Mycobacterium tuberculosis HBHA protein reacts strongly with the serum immunoglobulin M of tuberculosis patients

Identification and characterization of serologically active mycobacterial antigens are prerequisites for the development of diagnostic reagents. We examined the humoral immune responses of active tuberculosis (TB) patients against Triton-soluble proteins extracted from Mycobacterium tuberculosis by immunoblotting. A 29-kDa protein reacted with immunoglobulin M (IgM) in the pooled sera of the patients, and its N-terminal amino acid sequence matched that of the heparin-binding hemagglutinin (HBHA). Recombinant full-length HBHA was expressed in Escherichia coli (rEC-HBHA) and M. smegmatis (rMS-HBHA). In immunoblot analysis, the IgM antibodies of the TB patients reacted strongly with rMS-HBHA but not with rEC-HBHA, whereas the IgG antibodies of these patients reacted weakly with both recombinant HBHA proteins. In enzyme-linked immunosorbent assay analysis using rMS-HBHA and 85B as antigens, the mean levels and sensitivities of the anti-HBHA IgM antibodies of the TB patients were significantly higher than those of the anti-antigen 85B IgM antibodies, while the IgG antibodies showed the opposite results. Of interest in this respect, the pooled sera from the TB patients that contained anti-HBHA IgM antibodies neutralized the entry of M. tuberculosis into epithelial cells. These findings suggest that IgM antibody to HBHA may play a role in protection against extrapulmonary dissemination.     

Assessment of five antigens from Mycobacterium tuberculosis for serodiagnosis of tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major public health issue, particularly in developing countries, and thus effective diagnostic methods for TB remain a central theme in basic and clinical research. To evaluate five antigens (38-kDa protein [38kDa], Rv3621c, Rv3618, 38kDa-ESAT-6 [38E6], and Ag85B-HBHA [AH]) in serological tests for TB patients, we recruited 288 patients and 201 healthy controls. The median IgG reactivity to 38kDa, 38E6, and AH was higher than that to Rv3618 and Rv3621c in pulmonary TB. 38kDa and 38E6 provided high sensitivities in pulmonary TB but low sensitivities in extrapulmonary TB (EPTB). The specificities achieved by 38kDa and 38E6 ranged from 82.0% to 93.9% in patients with non-TB respiratory disease (PD) and in controls. 38kDa and 38E6 exhibited lower sensitivities and higher specificities than their combinations with Rv3618. These findings provide useful information on the relative importance of the above five antigens and suggest that combinations of Rv3618 with 38kDa and 38E6 can increase their sensitivities, but their specificities need to be further increased.     

Antimycobacterial antibody level in pleural, pericardial and cerebrospinal fluid of patients with tuberculosis

The goal of the study was to evaluate IgG, IgA and IgM mediated humoral immune response against 38kDa and 16 kDa or 38kDa and LAM mycobacterial antigens in pleural, pericardial or cerebrospinal fluid from patients with tuberculosis (TB) and to compare to non-tuberculous controls (NTB). 30 cerebrospinal fluids (CSF) (16 TB pts and 14 NTB pts), 17 pericardial fluids (6 TB and 11 NTB) and 20 pleural fluids (7 TB and 13 NTB) were examined. Commercially available ELISA-based assays (Pathozyme Tb complex plus, Myco G, A and M--Omega Diagnostic) were used. Tests were performed and cut off established according to manufacturer instruction. Mean IgG level against 38 + 16kDa was significantly higher in neurotuberculosis group compared to control (p<0.05). Sensitivity of the test in detecting neurotuberculosis was of 42% and specificity of 96%. Mean IgG, IgA and IgM against 38kDa + LAM level was higher in TB group compared to NTB in CSF. No difference was observed between TB and NTB group in pleural effusion. Antimycobacterial antibody levels were non-significantly increased in pericardial fluid in TB. The findings of the study indicate that TB is associated with the presence of detectable levels of antibodies in the CSF and pericardial effusion. Anti 38kDa + 16kDa IgG test can be used in combination with other diagnostic methods to increase diagnostic accuracy of neurotuberculosis.     

