AFB staining (direct) and AFB culture sensitivity was positive in 6(12%) cases Histopathology was positive for mycobacteria in all (100%) cases. PCR was positive in 49(98%) cases. All 27(100%) cases were serologically positive. Serological tests showed fall of IgM and rise of IgG titre at 3 months as compared to values at admission.

Discussion :

About 30 million people suffer from tuberculosis throughout the world. About 1-2% of these patients suffers from osteoarticular tuberculosis. Diagnosis of osteoarticular tuberculosis is difficult since the organism is fastidious and slow growing (2). AFB is difficult to isolate in osteoarticular tuberculosis, since it is a paucibacillary disease and since the lesion is deep seated; it is difficult to get the tissue (2).

The diagnosis of osteoarticular tuberculosis in endemic area is clinico-radiological. It is justified to treat the patients clinico-radiologically in classical lesions of the bone. The clinical response can be observed in 4-6 weeks. However, there would be certain doubtful diagnosis, where procurement of tissue is required to ascertain diagnosis. In the bone of appendicular skeleton, tissue may be procured by FNAC or core biopsy. Delay of few days in the treatment of the limb lesion does not give rise to severe consequences, as tuberculosis is a slowly progressive disease. Tuberculosis of the spine is a deep-seated lesion, which if not diagnosed promptly and treated adequately, then consequences would be hazardous, as patient may have grotesque kyphosis and neurological complication (paraplegia). The typical lesion can be diagnosed and treated clinico-radiologically with support of newer imaging modalities like C.T/ MRI, however tissue diagnosis is must, when there is slightest doubt clinico-radiologically. Tuberculosis of the spine needs to surgically decompress for certain indication. Osteoarticular tuberculosis, being a paucibacillary disease and some of the patients are already on ATT, hence no single modality like AFB culture sensitivity, AFB staining, histopathology are capable of ascertaining the diagnosis. The role of serological tests and PCR (molecular methods) are still being not defined in osteoarticular tuberculosis. That is why an attempt is made to evaluate these all-diagnostic modalities to ascertain their efficacy in establishing the diagnosis. Hence, we design this study. Sensitivity of AFB staining (direct) in various series was reported in the range of 25-75%(2). In our series, we got 8% (4 cases) positivity with direct AFB staining. Out of these four cases three patients were on ATT for more than 2 months before presentation and one patient was on ATT since 4 days. Out of these four cases in two cases, direct AFB staining was positive in pus aspirated from the cold abscess. Direct AFB staining was however negative in the tissue obtained during the surgery in both of them. No series to the best of our knowledge had evaluated direct AFB staining as a diagnostic modality in the diagnosis of tuberculosis of the spine. In pulmonary tuberculosis, direct AFB staining has an important role in the treatment of disease, as the conversion of sputum positive to sputum negative indicates the efficacy of the treatment. However in osteoarticular tuberculosis, whether the patient is sputum positive or negative does not indicate the efficacy of the treatment. In our series, we got 4% (2 cases) positivity with AFB culture sensitivity. Out of these two cases, one case had never taken ATT and one case was on ATT since 4 days. Overall, six (12%) cases were positive for AFB staining & culture sensitivity.  Lakhanpal et al (1976) reported 49.53% positivity by AFB culture sensitivity (2). Other workers have reported in a range of 48.6-80% [Dahl 1951,Dobson 1951,Holmdahl 1951,Wilkinson 1953,Weinberg 1957,Hald (Jr) 1964,and Kemp etal1973, Masood 1992] (2). Lakhanpal et al attributed the lower percentage to the possible effect of preoperative antitubercular therapy. Wilkinson also asserted the same point. In the previous series positivity by AFB, culture sensitivity is higher than in our series. This may be because more number of patients before presentation to the hospital is already on ATT for many days. However 16 (32%) cases in our series had never taken ATT. Out of these 16 cases only 2 cases were positive for AFB culture sensitivity. Therefore, this fact, that duration of ATT has any influence on the positivity of AFB culture sensitivity could not be substantiated in our study. Factors attributable for less positivity with AFB Culture sensitivity are paucibacillary disease (Number of AFB is about103 - 104 /ml), species present (M. tuberculosis is more likely to be positive than MOTT), patient already on ATT, Stain used, Observer's experience. The major limitations of AFB Culture & sensitivity are, it requires live organisms, long incubation period, low sensitivity in-patients already on ATT. Although the culture results are not available for up to four weeks, they prove the diagnosis of tuberculosis beyond doubt and the opportunity carrying out sensitivity test adds to its significance (2). Newer rapid culture techniques for diagnosis of tuberculosis like BACTEC and BACTEC-alert would be better alternative compared to conventional methods. In the need of avoiding culture techniques which has the hazard of handling live organisms various immunological methods have been developed to detect serum antibodies against M. tuberculosis. Of all techniques, ELISA is reported to be more sensitive. The problem of day-to-day variation and considerable overlap in distribution of antibody levels in active TB cases and controls were considered as major hurdles in using ELISA as diagnostic tool alone. Various authors have tested various antigens, which could be specific for M. tuberculosis (4,5). Serological positivity in our series was 100%. As per as the current record 16 cases are in chronic stage, six cases are in chronic active stage, and five cases are going into chronic stage from the acute stage. There was significant difference in the values of IgM and IgG at the time of admission and at 3 months postoperatively. These antibodies titre did not correlate with the recovery status of the patient, as patients did not show recovery proportionally to the declination of IgM titre. IgM titre was diagnostic of activity of the disease while IgG titre was diagnostic of the chronic disease. The IgG levels remain high even after full treatment. Although IgG level have no diagnostic value, it suggests that the patient has a chronic