Antimicrobial efficacy test

Medical devices such as catheters, implants, and prosthetics are continuously challenged in bacterial environments. Eor example, C VC s total number of days of insertion for all patients is estimated to be 15 million in intensive care unit in the United States every year [161], One potential risk of CVC utilization is catheter-related bloodstream 

The ZOI test, also widely known as the Kirby-Bauer disk diffusion test, is a fast in vitro but semiquantitative test [169], The original purpose of this test was to replace the MIC test for small molecule antibiotic efficacy [169], Soon, this method was adopted and modified to evaluate antimicrobial efficacy of silver and polymeric devices with eluting antimicrobial agents [170], Conunonly used eluting antimicrobial agents are zinc salt/particles [171-173], silver salt/particles [173-177], and chlorhexi-dine [178,179], These antimicrobial agents can be compounded/blended into polyurethanes or coated/adsorbed on polyurethanes. 

The ZOI test is designed for polymers with eluting antimicrobial kill mechanisms. However, when antimicrobial groups are covalently bonded to polymer chains, or antimicrobial agents are permanently trapped in polyurethane, the ZOI test is no longer suitable and will yield false negative results (no bacterial kill shown). 

HMDI-BD (30%), where HMDI = 4,4 -(methylene bis(p- clohexyl isocyanate)) and BD = 1,4-butane diol, HMDI-BD block is 30 wt% of PU composition 

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Many antimicrobial efficacy evaluations of topical antimicrobial products involve measurements of microbial population reductions at a specific time point after exposure to the product. To determine this accurately, the antimicrobial action of the product must be stopped at the time specified for sample, and it is for this action that neutralizer systems are employed. The validity of the neutralizer system must be established prior to performing the antimicrobial efficacy test. This concern for neutralizer validity has long been known, and a number of methods have been proposed for validating neutralizer systems [1-5]. Each of the methods focuses on two major concerns (1) the neutralizer system must demonstrably neutralize the antimicrobial properties of the product, and (2) the neutralizer system must be proven nontoxic to the test microorganism(s). Few validation methods apply techniques of statistical analysis to the determination of their validity... 

Topical antimicrobial efficacy tests comprise test methods similar to those outlined in the Food and Drug Administration s Tentative Final Monograph for Healthcare Antiseptic Drug Products time-kill kinetic studies effectiveness testing of a surgical hand scrub effectiveness testing of an antiseptic handwash or... 

All specific conditions of the antimicrobial efficacy test must be duplicated in the validation. All equipment, types of media, incubators, and temperatures should be identical to those applied in the antimicrobial efficacy test. Even the technician who will be performing the antimicrobial efficacy test should also perform the validation of the neutralization system. 

SVW Sutton. Neutralizer evaluations as control experiments for antimicrobial efficacy tests. In JM Ascenzi, ed. Handbook of Disinfectants and Antiseptics. New York Marcel Dekker, 1996, pp. 43-62. 

Where a large pack size is provided, it might be necessary to undertake more extensive in-use testing and/ or to restrict the in-use shelf life once the container has been opened. In addition to preservative efficacy testing the chemical stability of antimicrobial preservatives should also be investigated over the unopened and in-use shelf lives of the product. 

Aqueous products that are at greatest risk from microbial spoilage include solutions, suspensions, and emulsions for repeated oral, parenteral, or external use and include critical products such as multidose injections and eye drops. Unpreserved products without adequate antimicrobial efficacy should not be presented in containers intended for use on more than one occasion unless justified. When antimicrobial preservatives are used, their efficacy has to be demonstrated using the Ph Eur test for antimicrobial preservative efficacy. Factors to be taken into account in designing a preserved product include the nature of the preservative, its concentration in the product, the... 

A stability-indicating presrvative assay can be justified only at all time intervals as a substitute for the USP Antimicrobial Effectiveness Test by confirming preservative efficacy at 50, 75, and 100% of the lower shelf-life specification. 

In 1998, a CEFIC working group composed of industry experts from EPAS (European Producers of Antimicrobial Substances) and EPFP (European Producers of Formulated Preservatives) compiled a listing of over 200 relevant efficacy testing methods and around 100 of these were critically analysed in... 

Any differences between the release and shelf-life acceptance criteria for antimicrobial preservatives should be supported by the results of preservative efficacy testing. 

