Laboratory Diagnosis of Latent Tuberculosis Infection in Adults and Children

Tuberculosis (TB) remains a serious health concern, and approximately one-third of the global population has been infected with its causal pathogen Mycobacterium tuberculosis (M.tb). ¹ M.tb is transmitted from person to person via the airborne route. Several factors determine the probability of M.tb transmission:

• Infectiousness of the source patient—a positive sputum smear for acid-fast bacilli (AFB) or a cavity on chest radiograph being strongly associated with infectiousness
• Host susceptibility of the contact
• Duration of exposure of the contact to the source patient
• The environment in which the exposure takes place (a small, poorly ventilated space provides the highest risk)
• Infectiousness of the M.tb strain

Not everyone exposed to TB bacteria becomes sick. As a result, two TB-related conditions exist, Latent TB infection and TB disease.

Latent TB Infection (LTBI)

Many people, who are exposed to TB bacteria, will not develop TB disease. In these subjects, TB bacteria remain inactive for a lifetime without causing disease. They cannot spread TB bacteria to others and usually have a positive TB skin test reaction or positive TB blood test. The Centers for Disease Control and Prevention (CDC) estimates that up to 13 million people in the United States have LTBI.² 93% of TB disease in foreign-born persons in the US are attributable to reactivation of LTBI. While not everyone with LTBI will develop TB disease, about 5-10% of infected people will develop TB disease if not treated. This equates to approximately 650,000 to 1,300,000 people who will develop TB disease at some point in their life unless they receive adequate treatment for LTBI.³

TB Disease

TB bacteria overcome the defenses of the immune system and begin to multiply, resulting in the progression from LTBI to TB disease. Persons with TB disease are considered infectious and may spread TB bacteria to others. For people whose immune systems are weak (e.g., people with HIV infection), the risk of developing TB disease is much higher than for people with a healthy immune system.

Testing for LTBI

Identifying and treating those at highest risk for TB disease will help move toward the elimination of the disease. Primary care providers play a crucial role in achieving the goal of TB elimination because of their access to high-risk populations.The goal for LTBI testing is to identify those who will benefit from prophylactic therapy. The completion rates of the therapy are low; in one study 83% of those diagnosed with LTBI started treatment, and only 39% completed the treatment.⁵ Better testing strategies and treatment regimens will allow focusing on patients who are in need of evaluation and treatment of LTBI and result in increased therapy completion rates.Updated joint Official Clinical Practice Guidelines from the American Thoracic Society (ATS)/ Infectious Diseases Society of America (IDSA) / Centers for Disease Control and Prevention (CDC) ⁴ recommend performing an Interferon-gamma release assay (IGRA) rather than a tuberculin skin test (TST) for most groups.Individuals 5 years or older who meet the following criteria should be tested by IGRA rather than TST:

1. 1. 1. Likely to be infected with M.tba. Household contact or recent exposure of an active caseb. Mycobacteriology laboratory personnelc. Immigrants from high burden countriesd. Residents and employees of high-risk congregate settings
      2. Have a low or intermediate risk of disease progression (conditional recommendation)a. Low: no risk factors*b. Intermediate: Patients with diabetes, chronic renal failure, intravenous drug use
      3. It has been decided that testing for LTBI is warranted, that includes:a. Close contacts of persons known or suspected to have active TB, recent exposure to TB outbreaksb. Laboratory staff involved with TB specimen collection, transport, and processingc. Residents of correction facilities, long term care facilities, homeless shelters, etc.
      4. Either have a history of Bacillus Calmette–Guérin (BCG) vaccination or unlikely to return to have their TST read

TST can be an acceptable alternative in situations where IGRA is not available, too costly, or too burdensome.* Persons at low risk for M.tb infection and disease progression should not be tested for M.tb infection. However, such testing may be required by law or credentialing bodies.If diagnostic testing for LTBI is performed in individuals who are unlikely to be infected:

• Guidelines committee suggests performing an IGRA instead of a TST in individuals 5 years or older
• A second diagnostic test should be performed if the initial test is positive. The confirmatory test may be either an IGRA or a TST. When such testing is performed, the person is considered infected only if both tests are positive.

