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A New Approach for the Diagnosis of Tuberculosis


January 2010

Background

Tuberculosis (abbreviated as TB for tubercle bacillus or Tuberculosis) is a disease caused by the bacteria Mycobacterium tuberculosis. (Figure 1) These slow-growing bacteria are spread from an infected person to others by aerosol droplets produced by coughing and sneezing. If inhaled, the bacteria lodge in the lungs and cause infection. Globally, nearly one-third of the population has been infected with TB and new infections occur at a rate of 1 per second.1 However, not everyone infected with TB bacteria becomes sick with the disease. One of 3 scenarios is possible: 1) The immune system can clear the infection with no lasting effects; 2) The infection cannot be cleared by the immune system and an active infection ensues; or 3) The immune system controls the infection, but a latent infection results. (Table 1) Those who have latent tuberculosis infection (LTBI) do not have symptoms and cannot spread the disease to others. While the majority of people with LTBI never progress to active TB disease, some do, usually as the result of a weakened immune system as can be seen in the following situations: coinfection with human immunodeficiency virus (HIV) infection, chemotherapy treatment, and antirejection treatment for organ transplant. Although exact numbers are not known, an estimated 5% to 10% of people infected with the TB bacilli (without concurrent HIV infection) will become ill or infectious at some time during their life.1


Figure1

Figure 1. Scanning electron micrograph (SEM) depicts some of the ultrastructural details seen in the cell wall configuration of Mycobacterium tuberculosis bacteria.


  Latent TB Infection Active TB Disease
Symptoms None • Bad cough that lasts 3 weeks or longer
• Chest pain
• Coughing up blood or sputum
• Weakness or fatigue
• Weight loss
• Loss of appetite
• Chills
• Fever
• Night sweats
Infectious? No Yes
Positive skin test or interferon gamma release assay? Usually Usually
Chest x-ray Usually normal Abnormal
Sputum smear Usually negative Usually positive
Treatment Required to prevent active disease Required

Table 1. The difference between latent TB infection and active TB disease2


Diagnostic Testing

Detection and subsequent treatment of TB infection before it becomes contagious disease are essential in controlling TB and are the focus of public health prevention programs in developing countries. Prevention programs aim at stopping the rise in active TB cases, thus preventing deaths associated with TB and reducing the public health costs required to control the disease. At the center of TB control programs is reliable detection of Mycobacterium tuberculosis infection.

Tuberculin Skin Test

Until recently, the standard method for determining Mycobacterium tuberculosis infection has been the Mantoux tuberculin skin test (TST), used for over 100 years. The TST is performed by injecting a small amount of tuberculin purified protein derivative (PPD) into the forearm and examining the injection site at 48 to 72 hours after administration. (Figure 2) This skin test assesses an in vivo delayed-type hypersensitivity immune response to a polyvalent antigenic mixture in PPD. However, the TST is a subjective test that lacks sensitivity and specificity, and may result in false-positive and/or false-negative results. (Table 2) For example, false-positive results may appear in individuals who have received bacilli Calmette-Guérin (BCG) vaccination, as well as those exposed to mycobacteria other than Mycobacterium tuberculosis. The TST also has poor reproducibility and requires 2 office visits—one for the injection and a second, 2 to 3 days later, to read the skin test reaction. While reading a TST is often assumed to be an easy task, accurate measurement of the reaction requires highly trained medical personnel. Furthermore, even when performed by well-trained individuals, TST interpretation is still somewhat subjective and can be inaccurate. In addition, many patients do not return for the second visit, presenting patient follow-up concerns.


Figure 2

Figure 2. Injection of tuberculin material: correct placement of the intradermal injection is critical for accurate Mantoux tuberculin skin test results.


False-Positive Reactionsa False-Negative Reactionsb
Infection with nontuberculosis mycobacteria Weakened immune system and impaired cellular reactivity
Previous BCG vaccination Recent TB infection (within 8 to 10 weeks of exposure)
• Technical factors
• Incorrect administration of TST
• Incorrect interpretation of TST reaction
• Improper storage of PPD
Very old TB infection (several years)
  Very young age (<6 months)
  Recent live-virus vaccination (measles or smallpox)
  Overwhelming TB disease
  Some viral illnesses

Table 2. False-positive and false-negative reactions to tuberculin skin test3
a Some individuals may react to the TST, but are not infected with Mycobacterium tuberculosis
b Some individuals may not react to the TST even though they are infected with Mycobacterium tuberculosis


