BRPI0806169A2 - methodology for assessing the protective characteristics of personal protective equipment against biological agents - Google Patents

Abstract

EVALUATION METHODOLOGY OF PROTECTIVE CHARACTERISTICS OF PERSONAL PROTECTIVE EQUIPMENT AGAINST BIOLOGICAL AGENTS. The present invention relates to a new test methodology for the assessment of the protection of a Personal Protective Equipment (PPE) for use in the respiratory tract against biological agents, characterized in that different devices are used to reproduce the use of PPE, simulating a breath through a device known as the Sheffields head and a self-respirator. The apparatus consists of: a) a viral and / or bacterial aerosol generator; b) a test chamber containing the Sheffield's head device; c) a respirator simulating breathing and adjusting the rate of inspiration and exhalation; d) a suction system releasing air samples taken at different points to the bubblers to determine viral and / or bacterial concentrations.

Description

"EVALUATION METHODOLOGY FOR PROTECTIVE CHARACTERISTICS OF PERSONAL PROTECTIVE EQUIPMENT AGENTS"

Technical Field of the Invention

The present invention relates to a test methodology for evaluating the protection against biological agents of protective equipment for use in respiratory tractors, characterized in that different equipment is used to reproduce the actual use of protective equipment.

Antecedents of the Technique

There are several test methodologies for evaluating the effectiveness of respiratory tract protective equipment, in particular for calculating sealing loss (European Standard EN 13274-1) and respiratory resistance (European Standard EN 13274-3).

As is well known, there are several methodologies for calculating the viral removal efficiency of filter materials used as filtration membranes in laboratories, the pharmaceutical industry or medical devices. These methodologies use innocuous attack of aerosol and bacteriophages.

An example of such a methodology is described in the article "Efficacy of a pleated hydrophobic filter as abarrier to Mycobacterium Tuberculosis transmission within the system" (S. Speigh et al. - Center for Applied Microbiology & Research - Porton Down, Salisbury, Wilshire SP4 OJG, UK).

However, all of these methodologies have never been applied to respiratory tract protective equipment, in fact, to date, it has not yet been possible to test the effectiveness of bacterial and viral removal directly on personal protective equipment during a respiration simulation and actual application on the face of a patient. auser.

Ά In order to calculate the protective performance of personal protective equipment (such as filters for full face masks, half face masks, filtering face masks, etc.) against biological agents such as bacteria and viruses, analytical methodologies using powders and / or powders have always been used. or different types of chemical aerosols produced from non-vital substrates. However, all these test methodologies are not representative of the behavior of the microbial agent, nor its penetration properties in the barrier type, which is a protective device for the respiratory tract.

Description of the Invention

The present invention relates to a new test methodology for the assessment of protection of Personal Protective Equipment (PPE) for use in respiratory tractors against biological agents, characterized in that different equipment is used to produce the use of PPE, simulating respiration through the known device. Sheffield's head and a self-respirator. A particular embodiment of the present invention is the integral equipment used to assess the level of protection of PEP against biological agents, as well as the use of the Sheffield's head device and self-respirator to also assess the ability of PEP to protect against biological agents. Figure 1 highlights a schematic of the equipment used.

The equipment consists of:

a) a viral / bacterial aerosol generator;

b) a test chamber containing the Sheffield'shead device;

c) a respirator that stimulates breathing and adjusts the frequency of inspiration and exhalation; and

(d) a suction system which releases spray samples at different points to the bubblers to determine viral and / or bacterial concentrations.

This methodology consists mainly of feeding an aerosol from a test microorganism through the generator (a) and sending the aerosol medium to the test chamber (b).

Inside the test chamber is the Sheffield's head device on which the PPE is placed.

Air inside the test chamber is inhaled through the Sheffield ^ s head device through the self-respirator (c).

Some modifications to the Sheffield'shead device are made to allow inhalation through the mouth-nose region and exhalation from the back of the head to be sent to the self-respirator.

Thanks to placing PEP directly on the Sheffield 's head device, during implementation it is designed to verify the protective effectiveness of PEP, the inherent properties linked to PEP physical-mechanical and ergonomic characteristics (which are essential for PEP to be suitable for protective use). against biological agents) are also taken into consideration.

This means that possible contaminant passages due to loss of sealing due to poor use of PPE (and therefore independent of the filtration properties of the filter material itself) are also evaluated.

In order to calculate viral or bacterial PPE retention, two test analyzes are performed on the raid through suction system (d) and sent to some bubblers.

