Application of in-house nested polymerase chain reaction for rapid diagnosis of tuberculous pleural effusion
Keywords:
38kDa, BacT/Alert 3D system, Mycobacterium tuberculosis, nested PCR, Tuberculous pleural effusion
Abstract
Tuberculous pleural effusion (TPE) is a common manifestation of extrapulmonary tuberculosis and is the most common cause of pleural effusion in many countries. Conventional diagnostic tests for detection of Mycobacterium tuberculosis in pleural fluid or pleural tissue, have known limitations. Hence, there is need for a newer, rapid diagnostic tests. Molecular techniques, detecting DNA of M. tuberculosis in pleural fluid have better sensitivity and could be a potent tool for rapid diagnosis of tuberculous pleural effusion.
Objective: To evaluate Nested PCR protocol targeting 38 kDa gene for rapid detection of M. tuberculosis complex in clinically suspected cases of TPE.
Material and methods: A cross-sectional, prospective study was carried out at the tertiary care institute in a rural setup at western U.P. A total of 155 subjects with clinical suspicion of TPE enrolled during February 2015 to January 2016. About 10-20 ml of pleural fluid was collected and analysed for presence of M .tuberculosis by Z.N staining, culture on Lowenstein Medium (LJ), BacT/Alert 3D culture bottle and by Nested PCR targeting 38kDa gene of M. tuberculosis.
Result: Off the 155 patients enrolled, M. tuberculosis was detected by AFB staining, LJ culture and BacT/Alert 3D system staining in 13 (8.4%), 45 (29%) and 72 (46.5%) respectively. Diagnostic sensitivity of nested PCR (nPCR) was 60.6% and among smear positive and culture negative samples, sensitivity was 100% while in smear negative, culture negative it was 29.2%.
Conclusion: 38 kDa based nested PCR offers alternative robust approach for rapid and accurate detection of M .tuberculosis in paucibacillary tuberculous pleural effusion specimens.
Downloads
Download data is not yet available.
References
1. Global TB report 2015 [Internet]. Washington: World Health Organisation. 2015. [cited 2016 March 5]. Available fromhttp://www.who.int/tb/publications/ global_report /en/.
2. Vorster MJ, Allwood BW, Diacon AH, Koegelenberg CF. Tuberculous pleural effusions: advances and controversies. J Thoracic Dis. 2015;7(6):981-91.
3. Light RW. Update on tuberculous pleural effusion. Respirology2010;15(3):451-8.
4. Chakravorty S, Dudeja M, Hanif M, Tyagi JS. Novel multipurpose methodology for detection of extrapulmonary specimens by smear microscopy, culture and PCR. J Clin Microbiol. 2005;43(6):2697-702.
5. Makeshkumar V, Madhavan R, Narayan S. Polymerase chain reaction targeting insertion sequence for diagnosis of extrapulmonary tuberculosis. Indian J Med Res. 2014; 139(1):161-6.
6. Maurya AK, Kant S, Nag VL, Kushwaha R, Dhole TN. Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of
extrapulmonary tuberculosis. Indian J Med Microbiol. 2012;30(2):182-6.
7. Sekar B, Selvaraj L, Alexis A, Ravi S, Arunagiri K, Rathinavel L. The utility of IS6110 sequence based polymerase chain reaction in comparison to
conventional methods in the diagnosis of extrapulmonary tuberculosis. Indian J Med Microbiol. 2008;26(4):352-5.
8. Huyen MN, Tiemersma EW, Kremer K, de Haas P, Lan NT, Sola C, et al. Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam. Int J Tuberc Lung Dis. 2013; 17(11):1479-85.
9. Miyazaki Y, Koga H, Kohno S, Kaku M. Nested polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol.1993;31(8):2228-32.
10. Kulkarni S, Singh P, Memon A, Natraj J, Kanade S, Kelkar R, et al. An in-house multiplex PCR test for the detection of Mycobacterium tuberculosis, its validation
& comparison with a single target TB-PCR kit. Indian J Med Res. 2012;135(5):788-94.
11. Somerville W, Thibert L, Schwartzman K, Behr MA. Extraction of Mycobacterium tuberculosis DNA: a question of containment. J Clin Microbiol. 2005;43(6): 2996-7
12. Sjobring U, Mecklenburg M, Andersen AB, Miorner H. Polymerase chain reaction for detection of Mycobacterium tuberculosis.J Clin Microbiol.1990;28(10):2200-04.
13. Piersimoni C, Scarparo C, Callegaro A, Passerine Tosi CP, Nista D, et al. Comparison of MB/BacT ALERT 3D system with radiometric BACTEC system and Lowenstein-Jensen medium for recovery and identification of mycobacteria from clinical specimens:a multicenter study. J Clin Microbiol.2001;39(2):651-7.
