These are designed to give a "yes" or "no" answer to the question of whether a specific objectionable organism is detectable in a given sample. The word "detectable" is used rather than "present" because the situation here is similar to that in sterility testing. The possibility always exists that the organism of interest was present, but the conditions were not ideal, and it was not detected, so the mere passing of such a test does not guarantee that the organism was absent. The validation programme should confirm that the testing procedure will detect the organism of interest when it is present at a specified level in the product, but the sensitivities required by the EP and USP differ markedly in this respect.
The subject organisms of qualitative tests are described as "objectionable" and they are all potential pathogens, although they may also be significant as indicators of product quality. Currently, the EP and USP both describe tests for the absence of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Salmonellae in selected raw materials and finished product categories, and in addition, the EP has tests for Enterobacteriaceae and Clostridia. Other organisms have been suggested as candidates for inclusion in this category7,12 and the first of these two papers lists 20 such organisms relevant in eight different dosage forms (Table 5.2).
The exclusion tests are not applied indiscriminately to large numbers of materials that are the subject of pharmacopoeial monographs. Rather, they are invoked for raw materials in which the presence of the organism is a realistic (or historical) possibility, e.g., E. coli and Salmonellae in gelatin, and for product categories where they might represent a significant infection hazard, e.g., S. aureus and P. aeruginosa in topical products. Presence of the organism might also be indicative of quality since the most likely sources of E. coli and S. aureus are faecal contamination and manufacturing personnel respectively; the semiquantitative test for Cl. perfringens in the EP is explicitly stated as a quality criterion.
The principle of the methods used for all of the detection tests is similar: the sample is prepared as for a TVC, then a portion of it is incubated in a liquid-selective enrichment medium. Such media normally support the growth of the organism of interest and suppress the growth of others, so that the selected organism increases both in absolute numbers and in relative terms compared with the other constituents of the bioburden. This increase makes its detection more likely when the liquid medium is streaked onto a selective agar which, after incubation, is examined for the presence of characteristic colonies. An interesting and significant difference between the EP and USP protocols is noteworthy. The USP method directs, for all four objectionable organisms, that the first container of liquid medium containing the sample should be examined for the presence of growth after incubation, and the remainder of the process is only undertaken if growth (turbidity) is observed. The EP, on the other hand, does not provide this option, and the sample only passes the EP tests when the whole procedure is complete and no colonies observed. Thus it is not at all uncommon, when conducting an EP test, to transfer clear liquid from the primary to the secondary enrichment medium, and subsequently to streak multiple plates when there is already a strong indication that nothing will grow on them.
The detection of the target organism is dependent upon laboratory staff recognizing a typical colony when the only guide to appearance that they may have available is a description in a pharmacopoeia that can, at best, be described as vague, e.g., "well-developed colourless colonies" is the EP description of salmonellae growing upon deoxycholate citrate agar. It is an irony that as the quality of pharmaceutical products has improved, the pathogens in question very rarely arise in the bioburden. Increasing numbers of laboratory staff may be scrutinizing plates for organisms that they have never actually seen growing. This is a strong argument for ensuring that those same staff regularly conduct validation tests to confirm the nutritive properties of the medium, so that they have an opportunity to see the target organisms. This validation requirement that the test procedures should be capable of detecting culture collection strains of the objectionable organisms is also an ideal opportunity for a digital photographic record to be taken of the characteristic colonies on each of the media employed. Such a collection of photographs is outside the scope of this chapter, but appears elsewhere,14 together with an account of the characteristics and selectivity of the EP- and USP-recommended media and photographs of other organisms that might be the subject of false-positive identifications. Following the recognition of a suspect colony on selective agar, the organism may be subjected to a confirmatory test, e.g., indole test for E. coli, coagulase for S. aureus and oxidase for P. aeruginosa, although direct identification of the species using a commercially available product like API test strips or Vitek is more common. The confirmatory tests described in the pharmacopoeias are not necessarily absolutely specific for the respective organisms, although this is not always apparent from the text. The statement in the EP, for example, that "confirmation (of S. aureus) can be effected by suitable biochemical tests such as the coagulase test" disregards the fact that at least three other species of Staphylococcus are coagulase positive.15 The identity of suspect organisms is most convincingly confirmed using an appropriate commercial testing scheme in addition to the pharmacopoeial so-called confirmatory tests. However, this assumes that samples that are, in fact, contaminated, do actually give rise to suspect colonies on the selective agar media, and there are several potential problems that might prejudice this outcome. Recognition or adoption of the following points and strategies might reduce the chances of a false negative result in qualitative tests.