In particular, the microbial concentrations of the indoor air to the test chamber are calculated (in the blank test, the air is taken from the position corresponding to the right of the Sheffield's head device) and the air passed through the PPE (sample test, the air is drawn below corresponding to the mouth of the Sheffield's head).

Both tests, the blank test and the sample test, are performed simultaneously by boiling over a specific period of time, in a solution which has an appropriate composition and correct acidity / alkalinity (pH) through two separate tubes with dispersion of agents. in the test chamber (blank test) and in the air sample that crosses the EPP (sample test).

The two bubblers are connected to a suction system under constant flow.

At the end of the test, the solutions contained in the blowers are moved into appropriate sterile containers and then the collected microorganisms are counted.

The proportion of microorganisms blocked by PEP is determined as follows:

% (proportion of blocked microorg) = 100 - (Na / Nv χ 100) where:

Nv = concentration of test microorganism in aerosolateral posterior to the test chamber (blank test)

Na = concentration of test microorganism below filtration facial masks (sample test).

Figures 2-6 show in detail the process flow and its components.

The aerosol generator (Figure 2) consists of:

- peristaltic pump (1);

- nebulizer (2);

- nebulization line (3);

- dissecting tube (4) - circulation tube (5).

A suspension of a known amount of a microorganism is fed through a bombaperistaltic (1) into a nebulizer (2), where compressed air passing through the nebulization line (3) creates an aerosol medium.

This aerosol medium, once fed into the dissecting tube (4), is mixed with the dry compressed air, which arrives separately from the flow line (5).

The microbial aerosol droplets entering the dissecting tube evaporate rapidly and are moved to the test chamber under constant flow.

The test chamber (Figure 3) consists mainly of:

- a hermetically sealed container (6);

- a Sheffield's head device (7), placed inside the container with piping lines.

The shape and dimensions of the container (6) allow the Sheffield's head device to be installed and the container is made of a material capable of ensuring compressed air; To this end, the walls are sealed with gaskets and one of them can be opened to allow movements required by the methodology.

The Sheffield's head device (7) is placed inside the airtight container (6), and is completed with self-respirator connection tubing and tubing for taking air samples contained in the test chamber and air passing through the PPE. The airtight container is shaped like a parallelepiped, shaped to the size of a drawing table, with an opening / closing folding wall, typically being made of Lexan material or similar materials.

The Sheffield's head device, for example, is completed with an accessory featuring three concentric tubes, two of which are attached to an automatic respirator and the third tube collects air that has passed through the EPP in the mouth-nose region of the head; Another tube, the fourth tube, is placed at right eye level and removes air contained within the test chamber.

The respirator (figure 4) consists of a pump and an inverter which regulates the rate of exhalation / exhalation; The respiration rate can be adjusted as normal human respiration, typically between 20 and 40 cycles per minute, with an air volume ranging from 1.5 to 3.5 liters per cycle.

The suction system (Figure 5) consists mainly of:

- a vacuum pump (8);

- a flow regulator (9);

- a blank sample test inhalation tube (10);

- a blank sample test bubbler (11);

- a sample test inhalation tube (12); and

- a sample test bubbler (13).

The system sucks the dispersion of microorganisms into the test chamber to quantify them.

Suction occurs through a vacuum pump (8), which allows withdrawal under a constant flow, which is adjusted and controlled by flow regulating devices (9).

Both blank and sample tests are performed simultaneously across two different lines by bubbling microorganism dispersions into an appropriate pH controlled solution. The dispersion used to collect viral agents is typically a pH 6.8 solution, while that for collecting bacterial reagents, the pH is typically neutral.

The dispersion in the test chamber (blank test) is withdrawn at the right eye level of the Sheffield 'head device through the inhalation tube (10) and bubbles into the sterile glass bubbler (11).

The air sample that passes through the PPE (sample test) is drawn at the mouth level of the Sheffield's head device through the inhalation tube (12) and bubbles into the sterile glass bubble (13).

At the end of the test, the bubblers are disconnected, the solutions are released into sterile containers and the microorganism count is made in the solution.

A particular embodiment of the present invention is the apparatus used to verify the protective efficacy of PEP against biological agents, said apparatus comprising a viral and / or bacterial aerosol generator, a test chamber containing the Sheffield's head device, a respirator which stimulates respiration and adjusting the rate of inspiration and exhalation, a suction system that releases samples taken from different points to the bubblers to determine viral and / or bacterial concentrations, where the Sheffield's head device is fitted with tubes connected to a self- respirator to allow air to be inhaled and exhaled through the mouth-to-nose region and through the tubes to remove air contained within the test chamber and air passing through the EPP.

