Serological Diagnosis of Tuberculosis

A comparative study of the diagnostic value of the ICT-TB test and the TB-Dot test, based on laboratory examination, was carried out in 39 patients suffering from sputum positive pulmonary tuberculosis (25 males and 14 females, aged 16-50 years) and in 48 patients (27 males and 21 females, aged 17-55 years) suffering from non-tuberculosis pulmonary diseases, that had attended the Tembagapura Hospital and the TB Control Health Center Timika-Mimika, Papua. The diagnostic sensitivity of the ICT-TB test was 87.18%, the diagnostic specificity was 81.25%, the diagnostic positive predictive value was 79.07%, the negative predictive value was 88.64%, and the diagnostic efficiency was 83.91%. The diagnostic sensitivity of the TB-Dot test was 93.31%, the diagnostic specificity was 95.83%, the diagnostic positive predictive value was 94.74%, the negative predictive value was 93.85%, and the diagnostic efficiency was 94.25%. The results of the statistical analysis of the data obtained in this study revealed that the diagnostic specificity, the diagnostic positive predictive value and the diagnostic efficiency of the TB-Dot test were significantly higher (p< 0.05) than those of the ICT-TB test. However, the diagnostic sensitivity and the negative predictive value of both tests did not differ significantly (p>0.05). Viewed from the point of their practicability, it can be justified that the ICT-TB test is a very practicable test, which needs only 15 minutes and does not require special instruments to perform the test, but is more expensive than the TB-Dot test. On the other hand, though the TB-Dot test is not very practicable and relatively time consuming, it has a significantly higher degree of diagnostic value and is much cheaper when compared to the ICT-TB test. SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 142 Vol 36 No. 1 January 2005 chronic obstructive pulmonary diseases (COPD), and 3 patients with chronic allergic pharyngitis. All of them had negative sputum culture as well as negative smears for M. tuberculosis. None of the patients in the study had received corticosteroids or other immunosuppressive drugs previously. None suffered from diseases that might interfere with the humoral immune response. Each of the specimens of serum obtained from the patients in the study were tested with the local made TB-Dot (Mekar Jaya Diagnostika, Indonesia), and the ICT-TB (ICT Diagnostics, NSW, Australia) test that was imported. The procedures for the tests were followed in accordance with the manufacturer’s instructions. The ICT-TB test procedure The principles of this immunochromatografic test have been described previously (Cole et al, 1996). Five highly purified antigens (including one of the 38 kDa) secreted by M. tuberculosis during active infection were immobilized in four capture lines on the test strip. The test detects the presence of immunoglobulin G (IgG) to these antigens. A total of 30 μl serum was added to the blue pad and diffused along the test strip. The test card was closed and the conjugate (anti-human IgG labeled with colloidal gold particles) bound any human IgG on the capture lines, producing one or more pink lines of color. The presence of one or more pink lines on the test strips was considered a positive result. The TB-Dot test procedure The basic principle of this test is an indirect enzyme immunobinding assay using as the solid phase a piece (12x7 mm) of nitrocellulose paper (Padmidewi, 1996). The antigen used in this test is a polymerized peptide of the cytoplasm, cytoplasmic membrane and cell surface proteins of M. tuberculosis var bovis (ultrasonically disintegrated and ultracentrifuged). The conjugate used in this test was a goat antihuman IgG F(ab)2 fragment. The substrate used in this test was a 0.02% solution of H2O2 in substrate buffer (pH 5.6) and 3-amino9-ethylcarbozole (EAC) was used as the chromogen of the substrate. The nitrocellulose test strips, each containing one drop (1 μl) of the polymerized antigen, were distributed into the wells of the plastic tray provided by the manufacturer of the kit. One ml of diluted (1:3,200) sera (patient or control) was added into each well, and the tray was incubated at room temperature for 2 hours on a shaker (60 rotations per minute). Unbound sera components were removed with a micropipette and washed afterwards 3 times for 5 minutes with a washing buffer solution containing 0.15 M NETG (NaCl, EDTA, tris, and gelatin), and 0.05% Tween 20. Subsequently, 250 μl of diluted (1:2,500) conjugate solution was added into the wells and the tray was incubated at room temperature for one hour on a shaker. After another wash cycle, 250 μl of the chromogenic substrate solution was added to the wells. The colorimetric reaction proceeded in the dark for 8 minutes at room temperature. Finally, the reaction was stopped by rinsing the nitrocellulose test strips, twice, with aquadestilata and then the results of the tests were read with the naked eye. The result of the test was positive when a red dot appeared on the nitrocellulose paper and negative when no red dot was observed on the nitrocellulose test strips. In each series, positive and negative control sera were run. The results of the test were judged as valid if the positive control serum gave a positive result and the negative control serum gave a negative result. For both tests (ICT-TB and TB-Dot), 3 laboratory technicians were assigned to read each test. The reported end-results were those approved by at least 2 of the 3 readers. The diagnostic value of the ICT-TB and TB-Dot tests were assessed based on the determination of the diagnostic sensitivity, the diagnostic specificity, the diagnostic efficiency, diagnostic positive predictive value and negative predictive value (Krieg et al, 1975; Galen, 1982). If there were differences in the diagnostic values between the two tests, the McNemar’s test was used to recognize the significance (p<0.05) (Siegel, 1988).