Tuberculin Skin Testing Compared with T-Cell Responses to Mycobacterium tuberculosis-Specific and Nonspecific Antigens for Detection of Latent Infection in Persons with Recent Tuberculosis Contact

The tuberculin skin test (TST) is used for the identification of latent tuberculosis (TB) infection (LTBI) but lacks specificity in Mycobacterium bovis BCG-vaccinated individuals, who constitute an increasing proportion of TB patients and their contacts from regions where TB is endemic. In previous studies, T-cell responses to ESAT-6 and CFP-10, M. tuberculosis-specific antigens that are absent from BCG, were sensitive and specific for detection of active TB. We studied 44 close contacts of a patient with smear-positive pulmonary TB and compared the standard screening procedure for LTBI by TST or chest radiographs with T-cell responses to M. tuberculosis-specific and nonspecific antigens. Peripheral blood mononuclear cells were cocultured with ESAT-6, CFP-10, TB10.4 (each as recombinant antigen and as a mixture of overlapping synthetic peptides), M. tuberculosis sonicate, purified protein derivative (PPD), and short-term culture filtrate, using gamma interferon production as the response measure. LTBI screening was by TST in 36 participants and by chest radiographs in 8 persons. Nineteen contacts were categorized as TST negative, 12 were categorized as TST positive, and 5 had indeterminate TST results. Recombinant antigens and peptide mixtures gave similar results. Responses to TB10.4 were neither sensitive nor specific for LTBI. T-cell responses to ESAT-6 and CFP-10 were less sensitive for detection of LTBI than those to PPD (67 versus 100%) but considerably more specific (100 versus 72%). The specificity of the TST or in vitro responses to PPD will be even less when the proportion of BCG-vaccinated persons among TB contacts evaluated for LTBI increases.     

Prospective Evaluation of a Whole-Blood Test Using Mycobacterium tuberculosis-Specific Antigens ESAT-6 and CFP-10 for Diagnosis of Active Tuberculosis

A new immunodiagnostic test based on the Mycobacterium tuberculosis-specific antigens CFP-10/ESAT-6(QFT-RD1) has been launched as an aid in the diagnosis of latent tuberculosis (TB) infection (LTBI). The aim of this study was to evaluate this test for the diagnosis of active TB. Eighty-two patients with suspicion of TB and 39 healthy BCG-vaccinated persons were enrolled. Forty-eight had active TB, 25 did not, and 9 were excluded. Sensitivity and specificity of the test for active TB were evaluated in a prospective blinded manner in patients suspected of TB. The sensitivity of the QFT-RD1 was 85% (40/48; confidence interval [CI], 75 to 96), and it was higher than the sensitivity of microscopy, 42% (20/48; CI, 27 to 56; P = 0.001), and culture, 59% (27/46; CI, 44 to 73; P = 0.009). Of patients with extrapulmonary TB, 92% (12/13) were QFT-RD1 positive, whereas only 31% (4/13) were positive by microscopy and 42% (5/12) by culture (P < 0.05), and 87% (13/15) of those who were negative by both microscopy and culture were QFT-RD1 positive. By combining microscopy and culture with the QFT-RD1 test, sensitivity increased to 96% (CI, 90 to 102). Ten of 25 (40%) non-TB patients were QFT-RD1 positive, resulting in a specificity of 60%. However, 80% (8/10) of these had risk-factors for TB, indicating latent infection in this group. In healthy controls, only 3% (1/39) were QFT-RD1 positive. In conclusion, the QFT-RD1 test is sensitive for diagnosis of TB, especially in patients with negative microscopy and culture. The accuracy of the QFT-RD1 test will vary with the prevalence of LTBI. We suggest that the QFT-RD1 test could be a very useful supplementary tool for the diagnosis of TB.     

Comparative Evaluation of Profiles of Antibodies to Mycobacterial Capsular Polysaccharides in Tuberculosis Patients and Controls Stratified by HIV Status