Evaluation of Antigen-Specific Immunoglobulin G Responses in Pulmonary Tuberculosis Patients and Contacts

This study aimed to evaluate the serodiagnostic potential of immunoglobulin G (IgG) responses to Mycobacterium tuberculosis antigens in pulmonary tuberculosis (TB) patients, recent TB contacts with latent TB infection (LTBI), and healthy subjects. Infections were assessed using tuberculin skin tests, QuantiFERON-TB Gold In-Tube tests, drug susceptibility testing, and molecular genotyping of clinical isolates. Serum IgG responses to selective M. tuberculosis antigens, including the 38-kDa and 16-kDa antigens, lipoarabinomannan (LAM), and recombinant early secreted antigen target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10), were determined. We found that the serum IgG responses to all antigens might differentiate between active TB and LTBI, with LAM having the highest diagnostic value (area under the curve [AUC] of 0.7756, P < 0.001). Recurrent TB cases showed significantly higher IgG responses to 38 kDa, CFP-10 (P < 0.01), and LAM (P < 0.05) than new cases, and male patients had higher levels of antigen-specific IgG than females (P < 0.05). Conversely, drug resistance and patient body mass index did not affect IgG responses (P > 0.05). LAM-specific IgG responses differentiated between acid-fast bacillus (AFB) smear-positive and -negative patients (P < 0.01), whereas antigen-specific IgG responses did not vary with the M. tuberculosis genotype (P > 0.05). Significantly higher IgG responses to 38 kDa and 16 kDa were observed in AFB smear-negative patients than in controls. These results suggest that assessment of serum IgG responses to selective purified M. tuberculosis antigens may help improve the diagnosis of active TB, particularly for sputum smear-negative patients or recurrent cases, and these may also help to differentiate between active TB and LTBI.     

Comparison of antibody responses to a potential combination of specific glycolipids and proteins for test sensitivity improvement in tuberculosis serodiagnosis

The humoral response to different proteinaceous antigens of Mycobacterium tuberculosis is heterogeneous among patients with active disease, and this has originated in the proposal to use a combination of several specific antigens to find an efficient serodiagnostic test for tuberculosis (TB). However, to date, comparisons of antibody responses to several antigens in the same population have been carried out without consideration of antigenic cell wall glycolipids. In the present study the presence of immunoglobulin G (IgG), IgM, and IgA antibodies to M. tuberculosis glycolipids (sulfolipid I, diacyltrehaloses, triacyltrehaloses, and cord factor) was compared with the response to four commercially available tests based on the 38-kDa protein mixed with the 16-kDa protein or lipoarabinomannan. Fifty-two serum samples from TB patients and 83 serum samples from control individuals (48 healthy individuals and 35 non-TB pneumonia patients) were studied. Three relevant results were obtained. (i) Smear-negative TB patients presented low humoral responses, but the sera which did react principally showed IgA antibodies to some glycolipidic antigens. (ii) TB patients exhibit heterogeneous humoral responses against glycolipidic antigens. (iii) Finally, test sensitivity is improved (from 23 to 62%) when IgG and IgA antibodies are detected together in tests based on different antigens (proteins and glycolipids). We conclude that it is possible to include glycolipidic antigens in a cocktail of specific antigens from M. tuberculosis to develop a serodiagnostic test.     

