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Rapid drug sensitivity testing of M. tuberculosis based on culture on a porous ceramic support

Abstract number: p1120

Ingham  C., Ben Ayad  A., Hummelink  R., Mulder  B.

Objective: 

The growth of M. tuberculosis rate-limiting in many diagnostic procedures including drug sensitivity testing (DST). Attempts to speed up DST for this organism have included the detection of microbial metabolism and the optical imaging of microcolonies. Currently, there is no molecular method for determining the full drug resistance pattern required to guide effective treatment regimes. The objective of this work was to use a highly porous ceramic (Anopore) as a culture support to facilitate the rapid detection of M. tuberculosis growth, and to apply this capability to DST. This is a novel methodology, one that exploits the high porosity and excellent culture and imaging properties of Anopore.

Methods: 

Sterile anopore strips were placed upon Middlebrook 7H10 agar plates and inoculated with M. tuberculosis stains 445 (RIFS, INHS) and 473 (RIFS, INHR). The plates were incubated in a CO2 incubator at 37 degrees C. Cultures were then killed by placing on filter paper disks saturated with methanol or by heat treatment. This was followed by the transfer of the Anopore to a microscope slide covered with a film of solidified agarose containing 20 mM Syto16 dye for 45 min. Staining of cells on the Anopore surface was accomplished with minimal disruption of the microcolonies on the surface. Microcolonies were then imaged directly using an Olympus BX41 epifluorescence microscope equipped with Fluorotar lenses. Image capture used an 8-bit Kappa CCD camera and BMP files of images were analysed using ImageJ software.

Results: 

Colonies of M. tuberculosis were visible with 14 days incubation on Anopore strips placed on Middlebrook 7H10 agar plates and were similar in size and morphology to culture directly on the same agar base. Individual cells and microcolonies could be detected after Syto 16 staining. After only 2–3 days culture the increase in microcolony area was found to be significant (P < 0.001, n < 200, 2-tailed Mann Whitney U-test) indicating growth had occurred. DST testing against Isoniazid and Rifampin is in progress and the results will be shown.

Conclusions: 

Anopore is an effective culture support for M. tuberculosis. Imaging microcolonies in situ has been used to detect growth within a few days of inoculation and is now being applied to DST. The ability to kill, stain and image microcolonies in situ was facilitated by the porous, inert and rigid nature of Anopore.

Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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