Despite the complexity of tuberculosis (TB) serology, antibodies (Abs) remain attractive biomarkers for TB. Recent evidence of a mycobacterial capsule that consists mainly of the polysaccharides arabinomannan (AM) and glucan provides new options for serologic targets. For this study, Ab responses to AM and glucan for 47 U.S. TB patients (33 HIV negative [HIV⁻], 14 HIV positive [HIV⁺]), 42 healthy controls, and 38 asymptomatic HIV⁺ controls were evaluated by enzyme-linked immunosorbent assays (ELISAs). The results were compared with Ab responses to the mycobacterial glycolipid cell wall antigen lipoarabinomannan (LAM) and to the proteins malate synthase (MS) and MPT51. We found that the main immunoglobulin (Ig) isotype response to polysaccharides was IgG, predominantly of subclass IgG2. IgG responses to AM were significantly higher for HIV⁻ and HIV⁺ TB cases than for controls (P, <0.0001 and <0.01, respectively); significantly higher for HIV⁻ than for HIV⁺ TB cases (P, <0.01); and significantly higher in sputum smear-positive than smear-negative patients in both HIV⁻ and HIV⁺ cases (P, 0.01 and 0.02, respectively). In both TB groups, titers of Ab to glucan were significantly lower than titers of Ab to AM (P, <0.0001). IgG responses to AM and MS or to AM and MPT51 did not correlate with each other in HIV⁻ TB patients, while they correlated significantly in HIV⁺ TB patients (P, 0.01 and 0.05, respectively). We conclude that Ab responses to AM could contribute to the serodiagnosis of TB, especially for HIV⁻ TB patients. This study also provides new and important insights into the differences in the profiles of Abs to mycobacterial antigens between HIV⁻ and HIV⁺ TB patients.     

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.     

Frequencies of Region of Difference 1 Antigen-Specific but Not Purified Protein Derivative-Specific Gamma Interferon-Secreting T Cells Correlate with the Presence of Tuberculosis Disease but Do Not Distinguish Recent from Remote Latent Infections

The majority of individuals infected with Mycobacterium tuberculosis achieve lifelong immune containment of the bacillus. What constitutes this effective host immune response is poorly understood. We compared the frequencies of gamma interferon (IFN-γ)-secreting T cells specific for five region of difference 1 (RD1)-encoded antigens and one DosR-encoded antigen in 205 individuals either with active disease (n = 167), whose immune responses had failed to contain the bacillus, or with remotely acquired latent infection (n = 38), who had successfully achieved immune control, and a further 149 individuals with recently acquired asymptomatic infection. When subjects with an IFN-γ enzyme-linked immunospot (ELISpot) assay response to one or more RD1-encoded antigens were analyzed, T cells from subjects with active disease recognized more pools of peptides from these antigens than T cells from subjects with nonrecent latent infection (P = 0.002). The T-cell frequencies for peptide pools were greater for subjects with active infection than for subjects with nonrecent latent infection for summed RD1 peptide pools (P ≤ 0.006) and culture filtrate protein 10 (CFP-10) antigen (P = 0.029). Individuals with recently acquired (<6 months) versus remotely acquired (>6 months) latent infection did not differ in numbers of peptide pools recognized, proportions recognizing any individual antigen or peptide pool, or antigen-specific T-cell frequencies (P ≥ 0.11). The hierarchy of immunodominance for different antigens was purified protein derivative (PPD) > CFP-10 > early secretory antigenic target 6 > Rv3879c > Rv3878 > Rv3873 > Acr1, and the hierarchies were very similar for active and remotely acquired latent infections. Responses to the DosR antigen α-crystallin were not associated with latency (P = 0.373). In contrast to the RD1-specific responses, the responses to PPD were not associated with clinical status (P > 0.17) but were strongly associated with positive tuberculin skin test results (≥15-mm induration; P ≤ 0.01). Our results suggest that RD1-specific IFN-γ-secreting T-cell frequencies correlate with the presence of disease rather than with protective immunity in M. tuberculosis-infected individuals and do not distinguish recently acquired asymptomatic infection from remotely acquired latent infection.The immune response is responsible for both bacillary containment in latent tuberculosis infection (LTBI) and immunopathology in active tuberculosis (TB). Comparing key immune responses for these two states may therefore help us dissect which responses mediate or correlate with disease and protection. It is therefore of particular interest to compare the immune responses in individuals with latent infection, who have successfully achieved immune control, and individuals with active disease, in whom the immune response has failed to contain the bacterium but immunopathology is present.Gamma interferon (IFN-γ) is essential for controlling Mycobacterium tuberculosis infection (11, 17), and CD4⁺ IFN-γ-secreting T cells specific for mycobacterial antigens play a pivotal role (16, 31). Responses to region of difference 1 (RD1)-encoded antigens are of special interest because of the unique features of early secretory antigenic target 6 (ESAT-6) (1, 30) and culture filtrate protein 10 (CFP-10) (3, 5), which appear to be both virulence factors and putative targets of protective immunity.In a primary bovine TB infection, ESAT-6-specific IFN-γ production, although not a proliferative response, correlated with the severity of disease pathology (39). Likewise, increased ESAT-6 expression and CFP-10 expression in Mycobacterium bovis bacillus Calmette-Guerin (BCG) or Mycobacterium microti infections were associated with increased pathogenicity in susceptible mice and correlated with increased RD1-specific T-cell responses (9). However, RD1-specific responses induced by vaccination are associated with protective immunity against subsequent challenge with virulent organisms. Thus, mice infected with M. tuberculosis were resistant to reinfection (2), and protection correlated with accelerated accumulation of IFN-γ-secreting effector T cells responding to Ag85 and ESAT-6 (1). Mice (27) and guinea pigs (8) vaccinated with BCG::RD1 developed strong CD4⁺ IFN-γ and proliferative responses to ESAT-6. When challenged, these animals had superior protection compared with BCG-vaccinated or unvaccinated mice, as well as less severe pathology and reduced dissemination of the pathogen (32).However, the relationship between the magnitude of ESAT-6 responses and disease in humans is unclear; little is known about CFP-10, and almost nothing is known about the other RD1-encoded antigens. The chaperone protein α-crystallin (“Acr1,” “Rv2031c,” “HspX,” “16-kDa antigen”) is upregulated under oxidative stress conditions (41) and is important for growth in macrophages (12). Encoded by the M. tuberculosis “dormancy regulon” expressed during natural infection (25), this protein is upregulated during conditions of in vitro stress (35). For these reasons, it has been postulated that Acr1-specific T-cell responses may correlate with latency (12, 41).We recently recruited a cohort of 389 individuals suspected to have TB as part of a prospective study of the diagnostic utility of enzyme-linked immunospot (ELISpot) responses to RD1 antigens. (13). A total of 205 patients were given a definitive diagnosis of active or latent TB and had not received antituberculous chemotherapy. We enumerated IFN-γ-secreting T cells specific for ESAT-6, CFP-10, Rv3879c, Rv3878, Rv3873 (10), and purified protein derivative (PPD) and, in a subset, IFN-γ-secreting T cells specific for Acr1. We compared responses to these antigens in patients with active TB and patients with LTBI in a blinded, prospective manner to address the following questions. Are the frequencies of IFN-γ-secreting T cells specific for RD1 antigens, PPD, and Acr-1 different in patients with active TB and patients with LTBI? Do the breadth of the response to RD1-derived peptides in patients with active disease and the breadth of the response to RD1-derived peptides in patients with latent infection differ? Are the hierarchies of immunodominance similar? Do these responses vary with the tuberculin skin test (TST) results? Do responses differ between recently and remotely acquired latent infections?(Part of this work was presented at the winter meeting of the Acid Fast Club, United Kingdom, on 12 January 2007.)     

