Mycobacteriology
Evaluation of microplate Alamar blue assay for drug susceptibility testing of Mycobacterium avium complex isolates

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Abstract

Fifty-one clinical isolates and 5 clarithromycin-resistant mutants of Mycobacterium avium complex (MAC) were tested for their susceptibility to clarithromycin by microplate Alamar blue assay (MABA). The susceptibility results were compared with the results obtained by the BACTEC 460 method. All clinical isolates were susceptible, while all mutants were resistant to clarithromycin by BACTEC. Eighty-six percent of the clinical isolates were susceptible by MABA, and one of the resistant mutants was misclassified as susceptible by this method. The overall agreement between MABA and BACTEC was 86%, indicating the usefulness of MABA in drug susceptibility testing of MAC.

Introduction

Mycobacterium avium complex (MAC), which includes M. avium and M. intracellulare, are ubiquitous in the environment and are the most frequent isolates among nontuberculous mycobacteria found in clinical specimens (Portaels, 1995). In immunocompetent patients with predisposing lung conditions, nontuberculous mycobacteria pulmonary infections are primarily caused by members of MAC (Marras and Daley, 2002). In patients with advanced stages of human immunodeficiency virus (HIV) infection, disseminated MAC infection was shown to be the most common mycobacterial infection prior to MAC prophylaxis and the highly active antiretroviral treatment regimens (Nightingale et al., 1992).

Macrolides (clarithromycin and azithromycin) are the most active drugs against MAC and the effectiveness of these agents in the elimination of pulmonary MAC and MAC bacteremia have been demonstrated in controlled clinical trials (Chaisson et al., 1994, Dautzenberg et al., 1995). All wild-type MAC isolates are susceptible to macrolides, but they develop resistance after a few months of monotherapy or combination therapy (Heifets et al., 1996).

Susceptibility testing is thus necessary for initial isolates to establish baseline minimum inhibitory concentration (MIC) values and for isolates obtained during treatment to detect emergence of resistance. The second edition of the National Committee for Clinical Laboratory Standards (NCCLS) recommends susceptibility testing of clinically significant MAC isolates only for clarithromycin by broth-based macrodilution or microdilution methods (NCCLS, 2000).

In the BACTEC radiometric method, the number of concentrations used is limited because of cost considerations (Siddiqi et al., 1993). The nonradiometric system mycobacterial growth indicator tube (MGIT) is equally expensive for underdeveloped countries (Piersimoni et al., 1998). Other susceptibility testing methods, such as flow cytometry, luciferase reporter phage (LRP) assay, Etest (AB BIODISK, Solna, Sweden), and the macrophage system, have not been used in clinical laboratories for routine testing of the patient's isolates (Bownds et al., 1996, Cooksey et al., 1995, Fabry et al., 1996, Frehel et al., 1997). A molecular method, RNA/RNA duplex mismatch assay, which is reported to be both highly sensitive and specific for detecting mutations in the specific region of 16S and 23S rRNA genes, has been described (Nash and Inderlied, 1996).

However, alternate mechanisms are now shown to be involved in clarithromycin resistance (Jamal et al., 2000). Hence, a reliable and inexpensive method is needed in resource-limited countries for drug susceptibility testing of MAC. Microplate Alamar blue assay (MABA), a colorimetric drug-susceptibility testing method that uses a redox indicator that changes color from blue to pink to indicate bacterial growth, can be read visually without the need for expensive instruments (Collins and Franzblau, 1997). MABA has been used previously for drug-susceptibility testing of M. tuberculosis against first-line antituberculosis drugs and the second-line drug, kanamycin, and has also been used for screening 30 antimicrobial agents against M. tuberculosis and M. avium (Collins and Franzblau, 1997, Franzblau et al., 1998, Bastian et al., 2001).

In India, about one fourth of nontuberculous mycobacteria isolated from sputum samples belong to MAC and were found to be the most common in the environment (Paramasivan et al., 1985, Kamala et al., 1994). However, no data are available on the macrolide susceptibility pattern for these isolates. The present study investigated the reliability of the MABA for susceptibility testing of MAC to clarithromycin by comparing the results with those obtained by the BACTEC radiometric method.

Section snippets

Materials and methods

Fifty-one clinical isolates of MAC obtained from blood (n = 2), sputum (n = 46), stool (n = 1), urine (n = 1), and sternal aspirate (n = 1) were tested. Sixty percent of these isolates were from HIV patients, and nearly 50% of the sputum isolates were repeated excreters who met the American Thoracic Society criteria for clinical significance (Wallace et al., 1997).

All the isolates were identified by mycolic acid analysis by using high-performance liquid chromatography (HPLC) (Butler et al., 1999

Results

Results of MABA for most of the isolates were obtained after 8 days. Clarithromycin-resistant mutants grew very slowly, and the results were available only after 10 days. By the BACTEC method, the susceptibility results were available on day 7 for all the isolates. Figure 1 shows the results of clarithromycin susceptibility obtained by BACTEC and MABA. Of the 56 isolates tested, all the clinical isolates gave a MIC of ≤4.0 μg/mL, and all the resistant mutants gave a MIC of ≥64 μg/mL in BACTEC.

Discussion

An overall agreement of 86% (κ-0.43) was observed in this study between MABA and the BACTEC method. MABA has 86% sensitivity and 80% specificity. Extending the incubation time may increase the specificity of MABA as the mutants grew very slowly. Previous reports showed 93.6% overall agreement between MABA and BACTEC when M. tuberculosis isolates were tested against first-line drugs and 98.6% agreement between MABA and the proportion susceptibility method when M. tuberculosis was tested for

Acknowledgements

The authors thank D. Thangaraj and M. Anandan for technical assistance and Mrs. Fathima Rahaman for statistical analysis. The senior scholarship provided to J. Daisy Vanitha by the Council of Scientific and Industrial Research (CSIR) is gratefully acknowledged.

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