Use of multiepitope polyproteins in serodiagnosis of active tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of approximately 98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of approximately 93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Antigens of Mycobacterium tuberculosis Recognized by Antibodies during Incipient, Subclinical Tuberculosis

Serum samples obtained from human immunodeficiency virus (HIV)-infected tuberculosis (TB) patients months prior to clinical TB were used to delineate the profile of Mycobacterium tuberculosis culture filtrate proteins recognized during subclinical TB. A subset of ~12 antigens was recognized by antibodies in these serum samples. Antibodies to two of these antigens (81 [88]-kDa malate synthase [GlcB] and MPT51) were present in serum samples obtained during incipient subclinical TB in 19 (~90%) of the 21 HIV-infected TB patients tested. These antigens will be useful for devising diagnostic tests that can identify HIV-positive individuals who are at a high risk for developing clinical TB.     

Mass spectrometric identification of mtb81, a novel serological marker for tuberculosis

We have used serological proteome analysis in conjunction with tandem mass spectrometry to identify and sequence a novel protein, Mtb81, which may be useful for the diagnosis of tuberculosis (TB), especially for patients coinfected with human immunodeficiency virus (HIV). Recombinant Mtb81 was tested by an enzyme-linked immunosorbent assay to detect antibodies in 25 of 27 TB patients (92%) seropositive for HIV as well as in 38 of 67 individuals (57%) who were TB positive alone. No reactivity was observed in 11 of 11 individuals (100%) who were HIV seropositive alone. In addition, neither sera from purified protein derivative (PPD)-negative (0 of 29) nor sera from healthy (0 of 45) blood donors tested positive with Mtb81. Only 2 of 57 of PPD-positive individuals tested positive with Mtb81. Sera from individuals with smear-positive TB and seropositive for HIV but who had tested negative for TB in the 38-kDa antigen immunodiagnostic assay were also tested for reactivity against Mtb81, as were sera from individuals with lung cancer and pneumonia. Mtb81 reacted with 26 of 37 HIV(+) TB(+) sera (70%) in this group, compared to 2 of 37 (5%) that reacted with the 38-kDa antigen. Together, these results demonstrate that Mtb81 may be a promising complementary antigen for the serodiagnosis of TB.     

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.     

Assessment of three commercially available serologic assays for detection of antibodies to Mycobacterium tuberculosis and identification of active tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major world disease, with approximately 9 million new cases each year. Identification and treatment of active disease are essential for TB control. Serology may offer increased detection of active disease in patients with a positive tuberculin skin test (TST) or QuantiFERON-TB (QFT-G). The InBios Active TbDetect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), IBL M. tuberculosis IgG ELISA, and Anda Biologics TB ELISAs were evaluated for the ability to detect M. tuberculosis antibodies in patients with active disease. Agreement, sensitivity, and specificity for each ELISA were determined and compared to those for culture or amplified direct detection and M. tuberculosis low-risk control patients. The InBios Active TbDetect ELISA had an agreement of 96.2%, a sensitivity of 83.3%, and a specificity of 98.9%. The IBL M. tuberculosis ELISA had an agreement of 84.0%, a sensitivity of 5.6%, and a specificity of 100.0%. The agreement, sensitivity, and specificity of the Anda Biologics TB ELISA were 74.2%, 83.3%, and 72.0%, respectively. The sensitivity for detecting M. tuberculosis antibodies in human immunodeficiency virus-associated TB was 50% for both the InBios Active TbDetect ELISA and the Anda Biologics TB ELISA and 0% for the IBL M. tuberculosis ELISA. The positivity rates for InBios Active TbDetect ELISA, IBL M. tuberculosis ELISA, and Anda Biologics TB ELISA in latently infected individuals positive by TST and/or QFT-G were 5.1%, 0.0%, and 30.8%, respectively. It can be concluded that the InBios Active TbDetect IgG ELISA is superior to the other ELISAs in accurately detecting active TB.     

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.     

