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ORIGINAL ARTICLE
Year : 2005  |  Volume : 23  |  Issue : 4  |  Page : 245-248
 

Comparison of various microbiological tests including polymerase chain reaction for the diagnosis of osteoarticular tuberculosis


Microbiology Division, National Institute of communicable Diseases, New Delhi-110 054, India

Correspondence Address:
S Gupta
Microbiology Division, National Institute of communicable Diseases, New Delhi-110 054
India
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Source of Support: None, Conflict of Interest: None


PMID: 16327120

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 ~ Abstract 

Purpose: To evaluate the utility of the polymerase chain reaction(PCR) test for diagnosing osteoarticular tuberculosis (TB). Methods: Clinical samples (synovial tissue and synovial fluid) obtained from 23 cases of suspected osteoarticular tuberculosis were subjected to Ziehl Neelsen (ZN) smear examination, radiometric BACTEC culture and PCR test for tuberculosis by amplifying 65 kDa antigen coding region of Mycobacterium tuberculosis ( M.tb ) genome. Results: PCR test was found to be much sensitive than the ZN smear examination and BACTEC culture(p < 0.05) in the diagnosis of osteoarticular TB. In synovial fluid samples, PCR was positive in 73.9%, ZN smear examination in 17.39% and BACTEC culture in 39.13% of cases.The positivities were relatively lower with synovial tissue samples, the corresponding figures being 60.8, 8.6 and 26.08% respectively. Moreover, on combining the results of synovial fluid and tissues, the corresponding figures further increased to 78.2, 21.7 and 43.3% respectively. Further, sensitivity and specificity for PCR employing BACTEC culture as the "gold standard" was 100 and 83.3% respectively. Using BACTEC culture, the earliest positivity was seen in three days using synovial tissue specimen and 13 days with synovial fluid, the average detection times being 23.2 days and 32.6days respectively. On the other hand, PCR test gave a positive result within 24 hours.Conclusions: PCR test was shown to be much more sensitive than ZN smear examination and BACTEC culture test for diagnosing osteoarticular tuberculosis.


Keywords: Mycobacterium tuberculosis, Ziehl-Neelsen, BACTEC, PCR


How to cite this article:
Negi S S, Gupta S, Khare S, Lal S. Comparison of various microbiological tests including polymerase chain reaction for the diagnosis of osteoarticular tuberculosis. Indian J Med Microbiol 2005;23:245-8

How to cite this URL:
Negi S S, Gupta S, Khare S, Lal S. Comparison of various microbiological tests including polymerase chain reaction for the diagnosis of osteoarticular tuberculosis. Indian J Med Microbiol [serial online] 2005 [cited 2019 Aug 23];23:245-8. Available from: http://www.ijmm.org/text.asp?2005/23/4/245/17077


Osteoarticular tuberculosis (TB) is a relatively uncommon manifestation of extrapulmonary TB and is usually diagnosed late during the course of illness.[1] Tuberculous damage of bone and joint accounts for approximately 10 to 15% of all extrapulmonary forms of tuberculosis.[2] Early diagnosis is extremely important in order to initiate antitubercular chemotherapy to prevent further joint destruction. Radiological methods, which are useful for the early diagnosis of pulmonary tuberculosis, are not sensitive and specific enough to provide an early diagnosis of bone and joint tuberuclosis.[3] Even histology, is non conclusive in many of the cases besides requiring special technical expertise. Bacteriological diagnosis by conventional techniques,i.e., smear examination and culture lacks sensitivity and in addition, culture is time consuming which causes an inordinate delay in treatment of such cases.[4]

The technique of DNA amplification by polymerase chain reaction (PCR) has been used successfully to detect the presence of extremely small quantities of M. tb . in clinical samples.[2],[4],[5],[6],[7],[8]It is an extremely sensitive and specific technique available to many laboratories and has been suggested as a useful tool for diagnosis of extrapulmonary tuberculosis. [2],[3],[4],[5],[6]

In this study, synovial tissue and synovial fluid samples obtained from suspected cases of osteoarticular TB were examined by PCR test and the results compared with conventional techniques viz., smear examination and BACTEC culture for tuberculosis.


 ~ Materials and Methods Top


Clinical specimens

Between January 2003 to November 2003, 23 clinical samples of synovial fluid and tissue were received in Microbiology department from equal number of cases of monoarticular arthritis of any peripheral joint with strong clinical/radiological/histopathological evidence of tuberculosis from Lok Naik Jai Prakash Narain Hospital and Ram Manohar Lohia Hospital, Delhi. In addition eight samples (5 - synovial tissue, 3 - synovial fluid) from five patients with joint damage of nontuberculous origin were included as negative controls. Synovial fluid and tissues were obtained from these cases by carrying out joint aspiration and arthrotomy in the above mentioned hospitals. The samples were forwarded to the laboratory in sterile physiological saline. The inclusion and exclusion criteria are shown in [Table - 1].

