MXPA06003502A - Integator system and method for rapidly determining effectiveness of a germicidal treatment - Google Patents

Abstract

The effectiveness of an oxidative sterilization process is determined by exposing a substrate having a known amount of a primary amine or aldehyde indicator chemical to an oxidative germicide. The oxidative germicide reacts with the indicator chemical. The amount of indicator chemical remaining after exposure to the germicide is determined by reacting the indicator chemical with a dye precursor chemical to form a colored product. The amount of indicator chemical remaining on the substrate is determined from the intensity of the color of the colored product. The amount of indicator chemical remaining on the substrate is a measure of the effectiveness of the germicidal treatment. The dye precursor is an aldehyde when the indicator chemical is a primary amine and a primary amine when the indicator chemical is an aldehyde. An integrator for determining the effectiveness of the germicidal process includes a substrate and an indicator chemical, where the indicator chemical is a primary amine or an aldehyde.

Description

INTEGRATOR SYSTEM AND METHOD TO QUICKLY DETERMINE THE EFFICACY OF A GERMICIDAL TREATMENT FIELD OF THE INVENTION This invention relates to an integrating system and method for rapidly determining the effectiveness of a germicidal process of a medical device.

BACKGROUND OF THE INVENTION Medical devices are sterilized before being used in hospitals, clinics and other medical facilities. Steam, heat, ethylene oxide and hydrogen peroxide are commonly used as sterilizing agents. It is normal practice to include a sterility indicator in a load of articles to be sterilized in a sterilizer. The sterility indicator provides a measure of the effectiveness of the sterilization process of the articles in a particular load. If the sterility indicator shows that the sterilization process was not effective, the equipment load is rejected to use. Biological indicators are generally recognized as indicators of reliable sterility. The biological indicator includes a vehicle that has been inoculated with spores or other microorganisms. Spores are usually used in biological indicators because the spores are more resistant to sterilization than other microorganisms. The biological indicator is placed in the sterilizer with the equipment to be sterilized. At the end of the sterilization process, the biological indicator is removed from the sterilizer and the vehicle is immersed in a sterile culture medium. The culture medium and vehicle are incubated for a predetermined time at an appropriate temperature. At the end of the incubation period it is determined if any microorganism grew in the growth medium. If there is no growth of microorganisms in the growth medium, it is assumed that the equipment that was in the sterilizer was properly sterilized. If growth of microorganisms is observed, the sterilization process was not effective and the articles are rejected to use. The growth of microorganisms is determined by a signal, such as the generation of turbidity or a color change in a pH indicator, due to a pH change of the byproducts of cell growth in the medium. Biological indicators are described, for example, in the US patents. Nos. 5,552,320 and 6,436,659, both incorporated herein by reference in their entirety. Although biological indicators are accurate indicators of the effectiveness of the sterilization cycle, at least 24-48 hours are required to obtain results from the biological indicators. The equipment that was exposed to the sterilization process is normally kept in quarantine until the results of the biological indicator are available. Medical equipment is expensive and storage space in medical facilities is limited. Therefore, some hospitals use the equipment before having the results available. The storage of medical equipment in quarantine represents an inefficient use of resources. There is a need for a rapid test to determine the effectiveness of the sterilization process. Foltz et al. (U.S. Patent No. 6,355,448) describe a method for determining the effectiveness of a sterilization process using the activity of enzymes in place of spores. It is stated that the enzyme test procedure requires only a few minutes instead of several days as required to obtain the results of the biological indicators. The use of a plurality of enzymes instead of a single enzyme is described in the U.S. Patents. Nos. 5,486,459 and 6,528,277. It is believed that the use of a plurality of enzymes better mimics the response of a microorganism than a single enzyme. There is a need for sterilization indicators that provide sterilization results quickly.

BRIEF DESCRIPTION OF THE INVENTION One aspect of the invention includes a method for quickly determining the effectiveness of an oxidative germicidal process. The method includes providing a substrate having a known amount of a first reagent on the substrate, wherein the first reagent is selected from the group consisting of a primary amine, mixtures of primary amines, an aldehyde, and mixtures of aldehydes. The first reagent has a first color. The method also includes exposing the substrate and the first reagent to an oxidative germicide, thereby reducing the known amount of the first reagent to a final amount. The substrate having the final amount of the first reagent having the first color is contacted with a second reagent having a second color, thus generating a third reagent having a third color. The intensity of the third color is related to the final amount of the first reagent on the substrate. When the first reagent is a reagent selected from the group consisting of an aldehyde and a mixture of aldehydes, the second reagent is a reagent selected from the group consisting of a primary amine and mixtures of primary amines. When the first reagent is a reagent selected from the group consisting of a primary amine and a mixture of primary amines, the second reagent is a reagent selected from the group consisting of an aldehyde and a mixture of aldehydes. The method also includes determining the intensity of the third color and determining the efficacy of the germicidal process from the intensity of the third color. Advantageously, the effectiveness of the germicidal process is determined by correlating the intensity of the third color with the results of the biological indicators. In one embodiment, the oxidative germicide is a sterilant. In an alternative modality, the oxidative germicide is a disinfectant. The substrate can be an absorbent substrate.

