Towards a common susceptibility testing method?

I. M. Gould*

Department of Medical Microbiology, Aberdeen Royal Infirmary, Foresterhill, Aberdeen AB25 2ZN, Scotland, UK

Introduction

Antibiotic susceptibility testing in clinical diagnostic laboratories is a routine procedure of proven clinical benefit and is of increasing importance as we suffer under an increasing burden of antibiotic resistance.1 The problems of resistance are leading to an increasing emphasis on performance of susceptibility testing to generate resistance surveillance data. The need to compare this type of information across different centres and countries is necessitating a reassessment of methods in common use. Susceptibility testing is most commonly performed by agar or broth dilution with measurement of MICs or (most commonly in the UK) agar disc diffusion with measurement of zone sizes. Several authorities publish recommended methods.25 In the UK most laboratories still use a modification of Stokes' method.6 The BSAC has proposed another new method of agar disc diffusion which circumvents many of the criticisms of Stokes' method and are calling for this method to become the UK standard. The PHLS in England and Wales has accepted the new BSAC method as a standard operating procedure. It is timely to examine the pros and cons of this new method and debate the wider issue of whether we need a new standard method and, if so, which one we, in the UK, should adopt.

Problems with Stokes' method and the need for a change

Variations on the original Stokes' method are now common and diverse.7 Because of this, it cannot be used to compare accurately results from one centre with another. In addition, results are arbitrary and non-quantitative and it is not known how they relate to the MIC. A recent paper exploring this issue showed an unacceptable number of errors with a modified Stokes' method.6 Its quality and validity (and thus its usefulness) is being questioned increasingly and the BSAC has called for a standardized method across the UK.8 While a new method should bring clinical benefits, particularly if it can reduce the number of false susceptibility reports,8 the main driver of proposed new methodology is the need for comparability of data between different centres across the UK so that resistance surveillance data can be reliably compared. The consensus is that any new method should be quantitative so that trends in susceptibility can be compared over time and subtle changes noted.9 Finally, laboratory accreditation will also dictate a move away from Stokes' method.

The need for and the benefits of surveillance

There is much justified concern about antibiotic resistance even if research into its prevalence remains uncoordinated and hence is unlikely to lead to much success in finding solutions. Table IGo charts some of the changes necessary over the past few decades in first-line therapy for common infectious diseases, reflecting some of the practical implications of developments in antimicrobial resistance. With a dearth of new antibiotics entering clinical practice there are no easy answers to the questions regarding the next developments in therapy that will be needed for many of these infections.


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Table I. Common first line therapies for infection over time
 
There has, therefore, been an upsurge of interest in surveillance of resistance in order to allow informed development of new strategies to counter antibiotic resistance.8,10 One of the common themes running through various recent meetings held on the subject911 is that there must be better co-operation and co-ordination of the many surveillance projects presently being undertaken. Certainly communication between networks must be strengthened, quality assurance harmonized and adequate support given to training in microbiology and epidemiology.10

Agar disc diffusion or breakpoint MIC?

What is the best method for laboratory detection of resistance and is this necessarily the same when applied to the two very different problems of guiding an individual patient's therapy and providing long-term resistance surveillance data? The main factors thought to affect reproducibility of susceptibility testing include inoculum, media composition and depth, delay between application of the disc and incubation, temperature, atmosphere and duration of incubation, generation time, the antibiotic concentration of the disc and the method of reading zone size. Some of these are peculiar to agar disc diffusion, particularly the possibility of delay between application of the antibiotic disc and incubation and the generation time of the test bacterium;12 both of these may allow excess diffusion of antibiotic from the disc into the agar, falsely increasing zone size and leading to major errors in susceptibility testing.

It is no surprise that agar disc diffusion is considered less reproducible and, in practice, is less standardized. Even so, according to the original International Collaborative Study,13 reproducibility should be within ±2 mm when measuring zones of inhibition.

Agar disc diffusion, however, does have many advantages. It is more flexible and cheaper than MIC determination and, presumably because of these factors, it is the most frequently used method in Europe for routine clinical diagnostic work. For surveillance purposes, many authorities believe it is superior to dilution techniques if the zone of inhibition is measured carefully, since this gives a sensitive marker of subtle changes in susceptibility over time and zone sizes are valuable for plotting frequency distribution.9,14 Also, if the data produced for these distribution frequencies are representative of a wide spectrum of susceptibility (and resistance) then an MIC can be calculated by regression line analysis.15,16

Broth dilution MIC testing lends itself well to automated systems and is the most frequently used susceptibility testing method in North American diagnostic laboratories.17 It is said to be more reproducible and quantitative than agar disc diffusion although antibiotics are usually tested in doubling dilutions which produce inexact MIC data.17 In fact, the reproducibility of both methods is probably similar.18 Furthermore, broth dilution is not easily adapted for detection of new resistance mechanisms such as extended spectrum ß-lactamases (ESBLs) and vancomycin resistance in enterococci and, hence, needs to be complemented by agar disc diffusion in certain circumstances.

