in contact with it.
- Is virucidal for viruses, especially encapsulated viruses that come in contact with it.

[004] The inventors have developed surfaces that are capable of killing bacteria and virus and developed it into configurations that helps to get good filtration property along with bacterial and virus kill on both sides of the device.

[005] The device is been put to use with engineered fit for face to be covered in such a way that effective filtration without any dead zones of air leakages are achieved.

[006] The construction material used in the making of the respirator is a cloth which forms top and bottom layer. The fabric is treated to have properties of bacterial kill, virus kill and also resistance to fluid penetration along with good flammability property. The cloth may be woven, knitted or nonwoven fabric. In the embodiment it is knitted fabric which is giving better stretch and fit due to inherent characteristic of the knitted structure.

[007] The material of respirator may be cotton, polyester, Nylon, Viscose ,Acrylic, polypropylene or any synthetic fibre or natural fibres like silk , wool , Hemp , linen or the blend of these fibres or combinations of any of these yarns .The embodiment uses face as predominantly Polyester fabric knitted with elastomeric yarn like polyurethane that gives excellent stretch property and the back layer is polyester viscose blended yarn fabric to facilitate easy absorption and management of liquid/vapours coming out from exhaled air. This will ensure that the face is not laden with moisture and is comfortable during wear.

[008] The face layer of the respirator is hydrophilic, tightly constructed structure that is coated with antimicrobial solution and also hydrophobic cross linking polymer to achieve the duel function of the repellence and bacterial/Viral kill.

[009] The back layer is a absorbent layer since it is in contact with the mouth and must be capable of absorbing the vapours and liquid droplets from the exhaled air. In the embodiment it is Polyester viscose blended knitted fabric that is acting as an antimicrobial/ antiviral fabric.

[0010] The filtration assembly is trapped in between the two layers of cloth as described above. The assembly is made up of layers of spun bond fabric, needle punched, spun laced or melt blown fabrics as used in the embodiment. The fabric of filtration assembly is treated as well for the property of antimicrobial /antiviral property. The assembly may be in the form of single layer or multiple layers as per the requirements of pore sizes and filtration efficiency desired.The entire layers are treated with the chemistry of antimicrobial and antiviral property.

Fig 1: Arrangement of layers in embodiment .

[0011] The entire assembly is unified with proper edge bindings that can be heat sealed, adhesive bonded or ultrasound bonded or stitched at the edges as done in the case of embodiment. Thus the multilayer composite material now acts as a single entity imparting the property as listed above.

[0012] The property of antimicrobial/Antiviral nature is been introduced in the fabric by treating the fabric with non eluting compound. The compound is an quaternary ammonium salt that is represented by general formula 1 or formula 2.

Formula-1

Formula -2
Wherein:
R1 = hydrogen and/or C1 to C4 alkyl;
R2 = divalent hydrocarbon radical with C1 to C8 carbon atoms;
R3 = hydrogen or C1 to C4 alkyl;
R4 = hydrogen or C1 to C10 alkyl;
R5 = C8 to C22 saturated or unsaturated hydrocarbon radical; and
X = alkyl halide.

[0013] The compound used in the embodiment is a quaternary ammonium salt that is represented by general formula 1.

[0014] For the treatment of the material pad dry cure method is employed, wherein the material has to be dipped in the formulation containing 0.25 % to 5 % by weight of material and then dried and cured at 150 to 200 degree Celsius for 10 to 60 seconds residence times. A person from the field of the art would be able to know the details as described in this section of the art.

[0015] In an implementation, the microbicidal agent is one of a 3-(trimethoxysilyl)propyl-N-octadecyl-N,N-dimethyl ammonium chloride, 3-(trimethoxysilyl)propyl-N-tetradecyl-N,N-dimethyl ammonium chloride, 3-(trimethoxysilyl) propyl-N,N-didecyl-N-methyl ammonium chloride, 3-(trihydroxysilyl) propyl-N-octadecyl-N,N-dimethyl ammonium chloride. In an example, the microbicidal agent used is 3-(trimethoxysilyl) propyl-N-tetradecyl-N,N-dimethyl ammonium chloride.

[0016] For the Embodiment studied, the material is treated by pad dry cure method is employed, wherein the material has to be dipped in the formulation containing1.0 % by weight of material and then dried and cured at 150 to 180 degree Celsius for 40 seconds residence times.

