Abstract
BackgroundEarly detection of drug resistance is one of the priorities of tuberculosis (TB) control programs as drug resistance is increasing. New molecular assays are only accessible for a minority of the second line drugs and their availability in high endemic settings is also hampered by high cost and logistic challenges. Therefore, we evaluated a previously developed method for drug susceptibility testing (DST) including both first- and second line anti-TB drugs for use in high endemic areas.ResultsBaseline mycobacterial isolates from 78 consecutive pulmonary TB patients from Addis Ababa, Ethiopia who were culture positive for Mycobacterium tuberculosis at the end of a two-month directly observed treatment short course (DOTS) were included. The isolates were simultaneously tested for isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide and para-aminosalicylic acid susceptibility using the indirect proportion method adopted for 24-well agar plates containing Middlebrook 7H10 medium. Applying the 24-well plate assay, 43 (55.1%) isolates were resistant to one or more of the first line drugs tested (isoniazid, rifampicin and ethambutol). MDR-TB was identified in 20.5% of this selected group and there was a perfect correlation for rifampicin resistance with the results from the genotype MTBDRplus assay. All isolates were susceptible to aminoglycosides and fluoroquinolones in agreement with the genotype MTBDRsl assay. The only tested second line drug associated to resistance was ethionamide (14.1% resistant). The method was reproducible with stable results for internal controls (one multi-drug resistant (MDR) and one pan-susceptible strain (H37Rv) and DST results could be reported at two weeks.ConclusionsThe 24-well plate method for simultaneous DST for first- and second line drugs was found to be reproducible and correlated well to molecular drug susceptibility tests. It is likely to be useful in high-endemic areas for surveillance as well as for the detection of second line drug resistance in targeted groups such as in those who fail empirical MDR treatment.
Highlights
Detection of drug resistance is one of the priorities of tuberculosis (TB) control programs as drug resistance is increasing
All patients Mycobacterial culture and typing The Mycobacterial isolates were obtained by sputum culture processed according to standard methods [16] on LJ media (Sigma-Aldrich Chemical Co.) and were confirmed as Mycobacterium tuberculosis (Mtb) on DNA isolated from heat killed, culture positive samples using RD9 typing which relies on analysis of species specific genomic deletions [17] and spoligotyping
Considering the multi-drug resistant (MDR) clinical strain used as a control, there was a complete agreement in the MIC level of isonicotinic acid hydrazide (INH), AMK and OFL and drug susceptibility results in each test round (n = 11) including STM, ETH, CAP, AMK, p-aminosalicylic acid (PAS), KAN and EMB (Table 1)
Summary
Detection of drug resistance is one of the priorities of tuberculosis (TB) control programs as drug resistance is increasing. Various DST methods have been developed including phenotypic testing based on the growth of the bacilli on drug containing solid or liquid medium [5] and genotypic detection of resistance determining genes [6,7,8]. Molecular approaches such as line probe assays and the GeneXpert MTB/ RIF (Cepheid, Inc. USA) present a significant advantage mainly in terms of rapid diagnosis of MDR-TB and have been recommended as initial diagnostic tests in individuals at risk [7,9]. Conventional broth- and agar-based methods have been validated against clinical outcome at least for the first line drugs and are established for most second line drugs used the breakpoints to predict clinical susceptibility for some of those drugs are not as well characterized [10]
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