Preferably, the substrate is a non-absorbent substrate. In one embodiment, the oxidative germicide is a liquid, a vapor, or a gas. Advantageously, the intensity of the third color is determined visually. Preferably, the intensity of said third color is determined spectrophotometrically in the visible or ultraviolet region. In one embodiment, at least one of the first reagent or the second reagent is colorless. Advantageously, the oxidative germicide is selected from the group consisting of hydrogen peroxide, peracetic acid, ethylene oxide, ozone and chlorine dioxide. Preferably, the method also includes exposing the substrate and the oxidative germicide to plasma. In one embodiment, the percentage of completion of the germicidal process is determined by comparing the intensity of the third color with the color intensity of a standard. Preferably, the primary amine is glycine or histidine, and the aldehyde is ortho-phthalaldehyde or glutaldehyde. Another aspect of the invention includes an integrator for determining the efficacy of a germicidal process with an oxidative germicide. The integrator includes a substrate with a known amount of a first reagent on the substrate, wherein the first reagent is selected from. group consisting of a primary amine, mixtures of primary amines, an aldehyde, and mixtures of aldehydes. The substrate is in an enclosure. The first reagent is able to react with the oxidative germicide when exposed to the oxidative germicide. The integrator also includes a deposit of a second reagent, wherein the second reagent is a reagent selected from the group consisting of a primary amine and mixtures of primary amines, when the first reagent is a reagent selected from the group consisting of an aldehyde and a mixture of aldehydes, and the second reagent is a reagent selected from the group consisting of an aldehyde and a mixture of aldehydes, when the first reagent is a reagent selected from the group consisting of a primary amine and a mixture of primary amines. The second reagent is capable of reacting with the first reagent to form a third reagent having a color. The reservoir has a breakable barrier, which isolates the second reagent from the first reagent and the oxidative germicide during the contact of the first reagent with the oxidative germicide. The breaking of the breakable barrier of the deposit brings the second reagent into contact with the first reagent, thus forming the third reagent having the color. The deposit is in the enclosure. In one embodiment, the breakable barrier of the reservoir includes a frangible ampule of the enclosure. Advantageously, the integrator also includes a second barrier, wherein the second barrier is within the enclosure between the frangible ampoule and the first reagent. The second barrier of the enclosure is permeable to the second reagent. The second barrier prevents fragments of the frangible vial from contacting the first reagent. In one embodiment, the integrator also includes a window in the enclosure, where the window is permeable to the oxidative germicide. The window allows the oxidative germicide to penetrate the enclosure. Advantageously, the primary amine is selected from the group consisting of glycine and histidine and the aldehyde is selected from the group consisting of orthophthalaldehyde and glutaldehyde. Preferably, the integrator enclosure also includes a transparent window, wherein the color change in the substrate can be observed through the transparent window, visually or with a spectrophotometer.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic diagram of an integrator according to an embodiment of the present invention; Figure 2 is a schematic diagram of a compressible integrator system containing an integrator according to an embodiment of the present invention; Figure 3 is a schematic diagram of a slidable integrator system containing an integrator according to an embodiment of the present invention; Fig. 4 is a schematic diagram of the slidable integrator system of Fig. 3, after an outer cover has been moved over an internal cover of a slidable container lockable in the slidable integrator system.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term germicidal includes both sterilants and disinfectants. As used herein, the term germicidal process includes sterilization processes and disinfection processes. Sterilization indicators that use reagents to mimic the resistance of a biological indicator (Bl) have been called integrators. The integrators use an indicator reagent that responds to the germicide used in the germicidal process. The reagent reacts with the germicide in a repeatable manner and responds to factors that are important for sterilization or disinfection in the germicidal process. The reaction of the indicator reagent with the germicide is integrated with time, and the amount of indicator reagent remaining in the integrator correlates with the response of Bl. Integrators integrate the reaction of the reagent with time in response to critical parameters during a specified scale of sterilization cycles. The integrators and the method according to the embodiments of the present invention provide results quickly, reproducibly and accurately. The reagents used in the integrators are cheap and stable. The results obtained from the integrators and the method according to the embodiments of the present invention correlate well with the results of biological indicator tests. The integrator according to the modalities of the present invention has the purpose of imitating the resistance of a biological indicator (Bl) without using spores. The integrator according to one embodiment of the present invention includes an indicator reagent that reacts with an oxidative germicide. The integrator is suitable for oxidative germicides including hydrogen peroxide, peracetic acid, ethylene oxide, ozone and chlorine dioxide. The oxidative germicides may be in the form of a liquid, a vapor, or a gas. In one embodiment plasma can be used in combination with oxidative germicides to increase the reaction of the oxidative germicides with the microorganisms in the chamber and the indicator reagent in the integrator, or to inactivate the oxidative germicide after use. The use of plasma is optional. The results of the integrator according to the embodiments of the present invention are readily available, in about 30 seconds to about 5-6 minutes, depending on the reagents selected for the integrators, compared to the 24-48 hours normally required for the integrators. obtain results of a biological indicator. Although the integrator according to the embodiments of the present invention is described in the context of sterilization with a combination of hydrogen peroxide and plasma in the STERRAD® process, commercially available from Advanced Sterilization Products of Irvine, California, it can be used with a variety of germicidal processes. The description of germicidal processes such as sterilization or disinfection with hydrogen peroxide and plasma by the STERRAD® process is only illustrative and is not considered limiting. The integrator according to the embodiments of the present invention contains an indicator reagent. The indicator reagent reacts with the germicide and responds to the factors that are important for sterilization. The amount of indicator reagent remaining in the integrator after exposure to the germicide can be correlated with the response of the Bl's that are placed in the sterilization chamber along with the integrator. The response of a Bl is a generally accepted measure of the effectiveness of a germicidal process. The response of a Bl in the sterilization chamber can be correlated with the response of the integrators to "calibrate" the response of the integrators with the response of a biological indicator. In one embodiment primary amines or aldehydes are used as indicator reagents in the integrators according to the embodiments of the present invention. Oxidative germicides react with both primary amines and aldehydes. Both the primary amines and the aldehydes are suitable indicator reagents for the integrators according to the embodiments of the present invention. The amount of primary amine indicator reagent or aldehyde indicator reagent remaining in the integrator after the oxidative process, can be used to determine the effectiveness of the sterilization or disinfection process of the treated cargo in the sterilizer.

