Interferon Test

Etiology and Epidemiology

TB remains a significant public health threat worldwide. TB affects all age groups, though it primarily affects adults in middle age. Over 80% of cases and deaths are in low- and middle-income countries.[3] In 2023, an estimated 10.8 million people were infected with TB globally, including 6.0 million men, 3.6 million women, and 1.3 million children. A total of 1.25 million people died from TB, including 161,000 people with human immunodeficiency virus (HIV). Tuberculosis (TB) regained its position as the leading cause of death from a single infectious agent after a 3-year period during which COVID-19 was the most fatal infectious disease. TB also remained the leading cause of death among people with HIV and a major contributor to mortality related to antimicrobial resistance. In the same year, the most significant number of new TB cases occurred in the World Health Organization's South-East Asia Region (45%), followed by the African Region (24%) and the Western Pacific Region (17%).[4]

Several risk factors contributed to the global burden of TB in 2023, with an estimated 0.96 million new cases attributable to undernutrition, 0.75 million to alcohol use disorders, 0.70 million to smoking, 0.61 million to HIV infection, and 0.38 million to diabetes.[5][6] In the United States, 10,347 TB cases were provisionally reported in 2024, corresponding to a rate of 3 cases per 100,000 population. The percentage increase in both case counts (8%) and rates (6%) from 2023 to 2024 was smaller than the increases from 2022 to 2023 (15% in both case counts and rates).[7] TB has the potential to remain persistent or worsen due to the reactivation of latent TB infection (LTBI), particularly in settings with low incidence. Therefore, effective diagnosis and management of both active and LTBI are essential strategies for TB control.[8] Ending the TB epidemic by 2030 is one of the health targets outlined in the United Nations' Sustainable Development Goals.[9]

Pathophysiology

Bacilli tubers invade the upper respiratory tract and travel in small (5 to 10 microns) droplets to the alveolar spaces. When the host's immune system does not eradicate the bacteria, the bacilli propagate within macrophages and ultimately destroy the cells. Infected macrophages generate inflammatory cytokines and recruit additional phagocytes, forming a nodular, caseating granuloma known as a tubercle. The granuloma prevents the infection from spreading to other areas of the lungs.

If bacterial proliferation is not regulated, the tuber may expand, seeding infection to other areas, including the lymphatic system. The resulting inflammation leads to mediastinal lymphadenitis, a clinical manifestation of TB. The cyst, caused by the growth of the tubercle into lung tissue and resulting lymphadenopathy, is known as the Ghon complex and is a classic radiographic finding in primary tuberculosis infection.[10][11]

Interferon-γ is the primary cytokine released in response to infection by M tuberculosis bacteria. Macrophages are the first immune cells to respond to infection. Following phagocytosis, macrophages secrete cytokines to attract T helper cells, the primary mediators of interferon-γ. Interferon-γ further amplifies the activation of macrophages. This activation-reactivation process between macrophages and T helper cells occurs repeatedly as a positive feedback loop until the infection is eradicated or dormant. IGRAs quantify the immune reaction of T helper cells to detect latent TB. Fresh blood samples are mixed with antigens and controls to determine if infection is present.[12][13]

Diagnostic Tests

Testing for LTBI is reserved for those who are at increased risk for progression to active TB. Examples include people who were recently in close contact with an affected person with TB, immunocompromised individuals, healthcare workers, or workers/persons in correctional facilities. Testing should not be performed in individuals who are otherwise healthy and have a low risk of progression to active disease.  Because medications for treating TB can have adverse effects, clinicians must weigh the risk vs benefit of detection.[2]

TSTs are an in vitro method used to measure a type IV cell-mediated immune response to antigens of M tuberculosis bacteria. Antigenic peptides derived directly from the DNA of M tuberculosis are injected subcutaneously. These antigens are specific to M tuberculosis and are not present in the genome of Bacille Calmette-Guérin (BCG) vaccine strains or most species of non-tuberculosis mycobacteria. Exceptions include M marinum, M kansasii, M szulgai, and M flavescens, which have low prevalence in the general population.[14]

IGRAs are blood tests used to detect latent M tuberculosis infection. Two IGRA formats are available: the QuantiFERON-TB Gold Plus (QFT-Plus) and the T-SPOT.TB assay.[15] The QFT-Plus is an enzyme-linked immunosorbent assay test that uses whole blood and must be processed within 16 hours. This test measures interferon-gamma (IFN-γ) concentration in response to M tuberculosis-specific antigens early secreted antigenic target (ESAT)-6, culture filtrate protein (CFP)-10, and TB7.7. Interpretations are classified as positive, negative, or indeterminate based on predefined cutoffs in international units per mL.[16]

In contrast, the T-SPOT.TB is an enzyme-linked immunosorbent spot assay that incubates peripheral blood mononuclear cells with ESAT-6 and CFP-10 antigens. This assay measures the number of IFN-γ–producing T-cells (spots) and can be processed within 8 to 32 hours. Results are interpreted as positive, negative, borderline, or invalid, providing more information in ambiguous cases. Both assays have a specificity exceeding 95%, though T-SPOT.TB has higher sensitivity, approximately 90% compared to 80% for QFT-Plus.[17][18] While both assays are effective, the choice depends on the time frame, sensitivity, and patient-specific factors. IGRA tests benefit high BCG vaccination populations due to a low rate of false positives, unlike TSTs.[2]

Interfering Factors

When the Centers for Disease Control and Prevention recommends TST to diagnose latent tuberculosis, IGRAs may be used instead or as an adjunct in concordance with the established criteria. Several sources of variability can affect IGRA results, some of which are not yet fully understood. Variation in reliability can include statistical variability in obtaining analytical data, responsive variability dependent on the immune response, and manufacturing variability.

