Evaluation of a Novel Biphasic Culture Medium for Recovery of Mycobacteria: A Multi-Center Study

on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients.<0.001).

A rapid loop-mediated isothermal amplification assay targeting hspX for the detection of Mycobacterium tuberculosis complex. Jpn J Infect Dis

SUMMARY: A rapid, simple, and low-cost diagnostic tool for tuberculosis (TB) detection is urgently needed in countries with a high TB burden. Here, we report a novel loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene for the rapid detection of Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti. The specificity of this assay was evaluated using 4 reference strains of Mycobacterium tuberculosis complex (MTC), 22 species of non-tuberculous mycobacteria (NTM), 7 non-mycobacterial species, and 50 clinical M. tuberculosis isolates. All the reference MTC strains and M. tuberculosis clinical isolates were successfully detected by this method, and there were no false-positive results with NTM or non-mycobacterial species, which demonstrates the high specificity of this assay for MTC. The detection limit was 10 copies of MTC genome within 27 min, and the detection speed of this assay was higher than that of any other isothermal methods reported so far. Because of its speed, simplicity, sensitivity, specificity, and inexpensiveness, the TB hspX LAMP assay is a potential gene diagnostic method for TB detection in developing countries with a high TB burden

Comparison of culture in biphasic medium to culture in L-J medium for detection of mycobacteria.

<p>Pos: positive. Neg: negative. CI: confidence interval.</p

Rates of recovery of mycobacteria by biphasic medium, L-J medium and MGIT 960 culture system.

<p>Rates of recovery of mycobacteria by biphasic medium, L-J medium and MGIT 960 culture system.</p

Growth of mycobacteria in L-J and biphasic medium scenarios with acid-fast bacilli (AFB) smear microscopy and status of the source respiratory specimen.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036331#s3" target="_blank">Results</a> are shown for 830 AFB smear-negative respiratory specimens and 362 AFB smear-positive respiratory specimens that were found positive for <i>M. tuberculosis</i> by both L-J and biphasic medium (B–P). L-J, culture on L-J medium; B–P, culture in biphasic medium.</p

Growth of H37Rv culture on and in different culture media.

<p>For L-J (0.1), biphasic medium (0.1), and MGIT (0.1), a mean 0.1 ml of inocula were incubated. For biphasic medium (0.5) and MGIT (0.5), a mean 0.5 ml of inocula were incubated.</p

Box plot of median time required for growth of mycobacteria in each culture medium.

<p>Box lengths indicate interquartile range. Horizontal lines in each box represent the median of the distribution. Whiskers extend from the normal minimum value to the normal maximum value. For L-J, biphasic medium (B–P), and MGIT, the results of 121 smear positive sputum specimens and 298 smear negative sputum specimens are shown.</p

Flow chart.

<p>This study evaluated the biphasic medium (B–P medium) for the recovery of H37Rv and mycobacteria in clinical sputum specimens. A total of 1260 sputum specimens were decontaminated and cultured in the L-J slant and biphasic medium. Among them, the MGIT culture system was also used in 441 decontaminated specimens in addition to the presence of the L-J slant and biphasic medium.</p

H37Rv culture in biphasic medium.

<p>H37Rv ranging in concentration from 10⁷ cfu to 1 cfu per vial were incubated in biphasic medium at 37°C for 2 weeks. The numbers of H37Rv were 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10, 1, and 0 CFU/ml in tubes 1 through 8. Both white and black arrows indicate the locations of positive colonies of mycobacteria.</p