Introduction

Tuberculosis, caused by Mycobacterium tuberculosis, is an infection that occurs on a spectrum between latent and clinical disease. Clinical disease, termed active tuberculosis, is most frequently characterized by pulmonary disease, but extrapulmonary manifestations can also be observed.[1] After initial infection, patients can progress to active tuberculosis or eliminate the organism via the innate immune response or T-cell immunity. Most are noted with latent tuberculosis infection (LTBI) characterized by M tuberculosis in a dormant state with no evidence of any symptoms.[2][1] These patients have the highest reactivation risk within the first two to five years of initial infection, during which LTBI can progress to active tuberculosis.[1] Lifetime reactivation risk is estimated at 5% to 10% and increases with immunosuppression.[3] Therefore, those with LTBI are at risk for developing active tuberculosis and infecting others while risking personal morbidity and mortality. The global mortality of tuberculosis infection was estimated at 1.4 billion by the World Health Organization and 1.3 billion by the Global Burden of Disease study in 2013.[4]

As patients with LTBI do not exhibit any symptoms, LTBI incidence has to be estimated. In 2014, the global incidence of latent tuberculosis infection (LTBI) calculated via mathematical modeling was 23%.[5] An estimated 10.4 million new infections were noted in 2015.[6] Screening for M tuberculosis infection is essential for public health, as it allows patients with LTBI to receive preventative treatment. The goal of testing for LTBI is the identification of those at high risk of developing tuberculosis. The decision to test should presuppose a decision to treat if the result is positive.[7] Providers should be committed to following up on test results, offering tuberculosis-preventative treatment, and monitoring for adherence and tolerance of provided therapy.[7]

The tuberculin skin test (TST) and the interferon-gamma release assay (IGRA) are the current screening methods for measuring adaptive host immune response.[8] During initial infection, M tuberculosis is encountered by alveolar macrophages, and antigen is presented to T-cells.[9] Upon second exposure, TH1 cells release IGN-γ, which stimulates macrophages to release inflammatory cytokines.[9] The TST measures the indirect evidence of this inflammatory reaction triggered by a reaction to purified protein derivative (PPD), a solution of antigens derived from M tuberculosis. In contrast, IGRAs measure the amount of IFN-γ released.[9]

Specimen Collection

For interferon-gamma release assays, whole blood should be collected in evacuated, sterile tubes containing an anticoagulant and blood separation media.[10] The "T-Spot" TB test requires the specimen to be processed on the collection day.[11]

Procedures

Tuberculin Skin Test (TST)

The TST measures the delayed hypersensitivity reaction to the purified protein derivative (PPD), a heat-inactivated tubercle bacilli protein precipitate.[9][12][13]

Five tuberculin units (0.1 mL) are injected intradermally on the volar surface of the forearm to form a 10 mm wheal (Mantoux method).[14] This administration should be performed by skilled personnel. After placement, a follow-up visit in 48 to 72 hours is needed so the results can be interpreted.[14] The greatest diameter of induration (not erythema) is measured in millimeters.[14]

Interferon Gamma Release Assays (IGRAs)

Two assays are approved by the United States Food and Drug Administration and the WHO: the QuantiFERON-TB Gold Plus (QFT-Plus) test and the T-SPOT.TB (T-Spot) test.[15] The QuantiFERON-TB Gold Plus replaced its predecessor, the QuantiFERON-TB Gold In-Tube, which is no longer commercially available.[15]

The QFT-Plus test is an enzyme-linked immunosorbent assay measuring IFN-γ release in 4 tubes: a negative control (nil), a positive control (mitogen), a tube containing ESAT-6 and CFP-10 antigens (TB1 antigen) that measures mainly CD4 T-cell response, and a tube containing shorter ESAT-6 and CFP-10 antigens (TB2 antigen) that measures both CD4 and CD8 T-cell response.[15] Due to sharing ESAT-6 with M tuberculosis, there is cross-reactivity in patients infected with certain non-tuberculous mycobacteria, including M kansasii, M marinum, M szulgai, and M riyadhense.[16] Results are either positive, negative, or indeterminate.[17]

The T-Spot test is an enzyme-linked immunospot assay in which the patient's mononuclear cells are incubated in microwells on assay plates containing positive control, negative control, ESAT-6, or CFP-10 peptides.[18] Positive microwells, representing T-cells capable of releasing cytokine, are counted after incubation.[18] Positive ESAT-6 and CFP-10 microwells are subtracted from positive or negative control wells. Results are either positive, negative, borderline, or invalid.[19]

Since the immune reaction is performed ex vivo in the laboratory, only one visit is needed for a single blood draw, and the results are available in 24 to 48 hours. Also, cross-reaction with antigens of M bovis bacillus Calmette-Guérin (BCG) vaccination is not a consideration with these tests.[20][15] IGRA methods are preferred for individuals who may not return for a TST reading and patients younger than 5.[13]

In addition to the QFT-Plus and the T-Spot approved by the US Food and Drug Administration, 5 newer IGRAs are available: T-Spot.TB/T-Cell select, QFT-Plus CLIA, Standard E TB-Feron, QIAreach, and the TB-IGRA.[21] The TB-IGRA was found to have very similar sensitivity and specificity to approved IGRAs.[21] It has additionally been approved for use by the WHO.