The amount of primary amine indicator reagent or aldehyde indicator reagent remaining in the integrator can be measured in various ways, such as instrumental methods, chemical analysis, etc.

Any method of measuring the concentration of the primary amine indicator reagent or the aldehyde indicator reagent is suitable. In one embodiment, the completion of the germicidal process can be conveniently determined by observing a color change in the integrator. Many primary amines react with the aldehydes to form colored products. The amount of primary amine indicator reagent or the amount of aldehyde indicator reagent remaining in the integrator after exposure to the oxidative germicide can be determined from the color intensity of the product of the reaction of an aldehyde with a primary amine. As used herein, an aldehyde which is contacted with a primary amine indicator reagent, or a primary amine which is contacted with an aldehyde indicator reagent, is referred to as a "dye precursor", because the product of the The reaction of a primary amine with an aldehyde is colored, a "dye", even though neither the primary amine nor the aldehyde has color. The primary amine and the aldehyde can change functions, depending on the reagent that is used as the indicator reagent in the integrator. In an embodiment in which a primary amine is the indicator reagent, the dye precursor is an aldehyde. In an embodiment in which an aldehyde is the indicator reagent, the dye precursor is a primary amine. The color intensity of the color product originating from the reaction between the primary amine and the aldehyde can be used to determine the efficiency of the treatment of the charge with the oxidative germicide. In one embodiment, an integrator according to one embodiment of the present invention contains a first reagent having a first color, wherein the first reagent is an indicator reagent. The indicator reagent is selected from the group consisting of a primary amine, a mixture of primary amines, an aldehyde, and a mixture of aldehydes. The integrator containing the indicator reagent is placed in a sterilizer with a load of equipment to be treated. The charge and the integrator are contacted with an oxidative germicide in a sterilizer. The oxidative germicide reacts with the indicator reagent, reducing the amount of indicator reagent remaining in the integrator. The amount of indicator reagent remaining in the recorder after contact with the oxidative germicide is a measure of the efficacy of the germicidal treatment with the oxidative germicide. When the effectiveness of the treatment with the oxidative germicide is to be determined, the integrator containing the first reagent having the first color is contacted with a second reagent having a second color. The first reagent that has the first color is the indicator reagent. The second reagent having the second color is the dye precursor. The dye precursor is a reagent selected from the group consisting of a primary amine, a mixture of primary amines, an aldehyde, and a mixture of aldehydes. The primary amines may not be mixed with the aldehydes to form the dye precursor. In an embodiment wherein the first reagent, the indicator reagent, is a primary amine or a mixture of primary amines, the second reagent, the dye precursor, is an aldehyde or a mixture of aldehydes. In an embodiment wherein the first reagent, the indicator reagent, is an aldehyde or a mixture of aldehydes, the second reagent, the dye precursor, is a primary amine or a mixture of primary amines. The product of the reaction of the first reagent, the indicator reagent, with the second reagent, the dye precursor, is a third reagent having a third color. The intensity of the third color of the third reagent that originates from the reaction between the first reagent and the second reagent, can be used to determine the amount of the first reagent, the indicator reagent, that remains in the integrator. The amount remaining in the integrator of the first reagent, the indicator reagent, is a measure of the effectiveness of the oxidative germicide treatment. If only a small amount of the first reagent remains in the integrator, the indicator reagent, the intensity of the third color due to the third reagent is low. A low intensity of the third color is an indication that the treatment with the oxidative germicide was effective.

