Context.—Tuberculosis of the joints and bones is a significant worldwide problem, often leading to joint and bone destruction. The diagnosis of this disease manifestation is difficult.

Objective.—To assess the role of conventional diagnostics compared to polymerase chain reaction applied to samples obtained at arthroscopy.

Design.—This was an open observational study that was blinded to the microbiologist, histopathologist, and molecular biologist responsible for assessing the main outcome measures.

Patients.—Seven patients (8 samples) with joint and bone tuberculosis and 14 patients (16 samples) with nontuberculous joint and bone disease.

Intervention.—Arthroscopic examination and tissue sample collection.

Main Outcome Measures.Mycobacterium tuberculosis staining, culture, and histopathologic assessment of caseating granulomas vs polymerase chain reaction.

Results.—Polymerase chain reaction was positive in all cases of true tuberculosis and falsely identified 2 samples as positive, both however, in patients who had lung tuberculosis in the past.

Conclusions.—Conventional bacteriological methods for demonstration of M tuberculosis are not very sensitive and can be time-consuming. Polymerase chain reaction of arthroscopically obtained joint tissue biopsies appears promising in the early diagnosis of tuberculous arthritis.

The incidence of tuberculosis is increasing worldwide.1 In particular, some developing countries in Africa and Asia and some new states of the former Soviet Union have experienced dramatic increases in the number of pulmonary tuberculosis cases. Human immunodeficiency virus and tourism increase the burden and immigration aggravates the situation in developed countries.2–5 Therefore, an increase in the incidence and prevalence of extrapulmonary joint and bone tuberculosis can also be anticipated. Tuberculous damage of bone and joints accounts for approximately 10% to 15% of all extrapulmonary forms of tuberculosis.6 Its prevalence competes with urologic and lymphatic organ damage, and it is the third, if not the second, most common extrapulmonary manifestation of tuberculosis.7 Treatment of bone and joint tuberculosis is extremely difficult, slow, and expensive, and the final result is often permanent disability. The main reason for this poor outcome is delay in the diagnosis, which is common.8 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 tuberculosis. A need for a simple, accessible, rapid, and reliable low-cost method is evident. In this study, tissue and synovial fluid samples were examined by traditional staining and histologic and bacteriological methods. These results were compared with results from polymerase chain reaction (PCR) analyses used to detect DNA of Mycobacterium tuberculosis.

Patients and Tissue Specimens

Synovial fluid was collected in a sterile test tube by arthrocentesis before the arthroscopy. Cells were centrifuged to a pellet and stored at −70°C. Tissue samples were collected under direct visual control during the arthroscopy using a conchotome, snap-frozen in liquid nitrogen, and stored at −70°C. If enough biopsy material was collected, it was used for both PCR and histology, culture, and staining. If only relatively little tissue was available, it was used for the histology and M tuberculosis cultures and staining, and only synovial fluid was available for PCR. Altogether, synovial fluid and tissue samples were obtained at arthroscopy from 21 patients. Seven patients (8 samples) had tuberculosis of the examined joint, whereas 14 patients had joint damage of nontuberculous origin, although 3 of these patients had previously had tuberculosis localized to some other joint than those examined in this study. The material subjected to examination originated from 5 hips, 13 knees, 2 ankles, and 1 elbow. Altogether, 24 samples were studied, namely, 13 tissue samples and 11 synovial fluid samples. In 3 patients (1 in the tuberculosis group and 2 in nontuberculous group), both synovial fluid and tissue were collected (Tables 1 and 2). Joint tuberculosis is a chronic disease, and owing to delay in the working diagnosis and referral, most of the patients in the present series had relatively long disease duration at the time of examination.

The biopsies were taken using an arthroscopic set, including 4-mm forward and oblique-forward (30°) telescopes and 2-mm forward and straight-angle telescopes (Grinext-EKOMP, St Petersburg and Mogilev, Russia-Belorussia) coupled with an endoscopic video system MSV-800 (Applitec, Tel-Aviv, Israel). The Parker-Pearson biopsy needle was used for biopsies.

Staining, Histology, and Culture of Mycobacteria

The specimens designated for histology were fixed in 10% formalin, embedded in paraffin, cut to 4-μm-thick sections, and stained with hematoxylin-eosin before microscopic examination.

For bacteriological studies, all samples were divided into 2 parts, one of which was immediately frozen and stored at −20°C for PCR, whereas the other part was subjected to conventional bacteriological studies, namely, acid-fast bacilli staining and M tuberculosis and nonspecific bacterial cultures. For staining and cultures, the samples were decontaminated with 10% trisodium phosphate for 24 hours before centrifugation at 1500 rpm for 20 minutes and division into 2 parts. Acid-fast bacilli were stained with auramine O fluorescent stain. The other part of the sample was inoculated onto a Löwenstein-Jensen slope and Finn-II slopes (Kreidcom Laboratory Supply Co, Moscow, Russia). Cultures were incubated at +37°C and checked weekly for 8 weeks for mycobacterial growth.

