Tuberculin testing: Its basis, methods, test and comparative results

DOUGLAS P. HAGEN, D.O. Kirksville, Mo.

Several procedures for administering test has converted to positive. Several methods tuberculin for skin testing of performing tuberculin skin tests will be re- viewed, and the results of the methods will be are reviewed. The is compared. considered the most accurate, in that the test dose can be measured Basis for tuberculin testing accurately, the equipment and During the first infection with the tubercle materials required are available readily, bacillus, the host acquires to the organism. Hypersensitivity can be induced and the reactions can be read by the whole tubercle bacillus alone or a and recorded quantitatively. Its chief combination of the wax fraction of the or- disadvantage is the skill required ganism containing a lipopolysaccharide and to administer the test. Multiple-puncture tuberculoprotein but not by tuberculoprotein techniques need minimal skill alone. The reaction that occurs when an anti- for administration and false negative gen to is introduced intradermally provides a valuable testing procedure for both reactions are rare. However, the epidemiologic and case finding purposes. are not well standardized, Tuberculin testing material is basically of the dose of tuberculin two types, old tuberculin (OT) and purified retained in the skin is unknown, and protein derivative (PPD). Old tuberculin is false positive reactions may a concentrated filtrate of broth in which tu- occur. Because of the variables affecting bercle bacilli have grown for 6 weeks. Cultures of tuberculosis are heat-ster- the dose, quantitative measures ilized. The dead tubercle bacilli are filtered off, or reactions are only approximate guides and the filtrate is evaporated to 1/10 its orig- to degrees of sensitivity. inal volume. This material contains other con- stituents of tubercle bacilli and the growth medium. Efforts are being directed on a national basis Purified protein derivative consists of the toward elimination of tuberculosis from the active protein principle obtained from cultures United States. Tuberculin skin testing will of M. tuberculosis that have been grown play a part in achieving this goal. on synthetic media and extracted by neutral Tuberculin testing is performed for several ammonium sulfate precipitation. In 1952, a purposes. One is to aid in establishing the diag- large batch of PPD was produced as described nosis when signs and symptoms of the disease and adopted by the World Health Organization are noted. Another is for epidemiologic sur- (WHO) as the international standard tuber- veys of population groups to determine the culin, designated PPD-S.2 incidence and prevalence of the disease. A In about 1958 a batch of a new standard third is for case finding, which may involve PPD called RT 23 was prepared in Copen- case detection during surveys or follow-up of hagen for WHO. The batch was large enough contacts of patients in whom the disease has so it was anticipated that it would suffice as a been diagnosed recently or those whose skin standard until a PPD more specific than that

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presently available could be developed. This tration and tissue induration. The reaction new PPD RT 23 was assayed against PPD-S is not associated with circulating antibodies. and other batches of PPD used in international and cannot be transferred passively by means work. Two reasons for preparing the large of serum, although it sometimes can be trans- batch of PPD were to make it unnecessary ferred passively by means of cells. to standardize new batches and to increase the When tuberculin testing is carried out, there comparability of data obtained by tuberculin is no immediate reaction to the intracutaneous testing in different areas for different periods.3 injection of tuberculin. Induration and er- The amounts of tuberculin used have been ythema develop hours after the injection of measured both in tuberculin units (TU) and the material intracutaneously. The reaction by the weight of the material. Although the continues to increase in magnitude for from WHO committee has decided to abandon the 24 to 72 hours. tuberculin unit and substitute weight of ma- terial, doses are still referred to in the United Methods States as first strength (0.00002 mg./0.1 ml., Several testing procedures are available for 1 TU) ; intermediate strength (0.0001 mg./0.1 the administration of tuberculin for skin test- ml., 5 TU or 0.0002 mg./0.1 ml., 10 TU) ; ing. They include the tuberculin , the and second strength (0.005 mg./0.1 ml., 250 Mantoux test, the , and the injection TU) . gun test. The tuberculin tine test utilizes OT, The field of and hypersensitivity is and the Mantoux, Heaf tests and injection concerned with an individuals abnormal reac- guns utilize PPD. tions to a specific substance with which he has had previous contact. Allergic reactions Mantoux test are divided into two main groups, immediate The Mantoux test is a procedure by which 0.1 (anaphylactic) and delayed (tuberculin type) . ml. of a test solution of PPD antigen is placed An example of an immediate reaction is intradermally in the skin of the suspect. The anaphylactic shock, which begins immediately strength of the test solution can be varied or within minutes after contact with the al- considerably to compensate for the type of lergen and disappears within hours. The prin- response anticipated. For persons in whom the cipal pathologic change consists of dilation chances of active tuberculosis are high, it is of capillaries and arterioles, with prominent advisable to use a dilute (first strength) test erythema and edema but little cellular infiltra- dose of 1 tuberculin unit (TU) of antigen con- tion. The reaction is associated with circulat- taining 0.00002 mg. of PPD per 0.1 ml. of solu- ing antibodies and can frequently be trans- tion. The hypersensitivity reaction to this dose ferred passively by means of serum. is rarely severe. For those in whom the risk of Examples of the delayed, or tuberculin type, tuberculosis is average, the intermediate reaction are bacterial allergy and tuberculin strength (called intermediate test dose) is the hypersensitivity. The reaction begins within antigen of choice. This contains 0.0001 or several hours or days after contact with the 0.0002 mg. of PPD per 0.1 ml. of test solution. allergen and lasts for several days. The main These two doses correspond to 5 TU and 10 pathologic reaction consists of inflammatory TU, respectively. For persons in whom the change, with predominant mononuclear infil- diagnosis of tuberculosis is suspected but hi