Profiling antibodies to Mycobacterium tuberculosis by multiplex microbead suspension arrays for serodiagnosis of tuberculosis

Tuberculosis (TB) is a serious global disease. The fatality rate attributed to TB is among the highest of infectious diseases, with approximately 2 million deaths occurring per year worldwide. Identification of individuals infected with Mycobacterium tuberculosis and screening of their immediate contacts is crucial for controlling the spread of TB. Current methods for detection of M. tuberculosis infection are not efficient, in particular, for testing large numbers of samples. We report a novel and efficient multiplex microbead immunoassay (MMIA), based on Luminex technology, for profiling antibodies to M. tuberculosis. Microbead sets identifiable by unique fluorescence were individually coated with each of several M. tuberculosis antigens and tested in multiplex format for antibody detection in the experimental nonhuman primate model of TB. Certain M. tuberculosis antigens, e.g., ESAT-6, CFP-10, and HspX, were included to enhance the specificity of the MMIA, because these antigens are absent in nontuberculous mycobacteria and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. The MMIA enabled simultaneous detection of multiple M. tuberculosis plasma antibodies in several cohorts of macaques representing different stages of infection and/or disease. Antibody profiles were defined in early and latent/chronic infection. These proof-of-concept findings demonstrate the potential clinical use of the MMIA. In addition, the MMIA serodetection system has a potential for mining M. tuberculosis open reading frames (about 4,000) to discover novel target proteins for the development of more-comprehensive TB serodiagnostic tests.     

Humoral Immune Responses against the Mycobacterium tuberculosis 38-Kilodalton,MTB48, and CFP-10/ESAT-6 Antigens in Tuberculosis

The diagnosis of smear-negative and culture-negative patients with active tuberculosis (TB) is challenging. The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been an important diagnostic aid. However, detection of antibody responses to a single antigen usually has a low sensitivity for diagnosis of TB. In this study, humoral immune responses against recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 (culture filtrate protein 10/6-kDa early secreted antigen target of M. tuberculosis) antigens in 250 Chinese TB patients and 260 healthy subjects were evaluated by an enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against those antigens in TB patients, even in bacterium-negative ones, were significantly higher than those in healthy subjects (P < 0.001). The serodiagnostic sensitivities to detect antibodies against individual antigens, i.e., recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 antigens, in TB patients were 73.6%, 73.2%, and 60.4%, respectively, with specificities of 85.4%, 77.7%, and 73.8%, respectively. Importantly, the sensitivity to positively detect humoral responses to one of the antigens increased further. Our data suggest that the humoral immune responses to M. tuberculosis antigens in TB patients are heterogeneous. The 38-kDa, MTB48, and CFP-10/ESAT-6 antigens can be used as the cocktail antigens in the serodiagnosis of active TB, especially for smear- or culture-negative TB cases.The control of tuberculosis (TB) remains challenging in China (18). Currently, the diagnosis of active TB mainly relies on clinical symptoms, radiologic findings, and the detection of Mycobacterium tuberculosis in clinical samples using smear staining and mycobacterial culture. However, the diagnosis of TB in smear- and culture-negative TB patients is difficult. The detection of M. tuberculosis-specific antibodies in human sera has been an important aid in diagnosis of TB. Notably, several antigens have been demonstrated to have merit in TB diagnosis, including the 38-kDa protein, which is commonly used in serodiagnostic tests (4, 5, 8, 13, 19, 22, 23). Previous studies suggest that the antibody responses to M. tuberculosis antigens are heterogeneous among individuals (17) so that the detection of antibodies against a single antigen usually has a low sensitivity for diagnosis of TB, especially for bacterium-negative cases. Therefore, it may be valuable to evaluate antibodies against the 38-kDa antigen and other major antigens for the diagnosis of active TB (14, 15).Notably, the MTB48, CFP-10 (culture filtrate protein 10), and ESAT-6 (6-kDa early secreted antigen target of M. tuberculosis) genes are conserved in M. tuberculosis and Mycobacterium bovis isolates but partially deleted or absent in M. bovis BCG as well as in most nontuberculous mycobacteria (NTM) (1-3, 10, 16). Importantly, the proteins encoded by these genes are immunogenic (7, 9, 12, 16). In this study, we cloned the 38-kDa, MTB48, CFP-10, and ESAT-6 genes and generated recombinant 38-kDa, MTB48, and CFP-10/ESAT-6 fusion proteins in Escherichia coli. Subsequently, we developed an enzyme-linked immunosorbent assay (ELISA) for the characterization of serum antibodies against 38-kDa, MTB48, and CFP-10/ESAT-6 antigens in a population of 250 active TB patients and 260 healthy subjects. We found that characterization of antibodies against multiple M. tuberculosis antigens were valuable for the diagnosis of active TB.     

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

Purpose

Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed.

Materials and Methods

The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains.

Results

The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%).

Conclusion

The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.     

