Analysis of SEVA TB ES-31 antigen specific immunoglobulins IgM, IgA and IgG in sera of sputum and culture positive pulmonary tuberculosis

Tuberculosis remains major health problem in India and developing countries Immunodiagnosis has important role in screening, diagnosis and management of tuberculosis. SEVA TB ES-31 antigen has shown potential in detecting tuberculous IgG antibody in earlier studies from our laboratory. In the present study we have analysedSEVA TB ES-31 antigen specific immunoglobulinsIgM, IgA and IgG in clinically and bacteriologically confirmed pulmonary tuberculosis cases to determine the usefulness of specific immunoglobulin class in the diagnosis of patients attending the hospital. Of the 30 cases of pulmonary tuberculosis 25 (83.3%) were positive for IgG, 19 (63.3%) for IgM and 16 (53.3%) for IgA. On combining IgG and IgM positivity, sensitivity was increased to 93.3%. While combining IgG and IgA positivity, sensitivity increased to 90%. However specificity was decreased to 66.6% and 70% for both of these combinations respectively. It could be envisaged from this study that IgG antibody detection against ES-31 antigen showed acceptable sensitivity (83.3%) and specificity (86.6%) compared to IgM or IgA alone or in combination. When immune responses were analysed according to degree of sputum positivity, IgG response was observed to be predominant in all grades, compared to IgM or IgA antibody. The addition of IgM or IgA as an adjunct test increases the sensitivity but at the cost of specificity. Hence the detection of IgG alone is more useful compared to IgM or IgA assay, in detecting tuberculosis disease cases coming to the hospital.     

<Emphasis Type="Italic">In vitro</Emphasis> released antigens in diagnosis and immunomonitoring of filaria and tuberculosis

In vitro released antigens by living parasites or bacteria underin vitro maintenance or short term culture showing specific humoral immune response have been explored in development of immunodiagnostics for infectious diseases such as filariasis and tuberculosis in our laboratory. ELISA usingB. malayi mf ES antigen has been explored for detecting IgG antibody by Indirect ELISA and antigen by Inhibition ELISA and in immunomonitoring of carriers as well as clinical filarial cases. A ten year follow up of mf carriers with DEC therapy showed disapperance of antigen and antibody followed by reappearance in few cases in an endemic area. None of the cases followed developed clinical symptoms suggesting the need for long term monitoring and treatment of microfilaraemic carriers. Further immunomonitoring was found to be useful in confirming filaria aetiology in the absence of microfilaremia and determining appropriate period of treatment of acute, early clinical and occult filarial infections for clinical relief and cure.Indirect Stick Penicillinase ELISA system using Mtb EST-6 antigen for detecting tuberculous IgG antibody and a Sandwich Penicillinase ELISA system using affinity purified antibody for detecting circulating antigen were explored in tuberculosis. A combination of both the assay systems with a sensitivity of 70% and specificity of 98% was found to be promising in the precise diagnosis of pulmonary tuberculosis. Further antigen detection was found to be useful in bone and joint tuberculosis.     

Detection of tubercular antibody and antigen in sera of bone and joint tuberculosis

Trichloroacetic acid (TCA) solubilized and DEAE fractionatedMycobacterium tuberculosis H₃₇Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solubilized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone & joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity 83%), while circulating tubercular antigen was detected by sandwich ELISA in 27 patients (sensitivity 75%). Out of 34 non tubercular disease control cases, 10 patients showed positive reaction for antibody while only 4 patients showed positive reaction for antigen. In another group of 34 healthy subjects who were screened, 4 individuals showed positive reaction for tubercular antibody and 2 cases for antigen. This study shows that antigen detection assay using affinity purified anti Mtb EST antigen antibody is superior with overall specificity of 91% as compared to antibody detection assay with 75% specificity in bone & joint tuberculosis.     

Mycobacterium tuberculosis H37Ra ESAS-7—An excretory-secretory antigen fraction of immunodiagnostic potential in pulmonary tuberculosis

A mycobacterial excretory-secretory protein fraction ESAS-7 purified by 50% ammonium sulphate precipitation followed by SDS-PAGE fractionation was evaluated by penicillinase enzyme linked immuno-sorbent assay (ELISA) for its sensitivity and specificity in the diagnosis of pulmonary tuberculosis. At a “cut off” serum dilution of 600, 38 (90%) of 42 sera from bacteriologically confirmed tuberculosis cases, 15 (100%) of 15 sera from bacteriogically negative but anti tubercular therapy (ATT) responded cases, 3 (7%) of 43 sera from normal healthy subjects and 4 (8%) of 48 sera from non tuberculous disease control cases gave positive reaction for tubercular antibody to ESAS-7 antigen fraction containing predominantly 33-kDa protein with a sensitivity of 90% in bacteriologically confirmed cases and specificity of 92%. Further, this diagnostic assay using the ESAS-7 antigen is more sensitive requiring as little as one nanogram antigen per test compared to use of 100 nanogram EST-6 antigen reported earlier. Thus use of ESAS-7 antigen for antibody detection has good diagnostic potential with improved specificity in pulmonary tuberculosis.     

