Rifampin Stability In 7H9 Broth And L(?)wenstein-Jensen Medium

The objectives of this study were to monitor the stability of rifampin (RIF) in Lowenstein-Jensen medium (L-J medium) and 7H9 broth, in order to confirm whether or not drug decay in media was one of the reasons for incorrect drug susceptibility testing (DST) outcome of Mycobacterium tuberculosis. Rifampin degradation in stock solution, 7H9 broth, L-J medium and the inspissation process of L-J medium preparation was serially monitored by high-performance liquid chromatography (HPLC). DST was carried out on media stored at 4℃for different periods of time in order to verify the impact of RIF decay on DST outcomes. Our results showed that: 1) The RIF stock solution was stable for at least 3 months when kept at either 4°C or -20°C; 2) RIF in 7H9 broth and L-J medium was almost 50% decayed after 1 week of storage at 37°C, and rifampin could not be detected in L-J medium or 7H9 after 3 weeks or 6 weeks of storage at 37°C respectively. 3) As storage time increased,RIF concentration in L-J medium and 7H9 medium showed a clear downward trend when stored at 4℃. After 6 months storage,RIF in L-J medium was undetectable while 38% of RIF remained in 7H9 medium compared with fresh medium.4) During the inspissation process for L-J medium preparation, approximately 21%, 24%, 29%, and 35% RIF degradations were detected when the L-J medium was coagulated at 75°C, 80°C, 85°C, and 90°C for 50 min respectively. Approximately26%, 35 % and 45% RIF degradation occurred when the mixture was coagulated at 85℃for 50 min, 1.5 h and 2 h respectively .5) The DST outcomes when using L-J medium freshly made and stored for different periods of time at 4℃were consistent with the RIF concentration monitoring data. The longer the media was stored, the more possibility that RIF- Sensitive strains were mis-categorized as resistant strains. Our study concluded that: 1) RIF in stock solution is stable for at least 3 months at a reduced storage temperature.2) Media containing RIF should be prepared strictly according to validated standard operating procedures. 3) RIF containing L-J medium should not be older than one month .4)Drug degradation is a possible reason for false resistance categorizations of M. tuberculosis isolates in the clinical laboratory.