In one embodiment, the degree of completion of the oxidative germicide treatment can be determined from the intensity of the third color due to the third reagent, the product of the reaction between the first indicator reactive compound and the second reagent, the dye precursor. The indicator compound in the integrator, the first compound, reacts with the oxidative germicide in parallel with the germicidal treatment of the charge in the sterilization chamber. The intensity of the third color due to the third reagent that originates from the reaction of the first reagent, the indicator reagent, with the second reagent, the dye precursor, decreases as the amount of indicator reagent decreases, due to the reaction with the oxidative germicide . The intensity of the third color can be correlated with the results of the biological indicators that are placed in the sterilization chamber together with the indicators. The intensity of the third color can be correlated with the percentage of sterilization or the percentage of disinfection, determined by biological indicators. The percentage of sterilization or disinfection, therefore, can be determined from the intensity of the third color due to the third compound. In one embodiment, the first reagent, the indicator reagent, is placed on a substrate for ease of handling. The substrate can be of a variety of materials. The substrate can be an absorbent substrate or a non-absorbent substrate. Absorbent substrates absorb the germicide. Non-absorbent substrates absorb little or none of the germicide. The filter paper is an absorbent substrate because it absorbs the germicide. A glass filter disc is a non-absorbent substrate because it does not absorb significant amounts of the germicide, and is therefore preferred. The indicator reagent can be packaged in a water-soluble binder, such as an acrylic polymer or carboxymethylcellulose. The indicator reagent and the water-soluble binder can be applied to the surface of the integrator or sterility indicator, for example, by ink-jet printing of a solution of the indicator reagent and the water-soluble binder, on the surface of a material of inert support. The absorbent substrates absorb the germicide during the germicidal process. When the second reagent, the dye precursor, is contacted with the absorbent substrate, the germicide absorbed onto the absorbent substrate can react with the dye precursor. Oxidative germicides generally react with primary amines and aldehydes, the two forms of the dye precursor. Therefore, when the substrate is an absorbent substrate, it is generally advantageous to use an excess of the second primary amine dye precursor reagent or aldehyde, because the absorbed germicide reacts with the dye precursor when the dye precursor is contacted with the dye precursor. the absorbent substrate. In one embodiment, the integrator containing the indicator reagent is placed in the sterilizer with the equipment to be sterilized and exposed to the germicide. The indicator reagent reacts with the germicide, reducing the initial concentration of the indicator reagent from an initial value to a final value. After the germicidal process is completed, the integrator containing the first reagent, the indicator reagent, is exposed to the second reagent, the dye precursor. If some indicator reagent is still present, the contact of the indicator reagent with the dye precursor forms the third compound having the third color. If a significant color develops in the integrator, it is considered that the germicidal cycle has not been effective. It is generally preferred that the second reagent, the dye precursor, be contacted with the integrator after cycle completion, because the dye precursor reacts with the germicide. If the dye precursor is contacted with the integrator before the end of the cycle, the germicide will react with the second reagent, the dye precursor, and it may be necessary to add dye precursor to provide sufficient dye precursor and cause a color change of the reaction of the second reagent, the dye precursor, with the first reagent , the indicator reagent, forming the third reagent that has the third color. Therefore, generally the second reagent, the dye precursor, is contacted with the integrator at the end of the cycle. In one mode, the cycle can be a canceled cycle. In one embodiment the second reagent is isolated from the germicide until the end of the cycle. The isolation of the second reagent, the dye precursor, from the germicide, protects the second reagent from the reaction with the germicide and from being destroyed. The color change of the reaction of the first reagent, the indicator reagent, with the second reagent, the dye precursor, to form the third reagent having the third color, can be determined visually. As a visual change is a bit subjective, generally the color change is determined with an optical detector. The optical detector for the color change that originates from the reaction of the first reagent, the indicator reagent, with the second reagent, the dye precursor, can operate at wavelengths of the visible or ultraviolet spectrum. The primary amine may be any suitable primary amine. In one embodiment, the primary amine is an amino acid. In one embodiment, the primary amine is selected from the group consisting of arginine, histidine, and combinations thereof. Other suitable primary amines include the following amino acids: alanine, proline, amino-caproic acid, phenylalanine, tryptophan, methionine, glycine, serine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine. Peptides or polypeptides formed from any number or type of amino acids are also suitable primary amines. Arginine is an exemplary primary amine indicator reagent.