[0017] In order to neutralize the load of microbes and pathogen some or all of the layers of the mask may also use effective compounds like Chlorhexidine, ethanol, lysostaphin, benzoic acid analog, lysine enzyme and metal salt, bacitracin, methicillin, cephalosporin, polymyxin, cefaclor, Cefadroxii, cefamandole nafate, cefazolin, cefixime, cefinetazole, cefonioid, cefoperazone, ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin, cefpodoxime proxetil, ceftaxidime, ceftizoxime, ceftriaxone, cefriaxone moxalactam, cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium salt, cephalothin, cephalothin sodium salt, cephapirin, cephradine, cefuroximeaxetil, dihydratecephalothin, moxalactam, loracarbef mafate, silver and its salts or any combination of compounds that are proven antibacterial and or antiviral in nature.

[0018] The repellence can be achieved by either of paraffin repellent, steric acid melamine repellents, silicon water repellents, fluorocarbon based repellents or surface induced repellence by plasma or irradiation with high energy radiations or by surface grafting of polymers. This is again achieved by the pad –dry –cure method as explained in the previous descriptions.

[0019] In the embodiment the hydrophobicity of the surface is achieved by cross linking the fabric with fluorocarbon .These reduces the surface energy of the surface such that the water and oils find it difficult to wet the surfaces. Person skilled in the art understands the method of pad –dry and cure.

Fig 2: Wettability of surfaces and Contact angle .

[0020] Figure 1 above shows the contact angle and the phenomenon of hydrophobicity. The respirator produced as above can be fitted with one-way breathable valves or may be without out breathable valve. The valve may be single or multiple depending on the ease of exhalation pressure and comfort aspect of the wearer.

[0021] Here the respirator is designed in such a way that it not only protects the wearer from the infections but also ensures that the infection from the infected person does not leaks out which helps the surroundings to be kept non contaminated when the infected person exhales or coughs or sneezes. This makes it ideal for care givers and caretakers as well. In case of protecting the surrounding environment from infected person from cases like SARS CoV 2 Virus which is cause of pandemic like Covid 19,it is desirable that the mask is without a breathable valve. This is because then the exhaled air is also filtered effectively and nullified of the pathogenic load when it passes through the material of respirator.

Figure 3: Schematic diagram of working principal of the respirator

[0022] The respirator combination assembly thus formed is found to be effective against penetration of bacteria and virus and at the same time able to kill bacteria and nullify the viruses that comes in contact with it. The hydrophobic outer layer limits the liquids and aerosol sprayed through coughing or sneezing to be absorbed in the assembly and dries it off on the surface of the mask which is also carrying the compounds to nullify the effect of bacteria and Viruses. Thus the risk of seepage of pathogens from outside to inside in case of accidental spills of bio fluids of the patients or infected person is totally avoided.

[0023] The respirator thus constructed is effective in ensuring the passage of air which is cleaned of particulate matters along with load of bacteria and virus and also filters the air coming out as a result of exhalation of infected person and makes sure the load of virus and bacteria spreading out is kept in control. The bacteria and viruses that are trapped in the layers during inhalation or exhalation are continuously been killed and eradicated so that the risk of infection spread by touching the respirator is at the minimum. This also ensures the respirator can be reused unlike the conventional disposable respirators.

[0024] The respirator ensures the passage of air and vapours from inside but does not allow the passage of liquid from outside. This is needed for health care providers where body fluids like blood ,Spit or vomit can be splashed on the face of health care provider. The Protector does not allow the seepage of the such fluids inside, which can be a cause of infection to the healthcare provider.

[0025] To Quantify the particulate filtration efficiency of the respirator, ASTM F2299 is used. This is a Standard Test Method for Determining the Initial Efficiency of Materials Used in Medical Face Masks to Penetration by Particulates Using Latex Spheres. This test is commonly referred to as the Latex Particle Challenge. PFE is the effectiveness of a material to filter aerosol particles in the size range 0.1µm to 5.0µm. This is expressed as a percentage of a quantity that does not pass through the material.

[0026] The samples will be preconditioned at 30 – 50% relative humidity (RH) at 21oC ± 3oC, prior to testing. The PSL particles is combination of water and aerosol. The aerosol was passed through drying chamber diluted to the required concentration (10,000 to 15,000 particles to cubic foot) using HEPA filtered air and passed through convex side of a test sample properly sealed and placed into filtering folder. Particles were passed through a flow rate of 28.3L/min. PFE is calculated using given below formula.
PFE% = (C-T) X 100
C
Where, C is Average upstream counts T is Average downstream count
PFE result should range for 1 – 99.99% as these values are the low (<1) and high (>99.99%) detection limits.The embodiment showed 96.5 % Particulate filtration efficiency.

Table 1: Test Result of Particulate Filtration Efficiency
Test PFE %
Particulate Filteration Efficiency (PFE) 96.5 % (Avg. of 25 readings)

[0027] The BFE of the filter device was measured as described by ASTM F2101 method. The test samples should be preconditioned for 4hrs at 21oc and 85 ± 5% RH, prior to testing. Penetration was measured using bacteria S. aureus as our challenge organism. A suspension of S. aureus was aerosolized using a nebulizer to give a challenge level of 1700 – 2700 Colony Forming Units (CFU) per test as specified by ASTM F2101 Standard. The bacterial aerosol was a water droplet containing the bacteria and not an individual bacterial particle.

[0028] The Aerosol samples was drawn through test sample clamped with agar plat for collection of bacterial particles at a flow rate of 28.5 L/min for 1 min. All airborne particles greater than 0.65µm are classified aerodynamically. The flowrate of 28.5 L/min is similar to human breathing flowrate to obtain deposition of particles. A positive control without test filter sample was clamped into system was used in each test. A negative control with no bacteria in the airstream was performed to determine the background challenge in the glass aerosol chamber prior to testing. The residual negative control (<1%) was subtracted from the sample CFU. The positive control CFU was used to MPS of the test aerosol. The filtration efficiency was calculated from positive control CFU and the test sample CFU by using given below formula.

BFE% = (Positive control CFU – Test sample CFU) X 100
Positive control CFU
Where,
Positive control CFU – Average CFU with no filter
Test sample CFU – Average CFU with test filter

BFE result should range for 1 – 99.99% as these values are the low (<1) and high (>99.99%) detection limits.
The BFE of the embodiment was measured to be 99.2 %
Table 2: Test Result of Bacterial Filtration Efficiency
Test BFE %
Bacterial Filteration Efficiency (BFE) 99.2 (Avg. of 25 Readings)

[0029] Viral Filtration Efficiency (VFE) Modified ASTM F2101 Method was conducted. The test samples should be preconditioned for 4hrs at 21oc and 85 ± 5% RH, prior to testing. Penetration was measured using the bacteriophage phiX174 was aerosolized in a nebulizer and each test was performed with a challenge virus and Escherichia coli bacteria as the host. A suspension of phiX174 was aerosolized in a nebulizer and each test was performed with a challenge level of 1700-2700 Plaque Forming Unit (PFU) with a MPS of 3.0 ± 0.3 µm for 2 min. The virus aerosol is a water droplet containing water and not an individual virus particle.

[0030] The un-neutralized aerosol sample was drawn through a test sample clamped into sampler with agar plate inoculated with E. coli for collection. The flowrate was maintained at 28.3L/min. The PFU represents the number of viral aerosol particles or droplets. The total number of viral aerosols for test sample, and positive test without a test sample. A negative control with no virus in the airstream was performed to determine the background challenge in the glass aerosol chamber prior to testing.

VFE% = (Positive control PFU – Test sample PFU) X 100
Positive control PFU

Where,
Positive control PFU – Average PFU with no filter
Test sample PFU – Average PFU with test filter

VFE result should range for 1 – 99.99% as these values are the low (<1) and high (>99.99%) detection limits.
The VFE of the embodiment was 98.9 %.
Table 3: Test Result of Viral Filtration Efficiency
Test VFE %
Viral Filtration Efficiency (VFE) 98.9 (Avg. of 25 readings)

[0031] The breathing resistance of the respirators were evaluated by Breathability (Delta P) test as per ASTM F2100-19. The test samples were preconditioned for 4hrs at 21oc and 85 ± 5% RH, prior to testing. It is also known as Delta P or ?P. It is the pressure drop across a surgical mask under specific conditions of air flow, temperature and humidity. This is an indicator of the breathability of the mask.

[0032] Test specimens shall be complete masks or shall be cut from masks. Each specimen shall be able to provide five different circular test areas of 2.5 cm in diameter. The test specimen is placed across the 2.5 cm diameter orifice (total area 4.9 cm2) and clamped into place so that the tested area of the specimen will be in line and across the flow of air. The flow of air pump is adjusted to 8 litre/minute. Both manometers 1 and 2 are read and recorded. For each test specimen, calculate the differential pressure, ?P as follows:

?P = (Xm1 - Xm2) / 4.9
Where,
X m1 is water pressure, manometer 1, mean of five test areas, low pressure side of the material
X m2 is water pressure, manometer 2, mean of five test areas, high pressure side of the material
4.9 is area of the test material, in cm2
?P is pressure differential per cm2 of test material expressed as mm of water.
There are different standard for in which facepiece respirator was tested. The results with respective standard are tabulated below.
Table 4: Test Result of Breathability (Delta P)
Test Standard Delta P Pa/cm2
Breathability ASTM F2100-19. 58.198.9 (Avg. of 25 readings)

[0033] Breathing Resistance of the respirator was tested as per other requirements as below. The air exchange differential or breathability of respirators was measured for inhalation resistance. The differential pressure technique is a simple application of a basic physical principle employing a water manometer differential upstream and downstream of the test material, at a constant flow rate.

[0034] The inhalation resistance criteria as stated in 42 CFR Part 84.180 is an initial inhalation not exceeding 35 mm water column height pressure. The exhalation resistance criteria as stated in 42 CFR Part 84.180 is an initial exhalation not exceeding 25 mm water column height pressure. The result and respective standard are tabulated below. From the result it can demonstrate that the invention has passed the acceptance criteria as specified by the testing standards.
Table 5: Test Result of Breathing Resistance
Test Standard Inhalation Resistance Exhalation Resistance
Breathing resistance ASTM F2100-19. Acceptance criteria 35 mm H2O 25 mm H2O
Result 6.7 mm H2O 5.8 mm H2O

Breathing resistance EN 149-01 + A1 Acceptance criteria =0,7 for 30L/min (mbar) =2,4 for 95L/min (mbar) =3,0 for 160L/min (mbar)
Result 0.33 1.06 1.61

[0035] Flame Resistance or flammability of the respirator was tested by 16 CFR part 1610 test method. This test was performed to evaluate the flammability of plain surface of respirator by measuring the ease of ignition and the speed of flame spread. The parameter of time is used to separate materials into different classes, thereby assisting in a judgment of fabric suitability for clothing and protective clothing material.
Test criteria for specimen classification as per 16 CFR Part 1610.7 is mentioned below
Class Plain Surface Respirator
1 Burn time =3.5 seconds
2 Not applicable to plain surface textile sheets
3 Burn time <3.5 seconds

Test results of flame resistance tested in different labs are tabulated below. It can observed that, invention shows good flame resistance when tested as per the standards discussed in this section.
Table 6: Test Result of Flame Resistance
Test Standard Result
Flame Resistance 16 CFR Part 1610 Burns time >3.5 Seconds and hence meets the standards

[0036] The splash Resistance property of the respirator was evaluated ASTM F1862. This procedure was performed to evaluate surgical facemasks and other types of protective clothing materials designed to protect against fluid penetration. The purpose of this procedure is to simulate an arterial spray and evaluate the effectiveness of the test article in protecting the user from possible exposure to blood and other body fluids. The distance from the target area surface to the tip of the cannula is 30.5 cm. A test volume of 2 mL of synthetic blood was employed using the targeting plate method.
Test specimen was considered as passed when no penetration of fluid was observed when tested at 160mm Hg (21.3 kPa). Test results of splash resistance tested in different labs are tabulated below. The embodiment was seen to have no leakages as per the splash resistance test and hence can be safely used in healthcare setup where the risk of spillage of blood, spit or vomit exists while handling infected patients.

Table 7: Test Result of Splash Resistance
Test Standard Result
Splash Resistance ASTM F1862 No leak found

[0037] Antimicrobial Testing was done to evaluate the microbial kill rate of the respirator material using AATCC 100 test method. The objective of this test method is to detect microbicidal activity on respirator substrate. Specimens of the test materials and control swatches are inoculated with test organisms and initial microbial growth is noted by means of serial dilution agar plate method. After required incubation period, the number of microbes is eluted from the swatches by shaking in known amount of neutralizing solution. The number of remaining microbes is determined by serial dilution agar plate method. The percentage of microbial reduction is determined based on the obtained results. Specimen was tested against one strains of Gram +ve, one strains of Gram -ve, fungi and yeast. The results are tabulated below.The Embodiment shows the treated surface has a broad range of anti microbial property spanning gram positive , gram negative bacteria along with fungi and yeast. Thus the respirator is capable of reducing microbial load in the embodiment.

Table 8: Test Result of AATCC 100
S.NO Test Organisms 0 hour 24 hours
1. Staphylococcus aureus ATCC 6538 CFU/mL 100000 0
Percentage reduction (%) 0% 99.9999%
Log Value 5 <1
2. Listeria monocytogenes ATCC 19115 CFU/mL 100000 0
Percentage reduction (%) 0% 99.9999%
Log Value 5 <1
3. Enterococcus faecalis ATCC 29212 CFU/mL 100000 0
Percentage reduction (%) 0% 99.9999%
Log Value 5 <1
4. Escherichia coli ATCC 25922 CFU/mL 100000 0
Percentage reduction (%) 0% 99.9999%
Log Value 5 <1
5. Pseudomonas aeruginosa ATCC 15442 CFU/mL 100000 0
Percentage reduction (%) 0% 99.9999%
Log Value 5 <1
6. Klebsiella pneumoniae ATCC 4352 CFU/mL 100000 0
Percentage reduction (%) 0% 99.9999%
Log Value 5 <1
7. Candida albicans ATCC 10231 CFU/mL 100000 0
Percentage reduction (%) 0% 99.9999%
Log Value 5 <1
8. Aspergillus niger ATCC 6275 CFU/mL 100000 0
Percentage reduction (%) 0% 99.9999%
Log Value 5 <1
9. Positive Control CFU/mL 100000 0
Percentage reduction (%) 0% 99.9999%
Log Value 5 <1
10. Negative Control CFU/mL 100000 TNTC

[0038] Another method for Antibacterial evaluation of Respirator using JISL 1902: 2008 was utilized. As per the protocol, A previous NB inoculum was incubated for 24 h at 37±2°C. Then, 1.0±0.1 ml from the inoculum with 1×107 cells ml -1 was added to 15 ml of NA warmed at 45–46°C. This solution was disposed in a sterilized Petri dish. After agar solidification, the respirators samples were placed over the agar, and incubated for 24 h at 37±2°C (JIS L 1902 2008). The evaluation of the antimicrobial activity, for all methods, was made based on the measure of the halo formed around the edges of the samples and the bacteria growth under the samples.The results of the embodiment proves that the respirator material was able to reduce microbial load very effectively.

Table 9: Test Result of JISL 1902
Quantitative Assessment of Activity - JIS L 1902: 2008
Test Bacteria: Staphylococcus aureus ATCC 6538
UNTREATED FABRIC: Concentration of Inoculum (Ma): 1.06 x 105 Log = 5.02
UNTREATED FABRIC: Concentration after 18 hour incubation (Mb): 5.00 x 106 Log = 6.69
Growth Value (F= Mb-Ma): 1.67
Sample
Identification No. Bacteria
Recovered Log Bacteria
Recovered
(Mc) Bacteriostatic
Activity
(S) = Mb – Mc Percentage
Reduction of
Bacteria
TRPT16069851 – BLACK
COLOR FABRIC < 10 < 1 >5.69 >99.99
Test Bacteria: Klebsiella pneumoniae ATCC 4352
UNTREATED FABRIC: Concentration of Inoculum (Ma): 1.14 x 105 Log = 5.05
UNTREATED FABRIC: Concentration after 18 hour incubation (Mb): 5.80 x 106 Log = 6.76
Growth Value (F= Mb-Ma): 1.71
Sample
Identification No. Bacteria
Recovered Log Bacteria
Recovered
(Mc) Bacteriostatic
Activity
(S) = Mb – Mc Percentage
Reduction of
Bacteria
TRPT16069851 – BLACK
COLOR FABRIC < 10 < 1 >5.76 >99.99

[0039] One more antimicrobial test was conducted to evaluate antimicrobial property of the respirator. Standard ASTM E2315 was used for evaluation. The testing included initial innoculum concentrations of 1 x 106 CFU/mL and an exposure time up to 24 hours. Acceptance criteria was defined as suppression of growth (of the innoculum species) within the dressing at various time points representing an assessment of the antimicrobial function of the respirator. Quantitative Testing was conducted to determine the antimicrobial properties of the device compared to a negative control within a specified sampling time. Thus the embodiment tested proved that the treatment is effective in terms of the its microbicidal activity in short span of time and thus keeps the respirator as sterile as possible of the microbial loads.