Characterization of Mycobacterium tuberculosis isolated from cancer patients with suspected tuberculosis infection in Egypt: identification,prevalence, risk factors and resistance pattern

Data are sparse on Mycobacterium tuberculosis infection among patients with cancer in Egypt. We sought to detect the presence of tuberculosis (TB) disease among patients with malignant conditions and suspected TB and to study the main risk factors. Also, we compared different diagnostic procedures and detected the antimicrobial susceptibility of M. tuberculosis isolates against rifampin and isoniazid. One hundred patients were included in this study, all of them had malignant conditions and were suspected by the clinicians of having TB. Identification of M. tuberculosis in different specimens was performed by smear microscopy, followed by Lowenstein–Jensen medium and Mycobacterium growth indicator tube (MGIT) cultures and artus^® real-time PCR. In addition, an indirect MGIT anti-TB susceptibility test was carried out against rifampin and isoniazid. A total of 76% of studied cases were found to be TB positive. The frequencies of TB-positive cases in the bronchogenic, haematological and solid tumour malignancy groups were 21%, 25% and 30%, respectively. Significant differences between pulmonary and extrapulmonary TB in different malignancy groups were recorded. Real-time PCR showed the highest overall diagnostic efficiency. Multidrug-resistance of M. tuberculosis to both rifampin and isoniazid was detected in 28.6% of examined isolates. Infection in cancer patients with TB was significantly more often recorded among elderly patients and those suffering from poverty. Pulmonary TB is more common than extrapulmonary TB in patients with malignancy. Real-time PCR is the most accurate and rapid method for TB diagnosis. MGIT-rifampin resistance may be used as a reliable marker for detection of multidrug-resistant TB. Diagnosis and instituting treatment course for active or latent TB infection are crucial before starting anticancer therapy.     

Gamma Interferon Responses Induced by a Panel of Recombinant and Purified Mycobacterial Antigens in Healthy, Non-Mycobacterium bovis BCG-Vaccinated Malawian Young Adults

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-γ) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.     

Analysis of Immune Responses against a Wide Range of Mycobacterium tuberculosis Antigens in Patients with Active Pulmonary Tuberculosis

Characterizing host immune responses to molecular targets of Mycobacterium tuberculosis is essential to develop effective immunodiagnostics and better vaccines. We investigated the immune response against a large series of M. tuberculosis antigens, including 5 classical and 64 nonclassical (39 DosR regulon-encoded, 4 resuscitation-promoting factor [RPF], and 21 reactivation-associated) antigens in active-pulmonary-tuberculosis (TB) patients. Whole blood from TB patients (n = 34) was stimulated in vitro with M. tuberculosis antigens. Gamma interferon (IFN-γ) was measured after 7 days of stimulation, using an enzyme-linked immunosorbent assay (ELISA). The majority of the study participants responded to the classical M. tuberculosis antigens TB10.4 (84.8%), early secreted antigenic target-6 kDa (ESAT-6)/CFP-10 (70.6%), and purified protein derivative (PPD) (55.9%). However, only 26.5% and 24.2% responded to HSP65 and Ag85A/B, respectively. Of the 64 nonclassical antigens, 23 (33.3%) were immunogenic (IFN-γ levels, >62 pg/ml) and 8 were strong inducers of IFN-γ (IFN-γ levels, ≥100 pg/ml). The RPF antigens were the most immunogenic. In addition, we observed distinct cytokine expression profiles in response to several M. tuberculosis antigens by multiplex immunoassay. Tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10), and IL-6 were commonly detected at high levels after stimulation with 4/15 latency antigens (Rv0081, Rv2006, Rv2629, and Rv1733c) and were found especially in supernatants of the three strong IFN-γ inducers (Rv2629, Rv1009, and Rv2389c). IL-8, IL-6, and IL-17 were exclusively detected after stimulation with Rv0574c, Rv2630, Rv1998, Rv054c, and Rv2028c. In conclusion, in active-pulmonary-TB patients, we identified 23 new immunogenic M. tuberculosis antigens. The distinct expression levels of IFN-γ, TNF-α, IL-6, and IL-10 in response to specific subsets of M. tuberculosis antigens may be promising for the development of immunodiagnostics.     

IgA Response to ESAT‐6/CFP‐10 and Rv2031 Antigens Varies in Patients With Culture‐Confirmed Pulmonary Tuberculosis,Healthy Mycobacterium tuberculosis–Infected and Non‐Infected Individuals in a Tuberculosis Endemic Setting,Ethiopia