Comparison of Antibody Responses to a Potential Combination of Specific Glycolipids and Proteins for Test Sensitivity Improvement in Tuberculosis Serodiagnosis

The humoral response to different proteinaceous antigens of Mycobacterium tuberculosis is heterogeneous among patients with active disease, and this has originated in the proposal to use a combination of several specific antigens to find an efficient serodiagnostic test for tuberculosis (TB). However, to date, comparisons of antibody responses to several antigens in the same population have been carried out without consideration of antigenic cell wall glycolipids. In the present study the presence of immunoglobulin G (IgG), IgM, and IgA antibodies to M. tuberculosis glycolipids (sulfolipid I, diacyltrehaloses, triacyltrehaloses, and cord factor) was compared with the response to four commercially available tests based on the 38-kDa protein mixed with the 16-kDa protein or lipoarabinomannan. Fifty-two serum samples from TB patients and 83 serum samples from control individuals (48 healthy individuals and 35 non-TB pneumonia patients) were studied. Three relevant results were obtained. (i) Smear-negative TB patients presented low humoral responses, but the sera which did react principally showed IgA antibodies to some glycolipidic antigens. (ii) TB patients exhibit heterogeneous humoral responses against glycolipidic antigens. (iii) Finally, test sensitivity is improved (from 23 to 62%) when IgG and IgA antibodies are detected together in tests based on different antigens (proteins and glycolipids). We conclude that it is possible to include glycolipidic antigens in a cocktail of specific antigens from M. tuberculosis to develop a serodiagnostic test.     

Immunoglobulin A (IgA) and IgG Immune Responses against P-90 Antigen for Diagnosis of Pulmonary Tuberculosis and Screening for Mycobacterium tuberculosis Infection

The purpose of the present study was to evaluate the usefulness of detection of serum immunoglobulin A (IgA) and IgG antibodies directed against the mycobacterial P-90 antigen for the diagnosis of active pulmonary tuberculosis (PTB) among symptomatic individuals and for the detection of Mycobacterium tuberculosis infections among close contacts of PTB patients. Two commercially available enzyme immunoassay (EIA) kits (IgA EIA-TB [EIA-IgA] and IgG EIA-TB [EIA-IgG]; Kreatech Diagnostics) were evaluated in a blinded fashion by using stored serum samples from 268 individuals, including 69 patients with PTB, 41 patients with diseases other than tuberculosis (TB), 12 subjects with healed PTB, 39 close contacts of PTB patients, and 107 healthy volunteers. For the EIA-IgA, the sensitivity was 74% and the specificity was 68% when a cutoff determined by a receiver operator characteristic curve was used. For the EIA-IgG, the sensitivity was 69% and the specificity was 64%. The EIA-IgA was positive for 54% of healthy close contacts of PTB patients but only 8% of healthy controls without contact with a PTB patient or a prior personal history of TB (P < 0.001). The relatively low sensitivities and specificities of these serologic tests make them poor tools for the diagnosis of PTB among patients with suspected PTB. However, the relatively high prevalence of positive EIA-IgA results among healthy close contacts of PTB patients warrants further evaluation of this test with close contacts and other populations at risk for recent M. tuberculosis exposure and development of disease.     

Immunoglobulin A (IgA) and IgG immune responses against P-90 antigen for diagnosis of pulmonary tuberculosis and screening for Mycobacterium tuberculosis infection

The purpose of the present study was to evaluate the usefulness of detection of serum immunoglobulin A (IgA) and IgG antibodies directed against the mycobacterial P-90 antigen for the diagnosis of active pulmonary tuberculosis (PTB) among symptomatic individuals and for the detection of Mycobacterium tuberculosis infections among close contacts of PTB patients. Two commercially available enzyme immunoassay (EIA) kits (IgA EIA-TB [EIA-IgA] and IgG EIA-TB [EIA-IgG]; Kreatech Diagnostics) were evaluated in a blinded fashion by using stored serum samples from 268 individuals, including 69 patients with PTB, 41 patients with diseases other than tuberculosis (TB), 12 subjects with healed PTB, 39 close contacts of PTB patients, and 107 healthy volunteers. For the EIA-IgA, the sensitivity was 74% and the specificity was 68% when a cutoff determined by a receiver operator characteristic curve was used. For the EIA-IgG, the sensitivity was 69% and the specificity was 64%. The EIA-IgA was positive for 54% of healthy close contacts of PTB patients but only 8% of healthy controls without contact with a PTB patient or a prior personal history of TB (P < 0.001). The relatively low sensitivities and specificities of these serologic tests make them poor tools for the diagnosis of PTB among patients with suspected PTB. However, the relatively high prevalence of positive EIA-IgA results among healthy close contacts of PTB patients warrants further evaluation of this test with close contacts and other populations at risk for recent M. tuberculosis exposure and development of disease.     

