A fluconazole 25 ug disk diffusion test was used to test 2230 consecutively isolatedCandida strains from 42 different hospital laboratories in 23 countries. Ninety seven percent of 1634Candida albicans isolates and 83.4% of 596 non-Candida albicans isolates were susceptible to fluconazole, applying the proposed breakpoints (≥26 mm for susceptible strains and 18-25 mm for dosedependent susceptible strains). This is the first hospital laboratory study to evaluate a large number and wide range of sequentialCandida isolates from patients with all types of hospital infections. The fluconazole disk diffusion test appears to be a low-cost, reproducible, and accurate means of assessing the in vitro susceptibility ofCandida isolate

Bille, J.

Glauser, M.

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924 Notes Eur. J. Clin. Microbiol. Infect. Dis. Evaluation of the Susceptibility of Pathogenic Candida Species to Fluconazole J. Bille 1., M. E Glauser 2, and the Fluconazole Global Susceptibility Study Group A fluconazole 25 IJg disk diffusion test was used to test 2230 consecutively isolated Candida strains from 42 different hospital laboratories in 23 coun- tries. Ninety seven percent of 1634 Candida atbi- cans isolates and 83.4% of 596 non-Candida albi- cans isolates were susceptible to fluconazole, applying the proposed breakpoints (_>26 mm for susceptible strains and 18-25 mm for dose- dependent susceptible strains). This is the first hos- pital laboratory study to evaluate a large number and wide range of sequential Candida isolates from patients with all types of hospital infections. The fluconazole disk diffusion test appears to be a low-cost, reproducible, and accurate means of assessing the in vitro susceptibility of Candida iso- lates. The lack of a reliable standardised antifungal sus- ceptibility test method has complicated the inter- pretation of many study reports until recently. However, significant progress has been made in the past ten years in developing more practical methods for the susceptibility esting of yeasts. Efforts of the National Committee for Clinical Laboratory Standards (NCCLS) have focused on developing a reference macrobroth minimum in- hibitory concentration (MIC) method for testing yeast species (1). Work is still ongoing to improve that assay, which is relatively difficult o perform and read, particularly when large numbers of iso- lates are tested. However, it does provide a refer- ence for comparison with other methods. Micro- dilution plate methods have been developed that 1Institute ofMicrobiology and 2 Division of Infectious Disease, Department of Internal Medicine, Centre Hospitalier Univer- sitaire Vaudois, Rue du Bugnon 44, CH-1011 Lausanne, Swit- zerland. produce results comparable to those of the NCCLS method and are easier to perform and read (2-5). However, the reading of endpoints still poses problems in both the macrobroth and mi- crobroth assays. Agar gradient methods have also been devel- oped that appear to provide reproducible, accu- rate, and more easily read results. These include the E test (6) and disk diffusion test (7, 8). These assays are more practical for use in clinical abor- atories. In both the E test and the disk diffusion test, results are reproducible, the tests are easy to set up, they have adequate controls, and the results are read on a precise quantitative scale. The disk diffusion test has the advantages of lower mate- rial costs, and relatively easy to read results. Growth inhibition zone diameters in the disk diffusion test also correlate well with the NCCLS reference method MICs, and with the MICs deter- mined in the E test assay (8, 9). The purpose of this study was to evaluate the sus- ceptibility to fluconazole of a large population of pathogenic Candida isolates from a wide range of hospitals and countries using the only standardised disk diffusion test method available at the time the study was started (Unipath, Pfizer, UK). Materials and Methods. Forty-two hospital labor- atories used the agar disk diffusion test (Unipath) to test approximately 50 consecutively isolated yeast strains each, all strains being