Assay of tubercular antibody, circulating free and immune complexed antigen in the diagnosis of pulmonary tuberculosis

Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in confimed pulmonary tuberculosis sera by ELISA, using ES-31 antigen and affinity purified anti ES-31 antibody. Twenty three of 25 (92%) tuberculosis sera were positive for IgG antibody to ES-31 antigen. Using anti ES-31 antibody, free tubercular antigen could be detected in 20 of 25 (80%) cases whereas circulating immune complexed antigen (CIC-Ag) in 18 of 25 (72%) cases by sandwich ELISA. Of the two sera showing absence of antibody, one showed presence of free and CIC-Ag whereas the other showed the presence of free antigen. Thus antigen assay may be used as an adjunct tool for confirmation of pulmonary tuberculosis.     

Potential of mycobacterial excretory secretory protein antigens (sevatb ES-31, ES-43, EST-6 and ES-20) as biomarkers to detect <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> bacilli

There is a need for a simple and reliable method to identify Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM). The utility of mycobacterial ES-31, ES-43, EST-6 or ES-20 antigen as a biomarker for differentiation of Mycobacterium tuberculosis bacilli from nontuberculous mycobacteria was explored using Fluorescein isothiocyanate conjugated antibodies against these antigens. Detection of these antigens was done from M.tb H₃₇Ra and H₃₇Rv DSS antigen. The presence of antigen in bacilli using FITC labelled antibody was indicated by green fluorescence on the cell surface while, its absence by no fluorescence under microscope. In M.tb H₃₇Ra and H₃₇Rv bacilli, fluorescence was observed on addition of FITC labelled anti ES-31 and anti ES-43 antibody; whereas no fluorescence was observed in case of EST-6 and ES-20 antibody conjugates. However all the antigens were detected in detergent soluble sonicate antigen of tubercle bacilli on addition of FITC conjugates. Fluorescence was not observed for ES-31, ES-43, EST-6 and ES-20 antigen in any of the tested NTM as well as in Escherichia coli. SEVA TB ES-31 and ES-43 may be used as biomarkers to distinguish M.tuberculosis bacilli from NTM.     

Diagnostic potential of fractionatedMycobacterium tuberculosis H37Ra excretory-secretory (EST-DE1) antigen in pulmonary tuberculosis

Tuberculosis is emerging as a major public health problem in developing and developed world. Early and precise diagnosis is of prime importance in successful control of infection. Indirect ELISA with penicillinase as marker was developed using purifiedM. tuberculosis excretory-secretory (EST-DE₁) antigen for detecting IgG antibodies in pulmonary tuberculosis. The assay System gave a overall sensitivity of 82% for both smear positive and smear negative pulmonary tuberculosis cases with a specificity of 84%. The positive and negative predictive values were 75% and 88% respectivaly. Further studies with EST-DE₁ antigen revealed that, it contains two of the active antigen fractions of Mtb EST antigen i.e. Mtb EST-4 (56–68 KDa) and Mtb EST-6 (37–45 KDa), as demonstrated by inhibition ELISA. Reactivity with monoclonal antibodies HGT 3a showed the presence of 38 KDa molecule in EST-DE₁ antigen.     

Immunodiagnosis of tuberculosis: An update

Tuberculosis is still a major health problem in most developing countries and its incidence is rising in many developed countries. This resurgence has been attributed to the HIV epidemic and TB has been declared as a global health emergency by WHO in 1993. The diagnosis of tuberculosis mainly depends upon initial clinical suspicion and radiographic findings with subsequent bacteriological confirmation by sputum smear examination and culture. Lack of sensitivity in smear examination, non specificity of radiological findings, extended tum around time ofMycobacterium tuberculosis culture and difficulties in diagnosing paucibacillary, childhood and extrapulmonary tuberculosis has necessitated to explore the utility of immunodiagnosis of tuberculosis as a convenient and cost effective test to supplement clinical information for definite diagnosis. Many commercial tests are available in the market for diagnosis of TB. Most of these tests are based on the detection of IgG, IgA and IgM antibodies to specific mycobacterial antigen or mixture of antigens. Indigenous immunoassay systems have explored excretory-secretory ES-31 mycobacterial antigen for immunodiagnosis of TB. Many a time there is lack of consistent elevation in all the three Ig classes in active infection thus making it more important to determine the ideal antibody isotype assay for reliable diagnosis of tuberculosis and to save the costs of the patient for unnecessary investigations.     