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 ~98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ~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.     

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.     

Long-Incubation-Time Gamma Interferon Release Assays in Response to Purified Protein Derivative,ESAT-6, and/or CFP-10 for the Diagnosis of Mycobacterium tuberculosis Infection in Children

The diagnosis of childhood active tuberculosis (aTB) and latent Mycobacterium tuberculosis (M. tuberculosis) infection (LTBI) remains a challenge, and the replacement of tuberculin skin tests (TST) with commercialized gamma interferon (IFN-γ) release assays (IGRA) is not currently recommended. Two hundred sixty-six children between 1 month and 15 years of age, 214 of whom were at risk of recent M. tuberculosis infection and 51 who were included as controls, were prospectively enrolled in our study. According to the results of a clinical evaluation, TST, chest X ray, and microbiological assessment, each children was classified as noninfected, having LTBI, or having aTB. Long-incubation-time purified protein derivative (PPD), ESAT-6, and CFP-10 IGRA were performed and evaluated for their accuracy in correctly classifying the children. Whereas both TST and PPD IGRA were suboptimal for detecting aTB, combining the CFP-10 IGRA with a TST or with a PPD IGRA allowed us to detect all the children with aTB with a specificity of 96% for children who were positive for the CFP-10 IGRA. Moreover, the combination of the CFP-10 IGRA and PPD IGRA detected 96% of children who were eventually classified as having LTBI, but a strong IFN-γ response to CFP-10 (defined as >500 pg/ml) was highly suggestive of aTB, at least among the children who were <3 years old. The use of long-incubation-time CFP-10 IGRA and PPD IGRA should help clinicians to quickly identify aTB or LTBI in young children.     

Enhancement of human antigen-specific memory T-cell responses by interleukin-7 may improve accuracy in diagnosing tuberculosis

Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-γ) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-γ. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-γ message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-γ responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-γ responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R² = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-γ by 39% compared to the level with antigen alone. Increased production of IFN-γ induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-γ-based assays might improve TB diagnosis in those populations at highest risk for TB.     

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.     

Gamma interferon-based immunodiagnosis of tuberculosis: comparison between whole-blood and enzyme-linked immunospot methods

The gamma interferon secretion levels of 52 adults whose T cells had been stimulated with the Mycobacterium tuberculosis antigens CFP-10 and ESAT-6 were measured by enzyme-linked immunospot methods and a whole-blood-based assay. The test results correlated well (r = 0.689, P < 0.0001). Its simple format and use of only small volumes of blood make the whole-blood assay suitable for pediatric practice and field trials.     