14. Liu KT, Su WJ, Preng RP. Clinical utility of polymerase chain reaction for diagnosis of smearnegative pleural tuberculosis. J Chin Med Assoc.2007;70(4):148-51.
15. Light RW. Pleural diseases. Baltimore: Lippincott Williams and Wilkins; 2001. p. 182-95.
16. Nagesh BS, Sehgal S, Jindal SK, Arora SK.Evaluation of polymerase chain reaction for detection of Mycobacterium tuberculosis in pleural fluid. Chest. 2001; 119(6):1737-41.
17. Conde MB, Loivos AC, Rezende VM, Soares SL, Mello FC, Reingold AL. Yield of sputum induction in the diagnosis of pleural tuberculosis. Am J RespirCrit Care Med.2003;167(5):723-5.
18. Reechaipichitkul W, LulitanondV,Sungkeeree S, Patjanasoontorn B. Rapid diagnosis of tuberculous pleural effusion using polymerase chain reaction. Southeast Asian J Trop Med Public Health. 2000;31(3):509-14.
19. Shital P, Gajanan H, Rujuta A. Role of nucleic acid amplification tests (NAATs) in tuberculous pleural effusion: where it fits in routine diagnostic workup? J
Cell SciTher. 2014. 5: 169. doi:10.4172/2157-7013.1000169.
20. Diacon AH, Van de Wal BW, Wyser C, Smedema JP, Bezuldenhout J, Bollinger CT, et al. Diagnostic tools in tuberculous pleurisy: a direct comparative study. EurRespir J. 2003;22(4):589-91.
21. Hillemann D, Richter E, Rusch-Gerdes S. Use of the BACTEC Mycobacteria Growth Indicator Tube 960 automated system for recovery of mycobacteria from 9,558 extrapulmonary specimens, including urine samples. J Clin Microbiol. 2006; 44(11):4014-7.
22. Lu D, Heeren B, Dunne WM. Comparison of the automated Mycobacteria Growth Indicator Tube System (BACTEC 960 / MGIT) with Löwenstein-Jensen medium for recovery of mycobacteria from clinical specimens. Am J ClinPathol. 2002;118(4):542-5.
23. Kalaiselve G, Rajendran K. Rapid diagnosis of tuberculous pleural effusion by IS6110 sequence based PCR. IOSR Journal of Dental and Medical Science.2013;12(6):50-4.
24. Cheng VC, Yam WC, Hung IF, Woo PC, Lau SK, Tang BS, et al. Clinical evaluation of polymerase chain reaction for the rapid diagnosis of tuberculosis. J Clin Path. 2004;57(3): 281-5.
25. Moon JW, Chang YS, Kim SK, Kim YS, Lee HM, Chang J. The clinical utility of polymerase chain reaction for diagnosis of pleural tuberculosis. Clin Infect Dis. 2005; 41(5):660-6.
26. Lima DM, Colares JK, da Fonseca BA. Combined use of the polymerase chain reaction and detection of adenosine deaminase activity on pleural fluid improves the rate of diagnosis of pleural tuberculosis. Chest. 2003;124(3): 909-14.
27. Villegas MV, Labrada LA, Saravia NG. Evaluation of polymerase chain reaction, adenosine deaminase, and interferon-gamma in pleural fluid for the differential
diagnosis of pleural tuberculosis. Chest. 2000; 118(5):1355-64.
28. Srivastav R, Kumar D, Waskar MN, Sharma M, Katoch VM, Srivastav BS. Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis. J Med Microbiol. 2006; 55(Pt 8):1071-7.
29. Dale JW, Al-Ghusein H, Al-Hashmi S, Butcher P, Dickens AL, Drobniewski F, et al. Evolutionary relationships among strains of Mycobacterium tuberculosiswith few copies of IS6110.J Bacteriol.2003; 185(8):2555-62.
30. Thangappah RB, Paramasivan CN, Narayan S. Evaluating PCR, culture & histopathology in the diagnosis of female genital tuberculosis. Indian J Med Res. 2011;134:40-6.
31. Kulkarni SP, Jaleel MA, Kadival GV. Evaluation of an in-house developed PCR for diagnosis of tubercular meningitis in Indian children. J Med Microbiol. 2005;54(pt4):369-73.
32. Kumar P, Sen MK, Chauhan DS, Katoch VM, Singh S, Prasad HK. Assessment of n-PCR assay in diagnosis of pleural tuberculosis: detection of M. tuberculosis in pleural fluid and sputum collected in tandem. PLoS ONE 5(4): e10220. doi:10.1371/journal.pone.0010220
33. Negi SS, Anand R, Pasha ST, Gupta S, Blasir SF, Khare S, et al. Diagnostic potential of IS6110, 38 kda, 65 kda and 85B sequence based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical specimens. Indian J Med Microbiol. 2007; 25(1):43-9. 34. Parandaman V, Narayanan S, Narayanan PR. Utility of polymerase chain reaction using two probes for rapid diagnosis of tubercular pleuritis in comparison to conventional methods. Indian J Med Res. 2000; 112:47-51.
