Characteristic profile of antibody responses to PPD, ESAT-6, and CFP-10 of Mycobacterium tuberculosis in pulmonary tuberculosis suspected cases in Surabaya, Indonesia

Abstract Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low.

Diagnostic value of serological tests [IgA, IgG, IgM] against A-60 antigen in tuberculosis

Tuberculosis [TB] is a common infectious disease worldwide especially in developing countries. Diagnosis of TB is difficult and often needs paraclinical studies. Measuring immunoglobulin's against A-60 antigen of TB microorganism has been reported as a useful technique. The aim of the present study was to evaluate the diagnostic value of serologic test [IgA, IgM, IgG] against A-60 antigen in tuberculosis. For this case-control study, 176 TB patients [124 patients with smear-positive pulmonary TB and 52 patients with extra pulmonary TB] and 283 healthy controls were enrolled. Then, all subjects were tested for IgM, IgA and IgG against A-60 antigen using ELISA technique. Sensitivities of IgM, IgG and IgA test were 15%, 25% and 40%, respectively while the following specificities were also reported 100%, 75% and 90%, respectively. Positive predictive values [PPV] were 100%, 57% and 72%, however, negative predictive values [NPV] were 65%, 72% and 70%, respectively. Combination of immunoglobilins results increased sensitivity, for which the most sensitivity was observed in combination of IgG and IgA results. Our results revealed the usefulness of serologic testing in TB diagnosis. Although its sensitivity is not high enough, combination of immunoglobulin results may improve the sensitivity

Delayed hypersensitivity skin testing in the Thai adult population.

OBJECTIVE: The present study was carried out in healthy Thai subjects to determine the types and concentrations of standard antigens used in delayed-type hypersensitivity (DTH) skin testing as an evaluation of cell-mediated immunity. MATERIAL AND METHOD: One hundred subjects were tested with three antigens including tuberculin (purified protein derivative), tetanus toxoid, 1:10 and 1:100 dilutions, and Candida albicans, 1:10 and 1:100 dilutions. RESULTS: We found that 92% of the subjects responded to tuberculin and/or tetanus toxoid at a 1:10 dilution, 77% responded to tetanus toxoid at a 1:10 dilution, and 35% responded to a 1:100 dilution. There was only one subject who responded to Candida albicans at a 1:10 dilution, and none to a 1:100 dilution. The size of tuberculin reactions varied from 5 mm to over 20 mm without any evidence of active tuberculosis. CONCLUSION: Tuberculin and tetanus toxoid at a 1:10 dilution are probably sufficient to be used in DTH skin testing to evaluate T-cell function.

Prevalence of Antibodies to PPD and Lipoarabinomannan of Mycobacterium tuberculosis among Patients with an Indication of Fine Needle Aspiration Biopsy

Recent increase in the incidence of lung cancer often makes it difficult to differentiate between lung cancer and tuberculosis (TB), due to their radiologic similarities. Fine needle aspiration biopsy (FNAB) has been widely employed for the diagnosis of lung cancer and TB, but the diagnostic accuracy of TB is not high enough. As a rapid screening test for tuberculosis, we evaluated serological tests using Mycobacterium tuberculosis PPD and lipoarabinomannan (LAM) antigens. A total of 95 patients with indication of FNAB cytology from initial CT findings were enrolled. 25 patients had TB, 76 thoracic malignancy, and six (7.9%) of the lung cancer patients also had TB, indicating much higher prevalence of TB in thoracic tumor patients. Antibodies to PPD were elevated in 18 (72.0%) of 25 TB patients and in 22 (31.4%) of 70 patients with thoracic malignancy. In contrast, only 3 (4.7%) of 64 healthy controls aged 40 or above were seropositive to PPD antigen. The prevalence of anti-PPD antibodies in thoracic tumor patients was therefore significantly greater than that amongst the healthy controls (p 0.001, chi-square test). However, no significant difference in the prevalence of anti-LAM antibodies was found between study subjects and controls. This study demonstrates that thoracic tumor patients have significantly elevated antibodies to PPD; therefore, high anti-PPD seroreactivity in thoracic tumor patients should be cautiously interpreted. A longitudinal investigation on seropositive thoracic tumor patients is required to determine the role of the serological test for TB in lung cancer patients.

Tuberculin sensitivity and skin lesions in children after vaccination with two batches of BCG vaccine.

