Molecular Detection of Specific Gene Mutations Associated with Isoniazid or Rifampicin Resistance among Category II Pulmonary Tuberculosis using DNA Microarray

This study was conducted in Baghdad Reference laboratory in association with Department of pathway medicine and Scotish Reference Laboratory/ RoyalInfirmary ofEdinburgh/ UK. The patients enrolled in this study were category II tuberculosis cases (default, relapse and failure), who sent to check the drug sensitivity test (DST) at reference lab/ Center of Tuberculosis & Chest Disease in Baghdad. One hundred andforty one positive sputum smear were collected byZiehle-Neelsen stain(ZN) and Auramine stain (AO), over thirteen months from September 2008 to October 2009. All samples subjected to culture in solid media (Lowenstein Jensen media-LJ). One hundred and ten (78.2%) were culture positiveand 31(21.98%) were excluded due to contamination and negative results. This study includes cases from different origin both in term of age group and gender to represent broad background of the study, the male 75(68%) more than female 35(32%) and the ratio was 2.1:1. The overall mean age of 110 patients with positive culture at time of sampling was 35.8± 1.3years with a range from 11-70 years. It seems that the highest percentage of tuberculosis, were 33.6%, 30.9% at age group 21-30 years and 31-40 years respectively. Genomic DNA was extracted from 110 isolates. Purity range was from 1.6-2,and the concentration rangewas 22.5-657 ng/ul. Extracted Genomic DNA of` 110 isolates and of 16 positive control isolates were subjected to molecular characterization by PCR and DNA-Microarray method. PCR amplification using two biotin labeled primer sets, the Genus mix which targets the rRNAInternal transcribed spacerregion (ITS) of Mycobacteria, producing an amplicon of ~226bp, and the Mycobacteriumtuberculosiscomplex (MTUBC) mix which specifically amplify a 126bp fragment from the IS6110 in M. tuberculosis complex. Slight variations in fragment size indicate presence of a Mycobacteriaspecies other than M. tuberculosis. Agarose gel electrophoresisof biotinylated PCR products showed that all samples were of the Mycobacteria tuberculosis complex. Twenty one selected isolates were also analyzed by Capillary gel electrophoresis (Bioanalyzer 2100, Agilent) to identify the exact size of amplified fragments with primer A and B. Myco-Direct microarray was used to detect tuberculosis by hybridization theprelabeled PCR primer mixes products to genus and /species specific capture probes immobilized on the low cost density (LCD-Chip) surface. All selected (39) samples showed positive signals intensity with all capture probe Broad I, Broad II, Mycobacterium tuberculosis complex01(MTUB-complex01) and Mycobacterium tuberculosis complex02 (MTUB-complex02), only 2 strains showed weak hybridization with capture probe Broad I, Broad II andMycobacterium tuberculosis complex01(MTUB-complex01) but with strong hybridization with Mycobacterium tuberculosis complex 02 (MTUB-complex02). By Myco-direct microarray, all selected strains were diagnosed Mycobacterium tuberculosis complex (MTUBC) and differentiated from Mycobacterium other than tuberculosis (MOTT). Drug resistance detected by hybridization to myco-resist microarray chip of labeled PCR resulting from a multiplex PCR containingprimers for the inhA,katGas well asrpoB for detection of Isoniazid and Rifampicin respectively.The analysis of 110 isolates showed 69.08% of them contained resistance genotypes. Wild type isolates showed no mutations and considered as a susceptible isolates constituted 30.90% tested samples. Resistance to both tested drugs occurred in 40% of samples, where as isolates resistant to one drug occurred in 29.08% of samples comprising 22.72% rifampicin resistance and 6.36% isoniazid resistance. There were 69 (62.72%) isolates showed single nucleotide polymorphism with rpo gene, all with mutations in the 81-bp hot-spot region of the rpoBgene. Out of the 69 mutant