Comparisons of the in vitro susceptibility testing results for garenoxacin using six different national methods: report from the garenoxacin international bridging study

R. N. Jones1,2,*, K. A. Gordon1 and D. J. Biedenbach1

1 The JONES Group/JMI Laboratories, Inc., 345 Beaver Kreek Centre, Suite A, North Liberty, IA 52317; 2 Tufts University School of Medicine, Boston, MA, USA

Received 16 May 2003; returned 2 July 2003; revised 1 November 2003; accepted 8 November 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: Laboratories worldwide carry out MIC/disc diffusion (DD) tests using various national methods, often published by local organizations. Potentially different results may complicate drug development or registration between the US methods and those used in the European Union (EU). This study uses a new des-F(6)quinolone, garenoxacin, to compare in vitro results among the most utilized antimicrobial susceptibility testing methods.

Methods: Investigators in France, Germany, Spain, Sweden and the UK tested two bacterial collections designated, challenge (CC; n = 330) containing quinolone-resistant strains and national (NC; n = 540) that were recent clinical isolates (2000–2001). Results were compared with values derived from the National Committee for Clinical Laboratory Standards (NCCLS) methods, carried out by the US coordinating site. Discords (greater than four-fold) between MIC method results were repeated. Results were analysed for variation from the NCCLS results (±1–2 dilutions or greater than 3–6 mm) and by regression statistics. Ciprofloxacin was used as the control quinolone agent.

Results: CC and NC testing compliance averaged 98.4% and 86.4% among participating centres, respectively, and correlation (r) of the national method to the NCCLS MIC was: France (0.98), Germany (0.95), Spain (0.98), Sweden (0.96) and UK (0.95). CC produced MIC results that were identical to the NCCLS (Spain) to 0.6 x log2 greater (Germany), but the percentage of strains ±2 dilutions versus NCCLS MIC results was 98% (Germany)–100% (Sweden, UK). Similar patterns were observed for the NC (r = 0.93–0.98), and all nations had >99% of results within ±2 dilutions (±6 mm for disc tests) of NCCLS values. MIC results from four national methods were slightly elevated compared to the NCCLS result (average 0.2 x log2). Control ciprofloxacin MIC and DD test results showed similar patterns.

Conclusions: Inter-method garenoxacin susceptibility test results indicate that MIC and zone diameter endpoints derived from five EU methods compare favourably to the NCCLS method results, and generally were identical or a fraction of a log2 dilution step higher. In contrast, zone diameters for garenoxacin and ciprofloxacin were routinely larger for the EU methods. This unique inter-method ‘bridging’ experiment allows regulatory agencies to better correlate in vitro testing results derived from procedures that use different national methodologies.

Keywords: fluoroquinolones, NCCLS, inter-method comparisons


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The vast majority of antimicrobial susceptibility testing methods that have been developed by national organizations or professional societies have been based on the comprehensive studies summarized by Ericsson & Sherris in 1971.1 In that 90 page document, results from numerous investigations by a multitude of scientists were summarized leading to recommendations for the technical aspects of susceptibility testing for three methods (broth and agar dilution, disc diffusion). Several issues, however, remained to be resolved including (i) selections of media for testing some types of bacteria; (ii) reporting formats and interpretive guidelines; (iii) establishing statistical methods for comparing zone diameters to MIC results; (iv) developing methods for emerging problem isolates such as hetero-resistant strains; (v) expanded numbers of organisms and antimicrobials should be rigorously investigated; and (vi) methods to develop and compare new techniques of susceptibility testing should be finalized.1 Many of these tasks have been accomplished in the last three decades on a national or international basis, but the evolving problem of potential inter-method discords in technical details and interpretation of results among the various national methods has compromised international antimicrobial development.