IgG subclass distribution of antibody responses to protein and polysaccharide mycobacterial antigens in leprosy and tuberculosis patients

Immunoenzymatic assays were developed for the measurement of antibodies against mycobacterial lipoarabinomannan (LAM), a cell-free proteic extract (CFX) of Mycobacterium leprae, and the 38-kD protein antigen of M. tuberculosis. Sera from 108 leprosy patients, belonging to all clinical–immunological forms of the spectrum, and 81 patients with localized or disseminated tuberculosis (TB) were tested for antibodies of the four IgG subclasses. Standard calibration curves were used to allow comparisons between results of different isotypes and specificities. Mean concentrations of total IgG antibodies were higher in the overall leprosy population than in TB patients. In leprosy, levels of anti-CFX increased from tuberculoid toward lepromatous forms, with a clear switch from IgG1 to IgG2 subclass predominance. A similar IgG1 to IgG2 conversion was observed in anti-LAM antibodies, although total levels of anti-LAM were similar in patients with tuberculoid and lepromatous forms. In TB, antibodies against polysaccharide and protein antigens were both predominantly of IgG1 subclass, whatever the patient's clinical status, although lower in disseminated forms, probably due to concomitant HIV infection. A hypergammaglobulinaemia was also found in most leprosy and TB patients. In TB this was due to increased IgG1 and IgG3, especially in HIV co-infected patients. Based on the current knowledge of the influence of T cell-secreted cytokines on human immunoglobulin isotype expression, these results do not fit with a putative role of Th1 (such as found in TB and tuberculoid leprosy (TT)) and Th2 (such as found in leprosy lepromatous (LL) leprosy) environment in the isotypy of antibody responses in mycobacterial infections. Nor do variations of isotypy according to pathological conditions seem to be related to the biochemical nature of antigens, since antibodies to LAM and protein antigens had comparable evolutions of their subclass distribution. Other factors are to be investigated in order to understand better the significance and possible roles of antibodies in mycobacterial diseases.     

Glycolipids of Mycobacterium tuberculosis strain H37Rv are potential serological markers for diagnosis of active tuberculosis

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.     

IgG anti-multiantigen sonicate of Mycobacterium tuberculosis (Mts) measured by ELISA-Mts

Laboratory diagnosis of tuberculosis (TB) is often difficult. In recent years the detection of TB by serologic means has been the subject of a number of investigations. We studied the diagnostic utility of enzyme-linked immunosorbent assay in pulmonary tuberculosis. Two serological tests, commercial Pathozyme-Myco IgG (Omega Diagn.), and self-prepared ELISA-Mts using mycobacteria sonicate were evaluated simultaneously with 132 serum samples. Frequency and level of anti-mycobacterial IgG were compared in sera from 68 patients with TB and in sera from 64 healthy volunteers. The sensitivities of the tests with sera from TB patients were, respectively 55.9% and 70.6%. The specificities of these tests were respectively 98.5% and 84.4%. By combining two tests, a maximum sensitivity of 76.5% was obtained with reciprocal drops in specificity to 82.8%. Despite that, it has been suggested that combinations of different antigens were attempted in order to improve the diagnostic yield.     

Glycolipids of Mycobacterium tuberculosis Strain H37Rv Are Potential Serological Markers for Diagnosis of Active Tuberculosis

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.     

Detection of pulmonary and extrapulmonary tuberculosis patients with the 38-kilodalton antigen from Mycobacterium tuberculosis in a rapid membrane-based assay.