Evaluation of A60 antibodies in pulmonary and neurotuberculosis

Humoral immune response against PPD derived A60 antigen was evaluated by quantification of serum A60 antibodies in thrity healthy adults not exposed to tuberculosis (Group 1), in twenty seven healthy adults exposed to tuberculosis patients i.e. staff working in wards of tuberculosis hospital for one to thirty years (group 2), in twenty five pulmonary tuberculosis patients admitted to the Institute for Chest Diseases, Hyderabad (Group 3) and in sixty neurotuberculosis patients admitted to Neurosurgery department of our institute (Group 4). Highly significant elevation of A60 antibodies was observed in pulmonary tuberculosis patients (p<0.01) compared to healthy adult groups. A significant elevation in serum was also observed in case of neurotuberculosis group compared to both healthy groups (p<0.01). A test on A60 antibodies in serum gavv a sensitivity of 100%, specificity of 96.6%, positive predictive value of 81% and negative predictive value of 100% for pulmonary tuberculosis, whereas a sensitivity of 58%, positive predictive value of 79% and negative predictive value of 75% were noted for neurotuberculosis patients. Results of A60 antibodies in ten cerebrospinal fluids (CSF) obtained from non tuberculosis patients and thirty two CSF from patients of neurotuberculosis did not show significant elevation of antibodies. However the ninetyfive percentile value of CSF A60 antibodies was higher in neurotuberculosis (7.4 U/ml) group compared to nontuberculous group (3.8 U/ml) and the test showed a good positive predictive value (83%), very low negative predictive value (25%) and low sensitivity (63%). Serum A60 antibody assay appears to be a good serological marker available today for pulmonary tuberculosis and a supportive marker for neurotuberculosis.     

Diagnostic role of the antibody response to the 38kDa, 16kDa proteins and lipoarabinomannan of mycobacterium tuberculosis

The antibody response to the 38kDa, 16kDa and Lipoarabinomannan (LAM) antigens ofMycobacterium tuberculosis was evaluated using three different ELISAs based on these antigens. The study group included tuberculosis patients (n=52), patients with HIV and TB co-infection (n=10), other chest symptomatics (n=5), HIV infected individuals (n=10), leprosy cases (n=7) and healthy controls (n=75). The results indicate that the 38kDa and LAM based ELISA for IgM/IgG has a low specificity (ranging from 69–85%) and sensitivity (ranging from 55–78%). When three ELISAs are carried out on a single patient the probability of detection of tuberculosis was significantly increased to 95.2% indicating that a single ELISA test is of low sensitivity and that a combination of ELISA’s may be needed to be of any value as a diagnostic test for tuberculosis. Additionally, a western blot assay of the serum antibody response to protein fraction ofM. tuberculosis was analysed in 15 tuberculosis patients and five healthy controls. A multiple antibody response to various M.tuberculosis proteins was observed which varied from patient to patient as compared to controls who showed a single 38–39 kDa protein band positivity. These finding suggest that a western blot assay which determines the antibody response to a set of antigenic components ofM. tuberculosis could be a better serological test for the diagnosis of tuberculosis in our population.     

Extra Pulmonary Tuberculosis: A Diagnostic Dilemma

Tuberculosis remains a major public health problem globally, with India being one of the high burden countries. The common causative agent is Mycobacterium tuberculosis but in developing countries M. bovis is reported as a potential human pathogen. Almost 20% of all reported cases of tuberculosis are of extra pulmonary form of disease. Diagnosis of extra pulmonary tuberculosis (EPTB) is not always possible with conventional methods, due to the long time required and the paucibacillary nature of samples; hence the need of rapid molecular methods. A prospective study was conducted on 300 patients of EPTB over a period of 5 years. These patients were suspected cases of tubercular meningitis, tubercular ascites and tubercular lymphadenitis. Samples analyzed were cerebrospinal fluid, ascitic fluid and lymph node fine needle aspirate. A two step PCR targeting hup B gene was used. Clinical response to anti tubercular therapy (ATT) was taken as positive (gold standard). PCR for hup B gene was positive in 147 samples out of 155 ATT responders. Of these 85.71% were infected with M. tuberculosis, 9.52% with M. bovis alone and 4.76% showed co infection with both M.tb and M. bovis. The sensitivity and specificity of PCR was 90.32 and 94.48% respectively.     

