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

REAL-TIME POLYMERASE CHAIN REACTION FOR THE DIRECT
DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN CLINICAL SPECIMENS
Karen, Morgan
Joint Clinical Research Centre, Kampala, Uganda
PP-75
Introduction
The resurgence of tuberculosis is a leading cause of death worldwide. Since M. tuberculosis, is slow growing, methods
of diagnostic testing based on culture are delayed. This study describes the development of a real-time polymerase chain
reaction (RT-PCR) assay and subsequent clinical testing.
Methods
Patients were recruited from routine TB suspects in the Monterey County Public Health laboratory, CA, USA. Initially,
using Bactec MGIT 960 cultures as the gold standard, 231 respiratory specimens composed of 76 specimens from culture-confirmed
tuberculosis cases and 155 culture negative specimens were analyzed by RT-PCR. Over the next 2 years
206 respiratory patient specimens were tested from 81 flurochrome smear AFB positive and 125 negative sputa. DNA
extraction was performed directly from patient specimens by the modified Qiagen mini-Amp kit (Valencia, CA). The RT-
PCR was performed on the Roche LightCycler instrument (Mannheim, Germany). The assay was designed to target the
ITS region of the 16S rRNA gene of M. tuberculosis using Taqman hybridization probes for detection of amplicons.
Results
The developed RT-PCR assay yielded results in one day and achieved a sensitivity of 85.5% and specificity of 100%. In
subsequent clinical use over the two year period the RT-PCR assay achieved a sensitivity of 92.3% and specificity of 100%
in direct detection of MTB from 206 respiratory patient specimens,while in contrast fluorochrome smears achieved
only a sensitivity of 60.5%% for non-specific AFB detection in the same population. The RT-PCR assay detected MTB in
64.7% of the smear negative but culture confirmed patient specimens. The RT-PCR assay costs $14 per test, inexpensive
compared to commercial MTB assays ($60).
Conclusion
RT-PCR methodolgy can be used to detect MTB directly in patient specimens within eight hours, without waiting for
culture growth. This rapidly increases and enhances specific detection and treatment of MTB.
European Society of Mycobacteriology | 30 th Annual Congress | July 2009 | Porto - Portugal
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