A rapid membrane-based serologic assay using the 38-kDa antigen from Mycobacterium tuberculosis for the diagnosis of tuberculosis (TB) was evaluated with 201 patients with pulmonary TB, 67 patients with extrapulmonary TB, 79 Mycobacterium bovis BCG-vaccinated healthy controls, and 77 non-TB respiratory patients. The overall sensitivities, specificities, and positive and negative predictive values were, respectively, 92, 92, 84, and 96% for sputum-positive TB patients; 70, 92, 87, and 79% for sputum-negative TB patients; and 76, 92, 80, and 90% for extrapulmonary-TB patients. Only 2% (1 of 44) of the healthy control BCG-vaccinated subjects gave weak positive signals in the assay, indicating that this rapid serological assay is a valuable aid in clinical diagnosis for both pulmonary and extrapulmonary TB.     

Serodiagnosis of Epstein-Barr Virus Infection by Using Recombinant Viral Capsid Antigen Fragments and Autologous Gene Fusion

Using recombinant 15- to 30-kDa fragments and fusion with glutathione S-transferase (GST), we investigated the seroreactivity of three large structural proteins of Epstein-Barr virus (EBV), p150 (BcLF1, capsid), p143 (BNRF1, tegument), and gp125 (BALF4, membrane) in Western blots. None of 13 fragments tested, however, was qualified for diagnostic application. In contrast, the two small viral capsid antigens (VCA), p18 (BFRF3) and p23 (BLRF2), demonstrated sensitive (100%) EBV-specific immunoglobulin G (IgG) reactivities. While p18 additionally showed maximum sensitivity for IgM detection, the IgM sensitivity of p23 was restricted (44%). An autologous fusion protein, p23-p18, which consists N-terminally of full-length p23, followed by the carboxy half of p18, was constructed. This antigen was subjected to indirect VCA enzyme-linked immunosorbent assays (ELISAs), for IgG and IgM, as well as to a micro-capture (microc) IgM ELISA. All assays were found to be 100% specific when EBV-negative sera were tested. Using sera from previously infected individuals, the p23-p18 fusion revealed an improved IgG sensitivity of 99% compared to sensitivities of 97 and 93% for the single antigens p18 and p23, respectively. The sensitivity and specificity of the indirect IgM ELISA with samples of primary and past infections, respectively, were 100%. The microc principle for IgM overcame completely the interference by rheumatoid factors. Compared to the specificity of the indirect IgM version, the specificity with sera collected from rheumatoid arthritis patients increased from 48 to 100%. In summary, the p23-p18 IgG and microc IgM ELISAs showed excellent performances and are promising new diagnostic tests for the detection of EBV-specific antiviral capsid antibodies.     

High-throughput analysis of T cell–monocyte interaction in human tuberculosis

The lack of efficient tools for identifying immunological correlates of tuberculosis (TB) protection or risk of disease progression impedes the development of improved control strategies. To more clearly understand the host response in TB, we recently established an imaging flow cytometer-based in-vitro assay, which assesses multiple aspects of T cell–monocyte interaction. Here, we extended our previous work and characterized communication between T cells and monocytes using clinical samples from individuals with different TB infection status and healthy controls from a TB endemic setting. To identify T cell–monocyte conjugates, peripheral blood mononuclear cells (PBMC) were stimulated with ds-Red-expressing Mycobacterium bovis bacille Calmette–Guérin or 6-kDa early secreted antigenic target (ESAT 6) peptides for 6 h, and analyzed by imaging flow cytometer (IFC). We then enumerated T cell–monocyte conjugates using polarization of T cell receptor (TCR) and F-actin as markers for synapse formation, and nuclear factor kappa B (NF-κB) nuclear translocation in the T cells. We observed a reduced frequency of T cell–monocyte conjugates in cells from patients with active pulmonary tuberculosis (pTB) compared to latent TB-infected (LTBI) and healthy controls. When we monitored NF-κB nuclear translocation in T cells interacting with monocytes, the proportion of responding cells was significantly higher in active pTB compared with LTBI and controls. Overall, these data underscore the need to consider multiple immunological parameters against TB, where IFC could be a valuable tool.     