35. Jatana SK, Nair MN, Lahiri KK, Sarin NP. Polymerase chain reaction in the diagnosis of tuberculosis. Ind. Pediatr. 2000;37(4):375-82.36. Takagi N, Hasegawa Y, Ichiyama S, Shibagaki T, Shimokata K. Polymerase chain reaction of pleura biopsy specimens for rapid diagnosis of tuberculous pleurisy. Int J Tuberc Lung Dis.1998;2(4): 338-41.
2. Vorster MJ, Allwood BW, Diacon AH, Koegelenberg CF. Tuberculous pleural effusions: advances and controversies. J Thoracic Dis. 2015;7(6):981-91.
3. Light RW. Update on tuberculous pleural effusion. Respirology2010;15(3):451-8.
4. Chakravorty S, Dudeja M, Hanif M, Tyagi JS. Novel multipurpose methodology for detection of extrapulmonary specimens by smear microscopy, culture and PCR. J Clin Microbiol. 2005;43(6):2697-702.
5. Makeshkumar V, Madhavan R, Narayan S. Polymerase chain reaction targeting insertion sequence for diagnosis of extrapulmonary tuberculosis. Indian J Med Res. 2014; 139(1):161-6.
6. Maurya AK, Kant S, Nag VL, Kushwaha R, Dhole TN. Detection of 123 bp fragment of insertion element IS6110 Mycobacterium tuberculosis for diagnosis of
extrapulmonary tuberculosis. Indian J Med Microbiol. 2012;30(2):182-6.
7. Sekar B, Selvaraj L, Alexis A, Ravi S, Arunagiri K, Rathinavel L. The utility of IS6110 sequence based polymerase chain reaction in comparison to
conventional methods in the diagnosis of extrapulmonary tuberculosis. Indian J Med Microbiol. 2008;26(4):352-5.
8. Huyen MN, Tiemersma EW, Kremer K, de Haas P, Lan NT, Sola C, et al. Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam. Int J Tuberc Lung Dis. 2013; 17(11):1479-85.
9. Miyazaki Y, Koga H, Kohno S, Kaku M. Nested polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol.1993;31(8):2228-32.
10. Kulkarni S, Singh P, Memon A, Natraj J, Kanade S, Kelkar R, et al. An in-house multiplex PCR test for the detection of Mycobacterium tuberculosis, its validation
& comparison with a single target TB-PCR kit. Indian J Med Res. 2012;135(5):788-94.
11. Somerville W, Thibert L, Schwartzman K, Behr MA. Extraction of Mycobacterium tuberculosis DNA: a question of containment. J Clin Microbiol. 2005;43(6): 2996-7
12. Sjobring U, Mecklenburg M, Andersen AB, Miorner H. Polymerase chain reaction for detection of Mycobacterium tuberculosis.J Clin Microbiol.1990;28(10):2200-04.
13. Piersimoni C, Scarparo C, Callegaro A, Passerine Tosi CP, Nista D, et al. Comparison of MB/BacT ALERT 3D system with radiometric BACTEC system and Lowenstein-Jensen medium for recovery and identification of mycobacteria from clinical specimens:a multicenter study. J Clin Microbiol.2001;39(2):651-7.
14. Liu KT, Su WJ, Preng RP. Clinical utility of polymerase chain reaction for diagnosis of smearnegative pleural tuberculosis. J Chin Med Assoc.2007;70(4):148-51.
15. Light RW. Pleural diseases. Baltimore: Lippincott Williams and Wilkins; 2001. p. 182-95.
16. Nagesh BS, Sehgal S, Jindal SK, Arora SK.Evaluation of polymerase chain reaction for detection of Mycobacterium tuberculosis in pleural fluid. Chest. 2001; 119(6):1737-41.
17. Conde MB, Loivos AC, Rezende VM, Soares SL, Mello FC, Reingold AL. Yield of sputum induction in the diagnosis of pleural tuberculosis. Am J RespirCrit Care Med.2003;167(5):723-5.
18. Reechaipichitkul W, LulitanondV,Sungkeeree S, Patjanasoontorn B. Rapid diagnosis of tuberculous pleural effusion using polymerase chain reaction. Southeast Asian J Trop Med Public Health. 2000;31(3):509-14.
19. Shital P, Gajanan H, Rujuta A. Role of nucleic acid amplification tests (NAATs) in tuberculous pleural effusion: where it fits in routine diagnostic workup? J
Cell SciTher. 2014. 5: 169. doi:10.4172/2157-7013.1000169.