Since 1972, the National Committee for Clinical Laboratory Standards (NCCLS) in the USA has had an active standardization programme, initially focused on the disc diffusion method.2,3 Later committee activities expanded the consensus standards process to include: agar dilution, broth dilution, broth microdilution and reference methods for testing anaerobic bacteria (agar dilution and broth microdilution).47 Concurrently, similar organizations mostly found in several developed nations (Europe and Japan) formalized the standardization processes. With the birth of the European Union (EU) and the aim of common regulatory standards for developing new prescription drugs, the need for harmonization of in vitro antimicrobial susceptibility testing methods has assumed a level of high importance. Also via the collaboration of the European Society for Clinical Microbiology and Infectious Disease and the EU regulatory agencies, a newer pan-EU initiative called EUCAST (European Committee for Antimicrobial Susceptibility Testing) was organized to develop common methods, quality assurance and interpretive standards.8

Other statistics from these European programmes illustrate the complex problems encountered in this geographic region when trying to combat emerging antimicrobial resistances. The European Antimicrobial Resistance Surveillance System (EARSS) in their resistance monitoring projects, indicated that a wide variety of methods and interpretive standards were utilized.9 Bruinsma et al. noted that among 24 European nations participating in 2001, the primary guidelines for testing were from diverse sources such as the NCCLS, British Society for Antimicrobial Chemotherapy (BSAC), Deutsche Industrie Norm-Medizinische Mikrobiologie (DIN), la Societé Francaise de Microbiologie (SFM), Swedish Reference Group for Antibiotics (SRGA), Commissie Richtlijnen Geovoeligheidsbepalingen, and non-national methods like those of Rosco and Stokes.9 However, overall in Europe, the NCCLS guidelines were used by 73% of EARSS participants. Other guidelines only predominated within a single nation such as Germany (DIN), Denmark (SRGA), France (SFM), Sweden (SRGA) and UK (BSAC). Clearly the field of medical microbiology needs to achieve greater international standardization (harmonization) of antimicrobial susceptibility test methods to assist other functions such as relating antimicrobial use patterns to resistance trends.10

The EUCAST group has provided excellent progress toward harmonization of susceptibility testing methods used in the EU nations and have sought greater international consensus via open dialogues with the International Standards Organization (ISO) and the NCCLS. As new and novel antimicrobial agents are developed for clinical use, worldwide development standards for susceptibility testing and their relationship to pre-registration clinical trials will streamline the process, and hopefully reduce the confusion and escalating costs of the current practices in Europe, North America and elsewhere. To this end, an investigation was designed to use a novel des(6)fluoroquinolone, garenoxacin,1113 to study the inter-relationships of six national standards for antimicrobial susceptibility testing by dilution (MICs) and disc diffusion (zone diameters) methods. Results derived from the dominant methods utilized in the EU (BSAC, DIN, Mesa Españole de Normalización de la Susceptibilitad y Resistencia a los Antimicrobianos [MENSURA], SFM and SRGA) would be compared to the results of the most used method worldwide and in the EU, the NCCLS M2-A8 and M7-A6 (2003) methods.4,5,1418


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Investigators and methods

The national level investigators and the method used by each participating laboratory were: Claude J. Soussy, Cifu Cote de Nacre, Paris, France (SFM);17 Arne Rodloff, University of Leipzig School of Medicine, Leipzig, Germany (DIN);15 Rafael Canton, Hospital Ramon y Cajal, Madrid, Spain (MENSURA);16 Gunnar Kahlmeter, Central Hospital, Vaxjo, Sweden (SRGA);18 and Gary French, St Thomas’ Hospital Medical School, London, UK (BSAC).14 The coordinating centre, JMI Laboratories, North Liberty, IA, USA (Ronald N. Jones), carried out susceptibility tests by the NCCLS methods4,5 for all strains.

