An evaluation study on phenotypical methods and real-time PCR for detection of Mycobacterium tuberculosis in sputa of two health centers in Iran
Background and Objectives: For further confirmation of the previous in-house real-time PCR and CYP 141 target as a rapid and cheap diagnostic technique and a new target for direct detection of Mycobacterium tuberculosis, we evaluated and compared the results of smear, culture and real-time PCR in sputa that were collected from 2 health centers. Moreover we tried to evaluate the diagnostic accuracy of phenotypical methods for detection of tuberculosis that have been applied in two health centers of Iran.
Materials and Methods: Thirty two sputa (including 15 smear positive and 17 smear negative) and 53 Sputa (29 smear and culture positive and 24 smear and culture negative specimens) were collected from tuberculosis suspected patients from health center No. 1 and 2 respectively. A Taqman probe was used for direct detection of M. tuberculosis using the specific primers.
Results: Because of the results, data of health center No. 1 was not reliable. The average number of bacteria that was detected in health center No. 2 by real time PCR was 1.2E+003-7.3+004; 1.4E+004-1.29+005 and 8.27E+005-1.07+006 for one to three plus smear result groups, respectively. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of real-time PCR were 96.5% (28/29), 95.8% (23/24), (96.6%) and (96%), respectively.
Conclusion: Compared with the results of previous studies and being a good correlation between real-time PCR and phenotypic methods, emphasize that CYP141 is a good target for quantification of M. tuberculosis in sputa and real-time PCR can be a good method for evaluation of smear microscopy. Moreover, further surveillance is needed to evaluate the phenotypical methods and final decisions that are taken in health centers of Iran that can be observed and evaluated by the cheap molecular methods like in-house methods.
Neonakis IK, Gitti Z, Krambovitis E, Spandidos DA.Molecular diagnostic tools in mycobacteriology. J Microbiol Methods 2008; 75: 1-11.
World Health Organization. Global tuberculosis report (2016). file:
Shojaei H, Heidarieh P, Hashemi A, Feizabadi MM,Naser AD. Species identification of neglected nontuberculous mycobacteria in a developing country. Jpn J Infect Dis 2011; 64: 265-271.
Thierry D, Brisson-Noel A, Vincent-Levy-Frebault V,Nguyen, S, Guesdon JL, Gicquel B. Characterization of a Mycobacterium tuberculosis insertion sequence,IS6110, and its application in diagnosis. J Clin Microbiol 1990; 28: 2668-2673.
Kox LF, van Leeuwen J, Knijper S, Jansen HM, Kolk AH. PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples. J Clin Microbiol 1995; 33: 225-3233.
Springer B, Stockman L, Teschner K, Roberts GD,Bottger EC. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J Clin Microbiol 1996; 34: 296-303.
Kirschner P, Rosenau J, Springer B, Teschner K, Feldmann K, Bottger EC. Diagnosis of mycobacterial infections by nucleic acid amplification: 18-month prospective study. J Clin Microbiol 1996; 34: 304-312.
Garcia-Quintanilla A, Gonzalez-Martin J, Tudo G,Espasa M, Jimenez de Anta MT. Simultaneous identification of Mycobacterium genus and Mycobacterium tuberculosis c omplex i n c linical s amples b y 5 '-exonuclease fluorogenic PCR. J Clin Microbiol 2002; 40:4646-4651.
Darban-Sarokhalil D, Imani Fooladi AA, Bameri Z,Nasiri MJ, Feizabadi MM. Cytochrome CYP141: A new target for direct detection of Mycobacterium tuberculosis. Acta Microbiol Immunol Hung 2 011; 5 8:211-217.
Darban-Sarokhalil D, Imani Fooladi AA, Maleknejad P, Bameri Z, Aflaki M, Nomanpour B, et al. Comparison of smear microscopy, culture, and real-time PCR for quantitative detection of Mycobacterium tuberculosis in clinical respiratory specimens. Scand J Infect Dis 2013; 45: 250-255.
Farzam B, Imani Fooladi AA, Izadi M, Hossaini HM, Feizabadi MM. Comparison of cyp141 and IS6110 for detection of Mycobacterium tuberculosis from clinical specimens by PCR. J Infect Public Health 2015; 8:32-36.
Palomino JC. Nonconventional and new methods in the diagnosis of tuberculosis: feasibility and applicability in the field. Eur Respir J 2005; 26: 339-350.
Ait-Khaled N, Enarson DA. Tuberculosis a Manual for Medical Students. WHO, IJTLD. 2003.
Rieder HL, Chonde TM, Myking H, Urbanczik R,Laszlo A, Kim SJ, Deun AV, Trebucq A. The Public Health Service National Tuberculosis Reference Laboratory and the National Laboratory Network: minimum requirements, role and operation in a low-income country.
Paris, International Union Against Tuberculosis and Lung Disease.
Somoskövi A, Hotaling JE, Fitzgerald M, O'Donnell D,
Parsons LM, Salfinger M. Lessons from a proficiency testing event for acid-fast microscopy. Chest 2001; 120:250-257.
Steingart KR, Ng V, Henry M, Hopewell PC, Ramsay A, Cunningham J, Urbanczik R, Perkins MD, Aziz MA, Pai M. Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 2006; 10: 664-674.
Murray SJ, Barrett A, Magee JG, Freeman RJ. Optimization of acid fast smears for direct the detection of mycobacteria in clinical samples. J Clin Pathol 2003; 56: 613-615.
|Issue||Vol 9 No 1 (2017)|
|Mycobacterium tuberculosis Real-time PCR Cytochrome P450 cyp141|
|Rights and permissions|
|This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.|