Table 10: Test Result of ASTM E2315
S. NO Test Organisms 0th hour 1 hour 4 hours 24 hours
1. Staphylococcus aureus ATCC 6538 CFU/mL 1.0 x 106 250 223 3
Percentage reduction (%) 0% 99.99 99.99 99.9999
Log Value 6 <1 <1 <1
2. Listeria monocytogenes ATCC 19115 CFU/mL 1.0 x 106 205 187 1
Percentage reduction (%) 0% 99.99 99.99 99.9999
Log Value 6 <1 <1 <1
3. Enterococcus faecalis
ATCC 29212 CFU/mL 1.0 x 106 211 196 1
Percentage reduction (%) 0% 99.99 99.99 99.9999
Log Value 6 <1 <1 <1
4. Escherichia coli
ATCC 25922 CFU/mL 1.0 x 106 320 298 5
Percentage reduction (%) 0% 99.99 99.99 99.9999
Log Value 6 <1 <1 <1
5. Pseudomonas aeruginosa ATCC 15442 CFU/mL 1.0 x 106 220 204 1
Percentage reduction (%) 0% 99.99 99.99 99.9999
Log Value 6 <1 <1 <1
6. Klebsiella pneumoniae
ATCC 4352 CFU/mL 1.0 x 106 214 190 1
Percentage reduction (%) 0% 99.99 99.99 99.9999
Log Value 6 <1 <1 <1
7. Candida albicans
ATCC 10231 CFU/mL 1.0 x 106 100000 100000 1
Percentage reduction (%) 0% 90 90 99.9999
Log Value 6 <1 <1 <1
8. Aspergillus niger
ATCC 6275 CFU/mL 1.0 x 106 40000 40000 1
Percentage reduction (%) 0% 90 90 99.9999
Log Value 6 <1 <1 <1
9. Positive Control CFU/mL 1.0 x 106 0 0 0
Percentage reduction (%) 0% 99.9999 99.9999 99.9999
Log Value 6 <1 <1 <1
10. Negative Control CFU/mL 1.0 x 106 TNTC TNTC TNTC

[0040] To estimate the antiviral property of the respirator, the testing of viral efficacy based on the bacteriophage were done. The test was conducted by infecting the sample infected with 109 PFU/mL of bacteriophage phi X 174 for 24h at 37 Deg C. Viable bacteriophage was extracted from the sample by shaking at 200 rpm in PBS for about 3h. Filter the filtrate using syringe filter. In parallel, A549 cells was seeded in 96 and 24 well plate respectively in DMEM (10 %) for 24 h at CO2 incubator. Cells after reaching 90 % confluence was infected with bacteriophage (positive control) and different volume of filtrate. Continue the incubation in CO2 for 24h. At the end of incubation, morphology of cells infected with and without bacteriophage were observed under Inverted Phase Contrast Microscopy. MTT assay was performed to quantify the viable cells.The results are tabulated below
Table 11: Test Result of Bacteriophage Activity
Test groups Cytotoxicity Cell viability Virucidal activity
control 0 100 NA
Control Phage 109 95.4 4.6 Observed
Control Phage 105 81.6 18.4 Observed
Control Phage 101 59.8 40.2 Not observed
10 microL 31.8 68.2 Observed
20 microL 32.0 68.0 Observed
30 microL 28.6 71.4 Observed

[0041] Morphology of cell: Elongated cells are viable and healthy; round and cells with disturbed membrane are dead cells. Morphology of the cells infected with 10 to 30 microL of filtrate was comparable with control cells suggesting the virucidal activity or the available virus is not sufficient enough to infect A549 cells. But when the volume of filtrate increases, no. of viable cells decreased. This in turn suggest the ability of virus to infect the cells after incubating in fabric.
Similar trend was observed in MTT assay as well where the number of viable cells decreased when increasing the volume of the filtrate.

A549 cells - Control A549 cells infected with 101 phage

A549 cells infected with 105 phage A549 cells infected with 109phage

10 µL 20 µL

30 µL

Fig 4: Infection of bacteriophages on cells .

