Rapid, accurate and early diagnosis of tuberculosis is a major health concern especially in developing country like Egypt. This study aimed to evaluate Multiplex PCR (MPCR)using both MPB64 and IS6110 gene targets, and to compare it with conventional methods such as microscopy and mycobacteria growth indicator tube (MGIT)  culture and also to compare with single target PCR IS6110 or MPB64 for rapid diagnosis of Mycobacterium tuberculosis (M. tuberculosis) from clinical samples. Sixty samples were processed for Ziehl-Neelsen (ZN smear), MGIT culture and PCR for single target PCR IS 6110 target or MPB 64 gene target and MPCR (IS6110 & MPB 64 gene target). Our study showed that out of 60 samples processed, 32 (53.3%) were positive for AFB, whereas 42(70%) were positive for MGIT culture.  PCR targeting IS6110 showed positive results 45 (75%)  as compared to PCR assay targeting MPB64 by which 40(66.7%) samples showed positive results  while the higher proportion of positive results were detected with MPCR was 49 (81.7% ( samples. when clinical diagnosis was considered as the gold standard.  AFB smear examination had a sensitivity of 53.3% and a specificity of 100%. For MGIT culture test, sensitivity was 70% and specificity was 100%. In comparison, single target PCR test was found to have a sensitivity of 75% and 66,7%  for PCR 1 (IS 6110) and PCR 2 (MPB64) respectively And specificity was 90%   while Multiplex PCR has much higher sensitivity 81.7% and specificity 92.3%. Combined ZN smear & MIGT culture showed 33 (55%) positive smear and culture, 9(15%)  smear negative culture positive and 18 (30%)  smear negative culture negative samples. Of  the 33 samples PCR positivity was found to be 33(100),30(90.9) and  28(84.8) with multiplex PCR,  PCR 1 (IS 6110) and PCR 2 (MPB 64)  respectively. In the 9 smear negative culture positive samples PCR positivity was observed as 8(88.9)  in both PCR 1 (IS 6110) and multiplex PCR while PCR2 (MPB64)  positivity 7)77.8%. However the MPCR, PCR 1 (IS 6110) and PCR 2 (MPB 64)  positivity was 8(44.4%),  7(38.9%) and 5(27.8%) in smear negative culture negative samples. This study reveals that multiplex TB-PCR, amplifying two targets specific for M. tuberculosis has higher specificity and sensitivity and can complement the tests in TB diagnosis and allow an initial therapeutic approach.

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