disease or healed disease. Seven cases were negative for IgM. Out of these 7 cases 6 cases were on ATT for more than 2 months and the remaining one had never taken ATT. Serological tests results did not correlate with the duration of ATT intake. This means that ELISA values are dependent on time of taking sample and state or phase of disease. Whether patient is smear negative or have pulmonary or extrapulmonary TB cannot be distinguished by this method. These observations on serological test results are essentially the same as those in other series [Stroebel et al (1982), Bhattacharya. A. Et al, (1986)] (4,5). Large number of organisms required by all the above methods was major limitation in detection of M. tuberculosis. A single test, which would amplify the genome, even if single organism were present, was though to be ideal for detection of paucibacillary TB cases. PCR can analyze the expression of genes even from single cells. PCR. was positive in 49 (98%) cases in our series. One case who was PCR negative had histopathology report suggestive of tubercular osteomyelitis. Our results with PCR was comparable to other author series [Noordhock.G.T. (1995), Jatana.S.K. (2000), Van der Spoel van Dijk A et al (2000)] (6,13,14). A number of target genes of mycobacteria DNA have been evaluated for diagnosis by PCR. The most common target used in PCR is IS6110. This sequence is specific for M.tuberculosis complex and is present upto 20 times in the genome thus offering multiple targets for amplification. PCR detection of IS6110 in pulmonary and extra pulmonary tuberculosis, when compared to culture has a sensitivity, specificity and positive predictability of 83.5%, 99%, 94.2% respectively (15). However IS6110 is a member of IS3 family, the most widely spread group of bacterial insertion sequences. It is therefore not surprising that sequences homologous to IS6110 have now also been found in mycobacteria other than M.tuberculosis complex. This may perhaps account for the false positive results that were observed in several studies that used IS6110 as a target sequence that range from 2.3%-20%(15). In addition to being prone to false-positive results, this target may also yield false-negative results [40%]. Indeed, several M.tuberculosis strains that lack this insertion sequence have now been isolated. In the present study, we have used 16srRNA as target sequence as it is universally present and hence rules out the chances of false negative results. It is a genus specific marker, and present in both typical and atypical mycobacteria. Combination of both PCR [IS6110 and 16srRNA{multiplex}] is prefered. ADVANTAGES OF PCR 1. It is a highly efficient and rapid method for diagnosis of the disease [24hr]. 2. A PCR result is of great value in early diagnosis, particularly in infection of certain body systems where disease progression is very fast and detection by culture method is time consuming. 3. As PCR is a very sensitive technique and could detect as few as 1-2 mycobacteria in the specimen, treatment may be initiated based on this result, if clinically indicated. 4. It can differentiate typical and atypical mycobacteria. 5. PCR requires a very small quantity of specimen and therefore, even microliters of a fine needle aspirate can be tested. PCR can detect very low levels of AFB in the clinical specimens and results are available within 3 days.However misleading results can occur as even the smallest amount of contaminating DNA can be amplified. PCR test result needs to be interpreted in its clinical context. A PCR positive result does not always confirm to culture results. PCR is not a substitute for culture; it is an addition to the routine battery of laboratory tests for the rapid and definitive diagnosis of tuberculosis. DISADVANTAGE not able to differentiate live from dead organisms, as it is not dependent on bacterial replication. The positivity of histopathological reports has been reported in the range of 72-97%(2,16). Histopathological positivity in our series was 100%. All the cases had histological features suggestive of tubercular osteomyelitis, confirmed by presence of caseation necrosis, epithelioid cell granuloma and Langerhans giant cells. In our series positivity with histopathology was more as compared to other series [Gardner (1946), Hald .J (1964)] (2,16). Sometimes the results are inconclusive, as the patients were already on ATT for many months before reporting to the hospital. The advantage of histopathology lies in the early results obtained, enabling the surgeon to embark upon the appropriate treatment Silverman.Jan.F. Et al (1985) stated that FNA biopsy could provide diagnostic material for morphologic and microbiologic confirmation, while avoiding either core biopsies or open biopsies under general anesthesia. With the cytomorphologic, recognition of a granulomatous process presumptive diagnosis of skeletal tuberculosis can be made, thus expediting early appropriate antitubercular treatment and excluding other process (17). Mondal .A. (1994) stated that CT-guided fine needle aspiration cytology is a useful and minimally invasive method of ascertaining histopathological diagnosis of vertebral lesions (18). Combined efficacy of histopathology, culture sensitivity and guinea pig inoculation as reported by Lakhanpal et al was 100% positive results (2). Saxena P.S. et al reported the positivity of combined efficacy as 76.4%(16). Van der Spoel van Dijk A et al (2000) reported that detection using culture could confirm only three of the 26 clinically diagnosed tuberculosis cases while PCR detection confirmed disease in 15 cases. The use of PCR increased the confirmation of clinically probable tuberculosis from 14 using standard laboratory techniques and histology to 18 of 26 cases. Calculated sensitivity and specificity for PCR employing culture as the "gold standard" were 100% (with 95% CI 29.2; 100.0) and 71.4% (55.4; 84.3), which due to low detection levels, basically excludes culture as a standard for statistical analysis. Sensitivity and specificity for PCR using histology as the "gold standard" were 78.6% (49.2; 95.3) and 87.1% (70.2; 96.4) respectively with positive and negative predictive values of 73.3% (44.9; 92.2) and 90% (73.5; 97.9) respectively (14). Only one case (3.7%) was positive for PCR, Histopathology, AFB staining and AFB culture sensitivity and serological tests in our series. No series upto best of our knowledge had evaluated the various diagnostic modalities in tuberculosis of the spine.

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