Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 23, Num. 4, 2005, pp. 245-248

Indian Journal of Medical Microbiology, Vol. 23, No. 4, October-December, 2005, pp. 245-248

Original Article

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

Negi SS, *Gupta S, Khare S, Lal S

*Corresponding author (email:)
Microbiology Division, National Institute of communicable Diseases, New Delhi-110 054, India

Code Number: mb05073

Abstract

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

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

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°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° 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 25μL 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 1μM, respectively, 2.5 U of Taq polymerase (Perkin Elmer) in amplification buffer, 200μM (each) of the four deoxyribonucleoside triphosphate and 5μL of DNA. Positive control DNA from H37Rv strain of M.tb and negative control (dH₂O) 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 μL of known positive control (DNA from H37Rv) was mixed with 2.5 μL of our test samples and the resultant amplicon suggested absence of inhibitory factors. The temperature of the reaction mixture was first raised to 95°C for 120 seconds, and 72°C for 40 seconds for denaturation. Thereafter, 40 cycles of 93°C for 40seconds and 72°C for 40 seconds for extension. After final extension, the samples were immediately processed or kept at -20°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

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

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

References

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