20. Diacon AH, Van de Wal BW, Wyser C, Smedema JP, Bezuldenhout J, Bollinger CT, et al. Diagnostic tools in tuberculous pleurisy: a direct comparative study. EurRespir J. 2003;22(4):589-91.
21. Hillemann D, Richter E, Rusch-Gerdes S. Use of the BACTEC Mycobacteria Growth Indicator Tube 960 automated system for recovery of mycobacteria from 9,558 extrapulmonary specimens, including urine samples. J Clin Microbiol. 2006; 44(11):4014-7.
22. Lu D, Heeren B, Dunne WM. Comparison of the automated Mycobacteria Growth Indicator Tube System (BACTEC 960 / MGIT) with Löwenstein-Jensen medium for recovery of mycobacteria from clinical specimens. Am J ClinPathol. 2002;118(4):542-5.
23. Kalaiselve G, Rajendran K. Rapid diagnosis of tuberculous pleural effusion by IS6110 sequence based PCR. IOSR Journal of Dental and Medical Science.2013;12(6):50-4.
24. Cheng VC, Yam WC, Hung IF, Woo PC, Lau SK, Tang BS, et al. Clinical evaluation of polymerase chain reaction for the rapid diagnosis of tuberculosis. J Clin Path. 2004;57(3): 281-5.
25. Moon JW, Chang YS, Kim SK, Kim YS, Lee HM, Chang J. The clinical utility of polymerase chain reaction for diagnosis of pleural tuberculosis. Clin Infect Dis. 2005; 41(5):660-6.
26. Lima DM, Colares JK, da Fonseca BA. Combined use of the polymerase chain reaction and detection of adenosine deaminase activity on pleural fluid improves the rate of diagnosis of pleural tuberculosis. Chest. 2003;124(3): 909-14.
27. Villegas MV, Labrada LA, Saravia NG. Evaluation of polymerase chain reaction, adenosine deaminase, and interferon-gamma in pleural fluid for the differential
diagnosis of pleural tuberculosis. Chest. 2000; 118(5):1355-64.
28. Srivastav R, Kumar D, Waskar MN, Sharma M, Katoch VM, Srivastav BS. Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis. J Med Microbiol. 2006; 55(Pt 8):1071-7.
29. Dale JW, Al-Ghusein H, Al-Hashmi S, Butcher P, Dickens AL, Drobniewski F, et al. Evolutionary relationships among strains of Mycobacterium tuberculosiswith few copies of IS6110.J Bacteriol.2003; 185(8):2555-62.
30. Thangappah RB, Paramasivan CN, Narayan S. Evaluating PCR, culture & histopathology in the diagnosis of female genital tuberculosis. Indian J Med Res. 2011;134:40-6.
31. Kulkarni SP, Jaleel MA, Kadival GV. Evaluation of an in-house developed PCR for diagnosis of tubercular meningitis in Indian children. J Med Microbiol. 2005;54(pt4):369-73.
32. Kumar P, Sen MK, Chauhan DS, Katoch VM, Singh S, Prasad HK. Assessment of n-PCR assay in diagnosis of pleural tuberculosis: detection of M. tuberculosis in pleural fluid and sputum collected in tandem. PLoS ONE 5(4): e10220. doi:10.1371/journal.pone.0010220
33. Negi SS, Anand R, Pasha ST, Gupta S, Blasir SF, Khare S, et al. Diagnostic potential of IS6110, 38 kda, 65 kda and 85B sequence based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical specimens. Indian J Med Microbiol. 2007; 25(1):43-9. 34. Parandaman V, Narayanan S, Narayanan PR. Utility of polymerase chain reaction using two probes for rapid diagnosis of tubercular pleuritis in comparison to conventional methods. Indian J Med Res. 2000; 112:47-51.
35. Jatana SK, Nair MN, Lahiri KK, Sarin NP. Polymerase chain reaction in the diagnosis of tuberculosis. Ind. Pediatr. 2000;37(4):375-82.36. Takagi N, Hasegawa Y, Ichiyama S, Shibagaki T, Shimokata K. Polymerase chain reaction of pleura biopsy specimens for rapid diagnosis of tuberculous pleurisy. Int J Tuberc Lung Dis.1998;2(4): 338-41.
CITATION
DOI: 10.17511/ijmrr.2016.i05.10
Published: 2016-05-31
How to Cite
1.
Singh A, Kumar A, Chaturvedi V, Sharma P. Application of in-house nested polymerase chain reaction for rapid diagnosis of tuberculous pleural effusion. Int J Med Res Rev [Internet]. 2016May31 [cited 2024Nov.23];4(5):723-30. Available from: https://ijmrr.medresearch.in/index.php/ijmrr/article/view/547
Issue
Section
Original Article
OAI - Open Archives Initiative