Individuals 5 years age or less should be tested by a TST rather than IGRA when diagnostic testing for LTBI is warranted. This recommendation is based on limited direct evidence showing TST might be more sensitive than IGRA in young children and IGRA are may be more specific than TST, especially children given BCG vaccination. Because young children have a high risk for progression of active TB disease, sensitivity of the test is more important than specificity and supported by observations that potential consequences of delayed treatment are high. In situations where more specific testing for a diagnosis is needed, IGRA is preferred and some experts are willing to use IGRAs in children over 3 years of age.While both IGRA and TST testing provide evidence for infection with M.tb, they cannot distinguish active from latent TB. Therefore, the diagnosis of active TB must be excluded prior to embarking on treatment for LTBI. This is typically done by determining whether or not symptoms suggestive of TB disease are present, performing a chest radiograph and, if radiographic signs of active TB (e.g., airspace opacities, pleural effusions, cavities, or changes on serial radiographs) are seen, then sampling is performed and the patient managed accordingly.

DIAGNOSTIC TESTS FOR LTBI

Tuberculin Skin Testing (TST)The TST detects cell-mediated immunity to M.tb through a delayed-type hypersensitivity reaction using a protein precipitate of heat-inactivated tubercle bacilli. Until now, the TST has been the standard method of diagnosing LTBI.The TST is administered by the intradermal injection of 0.1 mL of purified protein derivative (PPD) (5 TU) into the volar surface of the forearm (Mantoux method) to produce a transient wheal. The test is interpreted at 48–72 hours by measuring the transverse diameter of the palpable induration. TST interpretation is risk-stratified. A reaction of 5 mm or greater is considered positive for close contacts of tuberculosis cases. A reaction of ≥10 mm is considered positive for other persons at increased risk of LTBI (e.g., persons born in high TB incidence countries and those with at risk of occupational exposure to TB) and for persons with medical risk factors that increase the probability of progression from LTBI to TB. A reaction of 15 mm or greater is considered positive for all other persons.

• Limitations of the TST - The need for trained personnel to both administer the intradermal injection and interpret the test, inter- and intra-reader variability in interpretation, the need for a return visit to have the test read, false-positive results due to the cross-reactivity of the antigens within the PPD to both BCG and nontuberculous mycobacteria, false-negative results due to infections and other factors, rare adverse effects, and complicated interpretation due to boosting, conversions, and reversions are major limitations for TST.

Interferon-Gamma Release Assay (IGRA)The IGRAs are newer tests to diagnose infection with M.tb. IGRAs are in vitro, T-cell–based assays that measure interferon gamma (IFN-γ) release by sensitized T-cells in response to highly specific M.tb antigens. These are more specific for M.tb infection than the TST, particularly in the setting of BCG vaccination.IGRA is a reflection of the cellular immune response and are primarily a reflection of a CD4+ T-cell immune response to TB antigens. This response is characterized by the release of cytokines, as well as further expansion of these cells.Currently, there are two commercially available IGRA platforms that measure IFN-γ release in response to M.tb-specific antigens:

• QuantiFERON TB Gold In Tube (QFT-GIT; Cellestis Limited, Carnegie, Victoria, Australia)
• T-SPOT.TB test (T-SPOT, manufactured by Oxford Immunotec Ltd, Abingdon, UK)

The QFT-GIT measures IFN-γ plasma concentration using an enzyme-linked immunosorbent assay (ELISA), while the T-SPOT assay enumerates T-cells releasing IFN-γ using an enzyme-linked immunospot (ELISPOT) assay.

QuantiFERON AssaysWhole blood is drawn directly into heparinized tubes coated with lyophilized antigen and agitated. In this case, peptides from ESAT-6, CFP-10, and TB7.7 are found within the same tube. Two additional tubes are drawn as controls (mitogen control and nil control). The mitogen control (phytohemagglutinin ) stimulates T-cell proliferation and ensures that viable cells are present. After incubation for 16–24 hours at 37°C, plasma is collected from each tube and the concentration of IFN-γ is determined for each by ELISA. The next generation of QFT (QFTPlus) has been introduced in Europe and is currently approved in the United States. QFTPlus contains a tube of short peptides derived from CFP-10, which are designed to elicit an enhanced CD8 T-cell response. There is no TB7.7 peptide.

T-SPOT.TB AssaysWhole blood is drawn into either a heparin or CPT Ficoll tube, and must be processed within 8 hours. More recently, this time has been extended to 32 hours if the “T-cell Xtend” additive is used and the blood kept between 10°C and 25°C. Peripheral blood mononuclear cells (PBMCs) are separated using density gradient centrifugation, enumerated, and then added to microtiter wells at 2.5 × 105 viable PBMCs per well that have been coated with monoclonal antibodies to IFN-γ (ELISPOT assay). Peptides derived from ESAT-6 and CFP-10 antigens are then added and the plate is developed following overnight (16–20 hours) incubation at 37°C. Cells are then washed away and “captured” IFN-γ is then detected via a sandwich capture technique by conjugation with secondary antibodies hence revealing a “spot.” These spots are then enumerated as “footprints” of effector T cells.