[0042] The test conditions of Viral Load of 109 PFU/mL is an extreme condition. Under normal use conditions, the viral load will be significantly less. The current test item has performed satisfactorily under the extreme conditions. Hence usage on the normal conditions will have a good viricidal activity on the Test Fabric.

[0043] Antiviral property of the respirator was evaluated by testing the respirator against SARS CoV-02 virus. Here the SARS Cov2 testing kit from ALDONA Germany was used. The test method utilizes the established RTPCR test and compares it with the standards given to evaluate the viral kill efficacy of the respirator.

[0044] The study of antiviral efficacy was performed as per ISO 18184:2019 (modified). This international standard specifies testing methods for determination of the antiviral activity of the submitted respirator samples. Antiviral Respirator samples are intended to be used. In this test, the antiviral activity of Antiviral Respirator was inoculated with E and S gene of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with 60 minutes of treatment time and the samples were evaluated by analyzing the reduction of viral load in percentage. The test method involves the inoculation of SARS-CoV-2 viral genes (E gene and S gene) on to the test sample (Antiviral Respirator). In 60 minutes of treatment time, viral load on the samples were observed at the time frequency of 0, 30, 45, 60 and 120 minutes. Proceeding with neutralization to retrieve the inoculated SARS-CoV-2 viral genes. The retrieved RNA content was evaluated by performing qRT-PCR technique for further confirmation of antiviral activity of the selected test sample.

[0045] Here all reagents and samples were thawed completely, mixed and centrifuged. The kit used for qRT-PCR contains a heterologous Internal Control (IC) which can either be used as RT-PCR Inhibition Control or as a control of sample preparation procedure and as RT-PCR Inhibition Control. Prepare the master mix with IC (Internal Control). If IC was added during the sample preparation procedure, set the Master mix according to the following pipetting scheme.
Number of Reactions 1 12
Master A 5 µl 60 µl
Master B 15 µl 180 µl
Volume Master Mix 20 µl 240 µl

[0046] Pipette Master mix into each required well of an appropriate optical 96-well reaction plate or an appropriate optical reaction tube Add sample (eluate from the nuclic acid extraction) or controls (Positive or Negative control). So, the reaction setup will be
Master mix + Sample or control = Total volume.
There should be one positive and one Negative control per run and thoroughly mix the samples and controls with the Master mix by pipetting up and down. Close the 96-well reaction plates and tubes with appropriate lids or optical adhesive film. Centrifuge the 96-well reaction plates in the centrifuge with microtiter plate rotor for required amount of time.

Table 12: Test Result tested against SARS CoV-02
Sl. No. Time (min) AVT001 UTT02
SARS-CoV-2
E Gene S Gene E Gene S Gene
Ct Value* Delta Ct** % Reduction in Viral load Ct Value Delta Ct % Reduction in Viral load Ct Value Delta Ct % Reduction in Viral load Ct Value Delta Ct % Reduction in Viral load
1 0 25 - - 24 - - 26 - - 24 - -
2 30 29 4 - 27 3 - 27 1 NS## 25 1 NS
3 45 31 2 >95 29 2 >95 28 1 NS 26 1 NS
4 60 ND# - 99 ND - 99 29 1 NS 27 1 NS
5 PC 29 - - 29 - - 30 - - 30 - -
6 NC ND - - - - - - - - - -
PC means Positive Control
NC means Negative Control
* means triplicates
** Delta CT of 3-4 corresponds to 1 log difference
#ND -Not Detected
## NS-Non-significant

Table 13: Test Sample against E Gene

Figure 5: Graphical representation of activity in Antiviral Respirator (E Gene)


Table 14: Test sample against S Gene

Figure 6: Graphical representation of activity in Antiviral Respirator (S Gene)

[0047] In the present study, SARS-CoV-2 specific RNA (E&S target gene) was not detected after 45 minutes of contact time, indicating rupturing of viral envelope with >95% of reduction in viral load in test sample. It has also significantly enhanced the antiviral log reduction and reduces viral infectivity by 99% reduction of viral load in 60 minutes of contact time. It indicates, test sample (Antiviral Respirator) shows effective antiviral activity against encapsulated SARS-CoV-2 virus within 45 minutes of contact time

[0048] Sodium Chloride (NaCl) Aerosol Test were performed to understand the fit and no leakage test when worn on the face. Here Prior to testing, respirators were taken out of their packaging and placed in an environment of 85 ± 5% relative humidity (RH) and 38 ± 2.5°C for 25 ± 1 hours.