Part of each specimen was homogenized and inoculated without decontamination to the nutritive media for nonspecific (ie, other than M tuberculosis) bacteria.

DNA Extraction

Nucleic acids for PCR were extracted using a modified guanidine method.9 Synovial fluid samples were centrifuged at 10 000g for 10 minutes, and the pellet was used for DNA extraction. Frozen tissue samples and pellets were homogenized in Eppendorf tubes with a sterile glass stick and mixed with 150 mL of alkaline buffer containing 0.5% sodium dodecyl sulfate and guanidine thiocyanate–EDTA–Triton X-100 solution before 10 mL of diatomaceous earth (Sigma Chemical Co, St Louis, Mo) was added to each tube. Tubes were incubated with periodic shaking at room temperature for 30 minutes. The mixture was centrifuged and the pellet was washed sequentially with guanidine thiocyanate–EDTA solution, propanol-2, 70% ethanol, and acetone. Each washing was performed several times until the supernatant became colorless.

Polymerase Chain Reaction

After the last washing, the pellet was dried and nucleic acids were eluted with 10mM Tris–hydrogen chloride (HCl) and 1mM EDTA, pH 7.5. Five milliliters of the eluate was subjected to PCR amplification of a segment of the MPB64 gene of M tuberculosis, using the commercial kit Amplitube (Litech, Moscow, Russia) in accordance with the manufacturer's instructions (thermocycler Thermocell, Hybaid, Middlesex, United Kingdom). Sample DNA was subjected to PCR amplification. Twenty-five microliter reaction solution contained M tuberculosis MPB64 gene-specific primers and 1 U nuclease-free Taq-polymerase (Silex, Moscow, Russia). The forward primer was 5′-TGT CCG GCC CCG CCT ACA ACA TCA ACA-3′ and the reverse primer was 5′-CTT GCA CAA TGG GGA AGA CGA CGT GCA-3′ (10 pmol each). The Taq-polymerase buffer contained 20mM Tris-HCl, pH 7.5; 60% glycerine; 1mM dithiothreitol; 50mM potassium chloride; 0.1mM EDTA; 0.2 mg/mL bovine serum albumin; and 0.5% NP-40 (Nonidet P-40). The reaction buffer contained 10mM Tris-HCl, pH 8.2; 1.5mM magnesium chloride; 0.01% (vol/vol) gelatin; 0.1% Tween-20; 50mM potassium chloride; and 0.25mM dNTPs. The PCR was performed as follows: initial denaturation at 95°C for 10 minutes, followed by 30 cycles of denaturation at 95°C for 40 seconds, annealing at 63°C for 30 seconds, and extension at 72°C for 30 seconds. Results were read after 30 cycles. The 365-bp-long PCR products were analyzed by electrophoresis on 2% agarose gels, and visualization of bands was performed using ethidium bromide staining. Identification was based on comparison of the position of the sample amplicon with the positive sample control. Positive and negative controls were included in each assay. The positive control (provided as part of the Amplitube kit) was DNA extracted from M tuberculosis suspension (3000 cells/mL) by a guanidine method. In addition, we prepared our own positive controls by extracting DNA using a modified guanidine method (the same as was used for clinical specimens) from M tuberculosis suspension (1000 cells/mL). The following 3 negative controls were used: (1) DNA extraction control, (2) additional diatomaceous earth control (as the sorbent purity is very important), and (3) amplification reagents control.

Bone and Joint Tuberculosis

All 7 patients (8 samples) with joint tuberculosis (Table 1) had a positive PCR reaction (Figure, A). In this same series, samples from 3 patients (4 samples) had positive acid-fast bacilli staining and 3 patients (4 samples) had positive M tuberculosis cultures. However, 2 of 8 cultures failed for technical reasons (samples 14 and 15). The histologic picture was characteristic for tuberculosis inflammation (caseating granulomas) in 5 of these patients (5 samples) (Figure, B). There were no positive results from the cultures for nonspecific bacteria in any of these 7 patients.

Nontuberculous Bone and Joint Disease

In the nontuberculous group, 12 patients (14 samples) had negative results in the PCR test for M tuberculosis (Table 2). There were 2 false-positive PCR test results. Those 2 patients with apparently false-positive test results had had lung tuberculosis in the past, as shown by the typical radiographic findings in the form of calcifications in lung tissue and mediastinal lymph nodes. In addition, one of the patients with a false-positive PCR test result (patient 2, sample 2) had left tuberculous coxitis 45 years earlier, and the other false-positive result occurred in a patient (patient 18, sample 20) with nonspecific gonitis caused by Staphylococcus aureus. There were no positive results in acid-fast bacilli staining, routine histology, or M tuberculosis cultures in these 14 nontuberculous patients. The 3 patients in whom both synovial fluid and tissues were examined had similar test results for both types of samples (patients 4, 7, and 20).

