Optimization of the microscopic observation drug susceptibility assay for four first-line drugs using Mycobacterium tuberculosis reference strains and clinical isolates.
OBJECTIVE
The aim of this study is to determine the appropriate cut-off value and turnaround time of the microscopic observation drug susceptibility assay (MODS) for isoniazid (INH), rifampicin (RMP), streptomycin (STR), and ethambutol (EMB).


METHODS
A total of 39 Mycobacterium tuberculosis strains with confirmed drug susceptibility (reference strains) were tested with a range of drug concentrations to determine the optimal cut-off values for INH, RMP, STR, and EMB by MODS. Standard drug susceptibility testing (DST) results were evaluated relative to the Löwenstein-Jensen (L-J) proportion method. Following which, the performance of MODS was evaluated again using 36 sputum samples from patients with tuberculosis (TB) using the cut-off values determined in the aforementioned process.


RESULTS
With 39 reference strains, DST identified the following cut-off values: 0.8μg/ml INH (sensitivity, 96.0%; specificity, 92.9%), 2.0μg/ml RMP (sensitivity, 100%; specificity, 95.5%), 4.0μg/ml STR (sensitivity, 90.5%; specificity, 93.8%), and 4.0μg/ml EMB (sensitivity, 100%; specificity, 91.7%). When these cut-off values were used to analyze the 36 clinical isolates, the sensitivity and specificity of MODS were 100% and 93.1% for INH, 100% and 93.8% for RMP, 87.5% and 96.4% for STR, and 100% and 88.2% for EMB, respectively. The turnaround time for these clinical specimens was 9.0days by MODS (95% CI: 5.3-12.7), compared with 11.7days (95% CI: 9.5-13.9) for smear negative specimens.


CONCLUSIONS
Our study identified the optimal cut-off values of the four first-line drugs for MODS based on a wide concentration range. With the optimal cut-off values determined in this study, MODS showed high discriminatory efficiency for DST. This study also demonstrated that MODS is useful for rapid diagnosis of drug-resistant TB even for a smear negative specimen, despite the fact that it generally uses smear positive specimens as direct DST.
Department of International Cooperation, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama Kiyose-shi, Tokyo 204-8533, Japan; Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama Kiyose-shi, Tokyo 204-8533, Japan. Electronic address: hnishiyama1@gmail.com.
Hiroyuki
H
Nishiyama
Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama Kiyose-shi, Tokyo 204-8533, Japan.
Akio
A
Aono
Department of International Cooperation, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama Kiyose-shi, Tokyo 204-8533, Japan.
Tetsuhiro
T
Sugamoto
Department of Clinical Microbiology, Fukujuji Hospital, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama Kiyose-shi, Tokyo 204-8533, Japan.
Kazue
K
Mizuno
Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama Kiyose-shi, Tokyo 204-8533, Japan.
Kinuyo
K
Chikamatsu
Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama Kiyose-shi, Tokyo 204-8533, Japan.
Hiroyuki
H
Yamada
Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama Kiyose-shi, Tokyo 204-8533, Japan.
Satoshi
S
Mitarai
D000995:Antitubercular Agents / Q000494:pharmacology*; D004977:Ethambutol / Q000494:pharmacology; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D013307:Streptomycin / Q000494:pharmacology; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000382:microbiology*
D000995:Antitubercular Agents; D004977:Ethambutol; D007538:Isoniazid; D012293:Rifampin; D013307:Streptomycin
Drug susceptibility testing; MODS; Smear negative; Turnaround time