Little attention has been given to the role of antibodies against Mycobacterium tuberculosis (Mtb) infection. We have compared the levels of IgA and IgG against ESAT‐6/CFP‐10 and Rv2031c antigens in sera of patients with culture‐confirmed pulmonary tuberculosis (PTB), healthy Mtb‐infected and non‐infected individuals in endemic TB settings. Venous blood samples were collected from 166 study participants; sera were separated and assayed by an enzyme‐linked immunosorbent assay (ELISA). QuantiFERON‐TB Gold In‐Tube (QFTGIT) assay was used for the screening of latent TB infection. The mean optical density (OD) values of IgA against ESAT‐6/CFP‐10 and Rv2031 were significantly higher in sera of patients with culture‐confirmed PTB compared with healthy Mtb‐infected and non‐infected individuals (P < 0.001). The mean OD values of IgG against ESAT‐6/CFP‐10 and Rv2031 were also significantly higher in sera of patients with culture‐confirmed PTB compared with healthy Mtb‐infected and non‐infected individuals (P < 0.05). The mean OD values of IgA against both antigens were also higher in sera of healthy Mtb‐infected cases compared with non‐infected individuals. There were positive correlations (P < 0.05) between the level of IFN‐γ induced in QFTGIT assay and the OD values of serum IgA against both antigens in healthy Mtb‐infected subjects. This study shows the potential of IgA response against ESAT‐6/CFP‐10 and Rv2031 antigens in discriminating clinical TB from healthy Mtb‐infected and non‐infected cases. Nevertheless, further well‐designed cohort study is needed to fully realize the full potential of this diagnostic marker.     

Improved sensitivity of diagnosis of tuberculosis in patients in Korea via a cocktail enzyme-linked immunosorbent assay containing the abundantly expressed antigens of the K strain of Mycobacterium tuberculosis

Tuberculosis (TB) is the leading cause of death from a single infectious agent in Korea. In this study, we compared the proteins present in culture filtrates from Mycobacterium tuberculosis strain K, which is the dominant clinical isolate in Korea, with those present in culture filtrates from M. tuberculosis H37Rv. Several differences in expression were detected between the two strains for those proteins with a molecular mass of <20 kDa. ESAT-6, HSP-X, and CFP-10 were found to be abundantly expressed in the strain K culture filtrates by liquid chromatography-electrospray ionization-time of flight mass spectrometry. The serodiagnostic potentials of recombinant antigens rESAT-6, rHSP-X, and rCFP-10 and two native antigens (Ag85 and PstS1) were evaluated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera collected from 46 TB patients with active disease and 46 healthy controls. As for our ELISA results, HSP-X was superior to the other antigens in terms of sensitivity when a single antigen was employed. The results of a receiver operator characteristic analysis revealed that a cocktail ELISA using all five antigens was significantly more sensitive (77.8%) than the use of a single antigen and offered equivalent specificity; moreover, it produced the largest area under the curve (0.91 versus 0.55 to 0.87). Therefore, a cocktail ELISA containing abundantly expressed antigens enhances the sensitivity of a single antigen and can be a useful diagnostic tool for the detection of active TB.     

Super-paramagnetic iron oxide nanoparticles for use in extrapulmonary tuberculosis diagnosis

The limited sensitivity of serological tests for mycobacterial antigens has encouraged the development of a nanoparticle probe specific for the extrapulmonary form of Mycobacterium tuberculosis (Mtb). We developed an innovative probe comprised of super-paramagnetic iron oxide (SPIO) nanoparticles conjugated with Mtb surface antibody (MtbsAb-nanoparticles) to provide ultrasensitive imaging of biomarkers involved in extrapulmonary Mtb infection. MtbsAb-nanoparticles were significantly conjugated with Mtb bacilli. The extent of contrast enhancement reduction on magnetic resonance imaging (MRI) for Mtb and human monocytic THP1 cells was proportional to the concentration of MtbsAb-nanoparticles. When MtbsAb-nanoparticles were intravenously injected into mice bearing Mtb granulomas, the granulomatous site showed a 14-fold greater reduction in signal intensity enhancement on T₂-weighted MR images compared with an opposing site that received PBS injection. Mtb sAb-nanoparticles represent a new non-invasive technology for the diagnosis of extrapulmonary Mtb.     

Antituberculosis IgG Antibodies as a Marker of Active Mycobacterium tuberculosis Disease