Discrimination between Active and Latent Tuberculosis Based on Ratio of Antigen-Specific to Mitogen-Induced IP-10 Production

Mycobacterium tuberculosis is the major causative agent of tuberculosis (TB). The gamma interferon (IFN-γ) release assay (IGRA) has been widely used to diagnose TB by testing cell-mediated immune responses but has no capacity for distinguishing between active TB and latent TB infection (LTBI). This study aims to identify a parameter that will help to discriminate active TB and LTBI. Whole-blood samples from 33 active TB patients, 20 individuals with LTBI, and 26 non-TB controls were applied to the commercial IFN-γ release assay, QuantiFERON-TB Gold In-Tube, and plasma samples were analyzed for interleukin-2 (IL-2), IL-6, IL-8, IL-10, IL-13, tumor necrosis factor-alpha (TNF-α), IFN-γ, monokine induced by IFN-γ (MIG), interferon gamma inducible protein 10 (IP-10), interferon-inducible T cell alpha chemoattractant (I-TAC), and monocyte chemoattractant protein 1 (MCP-1) by using a commercial cytometric bead array. The Mycobacterium tuberculosis antigen-specific production of most of the assayed cytokines and chemokines was higher in the active TB than in the LTBI group. The mitogen-induced responses were lower in the active TB than in the LTBI group. When the ratio of TB-specific to mitogen-induced responses was calculated, IL-2, IL-6, IL-10, IL-13, TNF-α, IFN-γ, MIG, and IP-10 were more useful in discriminating active TB from LTBI. In particular, most patients showed higher IP-10 production to Mycobacterium tuberculosis antigens than to mitogen at the individual level, and the ratio for IP-10 was the strongest indicator of active infection versus LTBI with 93.9% sensitivity and 90% specificity. In conclusion, the ratio of the TB-specific to the mitogen-induced IP-10 responses showed the most promising accuracy for discriminating active TB versus LTBI and should be further studied to determine whether it can serve as a biomarker that might help clinicians administer appropriate treatments.     

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.     

Use of Recombinant ESAT-6:CFP-10 Fusion Protein for Differentiation of Infections of Cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-γ)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-α), IFN-γ, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-γ and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-γ responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.     

Anti-CagA Immunoglobulin G Responses Correlate with Interleukin-8 Induction in Human Gastric Mucosal Biopsy Culture

Helicobacter pylori persists in the human stomach despite eliciting both cellular and humoral immune responses and inducing proinflammatory cytokines. To determine whether local humoral and cytokine responses are related to each other and to histologic responses, we studied 66 Japanese patients who underwent gastroscopy. Using specific enzyme-linked immunosorbent assays, we examined gastric antral mucosal-organ biopsy culture supernatants to assess interleukin-6 (IL-6) and interleukin-8 (IL-8) levels and antibody responses to H. pylori whole-cell antigens CagA, HspA, and HspB. Of the patients studied, 11 were H. pylori negative and 55 were H. pylori positive; by PCR, all strains were cagA⁺. As expected, compared to H. pylori-negative patients, H. pylori-positive patients had significantly higher humoral responses to all H. pylori antigens and had higher IL-8 (47.8 ± 3.5 versus 10.1 ± 4.3 ng/mg of biopsy protein; P < 0.001) and IL-6 levels (2.8 ± 0.3 versus 0.26 ± 0.2 ng/mg of protein; P < 0.001). Among the H. pylori-positive patients, supernatant anti-CagA immunoglobulin G (IgG) levels were significantly associated with H. pylori density (P < 0.005) and neutrophil infiltration (P < 0.005) scores. Anti-CagA immunoglobulin A levels were correlated with intestinal metaplasia (P < 0.05). Mononuclear cell infiltration scores were significantly associated with supernatant IL-6 levels (P < 0.005) and with IgG responses to whole-cell antigens (P < 0.05). Supernatant IL-8 levels were significantly associated with anti-CagA IgG (r = 0.75, P < 0.001). Anti-CagA responses correlated with neutrophil infiltration, intestinal metaplasia, H. pylori density, and IL-8 levels, suggesting that the absolute levels of these antibodies may be markers for gastric inflammation and premalignant changes in individual hosts.     

