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Rapid Diagnosis of Multidrug Resistant Tuberculosis (MDR-TB) using Molecular Line Probe AssayRuqaya Mustafa Ali Genetic Engineering and
Biotechnology
Introduction Tuberculosis (TB) remains a major global health
problem. It causes ill-health among millions of people each year and ranks as the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus (HIV).
Tuberculosis is a major public health problem in Iraq with an estimated prevalence of 45/ 100.000 and mortality rate of 12/ 100.000.
Multi – drug resistant Tuberculosis (MDR-TB), defined as Mycobacterium tuberculosis resistant to both Isoniazid and Rifampicin, is a world problem with an estimated 630000 cases in 2011. The rate of MDR-TB in Iraq is reported to be 3.4 % of the new TB cases.
Early detection of MDR- TB provides better treatment outcomes and reduces the transmission of MDR.
Nucleic Acid amplification test (NAAT) like Line Probe Assays have been recently approved for use in low income settings and can be used to screen smear – positive sputum specimens for diagnosis resistance to Rifampicin and Isoniazid in (1-2) days.
Objective: The specific objective of this study was to determine the type of MDR –TB and detection the mutation in rpoB, KatG and inhA genes from culture specimens .
Patients, Materials
and methods
PatientsDuring the study interval (April 2010 -
August 2011), the Institute of Chest and
Respiratory diseases in Baghdad received
2866 suspected TB patients with pulmonary
and extrapulmoary, 1754(61.2%) male and
1112 (38.7%) females, with age rang from
(7 month – 85 years), of which (51) patients
as MDR .
Direct examination by ZN andFlorescent stain
DNA extraction
SAMPLE COLLECTION
Processing / Decontamination
Culture
Staining and Biochemical Tests
PCR technique and Hybridization with MTBDR plus strips
Genotyping Methods :All genotyping methods were performed at the Emerging Bacterial Pathogens Unit, WHO / IUATLD Supra-National Reference TB Laboratory / San Raffaele Scientific Institute (HSR)- Italy .
Chromogen (MBT/BCIP)
Alkaline Phosphatase
Streptavidin
Biotin
Nitrocellulose strip
DNA-probe
Biotin-labelled single stranded amplified target
Colour reaction
Control of the conjugate -Amplification control -
Amplification control MTBC -
Control rpoB - rpoB Wild type 1 - rpoB Wild type 2 - rpoB Wild type 3 -rpoB Wild type 4 -rpoB Wild type 5 -rpoB Mut D516V -rpoB Mut H526Y -rpoB Mut H526D -rpoB Mut S531L -
Control katG -katG wild type -
katG S315T1 (ACC) -katG S315T2 (ACA) -
1 2 3 4 5 6 7 8
Results
The most common genetic mutation conferring RIF resistance was S531L of rpoB gene which detected in 33 (82.5%) resistant strains .
Other mutations in this gene were D516Vand H526Y which detected in single strain (2.5%) for each.
Isonaiazid (INH) resistance due to KatG mutation S315T1 was found in 17 (42.5%) strains of (INH) – resistant M. tuberculosis
The second most common site of mutation was in the upstream promoter sequence of inhA, which found in 15 (37.5%)
Results RMP+INH Resistance
51 MDR strains
(17) strains INHwith mutations in codon of katG
(34) strains RIF mutations in rpoB at
S531 L region
(1) Strain RIF mutation in rpoB at D516V
14 strains with a mutation in inhA
+(1) strains
with a mutation in KatG and inhA
9 strainswith no mutation in katG and inhA
6 strain not detected + 5 strains detected as
sensitive to RIF and INH BY MTBDR Plus
+
++
5 Strains resistance
with no mutation in rpoB
+
Rapid Diagnosis of (MDR-TB) using molecular Line probe Assay
Gene Band Gene region or mutation MDR strains RIF
monoresistant INH monoresistant
rpoB WT1 506-509WT2 510-513WT3 513-517WT4 516-519WT5 518-522WT6 521-522WT7 526-529WT8 530-533
MUT1 D516V 1MUT2A H526Y 1MUT2B H526DMUT3 S531L 27 6
KatG WT 315MUT1 S315T1 14 3MUT2 S315T2
inhA WT1 -15/-16WT2 -8
MUT1 C15T 14MUT2 A16G
MUT3A T8CMUT3B T8A 1
Conclusions
Line Probe Assay is an appropriate tool for rapid screening for MDR-TB and has the potential to substantially reduce the turnaround time of drug sensitive test (DST) results.
Time for the detection has a potential to reduce the extend of spread of resistant strains
Drug Susceptibility Testing
7 - 10 days
3 - 4 weeksSolid MediaLöwenstein-Jensen
Liquid MediaBACTEC 460 TBMGIT
Molecular based Methods
GenoTypeMTBD
Hours – 1day
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