Anti-Mycobacterium tuberculosis IgG antibodies may aid in the diagnosis of active M. tuberculosis disease. We studied whether anti-M. tuberculosis IgG antibodies are elevated in active M. tuberculosis disease and assessed factors contributing to false-positive and -negative results. A retrospective study of 2,150 individuals tested by the QuantiFERON-TB Gold In-Tube (QFT-GIT) assay was conducted at the University of Utah, ARUP Laboratories, November 2008 to December 2010. All samples were tested with the InBios Active TbDetect antituberculosis (anti-TB) IgG antibody assay. Of 1,044 patients with a positive QFT-GIT, 59 (5.7%) were positive for M. tuberculosis antibodies. Fourteen of 1,106 (1.3%) with a negative or indeterminate QFT-GIT were positive for M. tuberculosis antibodies. M. tuberculosis antibody tests were positive in 61.5% with confirmed active M. tuberculosis disease and other mycobacterial infections. Over half of the false-negative M. tuberculosis antibody tests occurred in patients ≥90 years of age. False positives were seen in 12.9% of autoimmune patients. The odds ratio of being positive by the QFT-GIT and the InBios TB IgG assay increased with confirmed M. tuberculosis disease or highly suspected M. tuberculosis disease and was 86.7 (95% confidence interval [CI], 34.4 to 218.5) in these two groups compared to patients negative by both tests. Although anti-M. tuberculosis antibodies can be detected in patients with active M. tuberculosis disease, caution should be used with patients where immunoglobulin levels may be decreased or patients with autoantibodies.     

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.     

Virologic and immunologic outcome of HAART in Human Immunodeficiency Virus (HIV)-1 infected patients with and without tuberculosis (TB) and latent TB infection (LTBI) in Addis Ababa,Ethiopia

Background

HIV/TB coinfection remains a major challenge even after the initiation of HAART. Little is known about Mycobacterium tuberculosis (Mtb) specific immune restoration in relation to immunologic and virologic outcomes after long-term HAART during co-infections with latent and active TB.

Methods

A total of 232 adults, including 59 HIV patients with clinical TB (HIV?+?TB+), 125 HIV patients without clinical TB (HIV?+?TB-), 13 HIV negative active TB patients (HIV-TB+), and 10 HIV negative Tuberculin Skin TST positive (HIV-TST+), and 25 HIV-TST- individuals were recruited. HAART was initiated in 113 HIV?+?patients (28 TB?+?and 85 TB-), and anti-TB treatment for all TB cases. CD4+ T-cell count, HIV RNA load, and IFN-γ responses to ESAT-6/CFP-10 were measured at baseline, 6 months (M6), 18 months (M18) and 24 months (M24) after HAART initiation.

Results

The majority of HIV?+?TB- (70%, 81%, 84%) as well as HIV?+?TB?+?patients (60%, 77%, 80%) had virologic success (HIV RNA?<?50 copies/ml) by M6, M18 and M24, respectively. HAART also significantly increased CD4+ T-cell counts at 2 years in HIV?+?TB?+?(from 110.3 to 289.9 cells/μl), HIV?+?TB- patients (197.8 to 332.3 cells/μl), HIV?+?TST- (199 to 347 cells/μl) and HIV?+?TST?+?individuals (195 to 319 cells/μl). Overall, there was no significant difference in the percentage of patients that achieved virologic success and in total CD4+ counts increased between HIV patients with and without TB or LTBI. The Mtb specific IFN-γ response at baseline was significantly lower in HIV?+?TB?+?(3.6 pg/ml) compared to HIV-TB?+?patients (34.4 pg/ml) and HIV?+?TST?+?(46.3 pg/ml) individuals; and in HIV-TB?+?patients compared to HIV-TST?+?individuals (491.2 pg/ml). By M18 on HAART, the IFN-γ response remained impaired in HIV?+?TB?+?patients (18.1 pg/ml) while it normalized in HIV?+?TST?+?individuals (from 46.3 to 414.2 pg/ml).

Conclusions

Our data show that clinical and latent TB infections do not influence virologic and immunologic outcomes of ART in HIV patients. Despite this, HAART was unable to restore optimal TB responsiveness as measured by Mtb specific IFN-γ response in HIV/TB patients. Improvement of Mtb-specific immune restoration should be the focus of future therapeutic strategies.

     

Evaluation of Two Line Probe Assays for Rapid Detection of Mycobacterium tuberculosis,Tuberculosis (TB) Drug Resistance,and Non-TB Mycobacteria in HIV-Infected Individuals with Suspected TB

Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB), nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individuals, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube (MGIT) liquid culture. HIV-positive (HIV⁺) individuals with suspected TB from southern Africa and South America with <7 days of TB treatment had 1 sputum specimen tested with Direct LPA, MTBDR-Plus LPA, smear microscopy, MGIT, biochemical identification of mycobacterial species, and culture-based drug-susceptibility testing (DST). Of 639 participants, 59.3% were MGIT M. tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-positive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensitivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens. Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Direct LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative specimens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus. LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are priorities.     

Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by “dormant” M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ⁺/TNF⁺) and IFN-γ⁺ CD4⁺ T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.