Identification of Immunological Biomarkers Which May Differentiate Latent Tuberculosis from Exposure to Environmental Nontuberculous Mycobacteria in Children

A positive gamma interferon (IFN-γ) response to Mycobacterium tuberculosis early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) has been taken to indicate latent tuberculosis (TB) infection, but it may also be due to exposure to environmental nontuberculous mycobacteria in which ESAT-6 homologues are present. We assessed the immune responses to M. tuberculosis ESAT-6 and cross-reactive responses to ESAT-6 homologues of Mycobacterium avium and Mycobacterium kansasii. Archived culture supernatant samples from children at 3 years post-BCG vaccination were tested for cytokine/chemokine responses to M. tuberculosis antigens. Furthermore, the IFN-γ responses to M. tuberculosis antigens were followed up for 40 children at 8 years post-BCG vaccination, and 15 TB patients were recruited as a control group for the M. tuberculosis ESAT-6 response in Malawi. IFN-γ enzyme-linked immunosorbent assays (ELISAs) on supernatants from diluted whole-blood assays, IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, QuantiFERON TB Gold-In Tube tests, and multiplex bead assays were performed. More than 45% of the responders to M. tuberculosis ESAT-6 showed IFN-γ responses to M. avium and M. kansasii ESAT-6. In response to M. tuberculosis ESAT-6/CFP-10, interleukin 5 (IL-5), IL-9, IL-13, and IL-17 differentiated the stronger IFN-γ responders to M. tuberculosis ESAT-6 from those who preferentially responded to M. kansasii and M. avium ESAT-6. A cytokine/chemokine signature of IL-5, IL-9, IL-13, and IL-17 was identified as a putative immunological biosignature to differentiate latent TB infection from exposure to M. avium and M. kansasii in Malawian children, indicating that this signature might be particularly informative in areas where both TB and exposure to environmental nontuberculous mycobacteria are endemic.     

Increased synthesis of anti-tuberculous glycolipid immunoglobulin G (IgG) and IgA with cavity formation in patients with pulmonary tuberculosis

Tuberculous glycolipid (TBGL) antigen is a cell wall component of Mycobacterium tuberculosis and has been used for the serodiagnosis of tuberculosis. We investigated correlations between the levels of anti-TBGL antibodies and a variety of laboratory markers that are potentially influenced by tuberculous infection. Comparisons between patients with cavitary lesions and those without cavitary lesions were also made in order to determine the mechanism underlying the immune response to TBGL. Blood samples were obtained from 91 patients with both clinically and microbiologically confirmed active pulmonary tuberculosis (60 male and 31 female; mean age, 59 ± 22 years old). Fifty-nine patients had cavitary lesions on chest X-rays. Positive correlations were found between anti-TBGL immunoglobulin G (IgG) and C-reactive protein (CRP) (r = 0.361; P < 0.001), between anti-TBGL IgA and soluble CD40 ligand (sCD40L) (r = 0.404; P < 0.005), between anti-TBGL IgG and anti-TBGL IgA (r = 0.551; P < 0.0000005), and between anti-TBGL IgM and serum IgM (r = 0.603; P < 0.00000005). The patients with cavitary lesions showed significantly higher levels of anti-TBGL IgG (P < 0.005), anti-TBGL IgA (P < 0.05), white blood cells (P < 0.01), neutrophils (P < 0.005), basophils (P < 0.0005), natural killer cells (P < 0.05), CRP (P < 0.0005), KL-6 (sialylated carbohydrate antigen KL-6) (P < 0.0005), IgA (P < 0.05), and sCD40L (P < 0.01). The observed positive correlations between the anti-TBGL antibody levels and inflammatory markers indicate the involvement of inflammatory cytokines and NKT cells in the immunopathogenesis of pulmonary tuberculosis.     

Antibodies against Immunodominant Antigens of Mycobacterium tuberculosis in Subjects with Suspected Tuberculosis in the United States Compared by HIV Status