NCCLS in comparison with the new BSAC method

Many countries, including France, Germany, Sweden, the UK and the USA each have their own methods of susceptibility testing, which have been overseen by their respective national societies of chemotherapy for some time. Recently, the BSAC has completed field trials and published tentative guidelines for the introduction of a new quantitative agar disc diffusion method for use in the UK.8 It is instructive to compare this proposed method with the largest and most widely adopted agar disc diffusion method (NCCLS) and to ask the question, do we need yet another method in this day and age when we should be looking for co-ordination and standardization of surveillance systems? My comparison of these two methods is summarized in Table IIGo.


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Table II. Comparison of the new BSAC susceptibility testing method with the NCCLS agar disc diffusion method
 
The NCCLS is a large, well-resourced organization. Its antibiotic susceptibility testing subcommittee consists of members drawn from academia, clinical laboratories, industry and the federal government. All these committee members work in North America. This committee is responsible for making final decisions and its members and an advisory board are appointed every 3 years. Twice a year it sits in public session for debate and discussion. NCCLS welcomes attendance and participation at these meetings by any interested parties and representatives from some European organizations also attend. Comprehensive descriptions of susceptibility test methods and their interpretations are published every 3 years with updates published annually as informational supplements.

The NCCLS is commonly criticized on three fronts, namely the setting of breakpoints, its use of Mueller– Hinton (MH) medium, and, finally, its use of a light-confluent inoculum. NCCLS breakpoints, whilst in general higher than those of the BSAC, do not cause significant clinical problems by reporting of false susceptibility (S. Alcock, personal communication). Table IIIGo compares the BSAC and NCCLS breakpoints for some ‘alert’ organisms reported weekly by most Scottish laboratories.19 The higher NCCLS breakpoint for gentamicin may well be more appropriate with the move to once-daily gentamicin prescribing. In fact, most NCCLS breakpoints are currently being revised along pharmacodynamic guidelines. Table IVGo shows how some of these new, tentative breakpoints compare with the existing NCCLS ones.20 There is now little room for criticism since they are now very similar to the breakpoints of the BSAC3 and other European breakpoint groups.5,21


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Table III. Scottish antibiotic resistance surveillance ‘alert’ organism MIC breakpoints by susceptibility testing method19
 

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Table IV. Comparative analysis of H. influenzae susceptibility using NCCLS and pharmacodynamic breakpoints20
 
The BSAC draught guidelines for agar disc diffusion are based on data from 250 diverse clinical isolates.22 The NCCLS23 and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)24 recommend that at least 500 isolates are tested for each antibiotic and where there are only one or two species involved, then 300 of each should be tested. Where there is not a good distribution of MICs amongst the test isolates then an error rate bounding on at least 2000 different strains is recommended by the NCCLS.

The NCCLS recommends major error rates of <1.5% and minor error rates of <3%23 although these limits are presently being reviewed. For BSAC the corresponding figures are 1% and 5%.22 However, for the new BSAC method, many of the development data exceed these guidelines, some by several fold.8,22

The NCCLS is often criticized for its use of a lightconfluent inoculum, the correctness of which is more difficult to assess than a semi-confluent one, when reading the plates. The NCCLS relies chiefly on process control (although inoculum preparation is less complicated than the BSAC method). With NCCLS, providing that zone sizes of control organisms are within the expected range (and the inoculum is confluent) then the results are assumed to be satisfactory. The BSAC method instructs that tests be repeated if a semi-confluent inoculum has not been achieved, i.e. they rely more heavily on outcome control. How often are tests repeated in a busy diagnostic laboratory, if the inoculum is judged too light/heavy? Other arguments in favour of a heavy inoculum include its greater similarity to the inoculum found in many life-threatening infections (where the results of susceptibility testing are most critical) and its tendency to lead to reporting of false resistance due to too heavy an inoculum, rather than false sensitivity due to too light an inoculum.

Finally, the widespread use of MH media for testing most organisms, as recommended by NCCLS, is undoubtedly a compromise, but simplicity is often the best way to gain acceptibility of a method in routine diagnostic laboratories. The medium is low in inhibitors of various antibiotics such as trimethoprim/sulphamethoxazole and cation-adjusted MH is now available. Early worries about batch variability no longer seem to be a problem.