considered pathogens in the patients from whom they were obtained. A total of 2230 isolates from 23 countries were tested. Each yeast isolate was from a differ- ent patient; isolates from all types of infections and body sites were included in the study. Data on the mycological nd clinical response of fluconazole- treated patients were collected when possible. In- formation on the duration of therapy, dose of flu- conazole, concomitant medication, primary diag- nosis, underlying diseases, and other data usually collected in clinical trials was not considered relevant in this laboratory study. Three quality control strains of Candida albicans (Y01-09, Y01-108, Y01-19) were included with each batch of yeast isolates tested. The agar diffusion test was performed exactly as described by the original manufacturer (data on site, Pfizer, USA) using 25 ~tg fluconazole disks on 4 mm deep High Resolu- tion (HR) agar medium in 90 mm petri plates. The yeast inoculum was prepared by adjusting the den- sity of yeast cells in a normal saline suspension, using a microscope and haemocytometer, to ap- proximately 20 cells/mm 2 in the grid.The final cell density was adjusted to 4 x 107 cells/ml. After Vol. 16, 1997 Notes 925 t60 [ 146 140 9 124 121 120 - 99 100 * N 80- ~6 Z G9 60 - 1 6 52 1 22 22 20- 16 12 9 9 9 6 $ 6 T $ 0-  6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 57 59 60 Diameter of inhibition ( ram) Figure 1: Susceptibility to fluconazole of 1634 isolates of Candida albicans as determined by the disk diffusion test on HR medium incubated at 28~ The proposed breakpoints are: resistant < 18 mm; dose-dependent susceptible 18-25 mm; sus- ceptible >- 26 mm. incubation for 24 h at 28~ the diameter of the zone of inhibition of yeast growth around the flu- conazole disks was measured and recorded. Quality control strains were tested each day the clinical isolates were tested and results recorded; the diameter of their inhibition zones had to fall within the ranges specified. Because the MICs were not systematically deter- mined for these isolates, MIC correlates cannot be derived from these data. The proposed break- points for the disk diffusion test are deducted from statistical analysis of the inhibition zone diameters measured. Results and Discussion. The results for 1634 Candida albicans isolates are shown in Figure 1. They indicate that 97.1% of the Candida albicans isolates were susceptible, or dose-dependent sus- ceptible, to fluconazole using a cut-off value of _> 18 mm. The isolates displayed a normal bell- shaped distribution with a normality value of 0.96, mean of 33.5 mm, mode of 35 mm, and stan- dard deviation (SD) from the mean of 7.6 mm. One SD from the mean included zone diameters from 26 to 41 mm, and two SDs included zone dia- meters from 18 to 49 mm; 95% of test results were between two SDs. These SD values provided asta- tistical basis for proposing _> 26 mm and < 18 mm as the susceptibility and resistance breakpoints, re- spectively. The preliminary resistance breakpoint of < 18 mm, as defined in the protocol, thus re- mained unchanged. However, the preliminary susceptibility breakpoint of -> 30 mm, as defined in the protocol, divided the normal susceptible population ear the mean of the zone diameter measurements. The proposed breakpoints separ- ated the susceptible population from the more re- sistant population, i. e. those falling outside of two SD and representing 2.9% of 1634 isolates. Thus, 97.1% of isolates were determined to be suscep- tible, or dose-dependent susceptible, applying in- terpretive criteria based on the proposed NCCLS breakpoints. About 8.4 % of the Candida albicans isolates was categorised as being dose-dependent susceptible, i.e. the isolates were susceptible to doses greater than 100 mg fluconazole daily. The susceptibility to fluconazole of 596 isolates of other Candida species is shown in Figure 2. As most of these isolates showed growth rates comparable to those of Candida albicans on HR medium, these tests were read after 24 hours of incubation at 28~ When the criteria developed for Candida albicans were applied to these isolates, 83.6 % were