A preferred embodiment of the present invention is the protective efficacy screening apparatus of the PPE against biological reagents, where the device is placed within a test chamber made of Lexan material, the apparatus having an accessory having three concentric tubes, two of which are attached to the automatic respirator. and the third tube collecting the air that has passed through the EPP in the mouth-nose region of the head, and with another tube, the fourth tube, which is placed at eye level and which removes the air contained within the detested chamber, said respirator comprising a piston pump and an inverter that regulates the inhalation / exhalation velocity.

Other particular embodiments of the present invention include the use of the modified Sheffield's head device, as described above, and the self-respirator, which is produced by a pump to assess the protective efficacy of EPP against biological agents. This methodology can be used to determine protection against biological agents and in particular contrabacteria and viruses.

The preparation of the attack and strike suspensions of the microorganisms may be carried out by any known process for such use; The processes typically are different for viruses and bacteria.

In order to further explain the present invention, the following are two examples of methodologies used for bacterial and viral agents, respectively.

Example 1: Methodology for Viral Agents

In the example described herein, the detesting microorganism used is the bacteriophage MS-2 (National Collection of Industrial Bacteria: NCIMB 10108), which is a polypyridic virus of size 0.02 μπι. The number of MS-2 bacteriophages active in the attack suspension and stored after processing is determined by establishing a dispersion methodology on an agar layer.

The counting methodology consists of removing fractions ranging from 0.1 mL or 1 mL of pH 6.8 "Phage Buffer" containing the bacteriophage MS-2 and mixed in one case with 2.5 mL. detripton soy agar containing about 0.5 ml of Escherichia coli, NCIMB 9481 (approximately 108 CFU / ml), stationary growth (4-6 hours after inoculation); in the second case, with 5 ml tryptone soy agar containing about 1.0 ml Escherichia coli, NCIMB 9481 (approximately 10 8 CFU / ml), growing steadily (4-6 hours after inoculation).

The agar-containing soft bed is then immediately poured onto the tryptone soy agar plates to produce a double layer. After 24 hours of incubation at 37 ° C, visible plaques of bacteriophages were counted. Plates showing Unvisible (PFU) are selected and multiplied by their dilution. The PFU thus determined will be equivalent to the number of bacteriophages MS-2 in the buffer solution.

Known titration attack suspensions are initially prepared by diluting the original suspension in a phage buffer. Dilutions are then prepared and by the "double layer" method the concentration is verified. Once the count is finished, the highest dilution plates showing confluence of lise should be selected. A proportion of the tipophage buffer is added to these plates and by use of a sterile spatula, the agar is broken and mixed with the buffer.

In a sterile container, the agar-containing buffer is stored and decanted, then shaken vigorously until the agar breaks. The resulting material is rotated and the agar waste will be deposited. The floating material is taken and filtered through a membrane and the fractions are stored at 4 ° C. A suspension thus prepared of known titration is inserted into the deaerosol generator of a defined volume.

The viral suspension is directed to the nebulizer (2) through the flow generated by the pump (1).

The aerosol generator then functions by depressing the aerosol spray (3) and the line-drying flow (5).

Homogeneous filling of the test chamber is then awaited and once the chamber is full, the suction system vacuum and suction pump are activated. Both tests, the blank test and the sample test are performed simultaneously through separate tubes, while bubbling into the withdrawn phage buffer. At the end of the test, both sprayers are disconnected, solutions are transferred to appropriate sterile containers and stored immediately at 4 ° C in order to inhibit any microbial growth.

The live MS-2 bacteriophages collected by the sampler are calculated using the double layer method described above.

Example 2: Methodology for Bacterial Agents

The efficacy of protection against bacterial agents was determined by a procedure similar to that of viral agents, however, the test microorganism was bubbled through a pH 7.0 diluent. The microorganism used for the test is Brevundimonasdiminuta (ATCC 19146), a bacterium the size of 0.3 μπι. Bacterial suspension was prepared as follows: some secondary cultures are prepared from a stock culture by curling on a tryptone soy agar plate and stored at 30-35 ° C for 18-24 hours. Thereafter, an additional secondary culture is prepared to be taken in the same manner and stored at 30-25 ° C for 18-24 hours. The second secondary culture is processing culture.

The processing culture is removed and placed in a pH 7.0 diluent in a container. The container is shaken with a mechanical stirrer and then the suspension is taken and placed in a test tube.