Indications

Screening should be performed on those at high risk for infection. Those at high risk include:

• Recent exposure to persons with active tuberculosis, including all household contacts.[22]
• Residents and employees of congregate facilities with high risk (eg, homeless shelters, correctional facilities, refugee centers, nursing homes).[22]
• Immigrants from tuberculosis-endemic regions (more than 20 cases per 100,000 persons).[13]
• Persons working with M tuberculosis in mycobacterial laboratories.[13]
• Healthcare workers that work with a high-risk population.[22]

Additionally, screening should be performed on individuals with conditions or other factors associated with progression to active disease (reactivation):

• High risk: certain immunocompromising conditions, such as patients with HIV/AIDS, preparing for or have undergone organ transplantation, preparing to receive or receiving tumor necrosis factor (TNF) inhibitor therapy or other biologics, with end-stage renal failure on dialysis, malignancy, or silicosis.[13][23] Additionally, those with recent tuberculosis infection in the past 2 years with radiographic evidence of prior healed tuberculosis or a history of untreated or inadequately treated tuberculosis are considered at high risk.[13][23]
• Moderate risk: chronic steroid use (greater than 15 milligrams per day of prednisone equivalent), diabetes mellitus, and those at a young age (<5 years) when infected.[13][23]
• Increased risk: cigarette smoking, drug use, alcohol use disorder, body mass index of less than 20, or solitary granuloma on chest radiograph.[13][23]

Screening should be done with the intent to treat if the result is positive.[13] Chest radiography should be obtained.[13] If there are findings that could be consistent with active pulmonary tuberculosis, such as cavitation, airspace opacities, pleural effusions, or fibrotic changes, active pulmonary tuberculosis should be ruled out with 3 serial acid-fast bacilli (AFB) sputum cultures collected 8 hours apart with one being an early morning sample.[13] If all 3 AFB sputum smears are negative, active tuberculosis has been ruled out, and testing and offering tuberculosis-preventative therapy for LTBI can be considered.[13] Ziehl-Neelsen stain is often utilized for this smear, which employs carbolfuchsin and methylene blue dyes.[24]

It is not recommended that patients with low risk for LTBI and no risk factors for disease progression be tested.[13] However, if required by the law or a credentialing body, an IGRA is recommended in those over 5 years old, and if positive, a confirmatory TST is recommended.[13] Patients should only be prescribed TB-preventative treatment if both tests are positive.[13]

Test Selection

Centers for Disease Control, The Infectious Diseases Society of America, and World Health Organization guidelines endorse using IGRA or TST to screen for tuberculosis.[13][23] In certain situations, one test is preferred over the other.

The TST is the test of choice for children younger than 5 or when there is a concern for a waning immune response, as the TST has a boosting effect.[13] The TST may be somewhat preferred for patients, such as healthcare workers, who need repeat testing annually because IGRA test results may be less reproducible.[13] Issues with the reproducibility of IGRAs stem from several factors, including specimen handling, manufacturing processes, and immunological considerations, such as prior boosting with a TST.[25]

IGRAs are recommended for patients who may not return for the TST reading (people who use drugs, those with housing insecurity), those who have previously received BCG vaccination, or individuals older than 5 who are likely to be infected with M tuberculosis and have a low or intermediate risk of disease progression.[13] The area of induration of the TST has been shown to increase proportionally to the number of BCG vaccinations.[26] However, using traditional cutoffs, the TST remains a valuable tool in diagnosing LTBI.[27]

It is unclear which test modality is preferable in individuals 5 years or older with a high risk of progression to disease. TST should be done when IGRA is unavailable, too costly, or difficult to perform.[13]

Simultaneous or sequential testing is not recommended, except in cases where the initial test is negative, indeterminate, or borderline if the clinical suspicion is high. In that case, repeat testing with TST or IGRA (depending on which test was done initially) can be done. Additionally, the TST or IGRA should not be repeated if they were previously positive or to follow therapy, as this positive result is expected to be retained, and differences in results are challenging to interpret.[13]

TST and IGRAs are used to diagnose LTBI and should not be used to exclude the possibility of active tuberculosis.[13] Both tests can be falsely negative in the setting of active disease.[28] Other testing modalities, such as chest imaging and sputum collection, should be employed if active tuberculosis is suspected.[13]

Normal and Critical Findings

Interpreting the TST

Interpretation criteria for a positive result of the TST can be found in the following table.[29]

 If a TST is positive in a situation of high to above baseline risk:

• The test should not be repeated.[13]
• A chest radiograph and clinical evaluation for the potential of active tuberculosis should be obtained.[13]
• If no evidence of active disease is noted by history, physical examination, or radiography, the patient should be deemed to have LTBI and given tuberculosis-preventative treatment.[13]
• If the chest radiograph or clinical evaluation suggests active tuberculosis, then active infection must be excluded with further testing.[13]

Evaluating the potential of active tuberculosis is imperative, as therapy for LTBI consists of 1 to 2 active agents, which is insufficient for treating active tuberculosis and can promote resistance.[13]

If the TST is negative, experts' opinion outlines that repeat testing with another TST or an IGRA is reasonable if the patient is at high risk of infection progression or recent infection for further evaluation for the possibility of a false negative test.[13] However, not all of these cases would be amenable to repeat testing, and decisions to retest should be made on a case-by-case basis. Attention should be drawn to situations in which repeat testing may be considered due to the increased risk of a false negative test:

• TST can be negative in cases where testing occurs within the first 8 weeks of exposure when the immune response has not developed yet or in patients with remote tuberculosis exposure where the tuberculin reaction might have waned.[13][30][31] At times, these 2 situations may be difficult to distinguish.