The details of each method4,5,1418 were provided by each participant on a comprehensive series of spreadsheets. These technical differences were used to explain any variations in results for the study comparisons to NCCLS values.4,5 Generally where technical details differed, they were in the areas of medium or inoculum concentrations utilized. These variations have predictable influences on the susceptibility endpoint that could be expected for the inter-method results based on the earliest of published experience.1,2

Organisms

The organisms were selected through collaboration of the participants, sponsor and the study monitor. The project was carried out in two phases related to organism processing (Table 1).


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Table 1. Composition of the organism collections utilized during the multi-centre inter-method comparison study
 
In Phase I, a ‘Challenge Collection’ (CC) of organisms was selected to provide a wide variety of pathogens and organisms that had uncommon resistance patterns in some nations monitored (Table 1). These organisms included samples of penicillin- and ciprofloxacin- (MIC >= 4 mg/L) resistant Streptococcus pneumoniae; oxacillin-resistant staphylococci, vancomycin-resistant (van A and van B patterns) enterococci, ß-lactamase-producing Haemophilus influenzae and Haemophilus parainfluenzae, fluoroquinolone-resistant Enterobacteriaceae species and ciprofloxacin-resistant non-fermentative Gram-negative bacilli.

This collection contained 330 strains that were initially isolated from the SENTRY Antimicrobial Surveillance Program19 at diverse international locations. Each strain was identified by the monitor, subcultured and delivered to participant locations for Phase I testing.

Compliance in testing CC organisms varied, mainly as a result of poor survival of some fastidious strains (streptococci or H. influenzae). The number of participant results received for comparison evaluation included: France (321; 97.3%), Germany (324; 98.2%), Spain (323; 97.9%), Sweden (325; 98.5%) and UK (325; 98.5%); overall compliance for Phase I was 98.4%.

In Phase II, a second group of strains, called the ‘National Collection’ (NC), was selected by each national investigator from those isolates referred by SENTRY Program participants (via national ‘Bridging’ monitor) for the 2001 study year (Table 1). These strains were selected based on target numbers of strains per species, and would total 540 organisms if fully compliant. All strains were representative of the susceptibility patterns (by organism species) that were endemic within that nation.

Some species were rarely isolated at some of the participant SENTRY Program institutions, and total compliance was compromised by the rarity of these organisms. Regardless, compliance in Phase II was high as follows: France (463; 85.7%) Germany (456; 84.4%), Spain (501; 92.8%), Sweden (494; 91.5%) and UK (458; 84.8%); all-nation compliance was 86.4%. The compliance to protocol numbers of organisms processed in all Phases was 90.9%. Quality control strains (Table 1) were also circulated and all results were within NCCLS20 or sponsor recommended11 limits for each quinolone tested.

Study design

The following sequence of events was initiated during the investigation: (1) Reagents were shipped to the five national study locations that included: garenoxacin and ciprofloxacin (control fluoroquinolone) laboratory standard powders (from Bristol-Myers Squibb [Princeton, NJ, USA]; Sigma Chemical Co. [St. Louis, MO, USA], respectively) and commercially validated 5 µg garenoxacin and ciprofloxacin discs (BD Microbiology Systems, Cockeysville, MD, USA); (2) CC organisms were shipped to participants; and (3) test results compared with NCCLS values and discords >2 log2 dilution steps or >6 mm were repeated. New subcultures of discordant strains were sent to each participant and repeat tested. These repeated test values (mode of triplicate determinations) became the result entered into the study record. For the NC strains, the organisms were processed at participant sites and forwarded to the monitor for testing in the SENTRY Program and by this protocol using NCCLS4,5 methods (see number 3 above). Repeated values became the result utilized in the study record and each result by national method was compared to the NCCLS value by regression analysis and by error-rate techniques.6 The target correlations between methods that would be considered acceptable were established at a r value of >=0.90; >=90% of MIC results within ±1 log2 dilution step (>=95% with ±2 log2 dilution steps); and >=90% categorical agreement between method results using the same quantitative MIC breakpoint, all as applied to garenoxacin and ciprofloxacin.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inter-method comparisons of garenoxacin MIC results

