QFT-GIT in TB Screening: Current Research and Applications

Tuberculosis (TB) remains a major global health concern, requiring accurate and efficient diagnostic tools for early detection. The QuantiFERON-TB Gold Plus (QFT-GIT) test has emerged as a widely used interferon-gamma release assay (IGRA) for detecting latent TB infection. Unlike the traditional tuberculin skin test, QFT-GIT offers higher specificity and is unaffected by prior Bacillus Calmette-Guérin (BCG) vaccination, making it particularly valuable in screening programs.

Understanding how QFT-GIT works and its role in clinical practice is essential as research continues to refine TB diagnostics.

Biological Components Of The Test

The QuantiFERON-TB Gold Plus (QFT-GIT) test relies on specific Mycobacterium tuberculosis antigens to detect latent infection. These proteins, absent in most non-tuberculous mycobacteria and the BCG vaccine strain, enhance the test’s specificity. The key antigens used in QFT-GIT are ESAT-6, CFP-10, and TB7.7, each playing a distinct role in tuberculosis detection.

ESAT-6

Early Secretory Antigenic Target-6 (ESAT-6) is a 6-kDa protein encoded by the esxA gene, located within the RD1 (Region of Difference 1) of Mycobacterium tuberculosis. This antigen is absent in all BCG vaccine strains and most environmental mycobacteria, making it a highly specific target for TB detection. ESAT-6 facilitates host cell membrane lysis, promoting intracellular survival and dissemination of the pathogen. Studies in The Journal of Infectious Diseases have shown that ESAT-6 elicits a strong T-cell response in individuals with latent TB infection, making it a crucial component of IGRA-based diagnostic tests.

CFP-10

Culture Filtrate Protein-10 (CFP-10) is a 10-kDa antigen encoded by the esxB gene, also within the RD1 genomic region. Like ESAT-6, CFP-10 is secreted as part of the ESX-1 secretion system, a key virulence mechanism of Mycobacterium tuberculosis. CFP-10 plays a role in bacterial survival by modulating host immune responses and facilitating macrophage infection. Research published in Clinical Microbiology Reviews highlights its ability to trigger strong T-cell activation. The combination of CFP-10 and ESAT-6 in IGRA tests enhances sensitivity by capturing a broader spectrum of immune responses.

TB7.7

TB7.7, encoded by the Rv2654 gene, is expressed by Mycobacterium tuberculosis but not by the BCG vaccine strain. While its immunogenicity is less extensively studied than ESAT-6 and CFP-10, research in Tuberculosis and Lung Disease suggests TB7.7 improves the sensitivity of IGRA tests. It may help detect latent TB in individuals with variable immune responses, ensuring a more comprehensive assessment of TB exposure.

Mechanism Of T-Cell Activation

T-cell activation in response to Mycobacterium tuberculosis antigens underpins the functionality of the QFT-GIT test. When an individual with prior exposure encounters the test’s specific antigens, antigen-presenting cells (APCs), primarily dendritic cells and macrophages, internalize and process these proteins. Peptides derived from ESAT-6, CFP-10, and TB7.7 are then presented via major histocompatibility complex (MHC) molecules—MHC class II for CD4+ T-helper cells and MHC class I for CD8+ cytotoxic T-cells. The dual activation of CD4+ and CD8+ T-cells enhances detection sensitivity.

Once APCs present these antigens, T-cells recognizing the peptides through their receptors (TCRs) become activated. This interaction is reinforced by co-stimulatory signals, such as CD28 binding to CD80/CD86 on APCs, ensuring a sustained immune response. The activation triggers intracellular signaling cascades, leading to the transcription of genes responsible for cytokine production, particularly interferon-gamma (IFN-γ), the primary readout in QFT-GIT testing.

By detecting IFN-γ responses from both CD4+ and CD8+ T-cells, QFT-GIT provides a more comprehensive assessment of TB infection. Research in The Journal of Immunology indicates CD8+ responses are more relevant in recent or active infection, while CD4+ responses reflect long-term immune memory. This distinction improves result interpretation, particularly in immunocompromised individuals, where CD8+ responses may compensate for weakened CD4+ activity.

Laboratory Process

The QFT-GIT test follows a structured laboratory workflow to ensure accuracy in detecting latent TB infection. Blood collection is performed using specialized QuantiFERON tubes pre-coated with Mycobacterium tuberculosis-specific antigens. These tubes include a nil control for background immune activity, a mitogen control to verify immune competence, and antigen-containing tubes to stimulate a response. Proper handling is crucial, as delays in incubation or exposure to extreme temperatures can compromise results. The Clinical and Laboratory Standards Institute (CLSI) recommends maintaining samples at 17–25°C before incubation.

Samples are incubated at 37°C for 16 to 24 hours, allowing leukocytes to interact with the antigens and release IFN-γ into the plasma. Studies in The Journal of Clinical Microbiology highlight the importance of proper mixing during this step, as insufficient agitation can lead to inconsistent IFN-γ release. Automated tube handling systems help minimize variability.

After incubation, plasma is separated via centrifugation to remove cellular debris, ensuring a clear sample for analysis. The plasma is then tested using enzyme-linked immunosorbent assay (ELISA) to quantify IFN-γ levels. ELISA plates are pre-coated with monoclonal antibodies specific to IFN-γ, ensuring precise detection. Sensitivity is influenced by reagent stability, incubation time, and optical density readings. Multi-point calibration curves using IFN-γ standards help maintain accuracy.

Interpreting The Response

QFT-GIT results are classified as positive, negative, or indeterminate based on measured IFN-γ levels. According to the Centers for Disease Control and Prevention (CDC), a positive result indicates IFN-γ production above the assay’s cutoff, suggesting prior Mycobacterium tuberculosis exposure. A negative result signifies an absence of significant IFN-γ release, implying no detectable infection. Indeterminate results occur when the nil control is excessively high, indicating nonspecific immune activation, or when the mitogen control fails to elicit a response, suggesting immunosuppression or technical issues.

Clinical context is crucial in interpreting results, as false positives and negatives can occur. Individuals with recent exposure or immunosuppressive conditions may produce borderline IFN-γ levels. Studies in The Lancet Infectious Diseases highlight test variability among immunocompromised patients, such as those undergoing chemotherapy or receiving tumor necrosis factor (TNF) inhibitors, where IFN-γ responses may be diminished. In such cases, repeat testing or supplementary diagnostic methods, such as chest radiography or nucleic acid amplification tests (NAATs), may be needed for confirmation.

Current Scope In TB Screening

QFT-GIT has become a critical tool in TB screening, particularly in identifying asymptomatic carriers at risk of developing active disease. It is widely used in healthcare settings, occupational health programs, and public health initiatives in high-burden regions. The test has improved detection rates in high-risk populations compared to traditional skin tests, especially in areas where exposure to nontuberculous mycobacteria may confound tuberculin skin test results. A study in The International Journal of Tuberculosis and Lung Disease supports IGRA-based testing as more effective in such populations.

Ongoing advancements in TB diagnostics aim to refine IGRA applications. Research in Nature Medicine suggests integrating IGRA results with transcriptomic signatures may help identify individuals at higher risk of disease progression, improving targeted preventive therapy. Technological improvements are also streamlining test logistics, reducing processing time, and increasing stability for use in resource-limited settings. While QFT-GIT remains a cornerstone of TB screening, future developments seek to enhance its predictive capabilities, ensuring latent infections are identified and managed before progressing to active disease.