357/68 whom skin tests of lesser strength have been subject should be advised to return in from negative, the second strength antigen is avail- 48 to 72 hours, depending on the procedure able. This dose form contains 0.005 mg. of being followed by the testing team. The Man- PPD per 0.1 ml. of test solution. This dose toux test is best evaluated at 72 hours. Al- corresponds to 250 TU. The use of the second though erythema is frequently present, the strength testing antigen can produce severe size of the area of palpable induration is the necrotizing cutaneous reactions as well as cel- significant reaction to be observed. The test lular changes at the site of tuberculous lesions. site is carefully palpated, and if an area of The Mantoux test is suitable for screening induration is present, its limits are deter- programs or for use in private offices for mined and the transverse diameter is mea- single patients. sured, the largest diameter at right angles to It is important that exactly 0.1 ml. of test the long axis of the arm being noted. The in- antigen be injected and that the material be duration may be more or less easily recognized. placed intradermally as superficially as pos- It may vary from a firm, well-circumscribed sible, with the bevel of the needle facing up- density in the skin to a soft, ill-defined swelling. ward. It is best not to give the test dose in a The latter type may easily escape notice un- spot known to have been used for a previous less the site is palpated with a light touch. test. When the material is placed intradermal- An inexperienced observer may tend to over- ly, a small white wheal, from 6 to 10 mm. in estimate a strong reaction by failing to include diameter, will be raised. Its superficial surface its softer, flatter marginal part. Gentle strok- will resemble an orange peel. After a few ing of the area of induration will allow its out- minutes the wheal disappears, and the site of line to be appreciated. Marking the points at the needle puncture is the only sign that the which induration becomes noticeable to pal- test has been done. pation will facilitate measurement of the in- If the antigen is placed subcutaneously, a durated area. In recording the findings, the false negative response may be obtained. If size of the reaction in millimeters and the it is observed that the material is being de- strength of the test antigen used should be posited subcutaneously, the needle should be noted, rather than an impression or negative withdrawn and the material injected at a dif- or positive. The facts should be recorded, ferent site, which must be intradermal. If a as, for example, 7 mm. induration with 5 TU subject who would normally exhibit a positive at 72 hours. This type of information can be Mantoux reaction receives the test antigen sub- kept for evaluation in the future if the need cutaneously, he may experience mild systemic should arise. symptoms, such as malaise, with no cutaneous Generally, subjects who have tuberculosis indication of a positive reaction. It is recom- will show areas of palpable induration larger mended that a second skin test dose not be than 5 mm. in diameter. The frequency of re- given when a full dose of tuberculin antigen actions less than 10 mm. in diameter among has been given subcutaneously. If the tuber- patients with tuberculosis is low. 4 Cross sensi- culin is lost outside the skin, the test should tivity with other strains of mycobacteria often be repeated about 1 inch from the original is the cause of a small reaction to the Mantoux site. A record should be made of the date, and other intradermal tests. 4 It should be placement, and strength of antigen used. The noted, however, that even among patients in

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whom tuberculosis has been diagnosed, from (1) glass tuberculin syringes which have 1 to 5 percent have had no reaction at all to not been used for other antigen testing pro- the 5 TU (intermediate) dose. Occasionally a cedures, or disposable syringes; tuberculous patient will have no reaction to (2) 26-gauge platinum alloy needles, as rec- even stronger doses. ommended by the WHO standard tuberculin The preferred site for tuberculin skin test- test guide;3 ing is the volar surface of the forearm. In (3) 20-gauge steel needles for filling sy- view of the fact that evaluation of the result ringes; of the skin test involves palpation of cutaneous (4) alcohol lamps to heat the platinum induration, it is wise to select a site away from needles; veins, which could serve to complicate the (5) tuberculin, preferably PPD of inter- process of evaluating the amount of palpable mediate strength, although there may be in- induration present. In testing a group of per- dications for the use of first strength tuber- sons, the same arm (the left, for instance) culin; should be used for each, in order to facilitate (6) a holder for syringes, alcohol sponges, administration and improve the accuracy of and the like. evaluation of test results. The test site can be Tuberculin syringes should be filled by found more quickly and, in addition, a posi- means of 20-gauge needles, since these ac- tive reaction cannot be hidden by showing complish filling more quickly than 26-gauge the opposite arm. needles. Moreover, the platinum needles are Follow-up on tuberculin skin reactions of dulled unnecessarily if they are used for filling 10 mm. or more should be given first priority, of syringes. because persons with such reactions are at The 26-gauge platinum needle should be greater risk that clinical tuberculosis will de- placed on the syringe with the bevel facing velop than are persons with reactions of less the side of the syringe on which the numbered than 10 mm. It has been recommended 2 that graduations are imprinted. This allows for in- reactions between 5 and 9 mm. should be con- sertion of the needle with the bevel up and sidered positive and that the patients should simultaneous observation of the volume of be retested using the same dose of tuber- antigen administered. culin. After the needle is placed on the syringe Mantoux testing of large groups of per- properly, it should be heated in the alcohol sons, such as residents of nursing homes and flame until it is red. A drop of tuberculin school populations, can be accomplished quick- should be ejected from the needle, and then ly and easily with tuberculin syringes con- the intradermal injection can be accomplished. taining up to ten doses and fitted with platinum After the needle is withdrawn from the skin needles. The cost of such testing programs is it should be heated again, a drop of tuber- sometimes reduced when this apparatus is culin again ejected from the needle, and again used. Many times the state or county health 0.1 ml. of tuberculin injected intradermally. department will furnish the materials for such The volume of PPD injected is important for testing programs. accurate interpretation of the final result. Materials needed for testing of large groups Therefore, it should be measured exactly and include: not estimated on the basis of the size of the

