Evaluation of the Etest for rapid susceptibility testing of ...

Journal of Antimicrobial Chemotherapy (1996) 37, 999-1003

Evaluation of the Etest for rapid susceptibility testing of Mycobacterium avium to clarithromycin Lia Lebrun', Catherine Onody*, V6ronkjue Vincent* and Patrice Nordmann'

"Service de Bacteriologie-Virologie, Hopital Antoine Beclere, Clamart, Faculte de Medecine Paris-Sud 157 rue de la Porte de Trivaux, 92141 Clamart Cedex; b Centre National de Reference des Mycobacteries, Institut Pasteur, 75015 Paris, France MICs of clarithromycin were determined by the Etest method for thirty clinical strains of Mycobacterium avium complex (MAC) and compared with MICs results as determined by the reference agar dilution method. Agreement (within ± 1 I0& dilution) between the Etest and the reference method was 70% for susceptible strains and 100% for resistant strains. No major errors resulting in misclassification in susceptibility or resistance categories were detected for the Etest MIC method. It is suggested that the Etest is easy to perform and is an accurate method for determining susceptibility of MAC strains to clarithromycin.

Introduction

MAC is widely recognized as an opportunistic pathogen in AIDS-infected patients. Clarithromycin which is a new macrolide, possesses high in-vitro activity against MAC. However, monotherapy lead to a partial clearance of bacterial infection followed by a relapse associated with selection of drug-resistant strains (Chaisson et al., 1994). Such clarithromycin-resistant strains have been also reported in patients treated with clarithromycin in combination with one or more antimycobacterial drugs (Dautzenberg et al., 1993). Therefore, there is a need for MIC determination of clarithromycin in MAC in order to make the best antibiotherapy choice. Susceptibility testing has recently been standardized with the Bactec radiometric method (Heifets, Lindholm-Levy & Comstock, 1992). This method takes only 6-8 days to be performed as compared to conventional techniques used for Mycobacterium tuberculosis. However, its high cost limits its use in a clinical laboratory. Therefore, the purpose of this study was to evaluate the Etest, a technique for MIC determination based on a pre-defined antibiotic gradient on a plastic strip which gives, after its diffusion, a continuous logarithmic MIC scale covering 15 two-fold dilutions (AB, Biodisk, Solna, Sweden). This method accurately determines MICs of numerous antimicrobials with a wide variety of microorganisms including those which are fastidious such as meningococcus and rapidly-growing mycobacteria (Abadi, Yakubu & Pennington, 1995; Biehle et al., 1995; Hoffner et al., 1994; Koontz et al., 1994; Wanger & Mills, 1994). To our Tel: +33 1 45 37 46 29; Fax: +33 1 46 32 67 96. 999 0305-7453/96/050999 + 05 $12.00/0

© 1996 The British Society for Antimicrobial Chemotherapy

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knowledge, there are no reports of the Etest for susceptibility testing of clarithromycin for MAC.

Materials and methods

Test strains and preparation of mycobacterial inoculum Thirty strains isolated from blood of AIDS-infected patients were selected. Strains were identified as MAC by using the Accuprobe culture confirmation kits (Gen Probe, San Diego, California) as previously described (Lebrun et al., 1992). Strains were firstly subcultured into 12B vials (Becton Dickinson, Le Pont de Claix, France). Vials were incubated at 37°C, tested daily on the Bactec 460 instrument to reach a growth index (GI) of 999 and then used for determine MICs by the radiometric Bactec method. Then, 12B vials were stored at ambient temperatures in air until reference agar dilution and Etest MICs were performed. Prior to testing, 0.1 mL of the first subculture was inoculated into another 12B vial and incubated at 37°C. Vials were tested on the Bactec 460 instrument and used at a 1:10 dilution when the GI reached 999 to inoculate Mueller-Hinton agar plates (BioMerieux, La Balme-les-Grottes, France) supplemented with 10% oleic acid-albumin-dextrose complex (OADC) (Difco Laboratories Detroit, MI, USA). For an inoculum to be considered satisfactory, inoculated 12B vials should have reached a GI of 999 within 48-72 h. In these conditions, the seeded vials contained approximately 107 cfu/mL as determined by serial ten-fold dilutions plating of three different strains on to Middlebrook 7H10 agar plates (Difco Laboratories). The inoculum size for agar dilution and Etest was approximately 10*cfu/mL.