isolates, there were 50 (72.46%) isolates with rpo531-Leu, followed by rpo-526Tyr, rpo- 533 pro each of which 6 (8.69%), then, rpo-val516 3(4.3%), while the rpo-526-Asp frequency 2 (2.89%) and the lowest frequencies with rpo-531-trp and rpo-509- 515 each of which 1(1.44%). It was found that 51(46.36%) isolates had single nucleotide polymorphism in the katG gene or the InhA regulatory region. Mutations in codon 315 of the katGgene were most frequently observed, with 29(56.86%), there were 2 type of mutation within Kat-G: the Kat-315-thr was 22(43.13%) and the kat-315-Asn was 5(9.80%), in addition to 2(3.92%) strains showed no conclusive - either kat-315-thr or kat-315-Asn. Of the total 51 INH-mutants isolates, 22 (43.13%) had mutations in the inhA regulatory region with the -15C> T mutation. Further, no samples had both mutations, i.e., katG-315andInhA -15C>T. Single Nucleotide Polymorphism frequency (2 mutations) among 44 strains showed that the highest frequency in InhA-15T + rpoB531-Leu20(45.45%), followed bykatG 315/Thr + rpoBmut 531 Leu10(22.73%), then katG 315/Thr +rpoBmut 533 Pro 3( 6.82%), then InhA-15 T + rpoB533- Pro, KatG 315 Asn + rpoBmut 526 Asp, KatG 315 Asn + rpoBmut 526 tyr and katG 315/no conclusive + rpoBmut 533 Pro each of which 2(4.5%).While the lowest frequency was in katG 315/Thr + rpoBmut 516/Val, KatG 315 Asn + rpoBmut 531 Leu and katG 315/Thr + rpoB no conclusiveeach of which 1(2%).
To characterize the genetic basis of drug resistance in Mycobacterium tuberculosis in iraq, mutations in Rifampicin (rpoB gene) and Isoniazid (katG gene and InhA) in 110 M. tuberculosis complex isolates were involved. Microarray assay showed that the most frequent rpoB gene mutationswasin rpoB mut-531Leu 50/110 (45.45%). Followed by katG 29/110(26.36)%, thenInhA mut-15T 22/110(20%). Frequencies of rpoBmut/526 Tyr, rpoBmut 533 Pro each of which 6/110(5.45%), then KatG315-Asn,rpoBmut 516/Val 5/110(4.54%), 3/110(2.72%), respectively.The lowest mutation frequency in rpoBmut 531/Trp and rpoBmut 526 Asp each of which 2/110(1.8%),and rpo509-515 was 1/110 (0.90%) while no signals at gene region rpoBmut 511/Pro, rpoBmut 516/Tyr, rpoBmut 526/Asn, rpoBmut 526/Arg,rpoBmut 512/Thr, rpoBmut 526/ Leu, InhAmut 8/C,T and InhAmut 17/ T,0(0%). The association of each mutation with single drug resistance and MDR as follows: there were significant differences in rpo531-leu frequencies, there were 20 withRifampicin mono-resistant and 30 were associated with multi drug resistance (MDR).Regarding InhAmutnt15, the frequencies were 22, all were associated with MDR. The katG 315/Thr showed 7 frequencies with Isoniazid mono-resistance and 15 with MDR, while KatG 315-Asn showed5 frequencies all associated with MDR. The rpoBmut/526 Tyr frequencies were 2 with Rifampicin mono-resistance and 4 with MDR. The rpoBmut 533 Pro showed 6 frequencies and KatG 315 Asn showed 5 frequencies all with MDR, the rpo-val-516 frequencies were 2 associated with Rifampicin resistance and 1with MDR. The rpo-trp-531 frequencies were 2, both associated with Rifampicin mono-resistance. TheMyco-resist Microarray results of UK positive controls showed that the DNA microarray was more accurate and reliable than MICtest because there were4 strains tested by MIC showed Isoniazid resistance while by microarray they were not, while by comparing the microarray to HAIN MTBDRplus there were agreement between them. The total results of positive control showed high percentage of the non mutated strains and no MDR strains, while the most gene region mutation frequency with rpo531-leu and KatG-315-threach of which 25%, then Inh-15-T with 18.75%, but rpo526-Tyr had the lowest frequency6.25%. In conclusion, the microarray was fast reliable technique in tuberculosis screening. It is very accurate and fast in detection the prevalence of tuberculosis resistance. Also to make real diagnosis for the best antituberculosis instead of usingthe non reliable drug or wait weeks to get sensitivity test results by routine methods.