14th ICID - Poster Abstracts - International Society for Infectious ...

When citing these abstracts please use the following reference:
Author(s) of abstract. Title of abstract [abstract]. Int J Infect Dis 2010;14S1: Abstract number.
Please note that the official publication of the International Journal of Infectious Diseases 2010, Volume 14, Supplement 1
is available electronically on http://www.sciencedirect.com
Final Abstract Number: 75.044
Session: Diagnostics
Date: Friday, March 12, 2010
Time: 12:30-13:30
Room: Poster & Exhibition Area/Ground Level
Type: Poster Presentation
Multiprobes real-time PCR direct detection of MDR-TB related genes in pulmonary samples
W. Watcharasamphankul, S. Foongladda
Mahidol University, Bangkok, Thailand
Background: Multidrug resistant Mycobacterium tuberculosis infection is a serious contagious
disease that needs rapid detection to control the disease. Direct detection of the responsible
genes using hybridization probes with melting temperature shift will confer information of the
drugs resistance.
Methods: Primers and probes for rpoB, kat G and regulatory region of inhA genes were designed
to perform in single tube real-time PCR of the LightCycler system. Three sets of hybridization
probes were synthesized; probes cover codon 511-534 of rpo B gene were label with Red 640
and Red 705, codon 304-318 of kat G with Red 610 and -18 regulatory regions to codon 9 of inhA
with Red 670. Reduction of melting temperature in the system analysis indicates mutation in the
specific region. Specificity of the designed probes and primers were in silico tested with nontuberculous
mycobacterias and in vitro assessed with reference strains. The method was
evaluated with 134 M. tuberculosis clinical isolates (41 susceptible, 18 INH resistant, 1 RMP
resistant and 74 MDR), and 134 decontaminated tuberculosis pulmonary samples (75 smearpositive
and 59 smear-negative) by comparing to proportion method results of their isolates.
Results: This multiprobs method shows 100% specificity to M. tuberculosis complex. All
susceptible isolates showed absence of mutation by this method. Mutation in rpo B gene was
detected in all of 75 resistances. Mutations of katG and inhA genes were detected in 79 and 13,
respectively, of 93 INH resistances.
Among 134 decontaminated samples, susceptibility results of their culture were 104 susceptible,
23 INH resistances, 1 RMP resistance, and 6 MDR. This method could detect mutation in rpoB
gene of all 7 RMP resistances. Mutation of katG or inhA genes were detected in 27/29 INH
resistances. Nonsense mutation was found in the regulatory region of inhA gene in one
susceptible sample. The specificity and sensitivity of the method for direct detection were 100%
for RPM; and 99.0% and 93.1%, respectively, for INH.
Conclusion: This method is competent to detect mutation of the genes associated with RMP and
INH resistant tuberculosis in pulmonary specimen in one tube reaction.