The number of cells in the suspension should reach a value between Ix107 CFU / mL and Ix1010 CFU / mL using dilution and unit quantity estimation through the McFarland index.

Thus, a bacterial suspension count is then performed.

The suspension should be stored in a cooler at 2-8 ° C for use during the day.

Counting of the bacterial suspension, tested and downstream tested, was performed as follows. Serial dilutions of the suspension to be counted are prepared using the diluent. A duplicate sample of each suspension is mixed and collected and the sample is transferred to Petri dishes. A certain amount of TSA is added in liquid form, kept in a water bath at 45 ° C with gentle shaking of the plate. The plates are incubated at 30-35 ° C for 24 hours. After counting the number of units for each plate, the plates are incubated for an additional 24 hours. The number of units cultured on each card is counted again, with no occurrence of those not properly separated.

The highest number of units for each sample is determined.

The CFU / mL number of the test suspension is calculated.

The examples of methodologies described above are for the sole purpose of further explaining the invention, not implying in any limitations.

Thus, for example, microorganisms having similar microbiological sizes and characteristics may also be used, as well as alternative test suspension preparations, different collection times and methodologies, and different counting modes.

The methodology can be used to evaluate the effectiveness against biological agents of any type of PEP for use in the respiratory tract, such as, for example, full face masks, face filtration masks, folded flat face or bowl shape masks and filters.

For example, the processing in Example 1 was used to calculate the effectiveness of a folded flat face filtering mask, as described in WO 2005/077214 A1, characterized in a filtration layer composed of borosilicate-glass microfibres joined by means of a vinyl acetate resin, the fibrous matrix being supported by a strong cellulose-based substrate and the structure being treated with a silicone-based coating.

The results are as follows:

<table> table see original document page 16 </column> </row> <table>

The object of the methodology of the present invention has been in accordance with the guidelines of the "Good Laboratory Practice (GLP)" Standard.

Methodology Validation

The methodology validation test took into account the efficiency of the test system (uniformity of distribution within the microorganism suspension buffers, viability of the microorganism during the test implementation, total method accuracy) and the analytical efficiency of the microbial agent count (repeatability, intermediate precision). , overall accuracy).

The validation test was performed for the viral agent method using the bacteriophage MS-2 and for the bacterial agent method using the diminished Brevundimonas.

Initially, the counting efficiency of the microorganism suspension was verified.

The concentration of a microorganism suspension was calculated by an appropriate counting methodology. For each dilution, the titration was checked several times and the test was repeated several times as well.

The results were used to calculate the repeatability of the analytical results, that is, appreciation, repeatedly verified in a homogeneous counted suspension sample.

For each dilution chosen, the mean and standard deviation were calculated. Then, the Percentage of Variation (% CV) was calculated, obtained from a percentage measurement between the standard deviation and the mean of measurements made.

% CV = sigma / Y χ 100,

Where:

sigma = standard deviation, Y = Mean value.

The CV value obtained for the different dilutions was less than 15% in both virus and debacterial performances, showing a correct repeatability of the count.

Using the results obtained from the six tests described above, the accuracy of the methodology was checked, that is, how the experimental values differ from the known theoretical data.

The evaluation criterion is based on the recovery percentage (%), which is the ratio between experimental data and known theoretical data.

% Recovery = Ns / N χ 100,

Where:

N = known theoretical data of viral suspension (pfu / mL);

Ns = experimental data obtained from viral suspension (pfu / mL).

The percentage recovery values obtained in the tests (12) were all included in the 70-130% range, in both cases of viruses and bacteria. The mean percentage recovery in both tests performed using virus and bacteria ranged from 80-120%.

Finally, repeatability tests were performed for two different operators on two different days. Thus, the average accuracy of the analytical results was also calculated, and is considered as a precision dependent on days and different people.

The CV value obtained from different dilutions by two operators on two different days was less than 15%, both for the virus inclusion test and for the bacterium inclusion test. Such result indicates satisfactory average accuracy.

Also, the efficiency of the test system has been modified. Thus, a processing culture was prepared as shown in the description of the method and the aerosol device was fed with a defined volume of the microorganism suspension. The aerosol generator was activated and the time was allowed to allow homogeneous filling of the test chamber. Then the vacuum pump of the suction system was triggered and subsequently both the vacuum pump and the deaerosol generator were deactivated.

Both bubblers were deactivated and the solutions were shaken in sterile containers and kept at 4 ° C to inhibit any microbial growth.

