https://imcjms.com Ibrahim Medical College Journal of Medical Science https://imcjms.com/registration/journal_full_text/154 Thu, 15 Dec 2016 17:01:15 +0000 Background and objective: Serological test has become an important tool in the diagnosis of TB cases. This study focused on the analysis and comparison of antibody response to two Mycobacterium tuberculosis (MTB) antigens namely Ag85 complex and culture filtrate protein (CFP) in patients with tuberculosis. Antibody response to specific antigen was utilized as a diagnostic tool to detect active tuberculosis (TB) cases. Results: The mean OD values of serum IgM and IgG antibodies against Ag85 and CFP were significantly (p<0.0001) higher in patients than that of healthy control individuals. Among the 30 tuberculosis patients, anti-Ag85 complex IgM and IgG was positive in 66.7% and 70.0% patients respectively. The seropositive rate of anti-CFP IgM and IgG was 33.3% and 56.7% respectively. The sensitivity and specificity of anti Ag85 and anti-CFP IgM and IgG ranged from 60.0% to 96.7%. IMC J Med Sci 2016; 10(2): 53-57. DOI: https://doi.org/10.3329/imcjms.v10i2.31111   Tuberculosis (TB) caused by Mycobacterium tuberculosis(MTB) is one of the leading cause of morbidity and death in the world. In 2015, there were about 10.4 million new TB cases worldwide and 1.4 million deaths from TB [1]. The estimated annual incidence of TB in Bangladesh is 0.362 million cases [1]. Early diagnosis and treatment of TB patients is crucial for the control of TB [2]. In developing countries, current diagnosis of TB largely relies on clinical symptoms, radiographic evidence supported by presence of acid fast bacilli in smear and isolation of MTB by culture from the clinical specimen. Direct smear is fast and cheap method but it lacks sensitivity. Culture method is more sensitive but it is cumbersome and requires longer incubation period. In the recent years, the detection of MTB-specific antibodies has become important diagnostic aid in the diagnosis of TB especially in smear-negative sputum cases [3]. It is particularly important for pediatric and extra pulmonary tuberculosis and in those unable to produce sputum. Thus the antibody assay might be of great benefit for early diagnosis and control of TB. Serological methods have been regarded as attractive tools for rapid diagnosis of tuberculosis due to their simplicity, rapidity and low cost. Serodiagnosis also does not require safety measures associated with handling of live bacilli as in culture and offer the possibility of detecting cases often missed by routine sputum smear microscopy. It is estimated that a rapid and widely available diagnostic assay with 85% sensitivity and 95% specificity would result in 400,000 fewer deaths each year and would greatly reduce the global health cost of TB [7]. Materials and methods Study population: Study included thirty adult confirmed active pulmonary tuberculosis cases. Active TB cases were confirmed by clinical features, positive AFB sputum and culture. Age and sex matched thirty healthy individuals were enrolled in the study as healthy control. All the healthy individuals were BCG vaccinated and neither they nor any of their family members had history or any sign symptoms of tuberculosis. About 5 ml of blood was collected from each participant with aseptic precautions by venipuncture. The serum was immediately separated and stored at -200C until used. Interpretation of the result: The concentration of antibody was expressed in OD at a particular dilution of both patient and healthy control samples. In order to diagnose active tuberculosis cases by detecting IgM and IgG antibody response against specific antigens, a cut off OD value was determined by the following formula: Cut off OD value= Mean antibody (IgM/IgG) titer of healthy participants against specific antigen +2xSD. Any OD value above the cut off was considered as positive for respective antibodies. Significance of difference of mean OD value of IgM and IgG antibodies against respective antigens was calculated by independent student’s t test.   Results Antibody response to Ag85 complex and CFP was determined in sera of 30 confirmed cases of tuberculosis and 30 healthy individuals. IgM and IgG response against specific antigens were determined by ELISA method and concentration of antibodies was expressed in terms of OD value. Table-1 shows the antibody response to two mycobacterial antigens. It was observed that mean OD values of serum IgM and IgG antibodies against Ag85 complex and CFP were significantly higher in patients (p<0.0001) than that of healthy control subjects.   Table-1: Antibody response to different MTB antigens in tuberculosis and healthy participants   1.18±0.10 0.78±0.10 0.56±0.03 0.39±0.03 0.0001 0.0004 Antigen Mean serum OD±SD of healthy individuals for Cut off OD value (Mean OD +2xSD) IgM IgG IgM IgG Ag85 complex 0.56±0.16 0.87±0.39 0.88 1.66 CFP 0.57±0.39 1.36 Note: Cut off OD values were calculated from antibody response in healthy individuals.   Table-3 shows the seropositive cases of tuberculosis patients by detecting IgM and IgG antibodies against Ag85 complex and CFP antigens. It was observed that among the total number of 30 tuberculosis patients, anti-Ag85 complex IgM was positive in 66.7% patients and its sensitivity and specificity was 75.0% and 96.7% respectively. The anti-Ag85 complex IgG was positive in 70.0% of patients and its sensitivity and specificity was 76.9% and 93.75% respectively. The seropositive rate of anti-CFP IgM and IgG was 33.3% and 56.7% while the sensitivity and specificity was 60.0% and 96.7% respectively.   Table-3: Serodiagnosis of tuberculosis patients by detecting IgM and IgG antibodies against Ag 85 complex and CFP antigens   Sensitivity (%) 1.  World Health Organization. Global tuberculosis report 2016. Geneva: World health organization; 2016. 214 p. 3.  Wu X, Yang Y, Zhang J, Li B, Lian Y, Zhang C, Dong M. Comparison of antibody responses to seventeen antigens from Mycobacterium tuberculosis. Clin Chim Acta 2010; 411(19-20): 1520–1528. doi: 10.1016/j.cca.2010.06.014 5.  Demissie A, Leyten EM, Abebe M, Wassie L, Aseffa A, Abate G, et al. Recognition of stage specific mycobacterial antigens differentiates between acute and latent infections with Mycobacterium tuberculosis. Clin Vaccine Immunol 2006; 13(2):179–186. 10.  Xueqiong Wu, Yang Y, Zhang J, Li B, Liang Y, Zhang C, Dong M, Cheng H, He J. Humoral immune responses against the Mycobacterium tuberculosis 38-Kilodalton, MTB48 and CFP10/ESAT-6 antigens in tuberculosis. Clin Vaccine Immunol 2010; 17(3): 372-375. 11.  Prabha C, Jalapathy KV, Das SD. Humoral immune response in tuberculous pleuritis. Am J Immunol 2005; 1(2): 68-73. 13.  Voller A, Bartlett A, Bidwell DE. Enzyme immunoassays with special reference to ELISA techniques. J Clin Path 1978; 31: 507-520. 14.  Suraiya S, Musa M, Suppian R, Haq JA .Serological diagnosis for active tuberculosis in Malayasian population: comparison for four protein candidate. Asian Pac J Trop Dis 2012; 2: S312-S315. 2026 Ibrahim Medical College. All rights reserved.