BCG is one of the vaccines used, as control arm, in an ongoing large scale comparative leprosy vaccine trial in South India. The objective of the present study was to examine, in the local population, the sensitizing ability, as measured by skin test reactions to tuberculin, and reactogenecity, in terms of skin lesions at the site of vaccination, for the two batches of BCG vaccine used in the above trial. The study was undertaken in 816 tuberculin-negative, previously not vaccinated school children, aged five to 14 years. Each child received one of the two batches of BCG vaccine or normal saline (control), by random allocation. At 12 weeks from vaccination, character and size of local response, at the vaccination site, were recorded. At the same time, the children were retested with tuberculin and post-vaccination reactions to the test were measured after 72 hours. At three years after vaccination all available children were re-examined for the presence and size of BCG scar at the site of vaccination. It was found that healing of vaccination lesions was uneventful, with both batches of BCG. The mean size of the lesion was similar for the two batches, the overall mean being 6.3 mm. The mean size of post-vaccination tuberculin sensitivity increased with age, and it was 14.5 mm and 15.6 mm. The sensitizing effect attributable to the vaccine was 11 mm and 12 mm, for the two batches of BCG respectively. This study showed that the two batches of BCG, in a dose of 0.1 mg, used in the ongoing leprosy vaccine trial were acceptable in terms of vaccination lesion and were highly satisfactory in terms of development of hypersensitivity.

Detection of anti-PPD IgG antibody and PPD-induced delayed type hypersensitivity in anterior uveitis patients.

Present study relates to the results of anti-PPD IgG, anti-A60 and antinuclear antibodies and PPD-induced delayed type hypersensitivity (DTH) in 17 anterior uveitis, (AU) patients. Results of anti-PPD IgG assay revealed detection of higher mean antibody level (O.D. 0.11 +/- 0.06) compared to healthy controls (O.D. 0.04 +/- 0.03), other eye disease controls (O.D. 0.05 +/- 0.003) and leprosy controls (O.D. 0.03 +/- 0.03). Anti-A60 IgM antibody assay revealed insignificant differences in mean antibody levels between various groups. Four of 17(23.5%) AU patients and 1(5.8%) subject each, belonging to other eye disease and healthy control groups had raised anti-nuclear antibody index. Findings of PPD skin test revealed detection of moderate to strong (2 to 4+) reactivity in 14 (82%). AU patients. Conversely, 13(76%) healthy controls and 8(47%) other eye disease controls gave mild (1+) reactivity. Results of this study suggested possible role of hypersensitivity to mycobacterial antigens in pathogenesis of anterior uveitis.

Evaluation of lymphocyte proliferation assay to purified protein derivative, enzyme linked immunosorbant assay, and tuberculin hypersensitivity in Eales' disease.

The purpose of this study was to evaluate the immunological responses against mycobacterial antigens in Eales' disease. Fifty six patients with Eales' disease and fifty age-and-sex-matched healthy volunteers with normal fundus findings taken as controls, were subjected to Mantoux test, using 2 TU/0.1 ml of purified protein derivative (PPD), lymphocyte proliferation assay to PPD, and ELISA to detect IgM and IgG antibodies against mycobacterial A-60 antigen. The results of Mantoux test and lymphocytes proliferation assay did not differ significantly in the two groups suggesting a similar cellular immune response. The number of individuals with recent exposure/reexposure to tuberculosis (IgM+) was significantly higher among patients. However the number of people with past exposure (IgM-IgG+) was significantly higher among controls. Our study indicates that there are no statistically significant differences in the humoral and cellular immune responses to mycobacterial antigens between the patients with Eales' disease and controls, except for a significantly higher IgM positivity among the patients.

Characterization of mycobacterial antigens by Elisa and immunoblot methods.

Antibodies to two mycobacterial antigens viz - culture filtrate antigen (CFA) and Mycobacterium tuberculosis antigen 5 were raised in rabbits. Enzyme-linked immuno sorbent assay (ELISA) and immunoblot methods were used for the evaluation of the specificity of the rabbit antibodies to M. tuberculosis. Immunoblot method is more sensitive than ELISA for the detection of antibodies to M. tuberculosis in the rabbit sera. It is being emphasised that characterisation of the mycobacterial antigens and evaluation of the specificity of the antimycobacterial antibodies are essential prior to their applications as an adjunct in the laboratory diagnosis of human mycobacterial disease.

Detection of tuberculous IgG antibodies using Mycobacterium tuberculosis H37Ra, excretory secretory antigen and tuberculin-purified protein derivative.