Arginine gives a strong and rapid color change when exposed to aldehydes. Argillin also reacts rapidly with germicides. Arginine is a water soluble solid that is conveniently weighed, dissolved in a solvent and applied to the substrate or other support. In other embodiments, other primary amines may be used, as will be apparent from the description and the examples given below. Arginine has structure I: I The NH2 groups are primary amino groups. The NH groups are secondary amino groups. Aldehydes often do not react with secondary amino groups. The aldehyde can be any aldehyde that reacts with the primary amines, but not with the secondary or tertiary amines, to produce a color. Aldehydes such as OPA (ortho-phthalaldehyde), glutaldehyde, and aromatic aldehydes are suitable. Other aldehydes are also suitable. Figure 1 shows a diagram of an integrating system 10 according to an embodiment of the present invention. The integrator system 10 of FIG. 1 includes integrative chemistry 14, located on an integrator strip 16, wherein the integrator strip 16 is an inert material for supporting the integrative chemistry 14. The integrator strip 16 is generally made of a material that does not react or adsorb the germicide. Integrative chemistry 14 includes a first reagent, the indicator reagent. The integrator strip 16 is a substrate for integrative chemistry 14. The peel-off label 18 is optionally located on the integrator strip 16. Sterilization cycle information can be written on the tear-off label 18, and the tear-off label 18 with the Information on the sterilization cycle can be placed in a sterilization log. The indicator reagent strip, 20, contains a reagent that undergoes a color change when exposed to the germicide. A color change on the indicator reagent strip 20 simply shows that the indicator reagent strip 20 has been exposed to the germicide. The Indicator 20 reagent strip is not an indicator of sterilization efficacy, but merely indicates whether the indicator reagent strip 20 has been exposed to the germicide. The color change on the indicator reagent strip 20 shows the operator that the integrating system 10 should not be used again. Bordeaux red changes color when exposed to hydrogen peroxide. Other colorants may be used on the indicator reagent strip 20 to indicate exposure to other germicides. Suitable colorants are described, for example, in the U.S. patent. No. 5,942,438, which is incorporated herein by reference in its entirety. After the cycle, the integrating chemistry portion 14 of the integrator strip 16 is exposed to the second reagent, the dye precursor. The second reagent, the dye precursor, reacts with the first reagent, the indicator reagent, in the integrating chemistry 14, to form the third reagent having the third color. The presence of a significant amount of color in the integrating chemistry 14 on the integrator strip 16, after exposure of the integrative chemistry 14 to the second reagent, the dye precursor, indicates that the cycle was not effective. Figure 2 shows a compressible integrating system 22. The compressible integrating system 22 of Figure 2 includes the integrative chemistry 14 on the substrate 44 located in the container 26. The gas permeable surface, 24, allows the germicide to enter the container 26. and make contact with the integrative chemistry 14. The dye precursor 28 is contained in the tank 30.

Supports 32 are located adjacent the reservoir 30. The reservoir 30 protects the dye precursor from being destroyed by reaction with the oxidative germicide during the germicidal cycle. After the cycle, the compressible integrating system 22 is crushed or compressed. The supports 32 pierce the reservoir 30 and the second reagent, the dye precursor 28, which was contained in the tank 30, makes contact with the integrating chemistry 14. The second reagent, the dye precursor, reacts with any first reagent, the indicator reagent, that remains after the cycle. If on the substrate 44 there is some of the first reagent, the indicator reagent, this indicator reagent in the chelator 14 reacts with the second reagent, the dye precursor, to form on the substrate 44 the third reagent having the third color, indicating that the germicidal process was incomplete. A lack of color in the integrative chemistry 14 indicates that the germicidal treatment was successful. Figure 3 shows a schematic diagram of a slidable integrator system 34. The desirable integrator system 34 includes a lockable slidable container, 36. The closable slidable container 36 is formed of the outer cover 38 and the inner cover 40. The outer cover 38 slides on the inner cover 40. Sliding of the outer cover 38 on the inner cover 40 opens the window 42 in the lockable slidable container 36. The window 42 allows the germicide to enter the interior of the lockable slidable container 36. The germicide can be a liquid, a vapor, or a gas. The lockable slidable container 36 contains the substrate 44 which supports the integrating chemistry 14. The chemistry recorder 14 includes the first reagent, the indicator reagent. The substrate 44 is located adjacent to the transparent window 46 in the inner cover 40. Any color change in the substrate 44 can be observed through the transparent window 46. The substrate 44 is the substrate for the indicator chemistry 14. The chemistry indicator 14 includes the first reagent, the indicator reagent. In one embodiment, the substrate 44 is a glass filter, a non-absorbent substrate. The second reagent, the dye precursor 24, is contained in the collapsible vial 48 within the closable slidable container 36. The collapsible vial 48 is made of a frangible material, such as glass. The collapsible ampule 48 protects the second reagent, the dye precursor 24, from being destroyed by the germicide during the germicidal process. The wedge 50 is attached to an interior of the outer cover 38 of the lockable slidable container 36. The wedge 50 is a projection on the inside of the outer cover 38. In one embodiment, the wedge 50 has a pointed edge to assist in penetrating the Flattened blister 48. The barrier 52 is located within the lockable slidable container 34, between the collapsible blister 48 and the substrate 44. The barrier 52 prevents fragments of the collapsible blister 48 from affecting the reading of the substrate 44. The barrier 52 is permeable to the second reagent, the dye precursor 24. When the collapsible blister 48 is crushed the second reagent, the dye precursor, is released and can flow through the barrier 52 to make contact with the first reagent, the indicator reagent, on the substrate 44. In one embodiment, the barrier 52 is a wire mesh. Figure 4 shows a schematic diagram of the slideable ntegrator system 34 and the lockable slidable container 36 of figure 3, after completion of the cycle. The outer cover 38 of the lockable slidable container 36 in Figure 4 has been moved to the left of Figure 4 by sliding the outer cover 38 onto the inner cover 40. The sliding of the outer cover 38 on the inner cover 40 has various effects, as shown in Figure 4. First, the sliding of the outer cover 38 on the inner cover 40 closes the window 42. The closure of the window 42 isolates the lockable slidable container 36, retaining the second reagent, the precursor of dye, inside the lockable slidable container 36. The second reactive, the dye precursor, can stain the hands of the operator. Secondly, the sliding of the outer cover 38 on the inner cover 40 forces the wedge 50 to make contact with the collapsible vial 48, pushing the collapsible vial 48 in contact with the barrier 52, crushing the collapsible vial 48. Crushing the collapsible vial 48 releases the second reagent, the dye precursor 24. The second reagent, the dye precursor 24 that is released, flows through the barrier 52 and makes contact with the substrate 44. If some of the first reagent remains on the substrate 44. , the indicator reagent, when the second reactant, the dye precursor 24, makes contact with the substrate 44, the second reagent, the dye precursor 24, reacts with the first reagent, the indicator reagent, in the integrating chemistry 14, to form the third reagent that has a third color. The third color is distinctive and is easily distinguished from the first color of the first reagent and the second color of the second reagent. The barrier 52 prevents fragments of the collapsible blister 48 from contacting the substrate 44 and affecting the determination. The color change in the substrate 44 can be observed through the transparent window 46.

