• Aucun résultat trouvé

Evaluation of the susceptibility of pathogenic Candida species to fluconazole

N/A
N/A
Protected

Academic year: 2021

Partager "Evaluation of the susceptibility of pathogenic Candida species to fluconazole"

Copied!
5
0
0

Texte intégral

(1)

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 testing 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 to 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 of Microbiology 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 labor- 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 and 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

(2)

Vol. 16, 1997 Notes 925 t60 [ 146 140 9 124 121 120 - 99 100 * N 8 0 - ~6 Z G9 60 - 1 6 52 1 22 22 2 0 - 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 ( r a m )

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, m e a n of 33.5 mm, m o d e of 35 mm, and stan- dard deviation (SD) from the m e a n of 7.6 mm. One SD from the m e a n 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 a sta- tistical basis for proposing _> 26 m m and < 18 m m as the susceptibility and resistance breakpoints, re- spectively. The preliminary resistance b r e a k p o i n t

of < 18 mm, as defined in the protocol, thus re- mained unchanged. However, the preliminary susceptibility b r e a k p o i n t of -> 30 mm, as defined in the protocol, divided the normal susceptible population near the m e a n 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 d e t e r m i n e d to be suscep- tible, or dose-dependent susceptible, applying in- terpretive criteria based on the p r o p o s e d N C C L S breakpoints. A b o u t 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 H R 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

(3)

926 Notes Eur. J. Clin. Microbiol. Infect. Dis. ~ 6 0 - 45 - 40-~ s 9 C , k r u s e i 3 5 - [ ] O t h e r s 3 0 - 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 i a m e t e r o f i n h i b i t i o n ( r a m )

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 to 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 albicans 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 albicans 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 species 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 of 75% and 54 %, respectively.

Clinical relevance is an important aspect of all drug susceptibility test results. Limited information has been published for any m e t h o d 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 M I C s _> 64 ~tg/ml (and smaller zone diameters) tended to respond

(4)

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 o u t c o m e in A I D S patients.

T h e interpretive guidelines defined in this study p r o t o c o l were preliminary and b a s e d on the lim- ited data available f r o m h u m a n 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). H o w e v e r , clinical efficacy protocols, by design, exclude m a n y patients and pathogens. Consequently, they m a y p r o v i d e a different pat- tern of results t h a n the non-selected sequential p a t h o g e n s tested in this hospital l a b o r a t o r y study which is the first to evaluate a large n u m b e r and wide range of sequential Candida isolates from pa- tients with all types of hospital infections. The fluconazole disk diffusion m e t h o d used in this study p r o v e d to be easy to set up and read, and p r o v i d e d test results that were reasonably repro- ducible in a diverse group of hospital laboratories, as d e t e r m i n e d by isolate and quality control strain performance. In particular, Candida albicans control strain Y01-19 ( A T C C 76615/NCPF 3726) yielded an acceptable p e r f o r m a n c e w h e n testing for small zone diameters. The disk diffusion test a p p e a r s to be a reproducible and accurate m e a n s of assessing the susceptibility of a wide range of yeasts in hospital laboratories (10). Materials are readily available at low cost, it is easy to establish controls, and the m e t h o d is similar to m e t h o d s c o m m o n l y 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 h a e m o c y t o m e t e r inoculum adjustment, and m a k i n g H R m e d i u m . Subsequent to this study, i m p r o v e m e n t s in the disk diffusion test that addressed these technical diffi- culties w e r e evaluated (8); 250 Candida species isolates w e r e t h e r e b y tested in parallel by the standard m a c r o b r o t h dilution assay, fluconazole E test, and 25 gg fluconazole disk diffusion test (using R P M I m e d i u m 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 m e t h o d s 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.

(5)

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 trial 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 testing: 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.

N o s o c o m i a l Outbreak of

Clostridium difficile

Diarrhea

in a Pediatric Service

A . F e r r o n i 1, J. M e r c k x 2, T. A n c e l l e 3, B. P r o n 1, E . A b a c h i n 1, E B a r b u t 4 , J. L a r z u l 1, R R i g a u l t 2, R B e r c h e ], J.-L. G a i l l a r d 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 to 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

diarrhea in pediatric wards (6). We describe an outbreak of diarrhea in a p e -

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.

Figure

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 &lt;  18 mm;  dose-dependent susceptible 18-25  mm;
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

Références

Documents relatifs

The unitary maps ϕ 12 will be very useful in the study of the diagonal action of U(2) on triples of Lagrangian subspaces of C 2 (see section 3).. 2.4

Multicenter com- parison of the etest and EUCAST methods for antifungal susceptibility testing of Candida isolates to micafungin... 1 Multicenter comparison of the Etest® and

My study will address this gap in the literature by looking at ethics consults that have been done by an ethics service for a health authority that includes acute care and

This was done by comparing the survival rates and tissue burdens for mice infected with CaENA21 transformants and control strains (Fig. This suggests that traits in addition to

To this aim, (1) the parameters of species’ responses to climatic variables were determined, in two types of models: specific statistical models and the generic

• Rare, la plupart des dyskinésies paroxystiques symptomatiques sont en effet liées à des.

In the present paper, we try to overcome the shortcomings associated with the use of multiple bibliometric measures by proposing a procedure to evaluate a global

Susceptibility to cell wall-perturbing drugs of Candida glabrata wild-type isolate 21231 or clinical isolates 21229 and 21230 and their complemented counterparts, isolates 21229C