1 Päijät-Häme Central Hospital, Department of Clinical Microbiology, Keskussairaalankatu 7, FIN15850 Lahti; 2 Keski-Suomi Central Hospital, Jyväskylä; 3 National Public Health Institute, Turku, Finland
Received 16 September 2003; returned 29 October 2003; revised 7 January 2003; accepted 8 January 2004
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Abstract |
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Methods: Three BLNAR and two ß-lactamase-negative ampicillin-susceptible isolates (BLNAS)originating from the American Type Culture Collection (ATCC) and UK National External Quality Assessment (UKNEQAS) schemeswere included in this study. Susceptibility tests for these isolates were performed in 26 clinical microbiology laboratories, in accordance with NCCLS guidelines. Additionally, low-strength discs for ampicillin (2 µg) and co-amoxiclav (3 µg) were tested.
Results: The low-strength discs for ampicillin and co-amoxiclav categorized more accurately BLNAR and BLNAS H. influenzae isolates than did the high-strength discs recommended by the NCCLS. In addition, the high-strength discs produced more major errors than the low-strength discs (22 versus six for ampicillin and 40 versus seven for co-amoxiclav). Great variation occurred in the method regardless of the antibiotic concentration of the discs.
Conclusions: The use of low-content ampicillin and co-amoxiclav discs is recommended for the susceptibility testing of H. influenzae. Interpretative criteria of S17 mm and R
13 mm for both discs are suggested.
Keywords: H. influenzae, BLNAR, susceptibility testing
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Introduction |
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Problems detecting BLNAR strains using the NCCLS disc diffusion method11 have been reported previously by several groups and different solutions have been proposed to correct this methodological difficulty. The use of a low-strength disc (oxacillin 1 µg) is acknowledged as a screening method to identify penicillin non-susceptible pneumococci. In cases of H. influenzae and ampicillin, the ability to discriminate BLNAR strains from ß-lactamase-negative, ampicillin-susceptible (BLNAS) strains was better using a 2 µg disc rather than the 10 µg disc recommended by the NCCLS.12
Here we report the results and conclusions of an evaluation of the NCCLS disc diffusion method for the susceptibility testing of H. influenzae in Finland. The main focus was to assess the method used to identify the BLNAR strains, and therefore discs with lower contents of ampicillin and co-amoxiclav were included. H. influenzae isolates from the ATCC collection and the UK NEQAS schemes with reference susceptibilities were used as challenge strains.
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Materials and methods |
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Results and discussion |
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Isolates 2, 3 and 5 were considered BLNAR strains resistant to ampicillin, co-amoxiclav and first and/or second-generation cephalosporins. The S/I/R classification is based on the interpretative criteria given by the NCCLS for ampicillin 10 µg and co-amoxiclav 30 µg discs. Interpretative criteria suggested by Zerva et al. (S17 mm, R
13 mm)12 were applied for both low-strength discs. Resistant and intermediate categories were combined for the analysis.
Both low-strength discs categorized more accurately the BLNAR isolates (Table 1). Sensitivities and specificities estimated for each test (disc) were as follows: ampicillin 10 µg: 71%/88%, ampicillin 2 µg: 92%/90%, co-amoxiclav 30 µg: 44%/96%, co-amoxiclav 3 µg: 91%/87%. Thus, the methods using low-strength discs were more sensitive for both antimicrobial agents. The specificity was acceptable for all tests (discs). Altogether, the NCCLS susceptibility method produced 22 major errors (false-susceptible results) for ampicillin and 40 for co-amoxiclav. The corresponding figures for the low-strength discs were six for ampicillin and seven for co-amoxiclav. In a three-centre, collaborative study in the USA, major errorsaccording to the NCCLSwere produced for two ß-lactamase-inhibitor combination discs (co-amoxiclav and ampicillin/sulbactam).13 However, in the same study, contrary to our results, ampicillin testingaccording to the NCCLSdid not produce major errors.
The variation in the zone diameters of the inhibition zones was considerable, as the ranges and standard deviations reveal (Table 1). Several of the 26 laboratories reported difficulties in determining the edge of the inhibition zones because of hazy limits of growth. This could, in theory, be due to two methodological factors: the medium or the inoculum density. Several reports have been published concerning the use of HTM in general, but we have found no evaluation of the Oxoid HTM. However, the composition of HTM by different manufacturers is identical and therefore we do not believe that the medium we used would be the main reason for the lack of good reproducibility in this study. Rather, the inoculum density may explain these difficulties. The inoculum density recommended by the NCCLS11 is McFarland 0.5, which corresponds to 14 x 108 cfu/mL. According to our unpublished data, however, the actual cfu/mL varies from 17 x 108, even though the density is adjusted to McFarland 0.5 using a photometric device. Thus, the use of too dense inocula may, in part, explain the great variability in the zone diameters. The worst results were seen for isolate 2, where the variation of zone diameters and misclassifications were greatest even with the low-strength discs. This is somewhat surprising, because the reference MIC values given by the supplier (UK NEQAS) were, out of all the isolates, highest for ampicillin (8 mg/L) and cefuroxime (64 mg/L) and one might assume that this strain would be the easiest to classify correctly. Most comments of hazy growth within the inhibition zones were for this isolate.
Although BLNAR H. influenzae isolates are uncommon in most western countries, their detection is important for two reasons. First, these isolates are resistant to first- and second-generation cephalosporins, and treatment failures have been reported with these antibiotics.4 Second, the BLNAR strains have become increasingly prevalent in certain countries. The widespread use of oral cephalosporins has been suggested as a reason for this increase, for instance in Japan.10 In Finland, the consumption of cephalosporins, all in all, has been considerably higher than in other Nordic countries (in 1995, three versus less than one DDD/1000 inhabitants per day). In outpatient care, oral cephalosporins have been particularly popular for the treatment of skin and middle ear infections.14
To conclude, in our opinion, low-strength ampicillin and co-amoxiclav discs should be added to the routine susceptibility testing of H. influenzae to detect BLNAR isolates. The sensitivity of the NCCLS method is not satisfactory. The high-strength discs do not seem to add value to the detection of BLNAR H. influenzae. However, ß-lactamase-positive isolates were not included in this study, and we do not know, at the moment, whether the detection of co-amoxiclav resistance among these isolates is accurate with the low-strength disc. The interpretative criteria used in this study are applicable for both low-strength discs, but require further validation. The suggested detection of BLNAR H. influenzae by the disc diffusion method should always be confirmed by MIC testing.
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Acknowledgements |
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Footnotes |
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The members of the FiRe network are listed in the Acknowledgements.
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References |
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