1. Ruqaya Mustafa Ali Genetic Engineering and Biotechnology

2. 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.

3.  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.

4. 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.

6.  Patients During 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.

7. Direct examination by ZN and Florescent stain DNA extraction SAMPLE COLLECTION Processing / Decontamination Culture Staining and Biochemical Tests PCR technique and Hybridization with MTBDR plus strips

8. 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.

9. Chromogen (MBT/BCIP ) Alkaline Phosphatase Streptavidin Biotin Nitrocellulose strip DNA-probe Biotin-labelled single stranded amplified target Colour reaction

10. 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

12.  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%)

13. Results RMP+INH Resistance 51 MDR strains (17) strains INH with 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 strains with 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 +

14. Rapid Diagnosis of (MDR-TB) using molecular Line probe Assay GeneBand Gene region or mutation MDR strains RIF monoresistant INH monoresistant rpoBWT1506-509 WT2510-513 WT3513-517 WT4516-519 WT5518-522 WT6521-522 WT7526-529 WT8530-533 MUT1D516V1 MUT2AH526Y1 MUT2BH526D MUT3S531L 276 KatGWT315 MUT1S315T114 3 MUT2S315T2 inhAWT1-15/-16 WT2-8 MUT1C15T14 MUT2A16G MUT3AT8C MUT3BT8A1

15. 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

16. Drug Susceptibility Testing 7 - 10 days 3 - 4 weeks Solid Media Löwenstein-Jensen Liquid Media BACTEC 460 TB MGIT Molecular based Methods GenoTypeMTBD Hours – 1day