  • In the former group, a second TST test placed after the first 8 weeks post-exposure will often be positive.[31]

• In the latter group, consisting primarily of older adults, a second TST ("booster") applied a couple of weeks later often will be positive.[31]

• A repeat positive second test increases the sensitivity for detecting LTBI in immunocompromised patients.[13] However, a negative test in this context can result from a lack of ability to mount an immune response rather than the absence of LTBI.[13] The likelihood of the diagnosis, considering potential exposure history, has to be used in this setting to decide whether or not to provide TB-preventative treatment, with a thorough consideration of the risks of offering and withholding TB-preventative therapy. A case-by-case evaluation is critical.

Alternatively, a false positive test should be suspected in low-risk patients for prior exposure or disease progression.[13] For this reason, these patients should not be tested unless otherwise mandated.[13]

In patients vaccinated with BCG and have a positive PPD reaction, the positive result is more likely due to tuberculosis exposure than the remote vaccination, and the immunization history should be disregarded when considering whether to offer TB-preventative therapy.[13] IGRA tests are advantageous in this situation, as these are unaffected by prior BCG vaccination.[13]

For healthcare workers and occupational exposure risk, the Centers for Disease Prevention recommend that United States healthcare workers receive LTBI screening, which should be repeated yearly and at hire.[32] As those with LTBI infection are most likely to progress to active infection within the first 2 years of disease, such screening optimizes the impact of LTBI treatment.[1]

Interpreting IGRA

Results are reported as indeterminate, positive, or negative. An indeterminate test signifies a weak response of TH1 cells to the positive control (low mitogen) or a strong response to the negative control (high nil).[33] Both values are reported so that it is known to which situation an indeterminate test can be attributed.[34] Indeterminate tests due to a strong response to the negative control occur in cases of high background immune activity with high initial levels of IFN-γ, such as in rheumatologic states, or weak response to the positive control, such as immunosuppression.[35] Indeterminate results are due to low mitogen response an overwhelming majority of the time.[35][17]

A meta-analysis involving 486,886 patients screened for LTBI noted that 3.9% had an indeterminate IGRA.[17] Immunosuppressed patients had a higher rate of indeterminate tests, and the rate of indeterminate tests increased as the CD4 count decreased in HIV-positive patients.[17] Additionally, higher rates were noted in children, and rates decreased as the age of children increased.[17] One study involving immigrants to the United States found that Asian race and the combination of anemia and hypoalbuminemia were independent risk factors for an indeterminate test.[35]

Interfering Factors

The varying causes of the TST's false positive and false negative results are outlined in the table below.[13]

Interreader variability should be highlighted as a significant cause of false-positive and false-negative results. Results have been shown to vary up to 15% between different personnel interpreting the test.[8] This figure increases when those with little prior experience read the test.[8]

In contrast, IGRAs are not affected by prior BCG immunization.[13] However, due to sharing ESAT-6 with M tuberculosis, there is cross-reactivity between previous exposure to M marinum, M kansasii, M szulgai, and M riyadhense.[16]

A meta-analysis of studies measuring the performance of the QFT-Plus performed in 2021 found that it had a sensitivity of 91.4% and a specificity of 97.8%.[36] The sensitivity of the TST is between 95% and 98%.[13]

Complications

Pain at the injection site and itching can rarely occur with the TST.[37] In healthcare workers, this was noted in 1.3% of tests.[37] As the TST requires that the patient return to have the test read by experienced medical personnel, loss to follow-up is a concern, as patients will not receive any benefits from the testing in this circumstance.[38] A study with a patient population consisting of those coping with housing instability and refugees noted that 79% of patients with an attempted TST returned to the clinic have it interpreted.[38]

Patient Safety and Education

Patients often have the misconception that a positive TST or IGRA indicates that they have active tuberculosis if thorough education is not provided. Patients should be educated regarding the rationale behind the testing ordered and counseled about what a positive result would mean in their specific context before the test is ordered. Patients should be thoroughly educated on the difference between LTBI and active tuberculosis, which can relieve significant emotional stress. If results are positive, patients should be educated about the importance of tuberculosis-preventative therapy in their specific context.

Clinical Significance

LTBI is prevalent, and screening and treating patients at high risk of infection or progression to active disease reduces tuberculosis's global burden. The TST and IGRAs are available to evaluate for LTBI, although some populations have minor differences in their performance. A decision to test should presuppose the intention to treat LTBI if detected.