Susceptibility testing Susceptibility testing to clarithromycin was firstly determined by the Bactec radiometric method using clarithromycin dilutions from 0.5 to 256 mg/L. Clarithromycin (Abbott Laboratories, Rungis, France), was dissolved in methanol and then diluted in phosphate buffer (pH 7.2). The MIC was determined as the lowest drug concentration that inhibited greater than 99% of the bacterial population within 8 days of culture as described by Heifets et al. (1992) i.e. resulting from comparison of GI values of a vial containing 1% of bacterial population to GI values of vials containing drug and 100% Table. Concordance of the Etest and agar dilution methods for clarithromycin MIC determination of thirty MAC strains

Strain category* (number) Susceptible (20) Resistant (10)

Number of Etest MICs within the indicated 1O& concentration of reference agar dilution MICs (% agreement) —2 —1 0 +1 +2 6 (30%) 0

11 (55%) 0

3 (15%) 10 (100%)

0

0

0

0

Agreement within ± 1 logj concentration 14/20 (70%) 10/10 (100%)

"Strains were previously classified as susceptible or resistant to clarithromycin according to the radiometric method result*.


Etest for clarithromycin MIC in M. avium

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of bacterial population. Twenty MAC isolates were categorized as susceptible (MIC 16 mg/L) according to the radiometric method results (Heifets, Mor & Vanderkolk, 1993). Then, isolates were further tested by the Etest method and the reference agar dilution method, with the same inoculum, onto 10% OADC-supplemented Mueller-Hinton agar at pH 7.4. For the reference agar dilution method, the inhibitory activity of clarithromycin was tested in two-fold serial dilutions ranging from 0.5 to 256 mg/L. 0.1 mL of the 1:10 diluted broth seeded vial culture was inoculated onto each plate. Plates were read after 8-10 days of incubation in plastic bags at 37°C in a 5% CO2 containing atmosphere (Generbag, BioMerieux). The MIC was denned as the lowest drug concentration of clarithromycin which inhibited visible bacterial growth as compared to the drug-free control plate. As suggested by Heifets et al. (1993), a MIC ^ 8 mg/L was considered as the breakpoint for determining susceptibility of MAC to clarithromycin and < 64 mg/L for determining resistance. For the Etest method, agar plates were flooded with the same inoculum and pre-incubated for 18 h at 37°C in a 5% CO2 containing atmosphere before putting the Etest strips which contained a gradient of clarithromycin ranging from 0.016 to 256 mg/L on the plates. Plates were then incubated in plastic bags at 37°C in a 5% CO2 containing atmosphere until growth was visible and the MIC was deduced after five to six days of incubation at the point at which the ellipse of inhibition crossed the strip. Results and discussion Concordance results between the Etest results and the reference agar dilution testing results is shown in the Table. Total concordance ranged from 15% for susceptible strains to 100% for resistant strains. For susceptible strains, concordance was 55% and 30% respectively within ± 1 Iog2 and ±21og2 dilutions. No discrepancies of > 2 logj dilutions were seen. Etest MIC values that differed were always lower than the reference MIC method values. MICs of clarithromycin ranged from 2 to 4 mg/L (mean: 3.0 ± 1.06) and 0.5-1.5 mg/L (mean: 0.96 ± 0.36) respectively for the reference agar dilution method and the Etest method. Agreement within ± 1 I0& dilutions was 70% for susceptible strains (14/20) and 100% (10/10) for resistant strains. Overall, the Etest method resulted in no major errors, all strains classified as susceptible ( ^ 8 mg/L) or resistant (;> 64 mg/L) with the reference agar method were classified into the same categories with the Etest method. An example of clarithromycin MIC determined by the Etest technique for a clarithromycin susceptible MAC isolate is given in the Figure. All resistant MAC isolates had a MIC ;> 128 mg/L either with the reference agar method or the Etest method. All these resistant strains were isolated from clarithromycin treated patients. As noted by Heifets et al. (1993) no resistant strain gave an intermediate MIC value by the radiometric broth method, or by the reference agar dilution and the Etest methods. Clarithromycin-resistant MAC will remain a therapeutic challenge in AIDS-infected patients (Chaisson et al., 1994). Therapy must be based on knowledge of in-vitro drug susceptibility test results and therefore requires a rapid and standardized method. The reference agar dilution method is time-consuming to prepare drug-containing plates. The radiometric method is expensive for testing several drug concentrations and requires radio-isotope use. Once subcultured into liquid media MAC strains grow better


