title: Comparison of smear microscopy, culture, and real-time PCR for quantitative detection of Mycobacterium tuberculosis in clinical respiratory specimens.
abstract: BACKGROUND
  As a rapid diagnostic technique, the real-time polymerase chain reaction (PCR) can detect Mycobacterium tuberculosis with a high sensitivity and specificity. However, further studies are needed to confirm it as a standard method. In this study, we evaluated the cyp141 gene for the detection and quantification of M. tuberculosis in respiratory specimens and compared the results with direct microscopy and culture.
  
  
  METHODS
  Sputum samples (n = 247) were collected from patients of the different provinces of Iran. DNA was extracted from clinical specimens and H37Rv strain. After measuring the standard strain DNA concentration by NanoDrop and using the Avogadro number, the DNA was diluted 6 times in order to obtain 1 × 10(6) to 10 template copies. A Taqman probe was designed for detection of the target in a real-time PCR using the specific primers.
  
  
  RESULTS
  Of 247 samples, 135 (55%) were culture-negative. Of 112 (45%) culture-positive samples, 88 were positive by both smear and culture and 24 were smear-negative but culture-positive. The real-time PCR enumerated 1.5E + 02 to 4.3E+ 03, 8.5E + 03 to 5.5E + 04, 7.2E + 04 to 1.1E + 06, and 1.2E + 06 to 8.1E + 07 M. tuberculosis cells in the specimens with smear-negative, 1-plus, 2-plus, and 3-plus codes, respectively. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the real-time PCR were 90.2% (101/112), 97.8% (132/135), 97.1%, and 92.3%, respectively.
  
  
  CONCLUSIONS
  The overall sensitivity and specificity, the results in comparison with those of the Xpert MTB/RIF kit, and the good correlation with molecular and phenotypic methods, show that cyp141 could be a good target for the quantification of M. tuberculosis in sputum and possibly other clinical specimens.
authors.affiliation: Department of Pathobiology, School of Medicine, Alborz university of Medical Sciences, Karaj, Iran.
authors.forename: Davood
authors.initials: D
authors.lastname: Darban-Sarokhalil
authors.forename: Abbas Ali
authors.initials: AA
authors.lastname: Imani Fooladi
authors.forename: Parviz
authors.initials: P
authors.lastname: Maleknejad
authors.forename: Zakaria
authors.initials: Z
authors.lastname: Bameri
authors.forename: Moloud
authors.initials: M
authors.lastname: Aflaki
authors.forename: Bizhan
authors.initials: B
authors.lastname: Nomanpour
authors.forename: Somayeh
authors.initials: S
authors.lastname: Yaslianifard
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Modarresi
authors.forename: Mohammad Mehdi
authors.initials: MM
authors.lastname: Feizabadi
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D001483:Base Sequence; D003627:Data Interpretation, Statistical; D006801:Humans; D007492:Iran; D008853:Microscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013183:Sputum / Q000382:microbiology*; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology*