Ruqaya 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/ and mortality rate of 12/  Multi – drug resistant Tuberculosis (MDR-TB), defined as Mycobacterium tuberculosis resistant to both Isoniazid and Rifampicin, is a world problem with an estimated cases in 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.

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 During the study interval (April 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 and Florescent 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 )

 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 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 Strains resistance with no mutation in rpoB +

Rapid Diagnosis of (MDR-TB) using molecular Line probe Assay GeneBand Gene region or mutation MDR strains RIF monoresistant INH monoresistant rpoBWT WT WT WT WT WT WT WT MUT1D516V1 MUT2AH526Y1 MUT2BH526D MUT3S531L 276 KatGWT315 MUT1S315T114 3 MUT2S315T2 inhAWT1-15/-16 WT2-8 MUT1C15T14 MUT2A16G MUT3AT8C MUT3BT8A1

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 days weeks Solid Media Löwenstein-Jensen Liquid Media BACTEC 460 TB MGIT Molecular based Methods GenoTypeMTBD Hours – 1day