The immunodominance of Mycobacterium tuberculosis proteins malate synthase (MS) and MPT51 has been demonstrated in case-control studies with patients from countries in which tuberculosis (TB) is endemic. The value of these antigens for the serodiagnosis of TB now is evaluated in a cross-sectional study of pulmonary TB suspects in the United States diagnosed to have TB, HIV-associated TB, or other respiratory diseases (ORD). Serum antibody reactivity to recombinant purified MS and MPT51 was determined by enzyme-linked immunosorbent assays (ELISAs) of samples from TB suspects and well-characterized control groups. TB suspects were diagnosed with TB (n = 87; 49% sputum microscopy negative, 20% HIV⁺) or ORD (n = 63; 58% HIV⁺). Antibody reactivity to MS and MPT51 was significantly higher in U.S. HIV⁺/TB samples than in HIV⁻/TB samples (P < 0.001), and it was significantly higher in both TB groups than in control groups with latent TB infection (P < 0.001). Antibody reactivity to both antigens was higher in U.S. HIV⁺/TB samples than in HIV⁺/ORD samples (P = 0.052 for MS, P = 0.001 for MPT51) but not significantly different between HIV⁻/TB and HIV⁻/ORD. Among U.S. HIV⁺ TB suspects, a positive anti-MPT51 antibody response was strongly and significantly associated with TB (odds ratio, 11.0; 95% confidence interval, 2.3 to 51.2; P = 0.002). These findings have implications for the adjunctive use of TB serodiagnosis with these antigens in HIV⁺ subjects.The detection and treatment of individuals who are at early stages of active pulmonary tuberculosis (TB) is critical for the successful control and elimination of TB (34, 38). Mycobacterium tuberculosis is a slow-growing pathogen, and it takes months to years for an infection (and, presumably, reactivation) to progress to clinical TB. In resource-limited countries, the microscopic examination of smears made directly from unprocessed sputum are used for diagnosis, resulting in the identification of only advanced TB patients with high bacillary burden. In contrast, in industrialized settings the combined use of the fluorescence microscopy of decontaminated and concentrated sputum, mycobacterial culture, and nucleic acid amplification technologies permits the identification of patients with much lower bacillary burden and, thus, in the early stages of TB. Still, only around 50% of TB cases are rapidly diagnosed by optimized microscopy (5, 18). While adjunctive amplification methods increase the yield of confirmed TB, albeit with added cost and delays, around 20% of TB cases remain without microbiologic confirmation (5, 18). Additional tests that can enhance the rapid identification of patients at early stages of TB are required to add to the armamentarium of TB diagnostic tests.The amplification power of immune responses potentially can detect TB at a low antigen threshold and without requiring a specimen from the site of infection. Assays that detect TB infection by measuring the gamma interferon release of circulating lymphocytes in response to M. tuberculosis-specific antigens (IGRAs) cannot distinguish active from latent TB infection (LTBI) (reviewed in reference 15) and have limited utility in patients with advanced HIV infection (3, 32). In contrast, positive antibody (Ab) responses to several new antigens during active TB but not LTBI have been reported. However, most studies have focused on patients with advanced TB, and the utility of these responses appears to be limited in patients with low mycobacterial burden (reviewed in references 29 and 30).Two immunodominant M. tuberculosis proteins, the 81-kDa malate synthase (MS; Rv 1837c, GlcB) and the 27-kDa MPT51 (Rv3803c), are reported to elicit Ab responses during early and advanced stages of TB in both HIV⁻ and HIV⁺ patients (1, 2, 10, 16, 24, 27, 37). This is important because HIV⁺ TB patients appear to develop Ab responses to a smaller repertoire of M. tuberculosis antigens than that of HIV⁻ TB patients (24, 25). In previous case-control studies with HIV⁻ and HIV⁺ patients, pulmonary and extrapulmonary TB patients from settings in which TB is endemic demonstrated the presence of anti-MS and/or anti-MPT51 Abs in about 80% of the TB patients but not in tuberculin skin test (TST)-negative and -positive volunteers (27, 37). Similar studies with U.S. patients demonstrated that while anti-MS and/or anti-MPT51 Abs were present in only ∼40% of the HIV⁻ patients at early stages of TB, ∼80% of the U.S. HIV⁺ TB patients were Ab positive (1). Nevertheless, combining serology with sputum microscopy improved the detection of TB in both groups compared to that of microscopy alone, and it led to the identification of 90% of HIV⁺ TB patients, compared to 60% by microscopy alone (1). These findings are of high clinical relevance, since the rapid identification and treatment of early TB is crucial for HIV⁺ patients, in whom the dual infection leads to the acceleration of both diseases (20, 35).While antigen discovery, selection, and validation initially relies on case-control studies comparing known TB cases to healthy controls, such comparisons result in the overestimation of accuracy (14, 23), and the real value of any antigen needs to be ascertained by cross-sectional studies in clinical settings where the TB suspects include patients with a variety of respiratory diseases. The goals of the current investigations were to (i) identify the range of Ab reactivities to MS and MPT51 in a cross-sectional study of U.S. TB suspects, (ii) compare Ab reactivities between U.S. HIV⁻ and HIV⁺ TB patients and to asymptomatic U.S. non-TB as well as endemic TB controls; and (iii) compare Ab reactivities of HIV⁻ and HIV⁺ TB suspects diagnosed to have respiratory diseases other than TB (ORD) to those of HIV⁻ and HIV⁺ TB patients.     