Evaluation of a commercially available recombinant-protein enzyme-linked immunosorbent assay for detection of antibodies produced in scrub typhus rickettsial infections

The 56-kDa major outer membrane protein antigen of Orientia tsutsugamuchi is the immunodominant antigen in human scrub typhus (ST) infections. An enzyme-linked immunosorbent assay (ELISA) using a recombinant 56-kDa protein (r56) to detect specific immunoglobulin M (IgM) produced in ST infections was developed, and its performance was evaluated using sera from patients with active ST (n = 59), spotted fever (SF) (n = 31), and murine typhus (MT) (n = 6) and from those without rickettsial infection (n = 52). The r56 ELISA was compared to an ELISA using native whole cell lysate of O. tsutsugamushi Karp or O. tsutsugamushi Gilliam as antigens. The performance of the assays using r56 was similar to that of those using native antigens. Using indirect immunoperoxidase (IIP) as the reference test, sensitivities were 86, 88, and 88% while specificities were 84, 90, and 87% in the three assays. Furthermore, cross-reactivity in confirmed cases of SF and MT was low (5.4, 2.7, and 2.7% respectively). The additional use of IgG in the r56 ELISA gave improved performance (sensitivity, 80%; specificity, 96%; cross-reactivity in SF and MT, 2.7%). The detection of high levels of IgG in some IgM-negative patients illustrates the importance of including a test for IgG in the detection of secondary or reactivated infections, since many of these patients were from regions in Thailand where these infections are endemic.     

The mycobacterial 38-kilodalton glycolipoprotein antigen activates the mitogen-activated protein kinase pathway and release of proinflammatory cytokines through Toll-like receptors 2 and 4 in human monocytes

Although the 38-kDa glycolipoprotein of Mycobacterium tuberculosis H37Rv is known to evoke prominent cellular and humoral immune responses in human tuberculosis (TB), little information is known about intracellular regulatory mechanisms involved in 38-kDa antigen (Ag)-induced host responses. In this study, we found that purified 38-kDa glycolipoprotein activates mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38) and induces tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in human monocytes. When the 38-kDa Ag was applied to monocytes from TB patients and healthy controls, the activation of ERK1/2 and p38 MAPK and the subsequent cytokine secretion were greater in the monocytes from the active pulmonary TB patients than in monocytes from the healthy controls. Additionally, neutralizing antibodies for Toll-like receptor 2 (TLR2) or TLR4 significantly reduced the ERK1/2 and p38 activation induced by the 38-kDa protein when the antibodies were applied to HEK293 cells overexpressing TLR2 or TLR4 as well as human primary monocytes. Furthermore, the inhibition of TLR2 significantly, and that of TLR4 partially, decreased the 38-kDa Ag-induced secretion of TNF-alpha and IL-6 in human monocytes. The intact protein moieties of the 38-kDa protein were responsible for biologic activities by this Ag. These data collectively demonstrate that the 38-kDa glycolipoprotein, acting through both TLR2 and TLR4, induces the activation of the ERK1/2 and p38 MAPK pathways, which in turn play an essential role in TNF-alpha and IL-6 expression during mycobacterial infection.     