determined to be susceptible or dose-dependent susceptible. Only 16.9% were categorised as dose-dependent susceptible with inhibition zones between 18 and 25 mm; these isolates were susceptible to doses greater than 100 mg fluconazole daily, except for Candida 926 Notes Eur. J. Clin. Microbiol. Infect. Dis. ~60-  45 - 40-~ s  9 C ,k ruse i  35-  [ ]  Others  30-  6 7 8 _ _ _ [ - -  9 1{) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 3{) 4{) >40 D iameter  o f  inh ib i t ion  ( ram)  Figure 2: Susceptibility to fluconazole of 596 Candida species other than Candida albicans including 139 isolates of Candi- da glabrata, 55 isolates of Candida krusei, and 402 isolates of other Candida species as follows: C. tropicalis (197; 6 resist- ant), C. parapsilosis (123; 2 resistant), C. guillermondii (22; 1 resistant), C. inconspicua (13; 1 resistant), C. famata (7; none resistant), C. humicola (6; none resistant), C. lusitaniae (6; 2 resistant), C. parakrusei (6; none resistant), others (22; 4 resist- ant). For method and breakpoints see legend to Figure 1. krusei isolates which were considered resis- tant to fluconazole regardless of the in vitro test result. Of the non-Candida albicans isolates, 32.6% (194/596) were Candida krusei or Candida glab- rata. Although only 16% (98/596) of all non-Can- dida albicans isolates were categorised as being re- sistant o fluconazole, 44% (24/55) of the Candi- da krusei isolates and 40% (56/139) of the Candida glabrata isolates were resistant (Figure 2). Thus, these two species represented 82% (80/98) of all fluconazole-resistant non-Candida lbicans isolates. Of 101 non-Candida albicans isolates categorised as dose-dependent susceptible, 17% were Candida krusei and 32% Candida gtabrata. Of the remaining 397 non-Candida lbicans iso- lates categorised as susceptible, only 3.5% were Candida krusei and 13% Candida glabrata. Thus the disk diffusion test breakpoints proposed for Candida albicans also appear to apply to these non-albicans pecies of Candida, although clinical studies and clinical experience with these isolates have been much less extensive than with Candi- da albicans (10). Candida species that do not pro- duce readily visible growth within the 24 hour in- cubation period should, for the meantime, be tested by a different method. Of the three Candida albicans quality control strains, Candida albicans Y01-19 (NCPF 3726) demonstrated the best reproducibility, 94% (163/174) of the inhibition zone diameter measure- ments falling between 15 and 21 mm. The two oth- er quality control strains, Y01-108 and Y01-09, yielded a wider range of inhibition zone diameter measurements, with a reproducibility of75% and 54 %, respectively. Clinical relevance is an important aspect of all drug susceptibility est results. Limited information has been published for any method on the corre- lation between clinical or mycological responses and test results (7, 9, 11). Rex et al. (9, 11, 12) re- ported that a strong correlation existed between clinical outcome and fluconazole MIC using the NCCLS M27-T broth method to test Candida iso- lates, and that higher doses could be used against isolates with higher MICs. Isolates with MICs < 64 ~g/ml tended to respond well to fluconazole therapy, whereas those with MICs _> 64 ~tg/ml (and smaller zone diameters) tended to respond Vol. 16, 1997 Notes 927 poorly. Troillet et al. (7) also showed good corre- lation between zone diameter results in the flucon- azole disk diffusion test and the clinical and my- cological outcome in AIDS patients. The interpretive guidelines defined in this study protocol were preliminary and based on the lim- ited data available from human and animal effi- cacy studies of fluconazole at the time the proto- col was written. Since then, test results for isolates in rigorously controlled clinical protocols with strict patient entry and exclusion criteria have pro- vided data consistent with the results of this study (9,12). However, clinical efficacy protocols, by design, exclude many patients and pathogens. Consequently, they may provide a different pat- tern of results than the non-selected sequential pathogens