A Novel Solid Medium for Culturing Mycobacterium tuberculosis Isolates from Clinical Specimens

Abstract

The laboratory diagnosis of tuberculosis usually relies on culture-based isolation of the causative Mycobacterium tuberculosis bacteria. We developed and evaluated the performance of MOD9, a new blood-free derivative of the MOD4 solid medium on which we previously reported for the isolation and culture of mycobacteria. First, inoculation of Lowenstein-Jensen medium with 21 M. tuberculosis isolates at 10 5 , 10 3 , and 10 CFU yielded colonies in 5.7 ؎ 1.5 days, 7.6 ؎ 0.8 days, and 10.8 ؎ 1.7 days versus 1.5 ؎ 0.4 days, 3.5 ؎ 0.6 days, and 4.9 ؎ 1 days in MOD9 (P < 0.05, Student's t test). Further, the time to detectable growth of M. tuberculosis was measured on MOD9 and Lowenstein-Jensen media after duplicate inoculation of 250 clinical specimens decontaminated with 0.7% chlorhexidine. The contamination rate was 1.6% (4/250) on MOD9 versus 4.4% (11/250) on Lowenstein-Jensen medium (P ‫؍‬ 0.11, Fisher's exact test). Chlorhexidine-MOD9 yielded 38/250 (15.2%) isolates versus 32/250 (12.8%) isolates for the chlorhexidine-LJ (P ‫؍‬ 0.5195, Fisher's exact test). All together, eight M. tuberculosis isolates were cultured solely from chlorhexidine-MOD9, and two M. tuberculosis isolates were cultured solely from chlorhexidine-LJ. The time to detection was 9.8 ؎ 3.9 (range, 5 to 18) days for chlorhexidine-MOD9 versus 17.4 ؎ 5.9 (range, 10 to 35) days for chlorhexidine-LJ (P < 0.05, Student's t test). These data indicate the superiority of the MOD9 medium over the standard LJ medium following chlorhexidine decontamination for the recovery of M. tuberculosis. P ulmonary tuberculosis caused by Mycobacterium tuberculosis complex (MTC) mycobacteria remains a global public health problem, causing on average 170 deaths every hour worldwide (1). Despite significant advances in the molecular diagnosis of tuberculosis over the past 2 decades (2), culture is still the universal gold standard for the laboratory diagnosis of tuberculosis, enabling complete postculture antimicrobial susceptibility testing and genotyping (3).MTC mycobacteria are fastidious organisms whose growth is routinely detected only after 7 to 12 days using advanced commercially available automated systems (4). These robots use various formulations of the Middlebrook liquid culture medium and indeed reduce the delay in growth detection compared to that with inoculation of standard solid culture media, such as Lowenstein-Jensen (LJ) medium (4). The World Health Organization (WHO) still recommends inoculating specimens in parallel into a liquid medium for accelerated diagnosis of high-titer specimens and onto a solid culture medium to increase the sensitivity of laboratory diagnosis of pulmonary tuberculosis (5).In an effort to conciliate speed and sensitivity of the culturebased diagnosis of tuberculosis, we developed new formulations of solid culture medium and new protocols for incubating and detecting colonies of M. tuberculosis. Recently, we showed that the MOD4 culture medium incorporating blood and egg lecithin, combined with an improved laboratory workflow, s...

Mycobacteria fecal shedding in wild boars (Sus scrofa), South-eastern France

“…However, contamination is a major limitation of culturing feces. Here, we applied to animal feces a 1% chlorhexidine decontamination and MOD9 medium culture protocol suitable for the recovery of Mycobacterium tuberculosis complex in patients on diagnosed with pulmonary tuberculosis 29,30 . Because the level of contamination of swine feces is very high, an efficient decontamination protocol is essential to isolate and culture mycobacteria from this type of sample 20 .…”

“…The MOD9 culture medium was prepared as previously described 29,30 . Briefly, MOD 9 culture medium incorporates for a total volume of 1.000 mL, 5 g of lecithin, 19 g of Middlebrook 7H10 powder, one gram of yeast extract, two grams of glucose, one gram of pancreatic digest of casein, 5 mL of glycerol and 2 mL of Tween 80.…”

Recent developments in the diagnosis and management of tuberculosis

“…40 Drug susceptibility testing on solid or liquid culture is essential to confirm a diagnosis of MDR-TB, but its availability in low-income and high-burden areas is far less than is needed. Molecular tests, such as GenoType MTBDRplus, a line probe assay, that rapidly detects TB bacteria and identifies resistances to the most important first-line drugs (rifampicin and isoniazid) and to some second-line molecules in its latest version, are often used as substitutes for conventional drug susceptibility testing, although their implementation is still challenged by high costs and complex technical requirements.…”

Rapid culture-based diagnosis of pulmonary tuberculosis in developed and developing countries

“…Comparison of MOD9 with LJ (chlorhexidine-0.7% decontamination) showed that MOD9 had a higher sensitivity, shorter time to detection of M. tuberculosis and lower contamination rate (95%, 10.5 ± 4.2 days and 1.6%) than LJ (80%, 18.1 ± 6.3 days and 4.4%; Asmar et al, submitted). Furthermore, the comparison of MOD9 medium (chlorhexidine-0.7% decontamination) with Bactec 960 MGIT (NALC–NaOH decontamination) showed that MOD9-chlorhexidine-0.7% had a higher sensitivity, shorter time to detection and a lower contamination rate (95.8%, 10.1 ± 3.9 days and 1.7%, respectively) than Bactec MGIT 960–NALC–NaOH (40.8%, 14.7 ± 7.3 days and 5.7%; Asmar et al, 2015 ). Combining MOD9 with scanning technology (Advencis-Bio-system) enabled the detection of M. tuberculosis colonies in 3.4 ± 2 days vs. 15 ± 6 for Bactec MGIT 960 (Ghodbane et al, 2014 , 2015 ; Table 1 ).…”