Different antigen preparations, viz. excretory-secretory antigen (ES Ag), phosphate buffer saline soluble antigen (PBS-S Ag) and sodium dodecyl sulphate soluble antigen (SDS-S Ag) of M. tuberculosis (M.tb) H37Ra strain along with tuberculin purified protein derivative (PPD) were employed in stick indirect ELISA to detect IgG antibodies in sera of sputum positive pulmonary tuberculosis cases. Sera from healthy individuals and individuals with diseases other than tuberculosis (cross-reacting diseases) were used as controls. ES antigen and PPD showed higher antibody titres in tuberculosis cases (GMT-1378 each) compared to PBS-S Ag (GMT-454) and SDS-S Ag (GMT-974). Thereafter, an extensive study was done analysing higher number of sera in each group for the detection of tuberculous IgG antibodies using ES Ag and PPD. The ES Ag showed better sensitivity (87%) and specificity (85%) compared to the sensitivity (73%) and specificity (78%) achieved with PPD. The ES Ag also showed higher IgG antibody titre (GMT-1068) than PPD (GMT-721). From the present study it can be envisaged that ES Ag has high diagnostic potential in tuberculosis.

Serum immunoglobulin E against PPD antigen in patients with pulmonary tuberculosis.

There is scant literature on the existence of IgE (type I allergic reactions) directed against Myocobacterium tuberculosis. To study this problem, total serum IgE (SIgE) and IgE specifically directed against the purified protein derivative of Myocobacterium tuberculosis (PPD-Sp IgE) was determined in 16 patients with AFB positive pulmonary tuberculosis (PTB) and their age and sex matched controls. Mean SIgE was significantly higher in PTB than in controls. Mild heating of the serum was necessary to detect the presence of PPD-Sp IgE which was positive in 81.3% PTB. One (6.3%) gave a class I, 9 (56.3%) gave class II and 3 (18.7%) gave class III RAST reactions. PPD-Sp IgE was absent in 3(18.7%). There were no class IV reactions. Control sera were negative for PPD-Sp IgE. Though the RAST test had a sensitivity of 80.1%, a specificity of 100% and a positive predictive value of 100%, we do not advocate this test as a diagnostic test. We have used this test to demonstrate the existence of PPD-Sp IgE and thus the existence of Type I allergic reactions against the PPD antigen of Mycobacterium tuberculosis. Its value in relation to clinical symptoms and its site of local production need further studies.

Evaluation of cellular immunity in diabetic uraemics by cutaneous response to recall antigens and 2:4 dinitrochlorobenzene and T-cell rosette formation.

Twenty diabetic uraemics and twenty two healthy matched controls comprised the material for this study. Cell mediated immunity was assessed by estimation of T-cell rosette percentage and cutaneous response to recall antigens--purified protein derivative, candida antigen and 2:4 dinitrochlorobenzene. Results analysis revealed depressed cell mediated immunity in diabetic uraemics in the form of impaired cutaneous response to recall antigens and reduction in T cell rosette percentage, in comparison to controls.

PPD-specific IgG and IgG subclasses in the sera of pulmonary tuberculosis patients

This study was performed to characterize the humoral immune responses with isotype profiles in Mycobacterium tuberculosis infection. PPD-Specific IgG and IgG subclasses were measured using ELISA in 212 patients with pulmonary tuberculosis. The values of PPD-specific IgG were significantly higher in pulmonary tuberculosis patients than those in the control group, and were correlated to the severity of illness (P < 0.01). The specificity and sensitivity of ELISA for IgG antibodies were 1.0 and 0.81, respectively as determined in 212 sera from tuberculosis patients and 44 from healthy controls. The positive predictive value was 1.0 (171/171), while negative predictive value was 0.52 (44/85). The values of PPD-specific IgG were significantly decreased after 2-4 months of treatment. Among the moderately and far advanced pulmonary tuberculosis patients, the values of PPD-specific IgG were significantly decreased in responders after 6 months of treatment. However, PPD-specific IgG in nonresponders was increased (P < 0.01). PPD-specific IgG subclass responses were evident to all four IgG subclasses. No changes of isotype response according to the severity of the disease were observed.

Bronchial challenge with purified protein derivative of Mycobacterium tuberculosis in asthma.