tested in this hospital aboratory study which is the first to evaluate a large number and wide range of sequential Candida isolates from pa- tients with all types of hospital infections. The fluconazole disk diffusion method used in this study proved to be easy to set up and read, and provided test results that were reasonably repro- ducible in a diverse group of hospital laboratories, as determined by isolate and quality control strain performance. In particular, Candida albicans control strain Y01-19 (ATCC 76615/NCPF 3726) yielded an acceptable performance when testing for small zone diameters. The disk diffusion test appears to be a reproducible and accurate means of assessing the susceptibility of a wide range of yeasts in hospital aboratories (10). Materials are readily available at low cost, it is easy to establish controls, and the method is similar to methods commonly used to test bacterial isolates. Techni- cal difficulties were experienced in conjunction with the 28~ incubation temperature, which is un- usual for hospital incubators, the haemocytometer inoculum adjustment, and making HR medium. Subsequent to this study, improvements in the disk diffusion test that addressed these technical diffi- culties were evaluated (8); 250 Candida species isolates were thereby tested in parallel by the standard macrobroth dilution assay, fluconazole E test, and 25 gg fluconazole disk diffusion test (using RPMI  medium with 2% glucose, swab inoculation of an inoculum of 0.5-2.5 x 103 cfu/ml, incubation at 35~ for 24 and 48 h). All three methods were in excellent agreement. Acknowledgement Members of the Fluconazole Global Susceptibility Study Group include: Dr. R. Negroni, Buenos Aires, Argentina; Dr. D. Ellis, Adelaide, Australia; Dr. F. A. Tosolini, Melbourne, Aus- tralia; Dr. Ch. Devroey, Brussels, Belgium; Dr. S. Barsanti Wey, Sao Paulo, Brazil; Dr. A. Campos Pignatari, Sao Paulo, Brazil; Dr. M. Miller, Montreal, Canada; Dr. A. Clark, Vancouver, Ca- nada; Prof. W. Shaoxu, Nanjing, China; Prof. L. Wang, Shang- hai, China; Dr. J. Moiler, Aarhus, Denmark; Prof. B. Couprie, Bordeaux, France; Prof. J. Wailer, Strasbourg, France; Prof. D. Cliabasse, Angers, France; Prof. D. Milatovic, Munich, Germa- ny; Dr. H. J. Tietz, Berlin, Germany; Dr. H. Dermoumi, Essen, Germany; Dr. U. Banerjee, New Delhi, India; Dr. R. Kelkar, Bombay, India; Dr. M.S. Mathews; Vellore, India; Prof. A. Mazzoni, Bologna, Italy; Prof. M. T. Montagna, Bari, Italy; Dr. S. Conti, Parma, Italy; Dr. S. Asari, Osaka, Japan; Prof. C.H. Pai, Seoul, South Korea; Dr. J. Ignacio Santos, Mexico, Mexico; Dr. J.W. van't Wout and Dr. H.EL. Guiot, Leiden, The Netherlands; Dr. E. Lingaas, Oslo, Norway; Dr. D. Dzierzanowska, Warsaw, Poland; Dr. G. Quindos, Vizcayo, Spain; Dr. J. Torres Rodriguez, Barcelona, Spain; Dr. M. Pereiro Miguens, La Corufia, Spain; Dr. R Wretlind, Damderyd, Sweden; Dr. R. Auckenthaler, Ge- neva, Switzerland; Dr. W. C. Hsieh and Dr. S. C. Chang,Taipei, Taiwan; Dr. M. Richardson, Dr. L. Milne, and Dr. L. Ganguli, Salford, UK; and Dr. M. Rinaldi, San Antonio, USA. References 1. National Committee for Clinical Laboratory Standards: Reference method for broth dilution antifungal suscep- tibility testing of yeast. Tentative standard M27-T. NCCLS, Villanova, PA, 1995. 2. Polanco AM, Rodriguez-Tudela JL, Baquero F, San- chez-Sousa A, Martinez-Suarez JV: Improved method of determining the susceptibility of Candida albicans to flu- conazole. Journal of Antimicrobial Chemotherapy 1995, 35: 155-159. 3. Fournier C, Gaspar A, Boillot F, Villard J: Evaluation of a broth microdilution antifungal susceptibility test with a pH indicator: comparison with the broth macrodilution pro- cedures. Journal of Antimicrobial Chemotherapy 1995, 35: 373-380. 4. Rodriguez-Tudela JL, Martinez-Suarez JV: Defining conditions for microbroth antifungal susceptibility tests: influence of RPMI-2% glucose on the selection of end- point criteria. Journal of Antimicrobial Chemotherapy 1995, 35: 739-749. 