Figure 1 shows the MIC scattergram comparing SFM and NCCLS method results for garenoxacin using CC organisms (321 strains). Excellent correlation was achieved (r = 0.98) and over 97% of isolates were within ±1 log2 dilution step. Table 2 illustrates that 51.1% of results were identical between the two methods with a slight skewing towards a higher MIC with the SFM method (Table 3). The inter-method results for the NC strains also showed high correlation (r = 0.96), although only 6.3% of all strains tested were beyond the ±1 log2 dilution limits (Table 2). No significant trend toward a higher SMF MIC result was noted among these results.



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Figure 1. Scattergram comparing MIC results for garenoxacin tested by NCCLS (y-axis) and the French SFM (x-axis) method using challenge organisms (321 strains).5,17

 

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Table 2. Variations of garenoxacin MIC results produced by five national methods against two collections/sets of organisms when compared to the MIC values obtained with the NCCLS M7-A6 broth microdilution method
 

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Table 3. Overall inter-method discrepancy rates when five national method results for garenoxacin by susceptibility category were compared to the results of the NCCLS MIC methoda
 
Among DIN and NCCLS method results for the CC organisms, the correlation coefficient was 0.95, but a pronounced trend toward a higher DIN MIC was noted with 40.3% of results two-fold higher than the NCCLS results (Table 2). The trend was equivalent to a 0.5 log2 dilution shift. Nevertheless, nearly 90% of DIN results were within ±1 log2 dilution step of the NCCLS MIC value. For the NC strains from Germany, a modal shift of +1 log2 dilution (45.0%) was noted. There was skewing of the DIN MIC toward an elevated MIC (0.6 log2 dilution) compared to NCCLS results.

For the Spanish method (MENSURA), very close correlation was noted (r = 0.98) for the CC strains. A total of 97.2% of garenoxacin MIC results were within ±1 log2 dilution step, and no trend toward higher or lower MIC values was noted when compared to the NCCLS MIC results (Table 2). For the NC strains, excellent correlation between methods was again demonstrated (r = 0.98). A total of 96.9% of the Spanish results were within ±1 log2 dilution.

The Swedish method results and those from the NCCLS method versus CC isolates (325 test pairs) showed a r value of 0.97, and 90.5% of results were within ±1 log2 dilution. However, a trend toward a higher SRGA MIC was observed (0.4 log2 dilution; Table 2). For the NC organisms, the inter-method correlation was high (r = 0.96). Agreement between methods at ±1 log2 dilution step was 96.3% and only a slight trend toward a higher SRGA MIC was detected at the level of 0.2 log2 dilution. Correlation between BSAC and NCCLS method results was excellent (r = 0.95), but a wide range of results was obtained (Table 2). Only 79.6% of comparison MIC values were within ±1 log2 dilution step, with 5.9% of results showing a four-fold lower BSAC result, and 14.5% of strains exhibited a four-fold higher BSAC MIC value compared to the NCCLS broth microdilution MIC. All of the BSAC and NCCLS comparisons were within ±2 log2 dilution, and a trend toward a higher BSAC MIC was only 0.1 log2 dilution for the CC set. The BSAC/NCCLS comparisons of the NC organisms show an even wider spread of results with only 76.6% of comparison MICs within ±1 log2 dilution (Table 2).

Taken as an entire comparison experiment (CC and NC organisms), 90.6% and 99.7% of results were found within ±1 log2 and ±2 log2 dilution steps, respectively (Table 2). The most marked trend toward a higher MIC when compared to the NCCLS MIC results, was observed for the DIN method. All other methods varied from the same MIC result to only +0.3 log2 dilution step (+0.2 log2 dilutions, overall).