For the T-SPOT.TB assay, a positive response is based on spot-forming units (SFU). The FDA has published revised criteria for T-SPOT.TB interpretation in the United States, in which a test is considered negative if there are ≤4 spots. Eight spots or greater is considered positive. Five, Six, and Seven spots are considered “borderline” and would be interpreted in conjunction with the subject’s pretest probability of infection with M.tb.

Indeterminate/Invalid IGRA Responses

IGRAs currently have a trichotomous outcome yielding a positive, negative, or indeterminate result (T-SPOT may also yield a borderline result). An indeterminate/invalid IGRA can result from either a high background (nil) response or from a poor response to positive control mitogen. Indeterminate IGRA results are associated with immunosuppression, although they may occur in healthy individuals. With regard to those with a poor response to the positive control mitogen, there are at least two possibilities. First, the test may not have been correctly performed. For example, errors in specimen collection, long delays in specimen processing, incubator malfunction, or technical errors might result in a poor mitogen response. Here, it is reasonable to simply repeat the assay. Second, a persistently diminished response to mitogen may be a reflection of anergy. Thus, the reproducibility and details regarding the reason for an indeterminate result may provide clinically useful information.

Reproducibility of IGRAs

Because IGRAs are predicated on in vitro release of cytokines from stimulated cells, there is likely to be more variability. There are at least four sources of variability which are inherent in the IGRA: (1) the type of measurement itself (i.e., ELISA or ELISPOT), (2) reproducibility of a complex biological reaction, (3) the natural variability of immune responses, and (<4) variability introduced during the course of test performance or manufacturing variances.

Boosting of IGRAs

Initial studies found that repeat TST testing did not alter the IGRA response. However, more recent evidence suggests that the prior placement of a TST can boost an IGRA, particularly in those individuals who were already IGRA positive to begin with (i.e., previously sensitized to M.tb or possibly other mycobacteria). Additionally, it was found that this could be observed in as little as three days post-TST administration, and that the boosting effect may wane after several months. While these data do not detract from the excellent overall agreement that has been reported, they suggest when dual testing is to be considered that the IGRA be collected either concurrently or prior to TST placement.

Benefits and Limitations of IGRAs

The benefits of IGRAs include the use of antigens that are largely specific for M.tb (i.e., no cross-reactivity with BCG and minimal cross-reactivity with nontuberculous mycobacteria), the test can be performed in a single visit, and both the performance and reporting of results in a laboratory setting fall under the auspices of regulatory certification.Limitations include cost, the need for phlebotomy (which may be particularly challenging in children), complicated interpretation due to frequent conversions and reversions and lack of consensus on thresholds, and inconsistent test reproducibility.In summary, individuals infected with M.tb may develop symptoms and signs of disease (TB disease) or may have no clinical evidence of disease (LTBI). IGRA tests are preferable over TST, and they offer improved detection in the diagnosis of LTBI. Persons at low risk for M.tb infection and disease progression are NOT to be tested for M.tb infection. Also, individuals who have received BCG vaccination, use of IGRA tests reduced false positives compared to TST and unnecessary treatment and accompanying risks can be avoided.L.V. Rao, PhD, FACB is Chief Scientific Officer, Quest Diagnostics, MA, LLC and Professor, Department of Pathology, UMASS Medical School

References:1. World Health Organization. Global Tuberculosis Report 2017. (2017) Available at: http://www.who.int/tb/publications/global_report/en(Accessed: 30th June 2016).2. Latent Tuberculosis Infection: A Guide for Primary Health Care Providers. https://www.cdc.gov/tb/publications/ltbi/intro.htm3. Latent Tuberculosis Infection: A Guide for Primary Health Care Providers. https://www.cdc.gov/tb/publications/ltbi/intro.htm4. Lewinsohn DM, Leonard MK, Philip A. LoBue PA, et al. Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children, Clinical Infectious Diseases, Volume 64, Issue 2, 15 January 2017, Pages e1–e33, https://doi.org/10.1093/cid/ciw6945. Horsburgh CR Jr, Goldberg S, Bethel J, et al. Latent TB infection treatment acceptance and completion in the United States and Canada. Chest. 2010, 137(2):401-409.