Some counting tests were then performed, with the necessary dilutions in the buffer solutions, using the appropriate method for the microorganism type.

The test was run several times in two different processing days.

The uniformity of the distribution of the microorganism suspension within the two bubblers (white test and sample test) was calculated, as well as at the two withdrawal points inside the chamber. For each test, the difference between the percentage of microorganisms collected in the two bubblers was calculated using the following formula:

Percentage of distribution difference (R%) = Na / Nv χ 100, where:

Nv = microorganism count performed in the bubbler (11) (pfu / mL);

Na = microorganism count performed in the bubbler (13) (pfu / mL)

No difference in distribution was within ± 5% for both the virus and bacterial tests. Therefore, the distribution of microorganisms at different points proved to be adequate.

The overall accuracy of the methodology was then assessed in terms of repeatability of analytical results.

Using the data produced in the previous tests for both bubblers, the percentage of CV was calculated using the following formula:

% CV = sigma / Y χ 100,

Where:

sigma = standard deviation;

Y = mean value for "n" samples.

The percentage of CV obtained in the two days was higher than 25% in both cases of virus and bacterium test, which indicates an adequate repeatability of the analytical results and, therefore, an adequate total methodology accuracy.

Finally, the viability of the organism during the test implementation was determined, ie the microorganism's ability to be viable for at least 30 minutes, allowing the test chamber to maintain a sufficient concentration in order to highlight, under the analytical aspect, the efficiency of EPP protection.

A count of the microorganism suspension having a known titration, between 1 χ 10 7 and 1 χ 10 8, was immediately performed after its preparation (T0). The same suspension was held 15, 30 and 45 minutes after preparation (Ti5, T30, T45).

The Ti5, T30 and T45 counts obtained were then compared with T0.

The test was repeated three times. The decrease in Ti5, T3o and T45 typing was lower than the logarithm of 2, compared to the viral titration at T0, both in the case of virus and in the bacterial test.

Therefore, microorganisms remained unpredictable during the test.

While particular embodiments of the present invention have been set forth in the foregoing description, it will be understood by those skilled in the art that any simple modifications and rearrangements of the invention will not depart from its essential features or attributes, which are defined in the appended claims.

Claims (15)