Distribution of more extreme inter-method MIC discords

The national methods having the largest number of four-fold or greater discords when compared to the NCCLS broth microdilution MIC were the BSAC and DIN methods. Each method contributed high numbers of inter-method discords (70.2% of all severe inter-method errors), with the DIN method having a four-fold higher rate. The SRGA test results were also noteworthy, where 15-fold more occurrences favoured a >=four-fold higher MIC versus the NCCLS MIC result. Overall, the most serious discords by the European methods (higher MICs), outnumbered those with lower MICs by approximately three to one (data not shown).

Categorical comparisons between methods

The susceptibility categories were arbitrarily assigned for comparison purposes only, as those consistent with the NCCLS criteria for the vast majority of other fluoroquinolones. Susceptibility was defined for garenoxacin as <=2 mg/L and resistance at >=8 mg/L, levels easily validated by prior pharmacokinetic/pharmacodynamic assessments.21,22 If the same criteria were utilized in Europe for each of these methods and also applied in nations using the NCCLS method (MENSURA, Spain), the inter-method discrepancy rates for MIC results would be calculated as follows: (1) serious discord = susceptible by one method and resistant by the other method; and (2) minor discord = intermediate by one method and susceptible or resistant by the other method.

The results in Table 3 illustrate very acceptable inter-method categorical agreement rates regardless of methods compared. The highest serious discord rates were detected with the DIN versus NCCLS at only 0.7–1.2%, and two methods (MENSURA and SRGA) had no serious inter-method categorical discrepancies for either collection (CC or NC) of organisms. The overall combined serious and minor discord rate was only 5.5% (0.3% serious), or an absolute categorical agreement of 94.5% between MIC methods. The component of the serious discord rates were contributed by the organisms having the most extreme (>=four-fold) inter-method MIC discrepancies (11 strains). In contrast, only 29 of 207 (14.0%) of the strains with minor discords were the result of >=four-fold inter-method variations.

The organisms producing the >=four-fold variations between tested methods and the NCCLS MIC results included 374 strains (9.5% of 3956 comparison tests). For the French organisms and methods, a wide range of species contributed to these discords that were very infrequent overall (37 occurrences; 4.7%). Similarly, the results with the Spanish MENSURA method produced only 23 discords (2.9%). Over one-third of the infrequent MENSURA/NCCLS discords were among fastidious streptococci. The Swedish SRGA method had a larger number of MIC discords at >=four-fold higher than the NCCLS MIC. Nearly one-half of these discordant SRGA MIC results occurred with Haemophilus spp. and fastidious streptococci and may have been the result of a possible medium effect (data not shown).

Lastly, the DIN and BSAC methods illustrated some organism-specific discrepancies that may be very important observations. For the DIN method, Klebsiella spp. strains were the most frequent cause of discords (13 occurrences), and 23 of 72 discords with elevated DIN MICs were recorded for fastidious species (H. influenzae, Streptococcus spp.) illustrating another possible medium effect. For the BSAC method, 178 high-level discords were reported with a very different pattern than for those observed in other national methods. These organisms were dominated by E.coli (21 of 67 strains) whereas those having >=four-fold higher BSAC MICs were generally fastidious species (48 Haemophilus and 40 streptococci). Again the latter could be explained by medium lot or product quality difference suspected by the BSAC laboratory (G. French and A. King, personal communications).

Organisms producing serious inter-method categorical MIC errors

Only 11 strains accounted for the 0.3% serious inter-method categorical errors. Non-fermentative Gram-negative bacilli were over-represented with four of 11 (36.4%) strains, causing errors between the NCCLS method and those of the SFM (two occurrences), DIN (one occurrence) and BSAC (one occurrence). Enterococci were also observed (three occurrences) followed by Serratia spp. (two strains in Germany), and single occurrences for coagulase-negative staphylococci and H. influenzae.

