Evaluation of Liquid Disinfectants

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Chapter: Pharmaceutical Microbiology : Laboratory Evaluation Of Antimicrobial Agents

Phenol coefficient tests were developed in the early 20th century when typhoid fever was a significant public health problem and phenolics were used to disinfect contaminated utensils and other inanimate objects.



a)    General


Phenol coefficient tests were developed in the early 20th century when typhoid fever was a significant public health problem and phenolics were used to disinfect contaminated utensils and other inanimate objects. Details of such tests can be found in earlier editions of this book. However, as non-phenolic disinfectants became more widely available, tests that more closely paralleled the conditions under which disinfectants were being used (e.g. blood spills) and which included a more diverse range of microbial types (e.g. viruses, bacteria, fungi, protozoa) were developed. Evaluation of a disinfectant’s efficacy was based on its ability to kill microbes, i.e. its cidal activity, under environmental conditions mimicking as closely as possible real life situations. As an essential component of each test was a final viability assay, removal or neutralization of any residual disinfectant (to prevent ‘carryover’ toxicity) became a significant consideration.


The development of methods to evaluate disinfectant activity in diverse environmental conditions and to determine suitable in-use concentrations/dilutions to be used led to the development by Kelsey, Sykes and Maurer of the so-called capacity-use dilution test which measured the ability of a disinfectant at appropriate concentrations to kill successive additions of a bacterial culture. Results were reported simply as pass or fail and not a numerical coefficient. Tests employed disinfectants diluted in hard water (clean conditions) and in hard water containing organic material (yeast suspension to simulate dirty conditions), with the final recovery broth containing 3% Tween 80 as a neutralizer. Such tests are applicable for use with a wide variety of disinfectants (see Kelsey & Maurer, 1974). Capacity tests mimic the practical situations of housekeeping and instrument disinfection, where surfaces are contaminated, exposed to disinfectant, re contaminated and so forth. The British Standard (BS 6907:1987) method for estimation of disinfectants used in dirty conditions in hospitals by a modification of the original Kelsey-Sykes test is the most widely employed capacity test in the UK and Europe. In the USA, effectiveness test data for submission must be obtained by methods accepted by the Association of Official Analytical Chemists, known collectively as Disinfectant Effectiveness Tests (DETs).


However, the best information concerning the fate of microbes exposed to a disinfectant is obtainable by counting the number of viable cells remaining after exposure of a standard suspension of cells to the disinfectant at known concentration for a given time interval— suspension tests. Viable counting is a facile technique used in many branches of pure and applied microbiology. Assessment of the number of viable microbes remaining (survivors) after exposure allows the killing or cidal activity of the disinfectant to be expressed in a variety of ways, e.g. percentage kill (e.g. 99.999%), as a log10 reduction in numbers (e.g. 5-log killing), or by log10 survival expressed as a percentage. Examples of such outcomes are shown in Table 18.1.


Unfortunately, standardization of the methodology to be employed in these efficacy tests has proven 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. Levels of killing required over a given time interval tend to vary depending on the regulatory authority concerned. While a 5-log killing of bacteria (starting with 106 CFU/ml) has been suggested for suspension tests, some authorities require a 6-log killing in simulated use tests. With viruses, a 4-log killing tends to be an acceptable result, while with prions it has been recommended that a titre loss of 104 prions should be regarded as an indication of appropriate disinfection provided that there has been adequate prior cleaning. With simulated use tests, cleaning followed by appropriate disinfection should result in a prion titre loss of at least 107.


b)   Antibacterial Disinfectant Efficacy Tests


Various regulatory authorities in Europe (e.g. European Standard or Norm, EN; British Standards, BS; Germany, DGHM; France, AFNOR) and North America (e.g. Food and Drug Administration, FDA; Environmental Protection Authority, EPA; Association of Official Analytical Chemists, AOAC) have been associated with attempts to produce some form of harmonization of dis-infectant tests. Perhaps the most readily accessible and recent guide to the methodology of possible bactericidal, tuberculocidal, fungicidal and viricidal disinfectant efficacy tests, is that of Kampf and colleagues (2002). This publication summarizes and provides references to various EN procedures (e.g. prEN 12054).


i)  Suspension tests


While varying to some degree in their methodology, most of the proposed procedures tend to employ a standard suspension of the microorganism in hard water containing albumin (dirty conditions) and appropriate dilutions of the disinfectant—so-called suspension tests. Tests are carried out at a set temperature (usually around room temperature or 20°C), and at a selected time interval samples are removed and viable counts are performed following neutralization of any disinfectant remaining in the sample. Neutralization or inactivation of residual dis-infectant can be carried out by dilution, or by addition of specific agents (see Table 18.3). Using viable counts, it is possible to calculate the concentration of disinfectant required to kill 99.999% (5-log kill) of the original sus-pension. Thus 10 survivors from an original population of 106 cells represents a 99.999% or 5-log kill. As bacteria may initially decline in numbers in diluents devoid of additional disinfectant, results from tests incorporating disinfectant-treated cells can be compared with results from simultaneous tests involving a non-disinfectant-containing system (untreated cells). The bactericidal effect BE can then be expressed as:


·                    Other than dilution.