Although assay manufacturers and test users can remove or mitigate systemic sources of variability through optimization, spontaneous sources of variance are inevitable and must be compensated for when obtaining data. The preanalytical or statistical variability interferes when the incubation period of blood samples is prolonged, or fluctuations in the accurate measurement of results interfere with the IGRA test readings. Immunological response variability rests on the host’s ability to form an adequate immune response to TB, which is problematic in immunocompromised individuals with HIV, chronic systemic steroid use, or malnutrition.[2][19]

Results, Reporting, and Critical Findings

Test interpretation is either positive or negative. Diverging outcomes are uninterpretable and have no clinical significance; therefore, they should be repeated. Indeterminate or borderline results, typically arising from either a failed control group or an inadequate immune response, are often uninterpretable and should be replicated. The statistical data regarding the transformation from positive to negative test results is unclear, although it may indicate resolution or regression of the disease.

Interpretation of TB blood test results depends on the specific IGRA used. For the QuantiFERON-TB Gold Plus, results are based on the amount of IFN-γ released in response to M tuberculosis antigens and control substances after incubation. In contrast, the T-SPOT.TB assay is interpreted by counting the IFN-γ–producing cells (eg, spots) after the blood is incubated with the antigens. Laboratories typically report both qualitative and quantitative results. The qualitative results for QFT-Plus are categorized as positive, negative, or indeterminate, whereas T-SPOT.TB results may be positive, borderline, negative, or invalid. Quantitative results include numerical values indicating responses to the TB antigens and 2 controls: nil (negative control) and mitogen (positive control). Although no standardized guidelines exist for interpreting the quantitative values alone, the numerical values can help interpret borderline or conflicting results, particularly when combined with clinical risk factors.[16][20]

A positive IGRA test result generally indicates TB infection, but further evaluation, including chest radiography and clinical correlation, is needed to exclude active TB disease. A negative test result suggests that TB infection is unlikely. However, individuals with symptoms of TB infection or those at high risk (eg, immunocompromised patients) may have an active infection and a negative test result. An indeterminate (QFT-Plus), borderline, or invalid (T-SPOT) result indicates that the test is inconclusive. Repeating the IGRA or performing a TST may be helpful.[21][22]

Clinical Significance

Diagnostic tests for latent TB have played a role in the decreasing prevalence of the disease over time. In addition, the development of vaccines for the prevention of TB in endemic areas and effective treatments for active TB have also contributed to a reduction in the incidence. The IGRA test has several advantages over other testing methods for diagnosing latent TB, including a single patient encounter for sample collection, the possibility of obtaining results within 24 hours, its usefulness in patients with prior BCG vaccination, and results that are not subject to reader bias. However, if the patient is immunosuppressed, they may have an inadequate immune response, resulting in a false negative. IGRA tests are not appropriate for children younger than 5 for the same reason. Additionally, IGRA tests are not useful for monitoring treatment response.[2][23][24]

Quality Control and Lab Safety

Quality control (QC) and quality assurance (QA) are critical components in ensuring the' accuracy, reliability, and consistency of IGRA tests, including QuantiFERON-TB Gold and T-SPOT.TB. Before patient testing, trained technologists must perform internal QC to verify the acceptability of assay performance. Patient samples are only processed after successful QC validation. [25]

Robust external quality assessment (EQA) programs can help to ensure QC in addition to internal QC programs. EQA strategies may include participation in proficiency testing schemes, which evaluate the laboratory's performance by comparing its results to those of reference laboratories. Retesting identical specimens in different laboratories is another valuable method for assessing interlaboratory consistency and is widely practiced in TB diagnostic networks.[26][27] Furthermore, on-site supervisory visits are essential to ensure adherence to protocols and technical competence, especially during the initial implementation of new technologies. Both internal QC and EQA are integral to the overall quality improvement framework and are guided by defined quality indicators and performance metrics.[27] 

Safety is fundamental in IGRA testing laboratories, protecting personnel, maintaining specimen integrity, and ensuring accurate results. Since IGRA tests involve handling human blood specimens, strict adherence to biosafety guidelines prevents exposure to potentially infectious material. All personnel must use appropriate personal protective equipment, including gloves, lab coats, and eye protection, and follow standard precautions when processing specimens.[2]

Specimen handling, incubation, and enzyme-linked immunosorbent assay procedures should be performed within designated biosafety cabinets where aerosol generation is possible. Clear labeling, safe centrifugation practices, and proper waste disposal protocols minimize risks. Regular staff training on biosafety, spill management, and equipment handling is critical, especially in high-volume settings. Additionally, temperature and environmental monitoring of incubators and analyzers should be conducted and documented routinely. Implementation of a biosafety manual, periodic audits, and incident reporting mechanisms is a key element of a comprehensive lab safety program in IGRA testing environments.[28]

Enhancing Healthcare Team Outcomes

In the context of IGRA testing, interprofessional collaboration is essential to ensure accurate diagnostics, patient safety, and optimal care outcomes. Nurses and phlebotomists are responsible for collecting specimens properly and transporting them in a timely manner to maintain sample integrity. Meanwhile, laboratory technologists ensure the rigorous implementation of internal and external quality control protocols. Clinicians must communicate clearly with the laboratory, providing relevant clinical context to support accurate interpretation of results and timely clinical decision-making. Pharmacists contribute by assessing and managing treatments when TB infection is confirmed.

All team members must uphold ethical considerations—such as informed consent, confidentiality, and equitable access to testing. Effective interprofessional communication, regular case reviews, and shared decision-making promote a patient-centered approach, thereby enhancing diagnostic accuracy and clinical outcomes. Coordinated care pathways, continuous quality improvement, and team training strengthen healthcare team performance and reduce the risk of diagnostic errors or delays in TB management.