Differential Live Mycobacterium tuberculosis-, M. bovis BCG-, Recombinant ESAT6-, and Culture Filtrate Protein 10-Induced Immunity in Tuberculosis

The high prevalence of Mycobacterium tuberculosis makes it imperative that immune responses to evaluate could be predictive of infection. We investigated live Mycobacterium- and recombinant antigen-induced cytokine and chemokine responses in patients with active tuberculosis (TB) compared with those of healthy controls from an area where TB is endemic (ECs). M. tuberculosis-, M. bovis BCG-, ESAT6-, and culture filtrate protein 10 (CFP10)-induced responses were determined in peripheral blood mononuclear cells from patients with pulmonary TB (n = 38) and ECs (n = 39). The levels of the cytokines gamma interferon (IFN-γ) and interleukin-10 (IL-10) and the chemokines CCL2, CCL3, and CXCL9 were measured. The levels of M. tuberculosis- and BCG-induced IFN-γ secretion were significantly reduced (P = 0.002 and P < 0.01, respectively), while the amount of IL-10 induced by both virulent (P < 0.01) and avirulent (P = 0.002) mycobacteria was increased in patients with TB. The ESAT6-induced IFN-γ responses were increased in the patients with TB (P = 0.013) compared with those in the EC group. When tuberculin skin test (TST)-negative (TST⁻; induration, <10 mm) and TST-positive (TST⁺) donors were studied separately, both TST⁻ and TST⁺ individuals showed increased IFN-γ responses to M. tuberculosis compared with the responses of the patients with TB (P = 0.037 and P = 0.006, respectively). However, only TST⁺ ECs showed reduced IFN-γ responses to ESAT6 (P = 0.008) compared with the responses of the patients with TB. The levels of M. tuberculosis-induced CCL2 (P = 0.006) and CXCL9 (P = 0.017) were greater in the patients with TB. The levels of CCL3 secretion in response to Mycobacterium and antigen stimulation were comparable between the two groups. While the levels of ESAT6-induced chemokines did not differ between the patients with TB and the ECs, the levels of CFP10-induced CCL2 (P = 0.01) and CXCL9 (P = 0.001) were increased in the patients. These data indicate differential host IFN-γ, CXCL9, and CCL2 responses to live mycobacteria and mycobacterial antigens and have implications for the identification of potential biomarkers of infection which could be used for the diagnosis of TB.Tuberculosis (TB) remains an important cause of mortality and morbidity worldwide, causing 2 million deaths annually. Vaccination with Mycobacterium bovis BCG provides protection mostly during childhood and primarily against the extrapulmonary forms of the disease (28, 45, 49). It is important to identify immune markers that may be reflective of natural infection in the host and that may therefore be useful in predicting the outcome of the disease.Host immune responses determine the outcome of M. tuberculosis infection. The balance between the key T-cell cytokines gamma interferon (IFN-γ) and interleukin-10 (IL-10), which are produced by macrophages and subsets of T cells, is key to the maintenance of effector responses against the mycobacterium (13, 34). This is further reflected by the relationship between IFN-γ and IL-10, which differs according to the severity of the disease, especially in patients with extrapulmonary M. tuberculosis infections (24).The formation and maintenance of granulomas are essential for the control of mycobacterial infections. Tumor necrosis factor alpha (TNF-α) is critical to an effective response against M. tuberculosis and for efficient granuloma formation (38). TNF^−/− mice show increased pathology and a more rapid progression of disease during infection with M. tuberculosis (6).IFN-γ- and macrophage-induced TNF-α activates and regulates the C-C chemokines CCL2, CCL3, CCL4, and CCL5 and the C-X-C chemokines CXCL8, CXCL9, CXCL10, and CXCL11 (2) (42). These chemokines have been shown to play an important role in the recruitment and activation of leukocytes at the site of granuloma formation.CCL2 is the most potent activator of monocytes and is essential for recruitment and migration to the lung. Studies with CCL2^−/− mice have investigated the pathology of M. tuberculosis infection (26) and have shown that CCCR2^−/− mice exhibit faulty granuloma formation and increased susceptibility to disease caused by M. tuberculosis (44). IFN-γ-activated chemokines, such as CXCL9 and CXCL10, are induced by M. tuberculosis antigens (1, 40).The circulating levels of cytokines such as IFN-γ, IL-10, transforming growth factor-β, and chemokine CXCL9 are found to be raised in patients with TB (17, 33). Patients with pulmonary TB also display increases in the level of monocyte-activating chemokine CCL2 (20). An increase in the CCL2 level is associated with monocyte migration to the lung and the reduced progression of disease (26, 43). Recent studies have identified molecular mechanisms which are deficient in patients with pulmonary TB. These include reduced IFN-γ responses at the disease site (21) and a reduced monocyte maturation ability concomitant with a reduction in the levels of markers of antigen presentation, such as major histocompatibility complex class II expression (36). In addition, it has been shown that granulomas differ in cavitary and noncavitary TB (48). This further emphasizes the need to understand the pattern of recognition of mycobacterial antigens in different forms of TB and in transitions from latent to active infection.Six-kilodalton early secreted antigenic target 6 (ESAT6) and culture filtrate protein 10 (CFP10) are both encoded by region of difference 1 (RD1), which is present in M. tuberculosis and M. bovis but which is absent from M. bovis BCG and most environmental mycobacteria (8, 16). ESAT6 is an immunodominant T-cell-stimulatory antigen and is recognized by specific IFN-γ-secreting T cells that are present in greater numbers in patients with active disease than in those who are uninfected (37, 47). The importance of these recombinant antigens is in their immune recognition by T cells specific to infected individuals. They are a primary component of new diagnostic assays and are meant to detect latent infections in individuals because of their lack of cross-reactivity with M. bovis BCG, which is used for vaccination (27). Commercial tests that use IFN-γ-release assays are increasingly available as diagnostic and predictive tests for newer-generation vaccines. ESAT6- and CFP10-induced IFN-γ responses have been shown to be useful in discriminating infected individuals from healthy controls (1, 9, 46). Demissie et al. have shown that immune responses to ESAT6 are more likely to be associated with active infection and that a dormancy-related antigen (HspX or α-crystallin) might be a better marker of latency in patients with TB, while cross-reactive antigens, such as the antigen 85A complex, do not distinguish between active and latent TB (11).Antigen recognition and binding to cell surface receptors result in cytokine activation and intracellular signaling cascades within the host cell (14, 39). Virulent M. tuberculosis organisms are efficient at evading macrophage host defenses, such as phagolysosome fusion; downregulation of the activation of proinflammatory cytokines, such as TNF-α; inhibition of antigen presentation; and also host apoptotic responses (19, 25). In contrast, avirulent mycobacteria, such as the M. bovis BCG strain, are processed along the endosomal pathway, activating stronger responses of proinflammatory cytokines, such as TNF-α, IL-2, and IL-6, than the responses activated by virulent mycobacteria (7). In addition, the uptake and processing of live and dead mycobacteria differ in host cells (18). Therefore, although both mycobacteria and mycobacterial antigens can stimulate host immunity, it is likely that their immunogenicities may differ due to processing and presentation of antigens.There is as yet a limited understanding of how host cell responses differ when the host is challenged with live mycobacteria as opposed to mycobacterial antigens. We investigated the differential cytokine responses to live mycobacteria (virulent M. tuberculosis and avirulent M. bovis BCG strains) and compared them with those to the M. tuberculosis antigens ESAT6 and CFP10. BCG vaccination is widespread in Pakistan and since 1981 has routinely been administered at birth through the Expanded Program for Immunization (51). Therefore, the levels of cytokine and chemokine secretion by peripheral blood mononuclear cells (PBMCs) in response to different stimuli were analyzed in patients with pulmonary TB and were compared with those in healthy vaccinated controls from areas where TB is endemic (ECs). Our results highlight the variability of host responses to mycobacteria and mycobacterial antigens.     