Method of using the sterilizer The integrator according to one embodiment of the present invention is placed in the sterilization chamber with the load to be sterilized, and the germicidal cycle is run. After finishing the sterilization cycle, the second reagent, the dye precursor, is contacted with the integrator. The second reagent, the dye precursor, reacts with any first reagent, indicator reagent, remaining in the integrator to produce the third reagent having the third color. The color produced depends on the structure of the first reagent, the indicator reagent, and on the second reagent, the dye precursor. The intensity of the color depends on the amount of the first reagent, the indicator reagent remaining in the integrator, and the concentration of the second reagent, the dye precursor. If color develops in the integrator within a predetermined period, for example approximately 5-6 minutes after the second reagent, the dye precursor, is added to the integrator, the sterilization cycle is considered to have been ineffective. The intensity of the color in the recorder can be judged visually by comparing it with a color standard, or the intensity of the color can be measured spectrophotometrically in the visible or ultraviolet region. Judging color intensity visually is more subjective than measuring the intensity of color with an instrument. The second reagent, the dye precursor, can be contacted with the integrator in several ways. In one embodiment, the second reagent, the dye precursor, is manually contacted with the integrator using a pipette, dropper or other suitable device. The manual addition of the second reagent, the dye precursor, is appropriate with an integrator as shown in Figure 1, where there is no method to protect the second reagent, the dye precursor, from being exposed and destroyed by the germicide during the sterilization cycle. Figures 2 and 3 show embodiments of integrators wherein the second reagent, the dye precursor, is present in the integrator during the sterilization cycle, but is protected from exposure to the germicide by being enclosed in a collapsible reservoir or ampule. In other embodiments, other means may be used to protect the second reagent, the dye precursor, from being exposed to the germicide. The following examples are intended to be illustrative only and not limiting of the scope of the invention.

EXAMPLES EXAMPLE 1 Test results with an integrator containing arginine as an indicator reagent, with varying volumes of hydrogen peroxide A series of integrators was prepared by contacting paper discs with an aqueous solution of arginine. The integrators were placed in a STERRAD® 50 sterilizer with a load of equipment to be sterilized and several biological indicators. Paper discs are absorbent substrates. The sterilizer was evacuated to 0.8 torr. Plasma was produced in the chamber for 15 minutes to condition the load. The sterilizer was further evacuated to 0.4 torr and hydrogen peroxide was injected, which made contact with the charge, integrators and biological indicators for 6 minutes. The sterilizer was ventilated with air for 2 minutes. The sterilizer was evacuated again at 0.5 torr and plasma was produced for an additional 2 minutes. The plasma power was 400 watts for both plasma exposures. The paper label discs were contacted with 100 μL of a 5% solution of the ortho-phthalaldehyde dye precursor (OPA) after the germicidal cycle, and the response of the integrators was measured visually after having contacted them. with the OPA. The results are shown in table 1 below.

TABLE 1 Results of the integrator test and biological indicator Cycles with 100 and 250 μL of hydrogen peroxide were ineffective, as shown by the positive results of the biological indicator. The results of the integrator were consistent with the results of the biological indicator because the integrators for the cycles with 100 and 250 μL of hydrogen peroxide had significant color in the course of 3-6 minutes after making contact with the OPA. The color is the result of a reaction between the unreacted arginine, the primary amine indicator compound and OPA, the aldehyde dye precursor. In the sterilization cycle with 400 μL of hydrogen peroxide, 10-15% of the integrators developed a faint color after 5-6 minutes. None of the biological indicators in this test was positive. The faint color that developed in the integrators after 5-6 minutes is a sign that the integrators provide a more rigorous measure of the degree of sterilization than the biological indicators. None of the biological indicators in the test with 500 μL of hydrogen peroxide was positive. None of the integrators had color.