Comparisons of inter-method results for the disc diffusion tests of garenoxacin (5 µg)

Table 4 shows the regression statistics (regression equation and correlation coefficient) comparing five national methods and NCCLS method disc diffusion zone diameters for garenoxacin. Garenoxacin correlation coefficients for the CC organisms ranged from 0.88 (Germany; DIN) to 0.95 (Spain; MENSURA). For the NC strains, the poorest r value was for the SRGA at 0.86 and the MENSURA method was best at 0.95.


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Table 4. Listing of regression equations and correlation statistics (r) for five national methods for disc diffusion testing using 5-µg garenoxacin discs and compared to the reference NCCLS zone diameter
 
The regression lines were ‘tipped’ toward a larger corresponding zone diameter with the European methods when compared to the NCCLS zone of inhibition (Table 4; Figure 2 [BSAC]). This was most evident for the SRGA method (+5.5 mm overall; data not shown) and least for MENSURA (+2.4 mm). The tabulation of the variations between zone diameters for each method (Table 5) showed larger zones overall ranging from +2.8 mm for the NCCLS correlate at 30 mm (CC organisms) to +4.6 mm for the NCCLS correlate at 25 mm (NC organisms).



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Figure 2. Scattergram comparing the NCCLS (y-axis) and BSAC (x-axis) method zone diameters around 5 µg garenoxacin discs using a collection of 478 strains isolated in the UK.4,14

 

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Table 5. Median 5 µg garenoxacin zone diameter for each national method for strains having zones of 25 and 30 mm by the NCCLS disc diffusion method for each organism sent
 
The ideal plate surface appearance of a disc diffusion test for the European-based disc diffusion test is so-called ‘semi-confluent or kissing colonies’, in contrast to the fully confluent growth recommended for the NCCLS disc method. This reduced inoculum density generally utilized in these European disc diffusion methods produces a 3–4 mm shift of the regression line toward large zone diameters when compared to the NCCLS M2-A8 (2003) method. This would require adjustments in the breakpoint zone diameters for each of these EU national methods to obtain comparable categorical results to the NCCLS M2-A8 (2003) if the same MIC breakpoint were applied. This shift was equivalent to a 1 log2 dilution step in the MIC, whereas the differences in the MIC results between methods was not significant for the five methods overall when compared to the NCCLS MIC results.

The examination of the scattergram (Figure 2; example for BSAC-NC strains) indicates that correlate breakpoints for each method could be established at zone diameters between 15 and 23 mm and minimize significant numbers of inter-method categorical discords. The exception would be the DIN method where an increased number of DIN-susceptible, NCCLS-resistant strains would occur using results from both organism collections (data not shown).

Summary of ciprofloxacin control results

Ciprofloxacin MICs and zone diameters (5 µg disc content) were used as internal fluoroquinolone susceptibility test controls. All strains tested with 5 µg garenoxacin discs were also tested with ciprofloxacin reference test reagents. Quality control (QC) guidelines for each method were applied locally as specified by the utilized European method. The monitor used the NCCLS methods, current QC ranges of MICs and zone diameters20 and proposed garenoxacin QC ranges suggested by Biedenbach et al.23 All QC results were reported by participants to be within specified ranges and the ciprofloxacin MIC correlations of each European method with the NCCLS results were very high (CC organisms, r = 0.95 [DIN] to 0.98 [SFM and MENSURA]); NC organisms, r = 0.94 [SFM] to 0.98 [MENSURA]).

Only nine MIC results were >2 log2 dilutions higher or lower when compared to the NCCLS broth microdilution MIC results (0.6% of 3955 comparisons). Nearly all of these aberrations occurred with the French SFM method. Overall correlations within ±1 log2 dilution step for each organism collection were 91.7–98.2% and 84.9–96.1% for the CC and NC organisms, respectively. The lowest inter-method accuracy observed for the BSAC method was associated with a two-fold higher ciprofloxacin MIC shift compared with the NCCLS and other methods.