Smear microscopy (ZN stain) and culture in Bactec 460 system

Synovial tissue samples were ground using sterile mortar and pestle, the material thus obtained was used for all the three tests mentioned above. The synovial fluid was used as such for all the tests. The smears prepared were stained by ZN staining as described elsewhere.[9] Each slide was examined microscopically for the presence of M. tb . bacilli. For BACTEC culture, 500mL of each sample was inoculated into a 12B vial as per the instructions given in the BACTEC 460TB system manual supplied by the Becton Dickinson and vials incubated for a maximum of six weeks at 37[0]C.[10] The results were interpreted as per instruction given in the manual.

DNA extraction and amplification of the 65kDa gene (165bp)of M.tb.

The One millilitre of ground synovial tissue and synovial fluid as such were used for DNA extraction. DNA was extracted using commerciallly available QIAmp DNA kit (QIAGEN company, Germany) with one initial additional step. The preliminary processed materials as described above were kept at 80[0] C for 10 minutes for inactivation of possible mycobacteria. The material was then further processed as per the guidelines of the manufacturer of the kit to obtain the DNA. A 165bp region of the 65kDa antigen coding gene of M.tb. was chosen as the primer target for DNA amplification.[8],[11] The sequence of the two primers used were: Forward primer 5' oligo: 5' CTA GGT CGG GAC GGT GAG GCC AGG 3' (91-114);Reverse primer 3' oligo: 5' CAT TGC GAA GTG ATT CCT CCG GAT 3' (254-231). DNA amplification by PCR was performed with a total reaction volume of 25mL by using a model PCR system 2700 thermal cycler (Applied Biosystems). The amplification reaction contained the forward and reverse primers at final concentration of 0.01 and 1mM, respectively, 2.5 U of Taq polymerase (Perkin Elmer) in amplification buffer, 200mM (each) of the four deoxyribonucleoside triphosphate and 5mL of DNA. Positive control DNA from H37Rv strain of M.tb and negative control (dH[2]O) were also used for amplification. For checking the PCR inhibition factor, with each set of experiment, we used spiked control along with test sample i.e., 2.5 mL of known positive control (DNA from H37Rv) was mixed with 2.5 mL of our test samples and the resultant amplicon suggested absence of inhibitory factors. The temperature of the reaction mixture was first raised to 95[0]C for 120 seconds, and 72[0]C for 40 seconds for denaturation. Thereafter, 40 cycles of 93[0]C for 40seconds and 72[0]C for 40 seconds for extension. After final extension, the samples were immediately processed or kept at -20[0]C till tested. PCR products were detected on 1.5% agarose gel. Samples showing the presence of 165bp band under ultraviolet transillumination were considered positive for the presence of M.tb [Figure - 1].

Statistical analysis

The difference between positivity rates for various clinical samples by different tests were compared using the Chi square test with Yates modification.


 ~ Results Top


A total of 46 clinical samples of synovial fluid and tissue were received and tested by different laboratory based diagnostic approaches including ZN stained AFB smear microscopy, radiometric BACTEC culture and molecular (PCR) test. Results obtained from these tests are given in the [Table - 2] and [Table - 3].

As is evident from [Table - 2], there was a better positivity seen in synovial fluid samples in all the three tests performed as compared to the synovial tissue samples, however, higher positivity was obtained when the result of both of these clinical samples were combined. Statistically, the detection rate of M.tb. by PCR was significantly higher than both ZN stained AFB smear examination and BACTEC culture(p < 0.05). In the group of five patients without bone tuberculosis, all eight samples showed negative result by PCR, smear examination and BACTEC culture depicting their specificity as 100%.

[Table - 3] gives a detailed comparison of PCR test results vis a vis results of other tests in isolation as well as when used in combination. As is evident, all the smear positive and BACTEC culture positive samples were also found to be positive by PCR test, whereas out of 40 smear negative samples, 14 turned out to be positive by PCR test. Even, out of 31 samples testing negative by BACTEC method, PCR test was able to detect 10 positives. So the calculated sensitivity and specificity for PCR with culture as "gold standard" was 100%.

PCR test proved to more sensitive even when both smear examination and BACTEC culture results were considered in conjunction. All the 13 samples which were negative by smear examination, but positive by BACTEC culture were also found to be positive by PCR test. Even, among the 26 samples which were negative by both the tests (smear examination as well as the BACTEC culture), PCR test was able to detect six positives [Table - 3].

Regarding the time taken for a positive result, it was seen that using BACTEC culture, the earliest positivity was found in three days with synovial tissue and 13 days with synovial fluid, the average detection times being 23.2 days and 32.6 days respectively. On the other hand, PCR test gave a positive or negative results within 24 hours.