5. Espinel-lngroff A, Rodriguez-Tudela JL, Martinez-Suarez JV: Comparison of two microdilution procedures with the proposed NCCLS reference macro-dilution method M27-P for in vitro testing of fluconazole resistant and sus- ceptible isolates of Candida albicans. Journal of Clinical Microbiology 1995, 33:3154-3158. 6. Colombo J, Barchiesi F, McGough DA, Fotherfill AW, Ri- naldi MG: Evaluation of the E-test system versus a mi- crotitre broth method for antifungal susceptibility testing of yeast against fluconazole and itraconazole. Antimicro- bial Agents and Chemotherapy 1995, 36:93-100. 7. Troillet N, Durussel C, Bille J, Glauser MR Chave JP: Cor- relation between in vitro susceptibility of Candida albi- cans and fluconazole resistant oropharyngeal candidia- sis in HIV-infected patients. European Journal of Clini- cal Microbiology & Infectious Diseases 1993, 12: 911-915. 928 Notes Eur. J. Clin. Microbiol. Infect. Dis. 8. Barry A, Brown S: Fluconazole disk diffusion procedure for determining susceptibility of Candida species. Jour- nal of Clinical Microbiology 1996, 34:2154-2157. 9. Rex J, Pfaller MA, Barry AL, Nelson PW, Webb CD: An- tifungal susceptibility testing of isolates from a random- ized multicenter t ial of fluconazole versus amphotericin B as treatment of non-neutropenic patients with candi- demia. Antimicrobial Agents and Chemotherapy 1995, 39: 40-44. 10. van't Wout JW: Fluconazole treatment of candidal infec- tions caused by non-albicans Candida species. European Journal of Clinical Microbiology & Infectious Diseases 1996, 15: 238-242. 11. Ghannoum M, Rex JH, Galgiani J: Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome. Journal of Clinical Microbiology 1996, 34: 489-495. 12. Rex J, Pfaller MA, Galgiani JN, Bartlett MS, Espinei- Ingroff A, Ghannoum MA, Lancaster M, Odds FC, Rinai- di MG, Walsh T J, Barry A: Development of interpretive breakpoints for antifungal susceptibility esting: concep- tual framework and analysis of in vitro - in vivo correla- tion data for fluconazole, itraconazole, and Candida in- fections. Clinical Infectious Diseases 1997, 24: 235-247. Nosocomial Outbreak of Clostridium difficile Diarrhea in a Pediatric Service A.  Fer ron i  1, J. Merckx  2, T. Ance l le  3, B. P ron  1, E.  Abach in  1, E Barbut  4 , J. Larzu l  1, R R igau l t  2, R Berche  ], J.-L. Ga i l l a rd  1. An outbreak of nosocomial diarrhea that occurred in a pediatric orthopedic service between 1 Decem- ber 1993 and 15 April 1994 is reported. A total of 37 patients (mean age, 9.6 years; range, 2 months-19.3 years) were involved in the outbreak, including six patients with bacteriologically docu- mented Clostridium difficile infection. A multivari- ate analysis identified lincomycin treatment for at least three days as the only significant risk factor. Stool samples from four asymptomatic patients were also positive for Clostridium difficile and its cy- totoxins. Isolates from all patients belonged to se- rogroup C, were highly resistant o lincomycin, and exhibited the same restriction pattern by pulsed- field gel electrophoresis. The outbreak ended after treatment with lincomycin was discontinued and hygiene control measures were implemented. Clostridium difficile is the most common agent of pseudomembranous colitis and antibiotic-asso- ciated diarrhea (1). It has been implicated in many outbreaks of diarrhea affecting hospitalized adult patients (2, 3). Hospital acquisition of Clos- tridium difficile has also been demonstrated in a pediatric population (4, 5). However, there have been few reports and no epidemiological investi- gations of nosocomial outbreaks of Clostridium difficile-associated iarrhea in pediatric wards (6). We describe an outbreak of diarrhea in ape- 1 Laboratoire de Microbiologie, and 2Service de Chirurgie Orthop6dique Infantile, H6pital Necker-Enfants Malades, 149 rue de S~vres, 75743 Paris Cedex 15, France. 3 Service de Parasitologie, H6pital Cochin, 75014 Paris, France. 4 Laboratoire de Microbiologie, HSpital Saint Antoine, 75012 Paris. France. 

Evaluation of the susceptibility of pathogenic Candida species to fluconazole

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Evaluation of the susceptibility of pathogenic Candida species to fluconazole

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