Interferon Gamma Release Assays

Interferon gamma release assays (IGRA) are a new class of assays that use a different test methodology to detect infections. IGRA assess a cell-mediated response in infected persons by determining the amount of interferon gamma (IFN-γ) released when sensitized lymphocytes are stimulated by recognizable antigen. During an infection, T cells are sensitized (via the major histocompatibility complex [MHC] pathway) to the antigens presented by the cells of the infecting organism. Once sensitized, T cells are able to bind to foreign infecting cells and, in the process, release the cytokine IFN-γ. (Figure 3) The presence of sensitized T cells in infected individuals will result in far higher levels of IFN-γ release than among uninfected individuals. Those infected with Mycobacterium tuberculosis complex organisms (ie, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti and Mycobacterium canetti) have T-cell lymphocytes in their blood that recognize specific mycobacterial antigens. The subsequently released IFN-γ is measured by enzyme-linked immunosorbent assay (ELISA), providing an indirect indication of Mycobacterium tuberculosis complex infection.


Figure 3

Figure 3. The stimulation of antigen-presenting T cells and subsequent quantification of the secreted IFN-γ is the basis of the QuantiFERON technology. Used with permission from Cellestis Inc, Valencia, CA


QuantiFERON Tests

QuantiFERON-TB was the original IGRA (approved for use by the US Food and Drug Administration [FDA] in 2001) developed for TB diagnosis. That novel in vitro test measured the release of IFN-γ when heparinized whole blood samples were mixed and incubated with PPD from Mycobacterium tuberculosis. After blood and antigens were incubated for 16 to 24 hours, the amount of secreted IFN-γ was measured by ELISA. In 2005, QuantiFERON-TB Gold (QFT-G) replaced the original QuantiFERON-TB assay. The QFT-G assay utilizes synthetic peptides that simulate proteins present in Mycobacterium tuberculosis–early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10)–to measure IFN-γ release. In 2007, the FDA approved the next generation of the assay–QuantiFERON-TB Gold In-Tube (QFT-GIT). With the addition of a third peptide, which simulates the protein TB7.7(p4), sensitivity is theoretically increased. The peptide cocktail simulating ESAT-6, CFP-10, and TB7.7(p4) proteins is coated on the inside of specialized blood collection tubes. Collection and processing of blood in this manner eliminates the previously required manual step of adding the peptides to the specimen to stimulate the lymphocytes. The advent of this assay allows for specimen collection, processing, and shipment of the specimens in the primary collection tubes and enables remote collection of specimens. QFT-GIT assay is offered by Mayo Medical Laboratories as #83896 Mycobacterium tuberculosis Infection Determination by QuantiFERON-TB Gold.

Advantages and Disadvantages of the use of the QuantiFERON assays

Because it is laboratory-based, the QuantiFERON test (QFT) is not subject to many of the problems associated with the TST, such as improper placement of the PPD and reading errors. However, QFT accuracy may be decreased by errors in sample collection, preparation, and transportation or in assay performance and results interpretation. The assay has very specific preanalytic requirements that must be followed explicitly. (Table 3)


Advantages of QFT-GIT Disadvantages of QFT-GIT
Results can be available within 24 hours, compared to 48 to 72 hours for the TST Blood samples must be processed within 16 hours after collection while white blood cells are still viable
Not subject to reader bias that can occur with TST Errors in collecting or transporting blood specimens, assay performance, and interpretation can decrease accuracy
Requires a single patient visit to draw a blood sample (TST requires 2 visits) Data is limited on the use of QFT in children <17 years of age, among persons recently exposed to Mycobacterium tuberculosis, and in immunocompromised persons (eg, impaired immune function caused by HIV infection or acquired immunodeficiency syndrome (AIDS), current treatment with immunosuppressive drugs, selected hematological disorders, specific malignancies, diabetes, silicosis, and chronic renal failure)
Not believed to boost responses measured by subsequent tests, which can happen with TST, causing incorrect interpretations Data is limited on the use of QFT to determine who is at risk for developing TB disease
Not affected by prior BCG vaccination  
Does not react to most of the nontuberculous mycobacteria, which are common causes of false-positive TST results  

Table 3. Advantages and disadvantages of the QuantiFERON-TB Gold In-Tube (QFT-GIT) test4


Intended Use

QFT is intended for use as a diagnostic tool to detect Mycobacterium tuberculosis complex infection, either in active TB disease or LBTI. In 2005, the US Centers for Disease Control and Prevention (CDC) published guidelines for prevention of TB transmission, which indicate that the QFT can be used in any instance in which the TST is used, but the guidelines impart caution when testing certain populations because of limited data.5 The QFT may be especially useful in the detection of LTBI in the following situations: preemployment and sequential testing of health care workers, investigations of persons who may have had contact with individuals with active TB disease, and screening of military personnel, students, and recent immigrants. A positive result warrants further medical and diagnostic investigation to confirm or exclude active TB disease. The diagnostic evaluation should include a complete medical examination including historical risk assessment, physical examination, radiography and, when indicated, microbiological and molecular examination of specimens for Mycobacterium tuberculosis complex.