Response to bronchial challenge (BC) with purified protein derivative of Mycobacterium tuberculosis (PPD), was studied in children with bronchial asthma and correlated with Mantoux test and serum immunoglobulin E (IgE) against PPD (PPD-Sp IgE). Nearly 59% patients with bronchial asthma and 12.2% patients with pulmonary tuberculosis gave positive BC. Children with extra-pulmonary tuberculosis and normal children did not show positive BC. In asthma, 25% gave early (EAR), 50% gave late (LAR) and 25% gave both early and late (DAR) asthmatic response. Forced expiratory flow volumes in 1 sec (FEV.1) of 13 age and sex matched asthmatic and normal children showed similar volumes before BC, however, the values were significantly lower in asthma at 20 min (p less than 0.005) and 24 h (p less than 0.005) after BC. There was no relationship between response to BC and the severity or chronicity of asthma. PPD-Sp IgE was estimated by the radioimmunoassay method (Pharmacia Diagnostics). It was detected in 75% with positive BC and none of the controls. The titre was of Phadebas RAST Class III in 66.7%, Class II in 22.2% and Class I in 11.1%. The presence of early Mantoux reactions, positive BC with PPD and serum PPD-Sp IgE suggest the existence of Type I or Arthus type of reactions to PPD, which could cause hyperreactive airways in some cases of asthma.

Hepatitis B virus replication and tuberculin reactivity

Both mycobacterium tuberculosis and hepatitis B virus [HBV] infections are common in Egypt. Among 43 HBsAg healthy carriers and 26 HBsAg [+ve] cirrhotic patients, there was an inverted relationship between HBeAg status and purified protein derivative [PPD] reactivity, but it was statistically significant in the group of carriers [P < 0.01]. When the state of Bacillus Calmette-Guerin [BCG] vaccination was considered, the percentage of PPD reactivity in vaccinated HBeAg [+ve] carriers was significantly lower than that in the vaccinated controls and HBeAg [-ve] ones 20% versus 69.7% and 66.7%]. HBeAg [+ve] cirrhotic patients did not react to PPD whether vaccinated or not. Regarding the HBeAg [-ve] cirrhotic ones, PPD reactivity was only demonstrated in the vaccinated group with a 50% confidence and failed to do so with the non-vaccinated ones. The inverse association of HBeAg and PPD reactivity may suggest that the host response to tubercle bacilli may inhibit the replication of HBV. Those who were vaccinated and positively reacted to PPD were more likely able to inhibit replication of HBV. A clinical trial with BCG vaccination to HBeAg [+ve] carriers may be necessary to support this hypothesis

ELISA of IgG antibody to Mycobacterium tuberculosis antigen-5, PPD in CSF in tuberculous meningitis patients.

IgG antibody to M. tuberculosis antigen-5 and tuberculin purified protein derivative (PPD) was measured by enzyme linked immunosorbent assay (ELISA) in cerebrospinal fluid (CSF) specimens of 55 patients with tuberculous meningitis (TBM) and 55 patients with non-tuberculous neurological diseases (control group). The geometric mean antibody titre in CSF specimens of TBM patients was 82.4 with antigen-5 and 96.5 with PPD. In the control group, the geometric mean antibody titres for these antigens in CSF specimens were 4.6 and 10.8 respectively. The antibody titres did not show any correlation with tuberculin reactor status, duration of chemotherapy and IgG levels in CSF specimens in patients with TBM. At dilution end point 1:80, specificity of the assay was 100 per cent with antigen-5 and sensitivity of the assay was 70.9 per cent. False positivity observed in the control group with PPD antigen could be eliminated in 1:80 dilution in the assay with antigen-5. Antigen-5 is more specific than PPD antigen for the diagnosis of tuberculous meningitis.

IgG antibody to Mycobacterium tuberculosis antigen-5 in cerebrospinal fluid and its diagnostic application in tuberculous meningitis.

IgG antibody to M. tuberculosis antigen-5 was detected by non-competitive ELISA in cerebrospinal fluid specimens (CSF), from 40 patients with clinical diagnosis of tuberculous meningitis and in 42 patients of non-tuberculous neurological diseases. The geometric mean antibody titer in CSF specimen for tuberculous and non-tuberculous groups were 156 and 8 respectively. The antibody titer in CSF specimens showed no correlation to IgG levels, tuberculin reactor status and duration of chemotherapy in patients with tuberculous meningitis. At a dilution end-point 1:40, the assay had a sensitivity of 84% and specificity of 92%. However at dilution end-point 1:80, the specificity of the assay could be increased to 100% but sensitivity of the assay decreased to 75%. IgG antibody detection against M. tuberculosis antigen-5 by non-competitive ELISA, described in this communication has potential application in the laboratory diagnosis of tuberculous meningitis, particularly in developing countries where the incidence and prevalence of tuberculous meningitis is still high. In culture-negative cases of tuberculous meningitis, non-competitive ELISA could be applied as an alternative diagnostic tool.