Evaluation of gamma interferon release assays using Mycobacterium tuberculosis antigens for diagnosis of latent and active tuberculosis in Mycobacterium bovis BCG-vaccinated populations

T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) using Mycobacterium tuberculosis-specific antigens have shown higher sensitivity and specificity than the routine tuberculin skin test (TST). However, the effects of Mycobacterium bovis BCG vaccination and anti-tuberculosis (TB) treatment on dynamic T-cell responses to M. tuberculosis-specific antigens in active TB cases have rarely been investigated in regions where TB is endemic. Eighty-nine patients with active pulmonary TB (ATB) and 57 healthy controls (HC) from China were recruited and tested by sputum smear and culture, TSTs, and IGRAs with M. tuberculosis-specific antigens ESAT-6 and CFP-10 (T-SPOT.TB) as well as purified protein derivative (PPD) stimulation. All 146 participants were screened by the T-SPOT.TB assay at recruitment. T-SPOT.TB-positive rates in ATB and HC groups were 87.6% (78/89) and 21.1% (12/57), respectively. Of 38 ATB patients who were both TST and T-SPOT.TB tested, the positive rates were 73.7% (28/38) and 94.7% (36/38), respectively (P = 0.0215), and those in the HC group were 62.3% (33/53) and 18.9% (10/53), respectively (P < 0.0001). The T-SPOT.TB-positive rates declined during TB treatment and were 94.4% (51/54), 86.4% (19/22), and 61.5% (8/13) for ATB patients receiving 0- to 1-month, 1- to 3-month, and 3- to 6-month anti-TB treatment, respectively. The IGRA is a most promising test for both active TB and latent TB infection (LTBI) diagnosis due to the improvement of its specificity and convenience, especially in the Mycobacterium bovis BCG-vaccinated population. Furthermore, the T-SPOT.TB assay using ESAT-6 and CFP-10 in ATB patients during anti-TB treatment could serve as a potential predictor of therapeutic efficacy.     

Human T-cell responses to secreted antigen fractions of Mycobacterium tuberculosis.

The T-cell response of human donors to secreted antigen fractions of Mycobacterium tuberculosis was investigated. The donors were divided into five groups: active pulmonary tuberculosis (TB) patients with minimal and with advanced disease, Mycobacterium bovis BCG-vaccinated donors with and without contact with TB patients, and nonvaccinated individuals. We found that patients with active minimal TB responded powerfully to secreted antigens contained in a short-term culture filtrate. The response to secreted antigens was mediated by CD4+ Th-1-like lymphocytes, and the gamma interferon release by these cells was markedly higher in patients with active minimal TB than in healthy BCG-vaccinated donors. Patients with active advanced disease exhibited depressed responses to all preparations tested. The specificity of the response to secreted antigens was investigated by stimulating lymphocytes with narrow-molecular-mass fractions of short-term culture filtrate obtained by the multielution technique. Considerable heterogeneity was found within the donor groups. Patients with active minimal TB recognized multiple secreted targets, but interestingly, six of eight patients demonstrated a predominant recognition of a low-mass (< 10-kDa) protein fraction which induced high levels of gamma interferon release in vitro. Only a few of 12 previously characterized secreted antigens were recognized by T cells isolated from TB patients, suggesting the existence of a number of as yet undefined antigenic targets among secreted antigens.     

Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis.

A serological survey was performed in groups of patients with active sputum smear-positive or smear-negative pulmonary tuberculosis, healthy household contacts, and controls. Sera were tested for titers of antibodies which bound to each of five purified mycobacterial antigens by enzyme immunoassay and for competition of binding to single epitopes, using six radiolabeled monoclonal antibodies directed toward corresponding molecules. The evaluation of diagnostic specificity was based on a positive score represented by titers above the cutoff point of 2 standard deviations above the mean titer of a control group. For smear-positive samples, the best sensitivity (83%) was achieved by exclusive use of the 38-kilodalton (kDa) antigen or its corresponding monoclonal antibodies. For smear-negative samples, levels of antibodies binding to the 19-kDa antigen showed a lower sensitivity of 62% compared with the control group or 38% compared with the contact group. Titers of antibody binding to the 14-kDa antigen were raised in Mycobacterium bovis BCG-vaccinated contacts, indicating that the greatest potential of this antigen may be in the detection of infection in a population for which tuberculin testing is unreliable. The results demonstrated the differing antibody responses to each of the tested antigens and distinct associations with the stage of infection or disease.