While recognizing that no system of susceptibility testing is perfect, on the basis that NCCLS has had the largest amount of development work and active, continued refinement, it certainly deserves its place as the world-wide reference method.

The current situation in the UK, Europe and the world

Currently most laboratories in the UK use a modified Stokes' method. Within Scotland there are automated systems such as Vitek (bioMérieux, Marcy l'Etoile, France) or zone readers such as Mast Elite (Mast, Bootle, UK) in six of the 10 largest of the 25 clinical diagnostic laboratories, all using NCCLS interpretations. In addition, one other large laboratory has been using the NCCLS agar disc diffusion method for more than 6 years and several others use it for clinical research.

Most European clinical diagnostic laboratories (excluding those in France, Sweden and Germany) use NCCLS routinely.25 Of the 47 European National Surveillance Systems described at the WHO Verona meeting, 33 use NCCLS.10 The great majority of pan-European, International and Pharmaceutical Surveillance Systems use NCCLS as do most laboratories in North and South America and also some in Japan and Australia (A. Coffey, personal communication)

It is likely that future EU guidelines will harmonize European and American practice regarding susceptibility testing, initially with a reference method. At the recent WHO meeting in Verona10 it was agreed that the emergence and growth of antimicrobial resistance could not be addressed effectively by any one country or group working in isolation. Europe-wide co-ordination and co-operation were deemed critical elements for any effective approach. It was concluded that further discussions are necessary, to develop collaboration between existing antimicrobial resistance surveillance programmes.10 It should be remembered that, in recent evidence to the House of Lords, the BSAC agreed that in order to perform surveillance of resistance effectively, it was important to develop links with other European countries as well as on a world-wide basis.25

Conclusions

If results achieved with different methods are to be compared, then comparability of results has to be shown and consensus on interpretation achieved. This was suggested as long ago as 1971 but has never been achieved.10 The alternative (and the ideal) is for all laboratories and networks to use the same methods and breakpoints for surveillance (and diagnostic work). In reality this has been achieved by NCCLS, if only by default and it is unnecessary and indeed, undesirable, to create new standards. The UK is part of Europe and the world. Multi-resistant bacteria recognize no national boundaries and nor should the BSAC!

Editorial note

Readers of this article are directed to the reply by Wise & Phillips in the Correspondence section of this issue.

Notes

* Fax: +44-1224-840632; E-mail: i.m.gould{at}abdn.ac.uk Back

References

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6 . Gosden, P. E., Andrews, J. M., Bowker, K. E., Holt, H. A., MacGowan, A. P., Reeves, D. S. et al. (1998). Comparison of the modified Stokes' method of susceptibility testing with results obtained using MIC methods and British Society of Antimicrobial Chemotherapy breakpoints. Journal of Antimicrobial Chemotherapy 42, 161–9.[Abstract]

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16 . D'Amato, R. F., Hochstein, L., Vernaleo, J. R., Cleri, D. J., Wallman, A. A., Gradus, M. S. et al. (1985). Evaluation of BIOGRAM antimicrobial susceptibility test system. Journal of Clinical Microbiology 22, 793–8.[ISI][Medline]

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19 . Gould, I. M. (1998). National surveillance of resistant organisms: a proposal for discussion. SCIEH Weekly Report 32, 66–8.

20 . Felmingham, D. & Grüneberg, R. N. (1998). Comparative analysis of H. influenzae susceptibility using NCCLS and pharmacodynamic breakpoints: the Alexander Project 1997. Clinical Infectious Diseases 27, 959 (Abstract 200).

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22 . British Society of Antimicrobial Chemotherapy report on the standardized disc testing method and ‘field trial’ on behalf of the Working Party on sensitivity testing. (1998) Newsletter of the British Society for Antimicrobial Chemotherapy 1–111.

23 . National Committee for Clinical Laboratory Standards. (1998). Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters: Tentative Guideline M23-T3 18 (5). NCCLS, Villanova, PA.

24 . European Society of Clinical Microbiology and Infectious Diseases. (1998). Determination of susceptibility test breakpoints. (EUCAST consultation document), pp. 8–10. European Society of Clinical Microbiology and Infectious Diseases, Munich.

25 . House of Lords Select Committee on Science and Technology. (1998). Resistance to antibiotics and other antimicrobial agents. HL Paper 81-II. The Stationery Office, London.

Received 21 June 1999; returned 28 October 1999; revised 6 December 1999; accepted 10 January 2000