Sensitivity and Specificity

Because no gold standard exists for the diagnosis of LTBI, assessing the sensitivity and specificity of the QFT has limitations. A recent review of 38 studies that assessed the performance of various IGRAs and the TST provides guidance in the utility of the test.6 Sensitivity was assessed in microbiologically confirmed TB cases and specificity was assessed in healthy, low-risk individuals without known exposure to TB. The reviewers found that the QFTs have excellent specificity that is unaffected by BCG vaccination. The pooled specificity of all studies was found to be 99% in non-BCG-vaccinated individuals and 96% in those who had been vaccinated with BCG, resulting in an overall specificity of 98%. TST specificity is high (97%) in non-BCG-vaccinated populations but low (average, 59%) and highly variable (46% to 73%) in BCG-vaccinated populations. The sensitivity of the QFT and the TST was not consistent across the various studies. Reasons for inconsistency could include the variety and severity of tuberculosis cases that were included, the varying rates of tuberculosis and HIV across countries, or the inherent differences among the various test formats. This review reveals that the pooled sensitivity of the TST (77%) and the QFT (76%) are quite similar.

Specimen Required

The accuracy of the QFT is dependent on the proper collection and incubation of the blood specimen. For each patient tested, 1 mL of blood is collected directly into each of 3 QFT blood collection tubes. Blood should fill the tube as close to the 1-mL mark as possible. Underfilling or overfilling the tubes outside the 0.8 to 1.2 mL range may lead to erroneous results. The tubes must be shaken vigorously for 5 seconds following collection. The vigorous mixing process ensures even distribution of stimulating antigens, allowing optimal processing and presentation of the antigens to T cells, which causes release of IFN-γ. Insufficient mixing may lead to inaccurate results. IFN-γ responses are also diminished or absent if the specimens are kept at room temperature for longer than 16 hours or stored outside of the recommended temperature range (17°C to 27°C) prior to incubation at 37°C. Optimally, the specimens should be incubated as soon as possible after collection. Incorrect incubation conditions may also lead to inaccurate results. The test requires that the blood be incubated with antigen for 16 to 24 hours at 37°C ± 1°C. Incubation for shorter periods, or outside the prescribed temperature, can result in diminished IFN-γ responses.

To ensure accurate results, instructions must be carefully and completely followed. A collection kit complete with collection tubes and instructions for collection and processing is available from Mayo Medical Laboratories. Complete instructions for collection, processing, and transportation are also included as an insert to this publication.

Results and Interpretation

The QFT is a qualitative, indirect assessment of infection and thus the results are reported as positive, negative, or indeterminate. Interpretation of results is based on IFN-γ concentrations present in test samples. (Table 4)


QFT Result Interpretation
Positive Mycobacterium tuberculosis infection likely
Negative Mycobacterium tuberculosis infection unlikely, but cannot be excluded. If TB disease is highly suspected, a negative result does not rule-out infection. False-negative results may be seen in immunocompromised patients.
Indeterminate Test not interpretable. Collection of a new specimen for testing is recommended, if clinically indicated.

Table 4. Interpretation of QFT results7


Cost Effectiveness of QFT

While the use of the QFT has definite advantages, the overall cost of performing this assay, compared to the TST, is somewhat difficult to assess. Many factors must be considered. QFTs are relatively costly, and testing must be performed in a clinical laboratory setting due to the requirement for specialized laboratory equipment and trained testing personnel. The PPD used in the TST is inexpensive, but costs of the follow-up visit for reading the TST reaction vary and are difficult to quantify. Furthermore, the TST lacks specificity, which may lead to unnecessary follow-up with costly associated expenses. The improved specificity of the QFT may lead to fewer patient follow-up visits with reduced laboratory and clinical costs, thereby making this testing strategy more cost-effective in the long term. A recently published study provides some insight into the cost-effectiveness of the QFT in the detection of LTBI in health care workers in the Veterans Health Administration (VHA) system.8 Direct and indirect costs and probabilities, based on manufacturer data, VHA records, and published literature were calculated to determine the cost of each strategy. The authors concluded that, in this setting, the QFT is a clinically and economically worthwhile alternative to the TST. Long-term studies are needed, however, to determine if these conclusions will generally apply.


Cautions and Limitations of QuantiFERON-TB Gold In-Tube (QFT-IT) Assay5

A negative QuantiFERON-TB Gold In-Tube result does not eliminate the possibility of Mycobacterium tuberculosis infection or TB disease. False-negative results can be due to stage of infection (ie, specimen obtained prior to the development of cellular immune response), comorbid conditions that affect immune function, or other individual immunological factors.

