Introduction

Since its discovery in 1882, tuberculosis (TB) has long been established as a potentially lethal infectious disease with severe complications to its host. While its eradication in developed countries proves to be substantial, TB is still a primary concern of significant morbidity and mortality in many parts of the world.

Caused by bacteria known as Mycobacterium tuberculosis, this bacterium works by damaging the lungs and, hence, affecting the breathing capacities of an individual. Most individuals who acquire infection by Mycobacterium tuberculosis, the bacteria species responsible for TB, will eliminate or contain the infection causing it to remain latent. Transmission occurs through respiratory droplets of affected individuals suffering from active disease.  Issues arise when reactivation of the latent disease occurs, causing significant symptoms to the patient and allowing the potential to spread.[1]

There are two testing methods currently used for the identification of latent tuberculosis infection: the tuberculin skin test (TST) and interferon-gamma release assay (IGRA). IGRAs diagnose tuberculosis infection by either calculating the concentration of interferon-γ generated ex vivo by the sufferer's immune cells or by counting the total number of interferon-γ secreting lymphocytes. The tuberculin skin test (TST) is performed by injecting a small amount of fluid called tuberculin purified protein derivative (PPD) into the forearm. A qualified health care professional then measures the induration within 48 to 72 hours of injection and takes into account the patient's background history for indications of a positive or negative test result.[2]

It should be noted that these testing methods are specific for latent tuberculosis infection, as they should not be used for testing active disease. The diagnosis of active TB is primarily based on clinical suspicion with confirmatory testing through sputum culture analysis.

Etiology and Epidemiology

TB remains a significant public health threat worldwide, causing an estimated 10.4 million new infections and 1.4 million TB deaths in 2015. Present epidemiological forecasts indicate only marginal improvement in the elimination of TB as measured by acute disease, fatalities, and antibiotic resistance. The downward trend in such indicators underwent an evaluation to determine if the original 2020 goals were achieved, which included estimations of strengthened attempts to enhance treatment plans and prevention of TB. The belief was that if the rate evolved in a positive direction, it could have met the 2020 targets of the End TB Strategy. In most cases, initial infection with Mycobacterium tuberculosis is prevented or suppressed by the host immune response, and the disease’s persistence is residual.[3][4]

Conversely, tuberculosis could continue growing as it has the potential to be reactivated with resultant active TB. Diagnosis and management of both active and latent TB can significantly reduce the risk of developing the disease and are essential TB control strategies, particularly in settings with low TB incidence where the reactivation of LTBI often accounts for the majority of pre-imported TB infection.

Pathophysiology

Bacilli tubers invade the upper respiratory tract and cause infection after they are carried in small (5 to 10 microns) droplets to enter the alveolar spaces. When the host's immune system fails to eradicate the bacteria, the bacilli propagate within macrophages and ultimately destroy the cells. Infected macrophages attempt to prevent this by generating inflammatory cytokines for the recruitment of other phagocytes to effectively create a nodular caseating granuloma known as a tuber. The granuloma serves to wall off the infection and prevents its spread to other areas of the lungs.

If bacterial proliferation is not regulated, the tuber may expand seeding infection by invasion to other areas, including the localized draining lymphatic system. This activity results in mediastinal lymphadenitis, a clinical embodiment of TB. The cyst, caused by the growth of the tuber into lung tissue and resultant lymphadenopathy, is called the Ghon complex and is a classic finding on radiographic imaging for primary tuberculosis infection.[5][6]

Interferon-γ is the primary cytokine released in response to infection by Mycobacterium tuberculosis. Macrophages are the first immune cells to respond and react to the site of infection, but require additional aid from other components of the immune system to effectively destroy intracellular pathogens. Following engulfment of foreign bacteria through phagocytosis, macrophages secrete cytokines to attract T helper cells, the primary mediators of interferon-γ. Interferon-γ, in turn, allows for greater activation of the macrophages by ramping up defense mechanisms designed to kill intracellular bacteria. This activation-reactivation process between macrophages and T helper cells occurs over and over as a positive feedback mechanism until the infection is eradicated or becomes dormant.