Ciprofloxacin disc diffusion control regression equations and correlation coefficients produced acceptable statistical results (r = 0.85–0.97). Scattergram plots for the disc diffusion tests showed wider variations for the following methods and organism collections: (1) SFM method versus NC strains; and (2) DIN method versus both organism collections. All other comparisons had very acceptable agreement with zone diameters correlating with applied MIC breakpoints for each method (data not shown). As noted with garenoxacin zone diameter results with the lower inoculum concentration, ciprofloxacin zones by the European methods were generally 2.2–4.4 mm larger compared with the NCCLS disc diffusion zone diameter. The study-wide performance of the ciprofloxacin control exhibited the same levels of accuracy and variations identified for the garenoxacin susceptibility test results.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Garenoxacin1113,23 MIC results obtained using the five tested European-based methods (BSAC, DIN, MENSURA, SFM and SRGA)1418 compare favourably with the MIC values generated by the NCCLS broth microdilution method.5,20 Correlation coefficients (r) averaged well over 0.90, inter-method serious categorical discord was at acceptable levels (target <=1.5%; actual 0.3%) and a modest, but acceptable, trend toward higher MIC results was observed for the European methods. The latter trend was greatest for the DIN and SRGA methods, but averaged only +0.2 x log2 dilution. Garenoxacin zone diameters generated around the 5 µg disc were routinely larger by 3–4 mm using the European disc diffusion methods when compared with the NCCLS M2-A7 method zones of inhibition.4,1418 However, correlation (r) between methods (zone versus zone in mm) remained high at >0.90 (average). Also the correlate zones of inhibition for a susceptible breakpoint could be selected for each method that would achieve excellent categorical agreement with the NCCLS results.4,5,7

Garenoxacin test results using the six national antimicrobial susceptibility testing methods for MIC and disc diffusion methods would yield comparable results if the interpretive criteria were both identical for the MIC breakpoints and correlate zone diameters for susceptibility and resistance were adjusted for medium and/or inoculum differences. Clinical trial susceptibility testing data from these assessed national systems during the development of garenoxacin appear to be closely related and compatible for the merging of in vitro data in preparation for new drug registration in the USA (FDA) and the EU. These unique collaborative studies should also lead to greater harmonization of susceptibility test methods within the EU nations, and between the laboratories using the NCCLS methods in Europe and elsewhere. Use of protocols similar to those described here will facilitate the movement toward a truly international antimicrobial susceptibility testing method and that will support the global development of new, orally administered drugs needed to address the emerging problem of resistance especially among S. pneumoniae.19,2427 The level of resistance to penicillin and/or macrolides among pneumococci has been predicted by a mathematical transmission model to be 46% in the USA by 1 July 2004,28 indicating the urgent need for more potent agents such as garenoxacin.


    Acknowledgements
 
The co-authors wish to thank the members of the Garenoxacin study group that includes R. Canton (Hospital Ramon y Cajal, Madrid, Spain); G. French and A. King (St Thomas’ Hospital Medical School, London, UK); G. Kahlmeter (Central Hospital, Vaxjo, Sweden); A. Rodloff (der Universitat, Leipzig, Leipzig, Germany); and C. J. Soussy (Hospital Henri Mondor, Creteil, France). We also acknowledge the support of a research/educational grant from Bristol-Myers Squibb.


    Footnotes
 
* Corresponding author. Tel: +1-319-665-3370; Fax: +1-319-665-3371; E-mail: ronald-jones{at}jmilabs.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Ericsson, H. M. & Sherris, J. C. (1971). Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathologica et Microbiologica Scandinavica Section B, Suppl. 217, 1–90.

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16 . Mesa Espanola de Normalizacion de la Sensibilidad y Resistancia a los Antimicrobianos (MENSURA). (2000). Recomendaciones del Grupo MENSURA para la seleccion de antimicrobianos en el estudio de la sensibilidad y criterios para la interpretacion del antibiograma. Revista Espanola de Quimioterapia 13, 73–86.[Medline]

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