1. Test methodology for the evaluation of the protection of a Personal Protective Equipment (PPE), used for respiratory tract against biological agents, characterized by the fact that: - the use of PEP is reproduced by stimulation of a real breath through the device known as Sheffield's head. Sheffield) of a self-respirator, thanks to placing PEP directly on the Sheffield ^ s head device and breathing stimulation, as well as PEP efficiency, the inherent properties linked to PEP physicochemical and ergonomic characteristics are taken into consideration, which is essential. to make PEP suitable for protection against biological agents - this means that possible contaminant passage due to seal loss / leakage and defects, and therefore regardless of the filtering properties of the filter material itself, are also taken into consideration. Methodology as claimed in claim 1, characterized in that: - generating a microorganism aerosol test by means of a viral or bacterial aerosol generator and aerosol movement to a test chamber in which a Sheffield's head device is provided. , on which a PEP is placed, the test chamber is connected to a breathing device and a sampling suction system, - the air inside the test chamber is inhaled by the Sheffield's head device through the respirator that stimulates actual breathing by adjusting of inhalation and exhalation frequency, the Sheffield's heads device has been modified to allow air to be inhaled through the mouth / nose area and exhalation from the back of the head to be sent to the self-respirator; samples of air taken at different points for some bubblers in order to calculate the m Microbial tests on the interior of the test chamber (blank sample test) and the air passed through the PPE (sample test) - both blank test and sample test are carried out simultaneously by bubbling over a specified period of time. time and under constant flow in a solution having a suitable composition and a suitable pH through two separate tubes where biological agents are dispersed in the test chamber and in the air sample passing through the PPE; solutions contained in the bubbles are moved into appropriate sterile containers and then the collected microorganisms are counted, the proportion of microorganisms blocked by the PEP is determined as follows:% (proportion of microorg. = 100 - (Na / Nv χ 100) where: Nv = concentration of test microorganism in aerosol posterior to test chamber (blank test) Na = concentration of test microorganism below filtration face masks (test sample ). Methodology as claimed in claim 2, characterized in that: a suspension of a known amount of a microorganism is fed through a bombaperistaltic inside a nebuliser, where compressed air passing through the nebuliser tube creates an aerosol; aerosol is fed into a desiccant tube, where it is mixed with dry compressed air that comes separately from a flow line, - microbial aerosol droplets entering the desiccant tube evaporate rapidly and are moved to the test chamber with a constant flow; - the test chamber is assembled in the form of a hermetically sealed container having sealed walls, including gaskets, one of which can be opened to allow operators to operate, - the Sheffield's head device is placed inside the airtight container, completing this step with pipe connection to self-respirator and with one pipe to remove air samples contained in the test chamber and air passing through the PEP - the respirator equipment consists of a pump and regulates the respiration rate according to typical human respiration - the suction system sucks the dispersion of microorganisms inside the quantification test - suction takes place through a vacuum pump that allows withdrawal under a constant flow, the withdrawal of which is controlled by flow regulators - both the blank sample test and the sample test are performed simultaneously by two separate lines by bubbling the microorganism dispersions in an appropriate pH-controlled solution, - this dispersion for the blank test is removed from the test chamber through a first suction line and bubbled into a first sterile glass bubbler; test sample is drawn from the air passing through the PEP through a of aerosol and bubble suction in a second sterilized glass bubbler - at the end of the test, the bubblers are disconnected, solutions are released into sterile containers and the microorganism counting in the solutions is performed. Methodology as claimed in claim 2, characterized in that: - the test chamber has Lexan walls, one of which has a dual open / close system, the Sheffield's head device is completed with an accessory having three concentric tubes , two of which being connected to the self-respirator and the third connected to the suction system, which collects air passed through the PEP at head level in the mouth / nose region; - a fourth tube, connected to the suction system, is placed at right eye level and removes air contained in the test chamber - the respirator consists of a piston pump, valves and an inverter which regulates the rate of exhalation / exhalation. Methodology as claimed in claim 2, characterized in that the respiratory frequency varies between 20 and 40 cycles / minute, the air volume varies between 1.5 and 3.5 liters per cycle and the suction system is activated a few minutes after the aerosol generator is activated to allow the detesting chamber to be homogeneously filled. 6. Apparatus for the evaluation of protection against biological agents in a Personal Protective Equipment (PPE) for use in the respiratory tract, characterized by the fact that it has: - a viral / bacterial aerosol generator, a test chamber containing a Sheffield's head device, a respirator that stimulates the respiration and adjusts the rate of inhalation and exhalation and a suction system that releases air samples taken at different points to the bubblers to determine viraise / or bacterial concentrations - where the Sheffield's head device is equipped with self-breathing tubes , to allow inhalation and exhalation of air through the mouth / nose area and with tubes drawn from the air contained within the test chamber and the arch passes through the PEP. Apparatus as claimed in claim 6, characterized in that: - the aerosol generator includes a pump, a spray line, a nebulizer, a dissecting tube and a flow line - the test chamber is comprised of a hermetically sealed container where the Sheffield's head device is completed with a connection to a self-respirator tubing and a tubing for sampling the air contained in the test chamber and the air passing through the PPE - the respirator equipment consists of a pump regulates the inhalation / exhalation rate - the suction system consists of a vacuum pump, flow regulators, a bubbler suction line for the blank test and a bubbler suction line for the sample test. Apparatus as claimed in claim 6, characterized in that: - the Sheffield 's head device is placed in a Lexan material test chamber and is completed with an accessory having three concentric tubes, two of which are attached to the self-respirator and the third collecting the air passed through the PEP at head level in the mouth / nose region - a fourth tube is placed at right eye level and the air contained in the test chamber is removed - the respirator consists of a piston pump, valves and an inverter, which regulates the rate of exhalation / exhalation. 9. Use of Sheffield's head device for the assessment of protection of Personal Protective Equipment, characterized in that its use is intended for respiratory tract against biological agents. Use of the modified Sheffield's head device as described in claim 8 for the assessment of the protection of Personal Protective Equipment, characterized in that said use is for the respiratory tract against biological agents. 11. Use of a piston pump complete with an inverter to stimulate respiration in the assessment of the protection of Personal Protective Equipment, characterized by the fact that said use is intended for respiratory tractors against biological agents. Methodology as claimed in claim 1 for the assessment of the protection of Personal Protective Equipment, characterized in that the said methodology is for use in the respiratory tract against viral agents. Methodology as claimed in claim 1 for the assessment of the protection of Personal Protective Equipment, characterized in that the said method is for use in the respiratory tract against bacterial agents. Methodology as claimed in claim 3, characterized in that the preparation of microorganism suspensions is carried out by any process known for such use. Methodology as claimed in claim 2, characterized in that the counting of microorganisms is carried out by any known process for such use.