Biopsy samples have been used extensively in the diagnosis of tuberculosis. Positive M tuberculosis cultures and/or a characteristic histologic picture have been the main criteria for diagnosis. In contrast to many other bacterial infections, M tuberculosis is often not easy to culture. Attempts to culture M tuberculosis from sputum, gastric lavage fluid, or synovial fluid may give false-negative results. Furthermore, such procedures are not fully developed routines in the diagnosis of extrapulmonary tuberculosis. Zvantseva10 reported sensitivities of 77.5% for synovial fluid samples and 89.8% for synovial tissue samples. Biopsy samples also have been used in the diagnosis of tuberculosis in sacroiliitis.11 Wallace and Cohen12 reported 2 cases of tuberculous arthritis. In their review of the literature, they noted that synovial fluid cultures are positive in almost 80% of proven cases and that open synovial membrane biopsies are positive by histology or culture in more than 90% of proven cases.

In 1992, Masood13 reported results of aspiration biopsies in 11 patients with tuberculosis of bones and soft joint tissues. Aspiration sites were spine, scapulae, chest wall, flank areas, tibia, and ring and index fingers. Granulomatous reaction, with or without caseation necrosis, was found in 73%. Acid-fast bacilli were found in 64%, and the M tuberculosis cultures were positive in 83% of all cases. Polymerase chain reaction analysis for M tuberculosis was not used. Computed tomography–guided aspiration biopsies of spinal vertebrae were described by Mondal14 in 1994 in 38 patients with spinal tuberculosis. The diagnosis was confirmed in 34 cases (89.5%) by microscopy (acid-fast bacilli staining and/or cytology) or M tuberculosis cultures. There were no complications related to the biopsy, and an open surgical biopsy could be avoided by using this approach.

Only a few publications have described the use of PCR in the diagnosis of tuberculous arthritis. In most of these articles, only 1 or 2 cases were reported.15–17 Li and coworkers18 reported that the sensitivity of the PCR test applied to synovial fluid was 57.7% in their series, that is, less than the sensitivity for sputum (81.0%) or pleural fluid (64.2%) samples in their study. They used the PCR method described by Hermans and coworkers.19 In addition, they extracted DNA fragments from M tuberculosis H37Rv IS986, which were amplified by PCR using INS-1 and INS-2 promoters. A 245-bp fragment was produced and labeled with digoxigenin. The specificity of the probe was tested by dot blotting. Li and his coworkers18 concluded that PCR together with Southern blotting help in the diagnosis of tuberculosis from synovial fluid, sputum, and pleural fluid samples. Van der Heijden and coworkers20 described results of PCR of joint samples (synovial fluid and synovial tissue samples). Two patients with septic mycobacterial arthritis, 18 with seronegative spondyloarthropathies, 21 with undifferentiated arthritis, and 40 patients with rheumatoid arthritis were analyzed using Mycobacterium-specific PCR. Mycobacterium tuberculosis was detected only in the 2 patients with septic mycobacterial arthritis.

Our results on the combined use of PCR and biopsy samples, which can in many cases be obtained by arthroscopy, are promising. Because M tuberculosis disseminates from the lung, a subclinical infection in the knee joint is possible and was perhaps detected as a false-positive PCR test result with the sensitive PCR method. Thus, these 2 false-positive results could perhaps be explained by a subclinical dissemination of M tuberculosis in the absence of clinically overt disease. This seems particularly likely in the patient who had an old, inactive hip joint tuberculosis and a false-positive PCR test result from the knee joint sample in this study. It is therefore unclear whether the questionable cases represent false-positive or true-positive results. Such a hypothesis has also been suggested by others.21,22 Polymerase chain reaction and arthroscopically obtained joint tissue biopsies appear promising in the early-stage diagnosis of bone and joint tuberculosis. This is of increasing importance owing to the human immunodeficiency virus pandemic23,24 and complications of total joint replacement surgery that sometimes result from reactivation of old bone and joint tuberculosis.25–27 

This research was supported by the Tampere Foundation Against Tuberculosis (Tampere, Finland), Pehr Oscar Klingendahl Foundation (Helsinki, Finland), and Viipuri Foundation Against Tuberculosis (Lappeenranta, Finland). We thank Maiju Kivistö for secretarial assistance.

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Author notes

Reprints: Yrjö T. Konttinen, MD, Biomedicum, PO Box 700, FIN-00029 HUS, Helsinki, Finland ([email protected])