Adenosine deaminase levels in cerebrospinal fluid as a diagnostic test for tuberculous meningitis in children

Adenosine deaminase activity (ADA) was estimated in cerebrospinal fluid (CSF) of 30 patients of tuberculous meningitis (TBM) and 10 patients each of partially treated pyomeningitis (PTM), aseptic meningitis (AM) and pyogenic meningitis (PM). Mean ADA levels in CSF of TBM patients were higher (18.22 U/L) as compared to 6.28 U/L, 3.43 U/L and 7.98 U/L in PTM, AM and PM respectively. This difference of ADA values in CSF between TBM and other types of meningitis was statistically significant (p<0.01) different. Sensitivity and specificity of ADA levels in CSF of children to diagnose tuberculous meningitis was 66.6% and 90% respectively at 10 U/L cut off of ADA levels in CSF. ADA levels in CSF could also differentiate PTM, AM and PM from TBM with a specificity of 90%, 100% and 80% respectively.     

Diagnostic Potential of Circulating Biomarkers in Adenosine Deaminase Diagnosed Pleural Tuberculosis Cases

Pleural tuberculosis accounts for nearly 20% of Extra pulmonary tuberculosis. Adenosine deaminase, commonly used biomarker for the diagnosis, is non specific and there is paucity of literature on its correlation with conventional or newer methods for the diagnosis of extra pulmonary forms of TB. The aim of the study was to assess diagnostic potential of T cell function markers [interferon (IFN-γ), interleukin (IL-2) and IFN-γ/IL-2 ratio]; macrophage activation marker [neopterin]; and oxidative stress markers [protein carbonyl and malondialdehyde (MDA)] in pleural tuberculosis. 26 pleural TB cases diagnosed on the basis of suggestive chest X-ray and raised serum ADA levels and healthy controls were included in the study. Pleural fluid specimens were subjected to Zeihl Neelsen staining and culture on Lowenstein Jensen medium. Serum IFN-γ, IL-2, neopterin and protein carbonyl levels detection were done by ELISA and MDA levels were determined by measuring the thiobarbituric acid reactive substances. Median serum levels of IFN-γ, IL-2, IFN-γ/IL-2 ratio, neopterin, protein carbonyl and MDA were significantly different between cases and controls. Levels of all biomarkers except IL-2 were significantly higher in cases with contact history. Mean levels of ADA and ESR were 46.27 U/L and 46.62 mm/hr in PTB cases. AUC for IFN-γ, IL-2, IFN-γ/IL-2 ratio, neopterin, protein carbonyl and MDA were significantly discriminative for cases and controls. IFN-γ/IL-2 ratio was best discriminatory biomarker with highest area under ROC curve. Though no correlation was seen between ADA and any of the six biomarkers, ESR levels correlated significantly with all biomarkers except IL-2 by spearman’s correlation coefficient. Though all the circulating biomarkers under study provide useful supportive evidence for the diagnosis of PTB, further studies involving diverse control groups particularly non-PTB effusion are needed to validate these results.     

Ophthalmic Presentation of Disseminated Tuberculosis with Relapse-Immunological Profile

TB as the cause of uveitis varies from 0.5 to 10.5%; low sensitivity of confirmatory laboratory investigations and inconsistency of diagnostic criteria leads to paucity of data. Diagnosis requires a high level of suspicion and is often presumptive based on indirect evidences. Interferon gamma, Interleukin-2 and Neopterin are key biomarkers in immuno-regulation of Mycobacterium tuberculosis infection. The relative shift from Interleukin-2 towards Interferon gamma (Interferon gamma/Interleukin-2) is more discriminatory for active tuberculosis. Protein carbonyl and Malondialdehyde, as oxidative stress markers, characterize active tuberculosis. A case of disseminated TB presenting with acute uveitis had a recurrent tubercular lymphadenitis after completing category I treatment under revised national tuberculosis control programme. The present study evaluates the potential utility of above mentioned biomarkers to predict atypical presentation in difficult cases of tuberculosis. Though tuberculous uveitis is amenable to treatment in early course of disease, the delay in diagnosis can have serious consequences for the patient.     

Molecular Mycobacteriology and Expansion in Disease Diagnosis

Molecular diagnostic tools for tuberculosis (TB) have evolved quickly with new innovations which can provide unprecedented opportunities for the rapid, sensitive and specific diagnosis of M. tuberculosis in clinical specimens and the status of its drug sensitivity. Microscopy and culture methods can not be replaced but the molecular assays can be applied in parallel with any new molecular tests for the diagnosis of TB. For extra pulmonary specimens, the use of the amplification methods is advocated, since rapid and accurate laboratory diagnosis is critical. Customization of the diagnostic usefulness of a molecular assay, according to the ease, reliability and need for health care sector is of immense value in a modern clinical mycobacteriology laboratory.     

