A candidate method for Clostridium difficile, purported to meet fitness for purpose for testing efficacy of liquids, sprays, and towelette formulations designed to treat surfaces contaminated with spores of C. difficile is being developed, with AOAC guidance, under a current EPA-AOAC contract on disinfectants. An expert review panel (ERP), chaired by Jafrul Hasan of the EPA-Office of Pesticide Programs (OPP), evaluated the most promising techniques and potential best candidate methods for generating spores of toxigenic strains of C. difficile of high titer and quality and testing those spores against products designed to treat surfaces contaminated with spores of C. difficile. AOAC Official Method^SM 2008.05 (Three-Step Method; TSM) and ASTM E 2197-02 (QCT) were chosen as candidate methods for further validation. The ERP presented Phase I of the project (proposal for spore production) to the AOAC Methods Committee on Antimicrobial Efficacy Testing at the AOAC Annual Meeting in Orlando, Florida, USA, in September 2010.

The objective for the ERP was to review relevant literature applicable to the fitness-for-purpose statement, with the ultimate goal of creating the best combination of methodologies for supporting the EPA guidelines for product registration. The group also determined that reproducibility of spore production should be given a first priority, involving strain selection, spore production, recovery/enumeration, harvesting and purification, and assessment.

By consensus of the panel, AOAC Official Method^SM 2008.05 [“Use of alternative carrier materials in AOAC Official Method^SM 2008.05, efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface, quantitative three-step method,” J. AOAC Int. (2010) 93, 259-276] satisfactorily demonstrates responsiveness and repeatability in regards to obtaining appropriate carrier populations as well as log reduction values associated with chemical treatments. Although the method addresses relevant procedures for preparing spore suspensions with respect to harvesting, concentrating, washing, staining, enumeration, and resistance testing, there is no mention of the use of toxigenic C. difficile strains or suitable anaerobic spore-forming surrogates. It does, however, describe an applicable quantitative method for evaluating sporicidal agents. A modification of this method is one of the four current acceptable methods outlined by the EPA interim guidance document.

The second method chosen as a prospective candidate method is ASTM (E 2197-02), a quantitative test method designed to conduct efficacy evaluation of liquid chemical germicides to inactivate vegetative bacteria, viruses, fungi, mycobacteria, and bacterial spores in the presence of a soil load on brushed disks of stainless steel as carriers. It is recommended by EPA for the efficacy evaluation of products with sporicidal claims against C. difficile. Although the method does not mention or specify conditions for generating C. difficile spores of high titer and does not provide protocols for product neutralization and confirmation, it is robust, simple, and well regarded to demonstrate control carrier counts >6 log spores/carrier.

To optimize a method for C. difficile, the candidate method must be appropriate to (1) culture and maintenance of C. difficile, (2) generation of high titer spore suspensions, (3) inoculation of carriers and enumeration of spore load, (4) product neutralization and confirmation, and (5) addition of soil load to the inoculum. Assays resistant to acid (e.g., as per AOAC Method 2008.05) or sodium hypochlorite and tolerance to heat may be used to qualify spore preparations for use.

For further validation of prospective candidate methods, the panel will begin securing volunteer laboratories to help collect data, with data collection planned to begin by the end of the year/early 2011.

For more information, contact Olayinka Ladeji, AOAC consultant, at oladeji@aoac.org or Maria Nelson, AOAC technical consultant, at mnelson@aoac.org.

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