European Society of Mycobacteriology - Instituto Nacional de Saúde ...

OP-24
Development of an automated culture system for
M. tuberculosis with autofluorescence detection
den Hertog, Alice 1 , Koeleman, Marc 1 , Ingham, Colin 2 , Fey, Frank 3 , Langerak, Edwin 3 , Klatser, Paul 1 , Anthony, Richard 1
KIT Biomedical Research, Amsterdam, The Netherlands
Microdish BV, Wageningen, The Netherlands
CCM, Nuenen, The Netherlands
Due to the unavailability of rapid sensitive diagnostic methods for tuberculosis (TB), culture remains one of the most
important diagnostic tools - even though it is technically demanding and slow. The time required for culture leads to a
delay in TB diagnosis and treatment with effective drugs. A standardized method for more rapid drug sensitivity testing
(DST) of TB would be valuable.
We are developing a system in which mycobacterial (micro-)colonies are detected by their autofluorescence based on
the MODS (microscopic observation of drug susceptibility) method, which is much more rapid than the traditional culture
methods, but is laborious.
By using microscopy with low-power magnification, colony growth could be determined much earlier than
when viewed by eye. Studying Mycobacterium smegmatis initially as a model organism, we have made sequential
brightfield and fluorescence microscopy photographs of colonies growing on movable solid supports
on media. These images were used to develop an image analysis protocol to determine the growth rate.
We found that the time to detection of microcolonies was less than half that required for visual detection. Furthermore,
as soon as the colonies could be visualized, further growth of colonies could be detected by the image analysis protocol
within 1-2 division times, from which the growth rate could be determined. As the bacteria were inoculated on movable
solid supports, exposing the microcolonies to selective media after their first detection could make DST of many individual
colonies possible after only a few additional division times, allowing the rapid determination of the proportion of
antibiotic resistant and sensitive mycobacterial colonies present in a sample.
After confirmation of the recently described autofluorescence of mycobacteria, this method was used for the detection
and enumeration of viable mycobacteria. The specificity of the autofluorescence may be used to confirm the presence
of mycobacteria.
The use of (semispecific-) autofluorescence in combination with growth rate will increase the feasibility of developing
an automated detection system based on this method, which is very challenging for light microscopy methods (such as
MODS). Additionally, the possibility of performing quick DST without re-inoculation may lead to a reduce time to correct
treatment.
European Society of Mycobacteriology | 30 th Annual Congress | July 2009 | Porto - Portugal
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