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Figure. Clarithromycin MIC determination of clarithromycin susceptible MAC isolate with the Etest method.

on solid media. Thus, in our conditions of subculture into I2B vials for standardizing the inoculum preparation, all isolates produced adequate growth for interpretation of MICs within 5-6 days of incubation. However, in our study, plates were not inoculated by swabbing as suggested by the manufacturer but by flooding to obtain an homogeneous inoculum. Moreover, before placing the Etest strip on the plates, a pre-incubation time of 18 h was necessary to obtain a clear Etest ellipse. Compared with agar dilution and radiometric broth dilution methods, the Etest is much easier to perform and less expensive than the radiometric broth method. This method has been already used for successfully studying MIC of ciprofloxacin for MAC (Wanger & Mills. 1994). Our data suggest that the Etest method may also be used for determining clarithromycin susceptibility of MAC isolates from an increasing number of clarithromycin treated-patients. References Abadi. F. J. R.. Yakubu. D. E. & Pennington, T. H. (1995). Antimicrobial susceptibility of penicillin-sensitive and penicillin-resistant meningococci. Journal of Antimicrobial Chemotherapy 35, 687-90. Biehle. J. R.. Cavalieri. S. J.. Saubolle. M. A. & Getsinger. L. J. (1995). Evaluation of Etest for susceptibility testine of rapidly growing mycobacteria. Journal of Clinical Microbiology 33, 1760-4. Chaisson. R. E.. Benson. C. A.. Dube. M. P.. Heifets. L. B.. Korvick. J. A.. Elkin. S. et al. (1994). Clarithromycin therapy for bacteremic Mvcobacterium avium complex disease. Annals of Internal

Medicine

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Dautzenbcrg, B., Saint Marc, T., Meyohas, M. C , Eliaszewitch, M., Haniez, F., Rogues, A. M. et al. (1993). Clarithromycin and other antimicrobial agents in the treatment of disseminated Mycobacterium avium infections in patients with acquired immunodeficiency syndrome. Archives of Internal Medicine 153, 368-72. Heifets, L. B., Lindholm-Levy, P. J. & Comstock, R. D. (1992). Clarithromycin minimal inhibitory and bactericidal concentrations against Mycobacterium avium. American Review of Respiratory Diseases 145, 856-8. Heifets, L., Mor, N. & Vanderkolk, J. (1993). Mycobacterium avium strains resistant to clarithromycin and azithromycin. Antimicrobial Agents and Chemotherapy 37, 2364-70. Hoffner, S. E., KJintz, L., Olsson-Liljequist, B. & Bolmstrom, A. (1994). Evaluation of Etest for rapid susceptibility testing of Mycobacterium chelonae and M.fortuitum. Journal of Clinical Microbiology 32, 1846-9. Koontz, F. P., Erwin, M. E., Barrett, M. S. & Jones, R. N. (1994). Etest for routine clinical antimicrobial susceptibility testing of rapid-growing mycobacteria isolates. Diagnostic in Microbiology and Infectious Diseases 19, 183-6. Lebrun, L., Espinasse, F., Poveda, J. D. & Vincent-Levy-Frebault, V. (1992). Evaluation of non-radioactive DNA probes for identification of mycobacteria. Journal of Clinical Microbiology 30, 2476-8. Wanger, A. & Mills, K. (1994). Etest for susceptibility testing of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare. Diagnostic Microbiology and Infectious Diseases 19,

179-81. (Received 3 October 1995; returned 20 November 1995; revised 2 January 1996; accepted 5 February 1996)