Heterophile (eg, human antimouse) antibodies in serum or plasma of certain individuals are known to cause interference with immunoassays. These antibodies or nonspecific IFN-γ production from other inflammatory conditions may interfere with specific responses to ESAT-6, CFP-10, or TB7.7(p4) peptides leading to indeterminate and unreliable results.

False-negative or indeterminate results can be caused by incorrect blood sample collection or improper handing of the specimen which may limit exposure of lymphocytes to the presenting antigen.

The effect of lymphocyte count on reliability of this assay results is unknown. Lymphocyte counts may vary over time for an individual and from person to person. The minimum number of lymphocytes required for a reliable test result has not been established and may also be variable.

A positive QFT-IT result can suggest and support the diagnosis of tuberculosis disease. ESAT-6, CFP-10, and TB7.7(p4) are present in Mycobacterium tuberculosis, but patients with infections by other mycobacteria (Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum) might also be responsive to these antigens as the genes encoding these proteins are also present in these mycobacteria. Therefore, a positive result should not be the sole or definitive basis for determining infection with Mycobacterium tuberculosis. Further medical investigation for active TB disease is needed (eg, acid-fast bacilli smear and culture, chest x-ray) for confirmation.

The performance of the test has not been extensively evaluated in certain populations. Increased risk for progression of Mycobacterium tuberculosis infection to active disease can be observed in the following conditions and circumstances:

1. Individuals who have HIV infection or AIDS
2. Those who have transplantation managed with immunosuppressive treatment or others who receive immunosuppressive drugs (eg, high-dose corticosteroids, tumor necrosis factor-alpha [TNF-a] antagonists, methotrexate, azathioprine, cancer chemotherapy)
3. Individuals with certain clinical conditions including diabetes, silicosis, chronic renal failure, hematological disorders (eg, myeloproliferative disorders, leukemia, and lymphomas), and other specific malignancies (eg, carcinoma of the head, neck, or lung)
4. Children aged <17 years
5. Pregnant women

Conclusion

The landscape of tuberculosis testing is changing. Recently introduced in vitro technology advances the field of TB diagnostics and the QFT may replace the TST in certain populations. Apart from the operational advantages over the TST, the QFT-G and QFT-GIT have excellent specificity in previously BCG-vaccinated individuals and do not react with most nontuberculous mycobacteria. QFT use increases the role of the clinical laboratory in TB control. While optimal quality control is difficult when using the TST, the QFT performed in a clinical laboratory will be tightly controlled and therefore may lead to a higher-quality assay and greater confidence in the results. While more studies are needed to assess the use of the QFT in all test populations, these assays hold promise for improved TB testing and control.

Authored by: Elizabeth Plumhoff, Dr. Matthew Binnicker, Dr. Jane Dale. Images provided by the CDC. Image 1: JH Carr, Image 2: G. Knoblach

References

  1. World Health Organization. Tuberculosis: Fact Sheet. Fact sheet N°104, revised March 2007. Available from: http://www.who.int/mediacentre/factsheets/fs104/en/index.html
  2. Centers for Disease Control and Prevention. Division of Tuberculosis Elimination. Questions and Answers about TB, 2009. Available from: http://www.cdc.gov/tb/faqs/qa_introduction.htm#Intro5
  3. Centers for Disease Control and Prevention. Division of Tuberculosis Elimination. TB Elimination: Tuberculin Skin Testing, May 2007. Available from: http://www.cdc.gov/tb/
  4. Centers for Disease Control and Prevention. Division of Tuberculosis Elimination. QuantiFERON®-TB Gold Test Fact Sheet. Reviewed 5/08/2008. Available from: http://www.cdc.gov/tb/pubs/tbfactsheets/QFT.htm
  5. Mazurek GH, Jereb J, Lobue P, et al: Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection. MMWR Morb Mortal Wkly Rep. 2005;54(RR-15):49-55
  6. Pai M, Zwerling A, Menzies D. Systemic Review: T-cell–based Assays for the Diagnosis of Latent Tuberculosis Infection: An Update. Ann Intern Med. 2008;149:177-184
  7. Clinical Experience with QuantiFERON®-TB Gold (In-Tube). Cellestis Inc, Valencia, CA. Available from: http://www.cellestis.com
  8. de Perio MA, Tsevat J, Roselle GA, et al: Cost-effectiveness of Interferon Gamma Release Assays vs Tuberculin Skin Tests in Health Care Workers. Arch Intern Ned. 2009;169(2):179-187

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