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.     

Serodiagnostic Efficacy of Mycobacterium tuberculosis 30/32-kDa Mycolyl Transferase Complex, ESAT-6, and CFP-10 in Patients with Active Tuberculosis

Elimination of tuberculosis (TB) largely depends upon definitive rapid diagnosis and treatment. Widely used diagnostic tests do not qualify for use in a developing country due to lack of either desired accuracy or their cost. In the present study an enzyme-linked immunosorbent assay was used to evaluate the diagnostic potential of an immuno-dominant 30/32-kDa mycolyl transferase complex (Ag85 complex) and Mycobacterium tuberculosis-specific proteins (ESAT-6 and CFP-10) of the RD1 region. Higher sensitivity (84.1%) with Ag85 complex was observed compared with ESAT-6 (64.9%) and CFP-10 (66%), with almost similar specificity (Ag85: 85.2%, ESAT-6: 88.9%, CFP-10: 85.2%), whereas the individual components of Ag85 complex, i.e. Ag85A, Ag85B, and Ag85C, showed sensitivities of 44.6, 34, and 80.9% and specificities of 55.6, 74.1, and 40.7% respectively. A cocktail of Ag85 complex, ESAT-6, CFP-10, Ag85A, Ag85B, and Ag85C antigens also could not help in increasing either sensitivity (51.1%) or specificity (85.2%). Furthermore, immunoblot analysis using clinical isolates as well as a standard strain (H37Rv) of M. tuberculosis also showed strong reactivity of sera from TB patients to Ag85 complex and, to a lesser extent, also to ESAT-6. To conclude, use of Ag85 complex along with ESAT-6 and CFP-10 seems to be promising in minimizing the heterogeneous sero-responses of adult TB cases.     

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.)     

Early Induction of Interleukin-10 Limits Antigen-Specific CD4+ T Cell Expansion,Function, and Secondary Recall Responses during Persistent Phagosomal Infection

Diverse pathogens have evolved to survive and replicate in the endosomes or phagosomes of the host cells and establish persistent infection. Ehrlichiae are Gram-negative, intracellular bacteria that are transmitted by ticks. Ehrlichiae reside in the endosomes of the host phagocytic or endothelial cells and establish persistent infection in their vertebrate reservoir hosts. CD4⁺ T cells play a critical role in protection against phagosomal infections. In the present study, we investigated the expansion, maintenance, and functional status of antigen-specific CD4⁺ T cells during persistent Ehrlichia muris infection in wild-type and interleukin-10 (IL-10)-deficient mice. Our study indicated that early induction of IL-10 led to reduced inflammatory responses and impaired bacterial clearance during persistent Ehrlichia infection. Notably, we demonstrated that the functional production of gamma interferon (IFN-γ) by antigen-specific CD4⁺ T cells maintained during a persistent phagosomal infection progressively deteriorates. The functional loss of IFN-γ production by antigen-specific CD4⁺ T cells was reversed in the absence of IL-10. Furthermore, we demonstrated that transient blockade of IL-10 receptor during the T cell priming phase early in infection was sufficient to enhance the magnitude and the functional capacity of antigen-specific effector and memory CD4⁺ T cells, which translated into an enhanced recall response. Our findings provide new insights into the functional status of antigen-specific CD4⁺ T cells maintained during persistent phagosomal infection. The study supports the concept that a better understanding of the factors that influence the priming and differentiation of CD4⁺ T cells may provide a basis to induce a protective immune response against persistent infections.     

Validation of a Single-Platform, Volumetric, CD45-Assisted PanLeucogating Auto40 Flow Cytometer To Determine the Absolute Number and Percentages of CD4 T Cells in Resource-Constrained Settings Using Cameroonian Patients' Samples

The study evaluated the single-platform, volumetric, CD45-assisted PanLeucogating Auto40 flow cytometer (Apogee Flow Systems Ltd., Hemel Hempstead, United Kingdom) for CD4 T cell numeration, compared to the reference FACSCalibur flow cytometer. Results of absolute counts and percentages of CD4 T cells by Auto40 and FACSCalibur of 234 tripotassium EDTA (K3-EDTA)-blood samples from 146 adults and 88 children (aged from 18 months to 5 years), living in Yaoundé, Cameroon, were highly correlated (r² = 0.97 and r² = 0.98, respectively). The mean absolute bias and relative bias between Apogee Auto40 and FACSCalibur absolute CD4 T cell counts were +9.6 cells/μl, with limits of agreement from −251 to 270 cells/μl, and +4.1%, with limits of agreement from −16.1 to 24.4%, respectively. The mean absolute bias and relative bias between Apogee Auto40 and FACSCalibur CD4 T cell results expressed as percentages were +0.05% CD4 (95% confidence interval [CI], −0.03 to 0.41), with limits of agreement from −6.0 to 5.9% CD4, and +1.0%, with limits of agreement from −32.3 to 34.4%, respectively. The Auto40 counting allowed identification of the majority of adults with CD4 T cell counts below 200 cells/μl (sensitivity, 87%; specificity, 98%) or below 350 cells/μl (sensitivity, 92%; specificity, 98%) and of children with CD4 T cell counts below 750 cells/μl (sensitivity, 82%; specificity, 98%) or below 25% CD4⁺ (sensitivity, 96%; specificity, 99%). The Auto40 analyzer is a reliable alternative flow cytometer for CD4 T lymphocyte enumeration to be used in routine immunological monitoring according to the WHO recommendations for HIV-infected adults as well as children living in resource-constrained settings.