Enzymes in biofluids for diagnosis of diseases

Alkaline phosphatase, alanine amino transferase, aspartate amino transferase and proteins were investigated in C.S.F from cases of pyogenic and non-pyogenic meningitis and controls; ascitic fluid from patients with cirrhosis of liver, tuberculous abdomen and malignancy; and pleural fluid. Same investigations were done in the corresponding blood-sera in respect of fluids other than C.S.F. Levels of proteins were elevated by 65.7% in C.S.F. in pyogenic meningitis and 27.7% in non-pyogenic meningitis compared to controls. Levels of alkaline phosphatase were found to be increased to 57% in pyogenic meningitis with a decrease of 17.8% in the non-pyogenic type while those of ALT and AST were found to be increased by 59% and 103% respectively in the non-pyogenic type. If the levels of the 3 enzymes of fluids other than C.S.F. were compared, alkaline phosphatase levels were almost double in ascitic fluid in cirrhosis and those of ALT and AST were greater in malignancy and tuberculous pleural effusion. Both alkaline phosphatase and ALT levels were low in ascites in tuberculous abdomen with the least ALT values.     

Direct diagnosis ofMycobacterium tuberculosis in blood samples of HIV infected patients by polymerase chain reaction

We have developed a simple, economical and reproducible method for processing blood samples from HIV infected patients for diagnosis of tuberculosis. The procedure was validated on 55 samples selected for tuberculosis based on clinical criteria. 52 patients had radiological changes indicative of pulmonary tuberculosis of which only 28 were positive for AFB in sputum (sensitivity 54%) and 27 for tuberculin (sensitivity 52%). 26 HIV positive patients who showed positive X-ray did not react to tuberculin. The genus PCR probe missed 3 samples (sensitivity 94%) compared to X-ray.M.tuberculosis was detected in the blood of all X-ray positive cases by PCR using TB400 probe (sensitivity 100%) and another probe forM. tuberculosis, IS6110, missed 6 of them (sensitivity 88% compared to X-ray and 89% compared to TB400). It is proposed that this simple sample processing method could be used to screen all blood samples quickly for mycobacteremia using the genus PCR and only those positive for mycobacteria need to be tested forM.tuberculosis. This would save the scarce resources and time by reducing significantly the number of samples to be screened for species confirmation.     

Levels of cerebrospinal fluid acetylcholinesterase in tuberculous and pyogenic meningitis in children

Total acetylcholinesterase (AChE) activity was estimated in cerebrospinal fluid (CSF) using colorimetric method in 64 cases with tuberculous meningitis, (TBM) (n=64), pyogenic meningitis (PM) (n=60) and in controls (n=39) of paediatric age group. Mean CSF—AChE values of both TBM and PM were significantly higher when compared with controls (p<0.001) but no significant change between TBM and PM was observed (p<0.01). It is suggested that inflammation of meninges due to the infection may attribute to a change in the function of blood brain barrier (BBB) causing higher CSF—AChE values in these diseases.     

Utility of serum LDH isoforms in the assessment of mycobacterium tuberculosis induced pathology in TB patients of Sahariya tribe

The present study was carried out in the Sahariya tribe of Central India, which reportedly have high prevalence of pulmonary tuberculosis. Total serum LDH and its tissue specific isoforms were estimated in TB patients and matched healthy controls to test the utility of LDH as diagnostic marker for tuberculosis. About 210 sputum positive cases and 328 age and sex matched sputum negative controls were recruited. The spectrophotometeric and densitometric analysis of each LDH isoform was carried out in both cases and controls. The mean values of serum LDH were estimated and compared for each class by t-test. The statistical comparisons were made between sputum negative controls and sputum positive cases by Mann-Whitney’s U test. The spectrophotometric estimation of serum LDH revealed significant (P=0.0016) increase in its level in cases (290 IU/L) as compared to controls (248 IU/L). The densitometric analysis of individual LDH isoforms in cases and controls demonstrated significant elevation in LDH1 (P>0.05), LDH2 (P>0.05) and LDH3 (P<0.005) in sputum positive cases in comparison to sputum negative controls. Our study revealed a positive correlation between serum LDH level and the presence of mycobacteria and their load, suggesting utility of LDH as an important diagnostic marker of tuberculosis induced stress, at least in tribal areas lacking access to modern clinical tests.