[0049] The filter tester used in testing was capable of efficiency measurements of up to 99.999%. It produces a particle size distribution with a count median diameter of 0.075 ± 0.020 microns (µm) and a geometric standard deviation not exceeding 1.86 µm. The mass median diameter was approximately 0.26 µm, which is generally accepted as the most penetrating aerosol size. The reservoir was filled with a 2% NaCl solution and the instrument was allowed with minimum warm-up time of 30 minutes. The main regulator pressure was set to 75 ± 5 pounds per square inch (psi). The filter holder regulator pressure was set to approximately 35 psi. The NaCl aerosol generator pressure was set to approximately 30 psi and the make-up airflow rate was set to approximately 70 litres per minute (L/min).

[0050] The NaCl concentration of the test aerosol was determined in mg/m3 by a gravimetric method prior to the load test assessment. An entire respirator was mounted on a test fixture, placed into the filter holder, and the NaCl aerosol passed through the outside surface of the test article at a continuous airflow rate of 85 ± 4 L/min.
The NIOSH N95 filter efficiency as stated in 42 CFR Part 84.181 is a minimum efficiency for each filter of =95% (=5% penetration).

Test Corrected, Initial Airflow Resistance (mm H2O) Maximum Particle
Penetration (%) Filtration Efficiency (%)
Nacl AEROSOL TEST For Fit 15.23
(Avg. of 3 readings) 3.20
(Avg. of 3 readings) 96.79
(Avg. of 3 readings)
Table 15: Nacl Aerosol Leakage test.
Thus the developed respirator is conforming to the face and has very minimal leakages and found effective towards the purpose of design.
From the above discussions and the descriptions given, it is evident that a Novel and innovative device has been invented that has multi functional properties of bacterial, viral kill along with bacterial, viral filtration, particulate filtration, it is shape conforming for minimal leakages as per Nacl Aerosol testing , and has splash proof property along with antiflamability that is a mandatory requirements for respirators in hospital setup. Such devices can be used to protect and prevent the spread of bacterial or viral infections at the time of outbreaks.
,CLAIMS:
We claim;
1] A face piece respirator made up of unified layers of reusable sheets of materials which has antimicrobial, antiviral properties imparted onto it by use of Silane and or siloxane based quaternary ammonium compounds along with liquid repellent property for splash proof surface and is able to filter viruses , bacteria and particulate matters, wherein the respirator is with shape conforming abilities to have proper fit onto the face of the wearer without any air leakages as per standard test methods, wherein the respirator is not only the physical barrier for pathogens like bacteria and virus but also is capable of actively eliminating the pathogenic load that is built up during the process of inhaling and exhaling.

2] The respirator as claimed in claim 1 has acquired the property of antimicrobial and antiviral kill by way of treating the fabric with non eluting quaternary compound that is represented by general formula 1 or general formula 2.

Formula -1

Formula – 2

Wherein:
R1 = hydrogen and/or C1 to C4 alkyl
R2 = divalent hydrocarbon radical with C1 to C8 carbon atoms;
R3 = hydrogen or C1 to C4 alkyl;
R4 = hydrogen or C1 to C10 alkyl;
R5 = C8 to C22 saturated or unsaturated hydrocarbon radical; and
X = alkyl halide.

3] The respirator as claimed in claim 1 may be constructed by sheets of material made by the process of weaving , fibre intermingling, knitting or combination of these processes in parts to achieve the desired functions.
4] The respirator as claimed in claim 1 may have the skin touching side as moisture absorbing layer for the aerosols from the exhaled breath.
5] The respirator as claimed in claim 1 may be made up of entirely or part of materials like polyester, Nylon, Viscose ,Acrylic, polypropylene, polyurethane or any synthetic fibre or natural fibers like cotton, silk , wool , Hemp , linen or the blend of these fibers or combinations of any of these yarns .
6] The repellency property for the respirator as claimed in claim 1 can be achieved by either of paraffin repellent, steric acid melamine repellents, silicon water repellents, fluorocarbon based repellents or surface induced repellency by plasma or irradiation with high energy radiations or by surface grafting of polymers.