 ~ Discussion Top


In the present study, we attempted to compare the utility of PCR test, ZN stained AFB smear examination and BACTEC culture for diagnosing osteoarticular TB. PCR showed a much higher sensitivity when compared with the other two tests(p < 0.05). The sensitivity of all the three tests was found to be much higher when the results of both synovial fluid as well as synovial tissue were considered together [Table - 1]. In a limited number of published studies, both smear examination and conventional culture have shown a low sensitivity in detecting this form of TB.[1],[2],[3],[4]Titov et al showed 50% sensitivity by smear examination and culture in diagnosis of bone and joint TB.[2]

In our study, the reasonable sensitivity of PCR test in smear negative samples and even BACTEC culture negative samples highlights the utility of PCR test in smear/culture negative samples [Table - 3]. The PCR test sensitivity of 78.2% was more than that shown in another study of Vanderspoel van dijk et al who depicted a sensitivity of 57.6% in skeletal tuberculosis[4] although sensitivity of PCR test using culture as the gold standard was found to be equal (100%) in both the studies. However, 78.2% sensitivity by PCR test in our study was less than that of Sun et al study who showed a 83% sensitivity in bone tuberculosis.[13] Titov et al showed PCR positivity in eight out of eight samples from bone and joint tuberculosis by amplifying 365bp region of MPB 64 gene.[2] Verettas et al showed a positive PCR test result in a study group of six patients of skeletal tuberculosis by targeting IS6110 gene.[13] The PCR test sensitivity in our study was found considerably higher than that of Singh et al study who depicted a sensitivity of 68% in lymph node biopsy samples by amplifying 513bp region of dev R gene.[6] Our PCR sensitivity was also higher than that of Tiwari et al who showed a 61.7% sensitivity by targeting 123bp region of IS6110 gene of M.tb. in different clinical samples of extrapulmonary TB.[5] PCR test was able to give a positive result in less than 24 hours as compared to an average time of 23.2 to 32.6 days taken for a positive BACTEC culture result further showing the utility of PCR in early detection of M.tb . Thus, we believe that the high sensitivity of PCR test should be useful for early and better detection of mycobacterial infections especially, in paucibacillary extrapulmonary tuberculosis.


 ~ Acknowledgement Top


The authors thank Mr. Udaiveer Singh for the technical assistance and Dr. Pankaj Aggarwal, LNJP Hospital, for providing clinical samples for the study.



 
 ~ References Top

1.Ellis ME, Ramahi KM, Al Dalaan AN. Tuberculosis of peripheral joints: a dilemma in diagnosis. Tuberc Lung Dis 1993; 74 :399-404.  Back to cited text no. 1    
2.Titov AG, Vyshnevskaya EB, Mazurenko SI, Santavirta S, Konttinen YT. Use of polymerase chain reaction to diagnose tuberculous arthritis from joint tissues and synovial fluid. Arch Pathol Lab Med 2004; 128 :205-9.  Back to cited text no. 2    
3.Sequeira W, Co H, Block JA. Osteoarticular tuberculosis: current diagnosis and treatment. Am J Ther 2000; 7 :393-8.  Back to cited text no. 3    
4.Vanderspoel van dijk A, Mcleod A, Botha PL, Shipley JA, Kapnoudis MA, Beukes CA. The diagnosis of skeletal tuberculosis by polymerase chain reaction. Cent African J Med 2000; 46 :144-9.  Back to cited text no. 4    
5.Tiwari V, Jain A, Verma RK. Application of enzyme amplified mycobacterial DNA detection in the diagnosis of pulmonary and extrapulmonary tuberculosis. Indian J Med Res 2003; 118 :224-8.  Back to cited text no. 5    
6.Singh KK, Muralidhar M, Kumar A, Chattopadhyaya TK, Kapila K, Singh MK, et al . Comparison of inhouse polymerase chain reaction with conventional techniques for the detection of M.tb. DNA in granulomatous lymphadenopathy. J Clin Pathol 2000; 53 :355-61.  Back to cited text no. 6    
7.Dwivedi A, Sarin BC, Mittar D, Sehajpal PK. Optimization of the 38 kDa based PCR assay for detection of M.tb. from clinical samples. Indian J Tub 2003; 50 :209-17.  Back to cited text no. 7    
8.Chiac P, Yen TS, You JB, Maa JS, Fiss EH, Chang CH. Detection and identification of Mycobacterium tuberculosis by DNA amplification. J Clin Microb 1990; 28 :1877-80.  Back to cited text no. 8    
9.Mycobacteria, chapter 13. In : Baron EJ, Finegold SM, editors. Bailey and Scott's Diagnostic Microbiology. 9th Ed. The CV Mosby Company: St Luis; 1994. p. 590-633.  Back to cited text no. 9    
10.Siddiqi SH. BACTEC 460 TB system, Product and procedure manual. Becton Dickinson Microbiology System, Sparks, Md 1996.  Back to cited text no. 10    
11.ShinnickTM. The 65kilodaalton antigen of Mycobacterium tuberculosis. J Bacteriol 1987;169:1080-8.  Back to cited text no. 11    
12.Sun Y, Zhang Y, Lu Z. Clinical study of polymerase chain reaction technique in the diagnosis of bone tuberculosis. Zhonghua Jie He He Hu Xi Za Zhi 1997; 20 :145-8.  Back to cited text no. 12    
13.Verettas D, Kazakos C, Tilkeridis C, Derman A, Petrou H, Galanis V. Polymerase chain reaction for the detection of Mycobacterium tuberculosis in synovial fluid, tissue samples, bone marrow aspirate and peripheral blood. Acta Orthop Belg 2003; 69 :396-9.  Back to cited text no. 13    


Figures

[Figure - 1]

Tables

[Table - 1], [Table - 2], [Table - 3]

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