MICROBIOLOGY NOTES

 

   

TUBERCULIN TEST

Historical introduction:
Robert Koch made the observation that when killed culture of Mycobacterium tuberculosis is injected sub-cutaneously to a guinea pig infected some weeks earlier with tubercle bacilli, a local inflammatory lesion develops followed by necrosis and ulceration. This is called Koch's phenomenon. Koch demonstrated that the same phenomenon could be demonstrated with a specific extract of tubercle bacilli, which he called tuberculin. Both old and new tuberculin were developed by him.

Tuberculin test is a delayed hypersensitivity (Type IV) to intradermal injection of purified protein derivative (PPD) of Tuberculin, to test for cell mediated immunity against Mycobacteria. The tuberculin test is based on the fact that infection with M. tuberculosis produces sensitivity to certain antigenic components of the organism that are contained in culture extracts called "tuberculins." Hypersensitivity to tuberculin, which is demonstrated by the development of a positive reaction to the tuberculin skin test, develops 2 to 10 wk after the initial infection.

Tuberculin antigen:
There are two preparations of tuberculin: Old Tuberculin (OT) and Purified Protein Derivative (PPD). OT is available only in multiple puncture devices whereas PPD is available for intradermal injection by the Mantoux technique and by multiple puncture devices. Old tuberculin is prepared by growing the bacilli in 5% glycerol broth for 6-8 weeks, concentrated by drying to 1/10 of its volume and then sterilized by heat. PPD is a precipitate obtained from filtrates of OT. The standard 5 Tuberculin Unit (TU) dose of PPD-S is defined as the delayed skin test activity contained in a 0.1 g/0.l ml dose of PPD-S. Products labeled 1 TU and 250 TU contain one-fifth and 50 times the concentration of antigen determined to be bioequivalent to 5 TU PPD-S. To prevent adsorption of tuberculoprotein to the glass surface, a small amount of detergent Tween 80 is added.

Immunologic Basis for the Tuberculin Reaction:
Sensitization during prior exposure to Mycobacterium tuberculosis results in production of memory Th1 cells to Mycobacterial proteins. When purified tuberculin is injected into the skin, memory Th1 cells secrete cytokines to attract macrophages and granulocytes and cause induration and erythema (firm red swelling). Delayed hypersensitivity reactions to tuberculin begin at 5 to 6 h and reach peak at 48 to 72 hours. Initial phase involving uptake, processing, and presentation by Langerhans-type dendritic cell in skin to T cells locally and in paracortical zones of nearby lymph nodes. T lymphocyte secretes cytokines, especially IFNg; macrophages are recruited and activated resulting in induration and erythema. The infiltrate of the tuberculin reaction is composed of monocytes (80-90%), CD4 T cells, CD8 T cells, and Langerhan cells. The lesion usually resolves within 5-7 days but if there is persistence of antigen, it may develop into a granulomatous reaction.

Types of tuberculin tests:
Mantoux test: 
5 TU, which equals 0.0001 mg of PPD, in a 0.1 ml volume is intradermally injected in the volar or the dorsal surface of the forearm. The use of a skin area free of any lesions and away from veins is recommended. The injection is made using a 27-gauge needle and a tuberculin syringe. The tuberculin should be injected just beneath the surface of the skin, with the needle bevel upward. A discrete wheal 6 to 10 mm in diameter should be produced when the injection is done correctly. The test is read within 48-72 hours. The diameter of induration should be measured transversely to the long axis of the forearm and recorded in millimeters. When hypersensitivity to tuberculin components are suspected in a person, 1TU is injected and upto 25 TU is used if the reaction is negative.

The Multiple-Puncture Test:
This test introduces tuberculin into the skin either by puncture with an applicator with points coated with dried tuberculin (Heaf's test) or by puncturing through a film of liquid tuberculin (Jelly test). Several types of applicators are available for multiple-puncture tests. All multiple-puncture tests use concentrated tuberculin, either OT or PPD. The quantity of tuberculin introduced into the skin using the multiple-puncture technique cannot be precisely controlled; hence multiple-puncture tests are not used as diagnostic tests. Multiple-puncture test reactions are measured at 48 to 72 hours as follows. (1) If the reaction is in the form of discrete papules, the diameter of the largest single papule should be measured. (2) If there is a coalescence of papular reactions, the largest diameter of coalescent induration should be measured. (3) If the reaction is vesicular, this is noted on the record.

Precautions: Tuberculin should be stored in the dark as much as possible and exposure to strong light should be avoided. It should never be transferred from one container to another and skin tests should be given soon after the syringe has been filled. The reading should be taken in bright light and area of erythema alone should not be measured.

Interpretation of Tuberculin test:

  • It is impossible to distinguish between present and past infection on the basis of a positive tuberculin test. A positive test does not indicate active disease, merely exposure to the organism.

  • An induration diameter of equal or greater than 10 mm suggests present or past infection. For all persons younger than 35 yr of age whose previous reaction was negative, a reaction size of 10 mm or more in diameter within a period of 2 yr would be considered a skin test conversion. For those older than 35 yr of age, an increase of 15 mm or more is considered a positive conversion, and they should be considered newly infected with M. tuberculosis.

  • In the absence of any other disease or disorders, a measurement of 5 - 9 mm is suggestive of atypical Mycobacteria sensitization. 
    False negatives are encountered in persons with HIV infection, particularly if the CD4+ cell count is < 200/L or in other conditions such as measles, mumps, chicken pox, typhoid fever, bruceIlosis, typhus, leprosy, pertussis, overwhelming tuberculosis, chronic renal failure, nutritional deficiency, Hodgkin's disease, lymphoma, chronic lymphocytic leukemia, sarcoidosis, treatment with corticosteroids or immunosuppressive agents. Such a condition is termed as "anergy".

  • False negative can also occur due to improper storage, improper dilutions, chemical denaturation, contamination, injection of too little antigen, error in recording etc.

  • False positives can occur in prior exposure or infection with other Mycobacteria or vaccination with BCG.
    A diameter of less than 10 mm may be significant in persons with HIV-infection, insulin-dependent diabetes mellitus, prolonged corticosteroid therapy, had undergone gastrectomy or end-stage renal disease.

  • All persons likely to be exposed to TB (eg, living or working in a nursing home or hospital or prison) should be tested initially with the 2-step Mantoux test. Persons who have no reaction to the first test will develop a significant reaction when the test is repeated 1 to 3 wk later. This is called a booster-positive reaction and has about the same significance as a test that is positive the first time.

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  Last edited on June 2006