Mycobacterium tuberculosis detection and drug susceptibility testing using AnyplexTM Real-time PCR in comparison with standard culture

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Somsak Sintu-urai
Kannaphat Kesornsukhon
Patthama Klomporn
Thanakorn Phowong
Ratchaneeporn Khummin
Suwannee Keeratiwasee

Abstract

Introduction: Bacterial cultivation is the gold standard method for diagnosis of tuberculosis (TB). However, technique is time consuming for 28-56 days of cultivation. Therefore, several rapid tests have been developed. Real-time PCR is one of the quick TB test currently used for identifying both drug-susceptible and multidrug resistant TB (MDR-TB). However, the efficiency of real-time PCR compared to conventional method has not been elucidated.

Objective: To compare the efficiency between real-time PCR and conventional methods including cultivation in solid media and mycobacteria Growth Indicator Tube (MGIT 960) system for detection of TB and MDR-TB.

Materials and methods: One hundred and forty-seven sputum specimens collected from hospital sectors of the Office of Disease Prevention and Control 2 in Phitsanulok were decontaminated and subjected to culture in LJ medium agar slant and MGIT 960 system. Bacterial DNA was extracted and real-time PCR was performed using the AnyplexTM MTB/NTM detection kit and AnyplexTM II MDR-TB detection kit.

Results: Samples (86.2%) were positive by real-time PCR and was 39.3% higher than conventional culture. Sensitivity, specificity, positive predictive value, and negative predictive value of real-time PCR for detection of multidrug-resistant tuberculosis were 96.82%, 28.05%, 49.17%, and 92.0%, respectively, as compared to MGIT 960 system. Time required for detection of TB between MGIT 960 system and real-time PCR is 13 and 2 days. However, total cost of MGIT 960 system is cheaper than that of real-time PCR (600 Baht vs 1,500 Baht, respectively).

Conclusion: Real-time PCR is the useful diagnostic test for rapid detection of MTB and MDR-TB. Real-time PCR provides higher sensitivity and specificity than convention culture methods. This method can be used as a screening test for TB infection where rapid treatment can be achieved. However, this method has to be further on evaluated for its cost effectiveness.


Bull Chiang Mai Assoc Med Sci 2016; 49(2): 218-226. Doi: 10.14456/jams.2016.22

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How to Cite
Sintu-urai, S., Kesornsukhon, K., Klomporn, P., Phowong, T., Khummin, R., & Keeratiwasee, S. (2016). Mycobacterium tuberculosis detection and drug susceptibility testing using AnyplexTM Real-time PCR in comparison with standard culture. Journal of Associated Medical Sciences, 49(2), 218. Retrieved from https://he01.tci-thaijo.org/index.php/bulletinAMS/article/view/59893
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Research Articles

References

1. World Health Organization [Internet]. Switzerland: World Health Organization; C2015 [updated 2016 Feb 1; cited 2016 Feb 9] Available from http://www.who.int/tb/publications/global_report/en/.

2. Global tuberculosis control : epidermiology, strategy, financing : WHO report 2009.

3. Ungkasrithongkul M. Situation of multidrug-resistant tuberculosis in Thailand: Fiscal year 2007-2009. Bureau of Tuberculosis, Department of Disease Control, Ministry of Public Health. (in Thai)

4. Guillerm M, Usdin M, Arkinstall J. Tuberculosis diagnosis and drug sensitivity testing, An overview of the current diagnostic pipeline. Campaign for Access to Essential Medicines. Paris: October 2006.

5. Hansen WL, Beuving J, Bruggeman CA, Wolffs PF. Molecular probes for diagnosis of clinically relevant bacterial infections in blood cultures. J Clin Microbiol 2010; 48: 4432-8.

6. Netto GJ, Saad RD, Dysert PA 2nd. Diagnostic molecular pathology: current techniques and clinical applications, part I. Proc (Bayl Univ Med Cent) 2003; 16: 379-83.

7. Ma X, Xu H, Shi L, Yang P, Zhang L, Sun X, et al. A Multiplex PCR assay for the detection of five influenza viruses using a dual priming oligonucleotide system. BMC Infect Dis. 2015; 15: 93-104

8. Chun JY, Kim KJ, Hwang IT, Kim YJ, Lee DH, Lee IK, et al. Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene. Nucleic Acids Res 2007; 35: e40

9. Singh HB, Singh P, Jadaun GP, Srivastava K, Sharma VD, Chauhan DS, et al. Simultaneous use of two PCR systems targeting IS6110 and MPB64 for confirmation of diagnosis of tuberculous lymphadenitis. J Commun Dis. 2006; 38(3): 274-9.

10. Turenne CY, Tschetter L, Wolfe J, Kabani A. Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J Clin Microbiol 2001; 39(10): 3637-48

11. Seegene, Inc. User Manual: AnyplexTM plus MTB/NTM Detection, MDR-TB Detection. 2011; Version 1.01: 16-26

12. Seegene, Inc. User Manual: AnyplexTM plus MTB/NTM Detection, MDR-TB Detection. 2011; Version 1.01: 29-37

13. Valvatne H, Syre H, Kross M, Stavrum R, Ti T, Phyu S, et al. Isoniazid and rifampicin resistance-associated mutations in Mycobacterium tuberculosis isolates from Yangon, Myanmar: implications for rapid molecular testing. J Antimicrob Chemother 2009; 64(4): 694-701

14. Ramirez MV, Cowart KC, Campbell PJ, Morlock GP, Sikes D, Winchell JM, et al. Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis. J Clin Microbiol 2010; 48(11): 4003-9

15. Kaul KL. Molecular detection of Mycobacterium tuberculosis: Impact on patient care. Clin Chem 2001; 47(8): 1553-8

16. Raqib R, Rahman J, Kamaluddin AKM, Kamal SM, Banu FA, Ahmed S, et al. Rapid diagnosis of active tuberculosis by detecting antibodies from lymphocyte secretions. J Infect Dis 2003; 188: 364-70

17. Mazurek G H, Jereb J, LoBue P, Iademarco MF, Metchock B, Vernon A. Guidelines for Using the QuantiFERON®-TB Gold Test for Detecting Mycobacterium tuberculosis Infection, United States. CDC. Guidelines for the investigation of contacts of persons with infectious tuberculosis: recommendations from the National Tuberculosis Controllers Association and CDC. MMWR 2005; 54(RR15): 49-55

18. Aung WW, Ei PW, Nyunt WW, Swe TL, Lwin T, Htwe MM, et al. Phenotypic and genotypic analysis of anti-tuberculosis drug resistance in Mycobacterium tuberculosis isolation in Myanmar. Ann Lab Med. 2015; 35(5): 494-9