Microbiological protection of multiple-dose presentations such as liquid inhalations, nasal sprays, oral liquids, creams, and lotions is more complex. Once opened they are susceptible to microbiological contamination. If they are aqueous-based, they are in principle susceptible to proliferation of these new contaminants. To avoid this, they are formulated with antimicrobial agents or preservatives and are expected to be able to comply with preservative efficacy standards specified in the pharmacopeias. Preservative efficacy tests (not harmonized) are described in Section 51 of the USP and Section VIII. 14 of the PhEur (Fig. 3). 

The test for antimicrobial effectiveness is used to demonstrate the effectiveness of any added antimicrobial preservative(s). Compendial references include USP 24 Chapter 51, Antimicrobial Effectiveness Test JP XIII, General Information 3, Preservatives-Effectiveness Tests and Ph. Eur. 3rd Ed., Biological Tests, 5.1.3., Efficacy of Antimicrobial Preservation. 

Unfortunately, standardization of the methodology to be employed in these efficacy tests has proved difficult, if not impossible, to obtain, as has consensus on what level of killing represents a satisfactory and/or acceptable result. It must be stressed however, that unlike tests involving chemotherapeutic agents where the major aim is to establish antimicrobial concentrations that inhibit growth (i.e. MICs), disinfectant tests require determinations of appropriate cidal levels. 

The multidose formulation will be tested to determine its efficacy according to the Antimicrobial Effectiveness Test required by the USP.39 If the results support the USP requirements, international requirements must then be met. International markets require different preservatives, different concentrations, and different excipients of the formulation. In addition, the period required for the inhibition and/or killing of the challenge microorganisms may be different. International regulatory requirements for compliance should be well researched and understood by the scientific and the management staff. 

The range of testing should cover not only chemical and biological stability but also loss of preservative, physical properties and characteristics, organoleptic properties and, where required, microbiological attributes. Preservative efficacy testing and assays on stored samples should be carried out to determine the content and efficacy of antimicrobial preservatives. 

Limits of acceptance should relate to the release limits (where applicable), to be derived from consideration of all the available stability information. The shelf life specification could allow acceptable and justifiable derivations from the release specification based on the stability evaluation and the changes observed on storage. It will need to include specific upper limits for degradation products, the justification for which should be influenced by the levels observed in material used in pre-clinical studies and clinical trials. The justification for the limits proposed for certain other tests such as particle size and/or dissolution rate will require reference to the results observed for batch(es) used in bioavailability and/or clinical studies. Any differences between the release and shelf life specifications for antimicrobial preservatives should be supported by preservative efficacy testing. 

Antimicrobial soap testing. An antimicrobial soap is a product that contains an active ingredient and has in vitro and in vivo activity against microorganisms residing on the skin. Therefore, the efficacy requirements must examine both the in vitro and in vivo efficacy characteristics of the product. The Antimicrobial I Panel did not provide specific recommendations for the assessment of these products but concluded that the Cade [20] and Quinn [24] handwashing procedures could be applicable to these products. A statement describing the efficacy requirement was not provided. 

In the smdy reported here, the objective was to evaluate the antimicrobial effectiveness of a 4% CHG used as a full-body shower wash by charting its effect on skin flora of two anatomical sites over the course of a 5-day application period. A test design was developed that would measure the immediate, persistent, and residual effects of the CHG product used in this study. The immediate antimicrobial effects are due to both the mechanical removal of washing and the immediate inactivation by the antimicrobial of microorganisms residing on the skin surface. The persistent antimicrobial effectiveness is a measure of the antimicrobial product s ability to prevent microbial recolonization on the skin surfaces, either by inhibition or by lethality. Finally, the residual antimicrobial efficacy is the measurement of the CHG product s cumulative antimicrobial properties after it has been used repeatedly [7],... 

The modified Cade handwash procedure is suboptimal, particularly in that the antimicrobial efficacy is evaluated primarily in terms of reductions in normal microbial flora, which is not appropriate for evaluating consumer antimicrobial hand soaps. Additionally, it measures the residual antimicrobial effectiveness of the test product, not the immediate effects. Finally, a control product is rarely used, so assuring the validity of tlie study is not possible. 

Although the residual efficacy test described above is an improved means of assessing the residual effect of antimicrobials, it is not optimized for use in consumer situations where high levels of bacteria are contacted from sources that are not perceived to be contaminated and hands are not washed frequently. For instance, in the kitchen, bacteria are commonly transferred to hands by use of sponges [6,7]. After using sponges, consumers often do not wash their hands. Therefore, the kitchen sponge is an ideal source of bacteria for a consumer-relevant test. 

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