Impact of Blood Volume,Tube Shaking,and Incubation Time on Reproducibility of QuantiFERON-TB Gold In-Tube Assay

Gamma interferon (IFN-γ) release assays (IGRAs) are functional assays used serially to measure the efficacy of novel tuberculosis (TB) vaccines and to screen health care workers for latent tuberculosis infection (LTBI). However, studies have shown nonreproducible IGRA results. In this study, we investigated the effects of blood volume (0.8, 1.0, and 1.2 ml), tube shaking (gentle versus vigorous), and incubation duration (16, 20, and 24 h) on the reproducibility of QuantiFERON-TB Gold In-Tube (QFT-GIT) results for 50 subjects (33 uninfected and 17 infected). The median IFN-γ TB response (TB antigen [Ag] minus nil value) was significantly higher with 0.8 ml blood (1.04 IU/ml) than with 1.0 ml (0.85 IU/ml; P = 0.002) or 1.2 ml (0.49 IU/ml; P < 0.001) for subjects with LTBI. Compared with 0.8 ml (11.8%), there were larger proportions of false-negative results with 1.0 ml (29.4%; P = 0.2) and 1.2 ml (41.2%; P = 0.05) of blood for infected subjects. Blood volume did not significantly change the proportions of positive results in uninfected controls. Compared with gentle shaking, vigorous shaking increased the median IFN-γ response in nil (0.04 versus 0.06 IU/ml; P < 0.001) and TB Ag (0.12 versus 0.24 IU/ml; P = 0.004) tubes and increased TB responses (TB Ag_vigorous minus nil_gentle) (0.02 versus 0.08 IU/ml; P = 0.004). The duration of incubation did not have a significant impact on the proportion of positive results in uninfected or infected subjects. This study identified blood volume and tube shaking as novel preanalytical sources of variability which require further standardization in order to improve the quality and reproducibility of QFT-GIT results.