D/E neutralizing media—adequate for QACs, phenols, iodine and chlorine compounds, mecurials, formaldehyde and glutaraldehyde (see Rutala, 1999).

·                    O ther appropriate enzymes can be considered, e.g. inactivating or modifying enzymes for chloramphenicol and aminoglycosides, respectively.

·                    F ilter microorganisms on to membrane, wash, transfer membrane to growth medium.

·                    R esins for the absorption of antibiotics from fluids are available.

·                    Tween 80 (polysorbate 80).




where NC and ND represent the final number of CFU/ml remaining in the control and disinfectant series, respectively.


Unfortunately, viable count procedures are based on the assumption that one colony develops from one viable cell or one CFU. Such techniques are, therefore, not ideal for disinfectants (e.g. QACs such as cetrimide) that promote clumping in bacterial suspensions, although the latter problem may be overcome by adding nonionic surface active agents to the diluting fluid.


ii)  In-use and simulated use tests


Apart from suspension tests, in-use testing of used medical devices, and simulated use tests involving instruments or surfaces deliberately contaminated with an organic load and the appropriate test microorganism have been incorporated into disinfectant testing protocols. An example is the in-use test first reported by Maurer in 1972. It is used to determine whether the disinfectant in jars, buckets or other containers in which potentially contaminated material (e.g. lavatory brushes, mops) has been placed contain living microorganisms, and in what numbers. A small volume of fluid is withdrawn from the in-use container, neutralized in a large volume of a suitable diluent, and viable counts are performed on the resulting suspension. Two plates are involved in viable count investigations, one of which is incubated for 3 days at 32°C (rather than 37°C, as bacteria damaged by disinfectants recover more rapidly at lowered temperatures), and the other for 7 days at room temperature. Growth of one or two colonies per plate can be ignored (a disinfectant is not usually a sterilant), but 10 or more colonies would suggest poor and unsatisfactory cidal action.


Simulated use tests involve deliberate contamination of instruments, inanimate surfaces, or even skin surfaces, with a microbial suspension. This may either be under clean conditions or may utilize a diluent containing organic material (e.g. albumin) to simulate dirty conditions. After being left to dry, the contaminated surface is exposed to the test disinfectant for an appropriate time interval. The microbes are then removed (e.g. by rubbing with a sterile swab), resuspended in suitable neutralizing medium, and assessed for viability as for suspension tests. New products are often compared with a known comparator compound (e.g. 1 minute application of 60% v/v 2-propanol for hand disinfection products—see EN1500) to show increased efficacy of the novel product.


iii)  Problematic bacteria


Mycobacteria are hydrophobic in nature and, as a result, exhibit an increased tendency to clump or aggregate in aqueous media. It may be difficult, therefore, to prepare homogeneous suspensions devoid of undue cell clumping (which may contribute to their resistance to chemical disinfection). As Mycobacterium tuberculosis is very slow growing, more rapidly growing species such as M. terrae, M. bovis or M. smegatis can be substituted in tests (as representative of M. tuberculosis). Recent global public health concerns regarding the increasing incidences of tuberculosis (including co-infections with HIV) in devel-oping, middletier and industrialized nations brings into sharp focus the necessity for representative evaluations of agents with potential tuberculocidal activity. This is particularly true given the high proportion of cases classified as multidrug resistant tuberculosis (MDR-TB). Apart from vegetative bacterial cells, bacterial or fungal spores can also be used as the inoculum in tests. In such cases, incubation of plates for the final viability determination should be continued for several days to allow for germination and growth.


Compared with suspended (planktonic) cells, bacteria on surfaces as biofilms are invariably phenotypically more tolerant to antimicrobial agents. With biofilms, suspension tests can be modified to involve biofilms produced on small pieces of an appropriate glass, metal or polymeric substrate, or on the bottom of microtitre tray wells. After being immersed in, or exposed to, the disinfectant solution for the appropriate time interval, the cells from the biofilm are removed, e.g. by sonication, and resuspended in a suitable neutralizing medium. Viable counts are then performed on the resulting planktonic cells. Reduction in biomass following antimicrobial challenge can be monitored using a standard crystal violet staining technique, however, viable counting permits evaluation of rate of kill. The Calgary Biofilm Device, permits the high-throughput screening of antimicrobial agents against biofilms grown on 96 polycarbonate pegs in a 96-well microtitre plate. Some important environmental bacteria survive in nature as intracellular parasites of other microbes, e.g. Legionella pneumophila within the protozoan Acanthamoeba polyphaga. Biocide activity is significantly reduced against intracelluar legionellae (see Figure 18.3). Disinfectant tests involving such bacteria should therefore be conducted both on planktonic bacteria and on suspensions involving amoebae-containing bacteria. With the latter, the final bacterial viable counts are performed after suitable lysis of the protozoan host. The legionella/protozoa situation may also be further complicated by the fact that the microbes often occur as biofilms.