The results of the biological indicator and the results of the integrator are consistent in showing that the sterilization cycle with 500 μL of hydrogen peroxide was effective. The data of the integrators according to one embodiment of the present invention were consistent with the data of the biological indicators. However, the results of the integrators were available in 5-6 minutes, compared to 24-48 hours for the results of the biological indicator.

EXAMPLE 2 Response of the integrator measured with a spectrometer Non-adsorbent fiberglass discs were impregnated with an aqueous solution of arginine. Fiberglass discs are non-absorbent substrates. The discs were placed in a STERRAD® 50 sterilizer and processed under the same conditions as in Example 1, with varying injection volumes of hydrogen peroxide. The amounts of hydrogen peroxide are shown in table 2 below.

The fiberglass discs were contacted with 50 μL of a 5% aqueous solution of the OPA dye precursor after finishing the germicidal cycle. The intensity of light absorption of the discs was determined with a color sensor TAOS evaluation module TCS230EVM (Parallax, Rockiin, California) at approximately 470 nm, approximately 550 nm and approximately 610 nm (wavelength red, green and blue ). Injection volumes of hydrogen peroxide of 50, 300 and 1000 μL were used. All biological indicators would be negative with a hydrogen peroxide injection of 300 μL in the STERRAD® 50 sterilizer. Table 2 summarizes the color intensities at a wavelength of 610 nm (blue). The answers of the integrator shown in table 2 are the differences between the initial reading and the final reading 30 seconds after the addition of OPA. Large numbers of the integrator response indicate more color and less effective sterilization.

TABLE 2 The small numbers of the integrator response indicate effective sterilization. The 5-17 scale of the integrator response in the samples that were exposed to 1000 μL of hydrogen peroxide is consistent with effective sterilization. The 28-47 scale of the integrator response in samples that were exposed to 300 μL of hydrogen peroxide is consistent with effective sterilization. The 58-85 scale of the integrator response in the samples that were exposed to 50 μL of hydrogen peroxide indicates significant color, showing ineffective sterilization. The results of Example 2 demonstrate that the results of the integrators according to the embodiments of the present invention can be effectively measured with a spectrophotometer, rather than visually as in Example 1.

EXAMPLE 3 Integrator tests with histidine as indicator reagent and OPA as a dye precursor Histidine was used in place of arginine as the primary amine indicator reagent to form a series of integrators in this example 3.

An aqueous solution of histidine was placed in a series of glass fiber discs to form the integrators according to an alternative embodiment of the present invention. The integrators were placed on a standard STERRAD® 50 validation load and the runs were run as described in Examples 1 and 2.

The cycles were run with 0 μL of hydrogen peroxide and 300 μL of hydrogen peroxide. The integrators were contacted with 50 μL of a 5% volume solution of OPA at the end of the sterilization process. The intensity of the third color of the third compound in the integrators was measured with the TAOS color sensor described in Example 2. A combination of red, green and blue wavelengths (RGB, 470, 550 and 610 nm) was used to measure the respo because the color of the product of histidine and OPA is different from the color of the product of arginine and OPA. An average square root (RMS) of the absorption measured by the sensor was calculated for the three wavelengths. The results of light absorption were consistent, the treatment with 0 μL of hydrogen peroxide being ineffective in sterilization, and treatment with 300 μL of hydrogen peroxide being effective in the sterilization. The example demonstrates that histidine can be used as a primary amine indicator compound instead of arginine. A wide variety of primary amines can be used as primary amine indicator compounds.

EXAMPLE 4 Integrator tests with histidine and glutaraldehyde A series of integrators with histidine was prepared as an indicator reagent on glass fiber discs as a substrate. The integrators were processed in a STERRAD® 50 sterilizer under the same conditions as in Example 3. The glutaraldehyde contacted the processed integrators in place of the OPA as the aldehyde dye precursor. The results are shown in table 4 below.

TABLE 4 Results of integrator tests with histidine and glutaraldehyde The small change in light absorption from the initial time to the termination time for the integrators that were exposed to 300 μL of hydrogen peroxide, demonstrates that sterilization with 300 μL of hydrogen peroxide was effective. The large change in light absorption from the initial time to the reading at 30 seconds for the installers who were not exposed to hydrogen peroxide (volume of 0 μL) shows that the sterilization was not effective. The results of Example 4 are a demonstration that glutaraldehyde can be used as a precursor of aldehyde dye in place of OPA.

EXAMPLE 5 Integrators with OPA as indicator reagent A series of integrators with OPA was prepared as indicator reagent. The integrators were placed in a sterilizer with a load to be sterilized and a series of biological indicators. The charge, the biological indicators and the integrators were contacted with 100-500 μL of hydrogen peroxide under the conditions described in example 1. The integrators with OPA as indicator reagent were contacted with an aqueous solution of arginine as dye precursor. The results of the integrators, with OPA as indicator reagent of aldehyde and arginine as precursor of primary amine dye, correlate well with the results of the biological indicators. The results of Example 5 demonstrate that aldehydes such as OPA can be used as an indicator reagent, with primary amines such as arginine as a dye precursor. The integrator according to the embodiments of the present invention allows to quickly determine the effectiveness of the sterilization process. Various modifications and alterations of this invention will be apparent to those skilled in the art without departing from the scope and spirit of this invention. It is understood that the invention is not limited to the embodiments described herein and that the claims should be interpreted as widely as the prior art admits.

Claims (20)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for quickly determining the effectiveness of an oxidative germicidal process, comprising: providing a substrate having a known amount of a first reactant on the substrate, wherein said first reagent is selected from the group consisting of a primary amine, mixtures of primary amines, an aldehyde, and mixtures of aldehydes; wherein said first reagent has a first color; exposing the substrate having the known amount of the first reagent to an oxidative germicide, thereby reducing the known amount of the first reagent to a final amount of the first reagent; contacting the substrate having the final quantity of the first reagent having the first color, with a second reagent having a second color, thus generating a third reagent having a third color, said third color having an intensity, wherein the intensity of said third color is related to the final amount of said first reagent on said substrate, wherein said second reagent is a reagent selected from the group consisting of a primary amine and mixtures of primary amines when said first reagent is a reagent selected from group consisting of an aldehyde and a mixture of aldehydes, and said second reagent is a reagent selected from the group consisting of an aldehyde and a mixture of aldehydes when said first reagent is a reagent selected from the group consisting of a primary amine and a mixture of primary amines; determine the intensity of said third color; and determining the efficacy of the germicidal process from the intensity of said third color.

2. The method according to claim 1, further characterized in that the step of determining the effectiveness of the germicidal process comprises correlating the intensity of said third color with results of biological indicators.

3. The method according to claim 1, further characterized in that said oxidative germicide is a sterilant.

4. The method according to claim 1, further characterized in that said oxidative germicide is a disinfectant.

5. The method according to claim 1, further characterized in that said substrate is an absorbent substrate.

6. The method according to claim 1, further characterized in that said substrate is a non-absorbent substrate.

7. The method according to claim 1, further characterized in that said oxidative germicide is a liquid, a vapor, or a gas.

8. The method according to claim 1, further characterized in that the intensity of said third color is determined visually.

9. The method according to claim 1, further characterized in that the intensity of said third color is determined spectrophotometrically in the visible or ultraviolet region.

10. The method according to claim 1, further characterized in that at least one of said first reagent and said second reagent is colorless.

11. The method according to claim 1, further characterized in that said oxidative germicide is selected from the group consisting of hydrogen peroxide, peracetic acid, ethylene oxide, ozone and chlorine dioxide.

12. The method according to claim 1, further characterized in that it comprises exposing said substrate and said oxidative germicide to plasma.

13. The method according to claim 1, further characterized in that a percentage of completion of the germicidal process is determined by comparing the intensity of the third color with the color intensity of a standard.

14. The method according to claim 1, further characterized in that the primary amine is selected from the group consisting of glycine and histidine, and the aldehyde is selected from the group consisting of ortho-phthalaldehyde and glutaldehyde.

15. An integrator for determining the effectiveness of a germicidal process with an oxidative germicide, said integrator comprising: a substrate with a known amount of a first reagent on the substrate, wherein said first reagent is selected from the group consisting of an amine primary, mixtures of primary amines, an aldehyde, and mixtures of aldehydes; wherein said substrate is in an enclosure; wherein said first reagent is capable of reacting with said oxidative germicide when exposed to said oxidative germicide; a deposit of a second reagent, wherein said second reagent is a reagent selected from the group consisting of a primary amine and mixtures of primary amines when said first reagent is a reagent selected from the group consisting of an aldehyde and a mixture of aldehydes, and said second reagent is a reagent selected from the group consisting of an aldehyde and a mixture of aldehydes when said first reagent is a reagent selected from the group consisting of a primary amine and a mixture of primary amines; in f wherein said second reagent is capable of reacting with said first reagent to form a third reagent having a color; wherein said reservoir has a breakable barrier that isolates the second reagent from the first reagent and from the oxidative germicide during contact of the first reagent with the oxidative germicide, wherein the breaking of said breakable barrier from the reservoir brings the second reagent into contact with the first reagent. reactive, thus forming said third reagent having the color; and where the deposit is in said enclosure.

16. The integrator according to claim 15, further characterized in that said breakable barrier of said deposit comprises a frangible ampule in said enclosure.

17. - The integrator according to claim 16, further characterized in that it comprises a second barrier, wherein said second barrier is within said enclosure, between said frangible ampule and said first reagent, wherein said second barrier of said enclosure is permeable to the second reagent, and wherein said second barrier prevents fragments of the frangible vial from contacting the first reagent.

18. The integrator according to claim 15, further characterized by comprising a window in said enclosure, wherein said window is permeable to said oxidative germicide, said window allowing said oxidative germicide to enter said enclosure.

19. The integrator according to claim 15, further characterized in that the primary amine is selected from the group consisting of glycine and histidine, and the aldehyde is selected from the group consisting of ortho-phthalaldehyde and glutaldehyde.

20. The integrator according to claim 15, further characterized in that said enclosure also comprises a transparent window, wherein a change of color in said substrate can be observed through said transparent window, visually or with a spectrophotometer.