• The major suppliers all produce culture media possessing the same names as those recommended in the pharmacopoeial tests, but in some cases, e.g., MacConkey's agar, there are differences in the formulation that might influence selectivity.
• Correct preparation and storage of media are essential for reliable results. The selectivity of some media might change quickly during storage, e.g., Bismuth sulphite, a USP medium for salmonellae, is most selective when freshly prepared, and becomes less so as it becomes green over 3 to 4 days storage at 4°C. This means the storage intervals during which the media permit reliable detection of low concentrations of the test organisms need to be validated. This may be of particular relevance because the ability of the media to detect low concentrations of the target organism depends not only upon its absolute numbers, but also on the concentration of other organisms that might be present. Thus, it may be easier to detect salmonellae at, say, 100 CFU/ml in a pure culture than to detect 10 or 100 times this concentration in the presence of a heavy load of other bacteria. Reduced selectivity as a result of prolonged or incorrect storage might result in the obscuring of a few salmonellae in the sample.
• The incubation conditions described permit relatively wide variations in both temperature and time, e.g., 30-35°C for 24 to 48 hours, and in some cases the appearance of the growing culture can change substantially within these limits. The hydrogen sulphide production that is typical of salmonellae, for example, might be apparent as a black colouration or precipitate during early growth in several of the common Salmonella media, but disappears after 48 hours at 35°C.14 Similarly, the typical pink colour of E. coli on MacConkey's agar may substantially diminish after the first day of incubation. Note that differences in the incubation temperatures recommended in the EP and USP represent another source of incompatibility between the two methods. The USP essentially uses only two incubation temperatures for both quantitative and qualitative testing: 30-35°C for bacteria and 20-25°C for yeasts and molds. In contrast, the EP tests additionally require incubators at 35-37°C, 41-43°C, 43-45°C and 45.5-46.5°C.
• It is useful to be aware of the degree of selectivity afforded both by the media and the confirmatory tests. Just as the confirmatory tests are not absolutely specific for their intended organisms, so, too, are the selective agars less than perfect. Thus, Proteus species might grow on cetrimide agar and Baird-Parker medium, and species of Edwardsiella and Citrobacter may exhibit the typical textbook appearance of salmonellae on XLD medium. There are many other examples!
• For most objectionable organisms of pharmaceutical interest there exist a few strains that do not conform to the standard description. Most strains of P. aeruginosa exhibit a green or blue pigment, but a few possess an orange or brown pigment and few are nonpigmented. Similarly, about 5% of E. coli strains do not ferment lactose, and about 5% of salmonellae produce little or no hydrogen sulphide when they might be expected to do so.
• The simultaneous testing of known positive and negative control organisms is recommended by the USP for the coagulase confirmatory test for S. aureus. This principle may usefully be applied, where possible, to other confirmatory tests and selective agars.
• There are commercially available test kits for the detection of certain organisms, e.g., kits based upon the coagulase test for S. aureus and salmonellae; these are generally more convenient to use and more reliable than traditional textbook descriptions of the test procedures.
• Salmonellae, in particular, may pose problems of recognition, because certain harmless organisms may mimic salmonellae in both the selective agars and the triple sugar iron agar that is recommended as part of the confirmation process. Even API and Vitek results do not always afford a high degree of confidence in detection based upon biochemical tests. Agglutination tests using Salmonella antisera may provide a definitive answer.
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