c)  Other Microbe Disinfectant Test


Suspension-type efficacy tests can also be performed on other microbes, e.g. fungi, viruses, using similar techniques to that described above for bacteria, although significant differences obviously occur in parts of the tests.


i)     Antifungal (fungicidal) test


In order for disinfectants to claim fungicidal activity, or for the discovery of novel fungicidal activities, a range of standard tests have been devised. Perhaps the main problem with fungi concerns the question of which morphological form of fungi to use as the inoculum. Unicellular yeasts can be treated as for bacteria, but whether to use spores (which may be more resistant than the vegetative mycelium) or pieces of hyphae with the filamentous moulds, has yet to be fully resolved. Spore suspensions (in saline containing the wetting agent Tween 80) obtained from 7-day-old cultures are presently recommended. The species to be used may be a known environmental strain and likely contaminant, such as Aspergillus niger, or a pathogen, such as Trichophyton mentagrophyes, other strains such as Penicillium variabile are also employed. Clearly the final selection of organism will vary depending on the perceived use for the disinfectant under test. In general, spore suspensions of at least 106 CFU/ml have been recommended. Viable counts are typically performed on a suitable media (e.g. malt extract agar, sabouraud dextrose agar) with incubation at 20°C for 48 hours or longer. EN 1275:1997 regulations for fungicidal activity require a minimum reduction in viability by a factor of 104 within 60 minutes; test fungi were Candida albicans and A. niger. Further procedures may be obtained by reference to EN 1650:1998 (quantitative suspension test for evaluation of fungicidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas) and AOAC Fungicidal activity of disinfectants (955.17).


     ii)  Antiviral (viricidal) test


The evaluation of disinfectants for viricidal activity is a complicated process requiring specialized training and facilities; viruses are obligate intracellular parasites and are therefore incapable of independent growth and replication in artificial culture media. They require some other system employing living host cells. Suggested test viruses include rotavirus, adenovirus, poliovirus, herpes simplex viruses, HIV, pox viruses and papova virus, although extension of this list to include additional blood-borne viruses

such as hepatitis B and C, and significant animal pathogens (e.g. foot and mouth disease virus) could be argued, given the potential impact on public health or the economy of a nation.


Briefly, the virus is grown in an appropriate cell line that is then mixed with water containing an organic load and the disinfectant under test. After the appropriate time, residual viral infectivity is determined using a tissue culture/plaque assay or other system (e.g. animal host, molecular assay for some specific viral component). Such procedures are costly and time-consuming, and must be appropriately controlled to exclude factors such as disinfectant killing of the cell system or test animal. A reduction of infectivity by a factor of 104 has been regarded as evidence of acceptable viricidal activity (prEN 14476). For viruses that cannot be grown in the laboratory (e.g. hepatitis B), naturally infected cells/tissues must be used. Further test procedures are detailed in British Standard BS EN 13610 (quantitative suspension test for the evaluation of viricidal activity against bacteriophages of chemical disinfectants used in food and industrial areas). The use of bacteriophage as model viruses in this procedure most likely reflects their ease of growth and survivor enumeration via standard plaque assay on host bacterial lawns grown on solid media.


i       iii)  Prion disinfection test


Prions are a unique class of acellular, proteinaceous infectious agent, devoid of an agent-specific nucleic acid (DNA or RNA). Infection is associated with the abnormal isoform of a host cellular protein called prion protein (PrPc). Prions exhibit unusually high resistance to conventional chemical and physical decontamination methods, presenting a unique challenge in infection control. Although numerous published studies on prion inactivation by disinfectants are available in the literature, inconsistencies in methodology make direct comparison difficult. For example, strain differences of prion (with respect to sensitivity to thermal and chemical inactivation), prion concentration in tissue homogenate, exposure conditions and determination of log reductions from incubation period assays instead of end-point titrations. Furthermore, since most studies of prion inactivation have been conducted with tissue homogenates, the protective effect of the tissue components may offer some protective role and contribute to resistance to disinfection approaches. Despite this, a consistent picture of effective and ineffective agents has emerged and is summarized in Table 18.4. Although most disinfectants are inadequate for the elimination of prion infectivity, agents such as sodium hydroxide, a phenolic formulation, guanidine thiocyanate and chlorine have all been shown to be effective.


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