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

Thanks