Rapid Direct Testing of Susceptibility of Mycobacterium tuberculosis to Isoniazid and Rifampin on Nutrient and Blood Agar in Resource-Starved Settings

Abstract

In this study, we evaluated the performance of blood agar (by macroscopic growth) and nutrient agar (by a microcolony detection method) for drug susceptibility testing of Mycobacterium tuberculosis against rifampin (RIF) and isoniazid (INH), using 67 smear-positive sputum specimens. The direct proportion method on Lowenstein-Jensen (LJ) medium was used as the "gold standard." Compared with LJ medium, results for both media were in 100% agreement for RIF, while for INH the agreement levels for blood agar and nutrient agar were 98% and 95%, respectively. Within 2 weeks, 100% of specimens yielded results on blood agar, while 96.8% of specimens yielded results on nutrient agar. Our study showed that blood agar and nutrient agar can be used as alternative media for direct susceptibility testing of RIF and INH, especially in resource-poor settings. Despite recent advancements in the field of mycobacteriology, multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) still remain public health nuisances (9,19). Conventional culture methods are slow, requiring about 6 to 8 weeks for diagnosis and subsequent drug susceptibility testing (DST). In the last decade, the Bactec MGIT 960 automated system was introduced for early diagnosis and DST of Mycobacterium tuberculosis from various clinical specimens. However, this system is costly, its consumables are not easily available, and it requires technical expertise. Parallel to the progress in automated systems, much work has also been published on the development of rapid but economical methods which are equally effective and comparable to automated systems, especially for resource-limited countries (1,11,12,15,18).Drancourt et al. were the first to report the incidental growth of M. tuberculosis colonies on blood agar and termed this finding the "end of dogma" (7). Their report led to further research on blood agar for cultivation of M. tuberculosis (2, 13). In addition, it was demonstrated that blood agar can be used instead of Middlebrook 7H10/11 agar or Lowenstein-Jensen (LJ) medium for susceptibility testing of M. tuberculosis (3-6). In this study, we evaluated for the first time the performance of blood agar and nutrient agar for DST of M. tuberculosis against rifampin (RIF) and isoniazid (INH) directly on smear-positive sputum specimens. MATERIALS AND METHODSSetting. This comparative study was carried out at the Department of Pathology, Combined Military Hospital, Dera Ismail Khan, Pakistan. A total of 70 fresh smear-positive sputum specimens were obtained from the Tuberculosis Control Program health setting.Specimen processing. All of the sputum specimens were from patients diagnosed clinically and radiologically along with having a positive acid-fast bacillus (AFB) smear. Informed consent was obtained from all patients, as approved by the hospital's ethical committee. In the protocol followed, all of the clinical specimens (one per patient) were homogenized and decontaminated by a standard sodium hydroxide-N-acetyl-L-cysteine met...

Evaluation of Culture Media for Isolation of Mycobacterium Species from Human Clinical Specimens

“…Among 100 smear-positive pulmonary samples, only 84% showed growth on the LJ medium, which was lower than previous reports by Tarshis, et al (89.9%), Tarshis, et al (90.1%), Satti, et al (95.7%), and Mathur, et al (97.1%) [ 8 - 9 , 12 , 14 ]. Only 75% smear-positive cases revealed growth on BA, which was lower than that reported by Tarshis, et al (93.6%), Tarshis, et al (94.2%), Maccabe, et al (78.26%), Dunlop, et al (87.5%), Satti, et al (97.9%) [ 12 ], and Mathur, et al (94.2%) [ 8 - 12 , 14 ]. Low isolation rates in our study could be attributed to sample decontamination with 4% NaOH or prior anti-tubercular treatment [ 16 ].…”

“…In the present study, we have used blood agar to isolate M. tuberculosis from both pulmonary as well as extrapulmonary samples. The culture sensitivity of BA was 89.3% for pulmonary isolates, which was lower than those reported by Drancourt, et al (98.9%), Satti, et al (90.9%), Mekasha (98%), and Mathur, et al (94.2%) [ 7 , 12 - 14 ]. This can be explained by the fact that we had used RNTCP-advocated modified Petroff’s method (4% NaOH) for sample processing while, in others studies, the NALC-NaOH method was used [ 3 ].…”

“…Recently, there has been a lot of interest in the use of blood agar for isolation and DST of M. tuberculosis . The studies conducted previously by Drancourt, et al, Satti, et al, and Mekasha have positively evaluated the utility of BA for primary isolation as well as DST of M. tuberculosis [ 7 , 12 - 13 ]. An Indian study reported by Mathur, et al also used BA for primary isolation of M. tuberculosis from pulmonary samples [ 14 ].…”

“…On the other hand, culture positivity on LJ media ranged from 10-41 days with a mean time of 22.7 days (20.8 - 24.6 days, 95% CI). Satti, et al reported time to detect growth on BA as seven days [ 12 ]. Our findings were in contrast to those reported by Dunlop, et al who observed average growth time as 10 days on BA and 9.3 days on LJ medium [ 11 ].…”

“…At this stage, the colony was about 2 mm in diameter [ 10 ]. Mathur, et al and Satti, et al described colonies of M. tuberculosis on BA as light grayish, glistening, and easily recognized against a red background of BA as compared to rough, cream-colored colonies on LJ medium against bluish-green backdrop [ 12 , 14 ].…”