Interferon-γ release assays (IGRAs) depend on this immune reaction involving T helper cells to detect latent TB through quantification. Fresh blood samples are mixed with antigens and controls to test to determine if an individual has indeed contracted an infection.[7][8]

Specimen Requirements and Procedure

Testing for latent tuberculosis infection (LTBI) should be 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 of correctional facilities. Testing should not be performed in individuals who are otherwise healthy with little to no risk for progression to active disease. Also, it should be taken into consideration whether detection would be beneficial in the specific patient, as medications for the treatment of TB can come with adverse effects.[2]

IGRAs are an in vitro method aimed at measuring a type IV cell-mediated immune response to antigens of M. tuberculosis. Antigenic peptides derived directly from the DNA genome of M. tuberculosis are used to perform testing. These antigens are specific to M. tuberculosis and are not encoded in the genome of BCG vaccine strains or most species of non-tuberculosis mycobacteria. Exceptions and overlaps include M. marina, M. kansasii, M.szulgai, and M. flavescens, which have little to no prevalence in society. [9]

IGRAs are available as two different tests: The QFT-GIT assay and the T-SPOT.TB assay. The QFT-GIT assay is an ELISA test that uses whole blood and combines it with antigens of M. tuberculosis, namely ESAT-6, CFP-10, and TB7.7, into a test tube. Measurement of the IFN-γ is reported in international units (IU) per milliliter and is considered positive when ranging above the test cutoff. The T-SPOT.TB assay is an ELISPOT assay where ESAT-6 and CFP-10 are incubated with peripheral blood mononuclear cells. The specificity of both exceeds >95%, however the T-SPOT.TB has greater sensitivity than QFT-GIT at 90% rather than 80%.[10][11][12]

IGRAs are beneficial in the setting of high BCG vaccination populations because it does cause false positives in these individuals, unlike TST.[2]

Interfering Factors

In all cases where the CDC recommends tuberculin skin testing (TST) as a tool in the diagnosis of latent tuberculosis, IGRAs may be used instead, or as an adjunct in concordance with special requirements and criteria. Several sources of variability can affect IGRA results, not all of which are currently understood. Variation in reliability can involve multiple forms, including statistical variability when it comes to obtaining analytical data, responsive variability dependent on immune response, and manufacturing variability.

Although the assay manufacturers and the test users can remove or mitigate systemic sources of variability by optimization, spontaneous sources of variance are inevitable and must be compensated for when obtaining data. The pre-analytical or statistical variability serves as an interfering factor when the incubation period of blood samples becomes prolonged, or fluctuations in the accurate measurement of results may cause issues in the IGRA test and readings. Immunological response variability rests on the host’s ability to form an adequate immune response to TB, such as would be problematic in immunocompromised patients with HIV, chronic systemic steroid use, or malnutrition.[2][13]

Results, Reporting, and Critical Findings

Test interpretation is positive or negative in a binary fashion. Verging outcomes are uninterpretable and have no clinical significance, and therefore should be reiterated. Indeterminate or borderline results, typically arising from either a failed control group and more likely in the setting of the weakened immune system, are often uninterpretable and should be replicated as well. The statistical data of positive to negative reversal is unclear, while it may indicate the voluntary resolution of disease or reduced/excluded regression threat.

Clinical Significance

Diagnostic tests for latent TB have largely played the most influential factor in fighting the prevalence of this disease over time. Modern measures of testing through IGRAs and TST have proven paramount in decreasing the incidence of illness and controlling the spread. In addition, the development of vaccines for the prevention of TB in endemic areas and effective therapeutic regimens for the treatment of active TB have helped to reduce progression as well.

There are multiple facets of clinical significance in using interferon-gamma release assay over other testing methods for the diagnosis of latent TB. Factors to consider include: single patient visit at the time of sample collection, the possibility of collection results within twenty-four hours, prior BCG vaccination history, results not subject to reader bias, and reduced staff time with avoidance of unnecessary follow-up. However, in cases of immunosuppression, IGRAs should be approached with caution as the patient may not be able to develop an adequate immune response to latent TB for the test, resulting in false negatives. IGRAs should also not be used in children under five years of age for this same purpose. Additionally, IGRAs should not be used to monitor treatment response for TB.[2][10][14]