359/70 wheal produced by the injection. Tissue turgor and skin testing be done with a platinum nee- and many other related factors influence the dle. size of the wheal. Another difference was the recommendation In the process of performing the tuberculin that 25- or 26-gauge steel needles be used for test, the outside of the needle becomes con- intradermal testing, and that the needles be taminated with tissue juices. Therefore, the heated in a living flame only to the point of needle should be sterilized by heating in a emission of steam from the needle rather living flame for each test. until dull red coloration appears as the result The safety of the multidose syringe tech- of heat. The result of heating steel needles to nique may be questioned. However, after 10 the point of redness is oxidation of the sur- years experience during which millions of face of the needle, which makes them rough intradermal tests were performed by federal and difficult to insert intradermally. They also and state agencies, no clinical or epidemiologic lose their temper and bend easily. evidence was found that homologous serum The dorsal aspect of the forearm rather than jaundice or other infection has occurred with the volar surface was the site suggested by any greater frequency in those who received WHO for tuberculin testing. Both publications intradermal tests than in those who did not. agreed, however, that areas previously known In a college program involving more than to have been used for tuberculin skin testing 5,000 skin tests, not one case of serum jaundice should be avoided, since reactions appear developed.° sooner, fade sooner, become larger, and more Persons with acute illnesses, colds, fever, frequently produce bullae in such areas than skin conditions that cover more than half of in areas not previously used for tuberculin the forearm, or fresh vaccinations should not testing. be tested. Prior to the removal of the platinum Since many antigens adsorb to syringes and needle from the syringe for refilling, the en- may be dislodged during testing causing un- tire needle should be heated. reliable results, the following procedure for In the section on skin testing procedures cleaning and sterilizing the equipment should in the WHO guide,3 there are several differ- be followed: All the parts, needles, and clips ences in technique for multiple-tuberculin dose should be separated and rinsed individually skin testing from the techniques just described. in tap water. A cleansing solution of 1 tea- That publication recommended that the plat- spoonful of sodium hydroxide or trisodium inum needle used for intradermal injection phosphate—or a comparable dishwashing de- not be removed from the syringe for refilling tergent—to a pint of water should be pre- or any other reason and that at the time of pared and all the items placed in a stainless refilling, the total length of the platinum needle pan and covered with the cleansing solution. should be heated until it is red, and then The solution should be brought to a boil and refilling should be accomplished with the plat- kept boiling for 15 minutes, the parts being inum needle. If it is necessary to change a sy- covered with solution during the boiling. Then ringe or a needle, WHO recommended that they should be removed from heat but not both should be changed. This is in contrast drained and the pan with contents placed under to the USPHS recommendation that the filling running tap water and each item flushed ten be accomplished with a 20-gauge steel needle times. Then the parts should be covered in the

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pan with distilled water and returned to heat, that were somewhat softer than those follow- brought to a boil and kept boiling for 5 min- ing 0.06 microgram of PPD-S. Therefore, in utes, after which they are removed from the some programs 0.4 microgram, a higher dose heat and rinsed three times with copious than the standard, has been used, so that reac- amounts of distilled water, with soaking for tions will be not only larger but firmer and from 15 to 30 minutes between rinses. Then easier to read. parts are dried, reassembled, and prepared for sterilization in the usual manner. Tongs or Multiple-puncture methods forceps should be used to remove items from Among other means of intracutaneous tuber- cleansing solution, and these should be rinsed culin testing are the multiple-puncture meth- after each use to prevent corrosion. Syringes ods, including the tuberculin tine test (Rosen- used for antigens other than tuberculosis thal) and the Sterneedle (Heaf) test. should not be used for tuberculin testing. The tine test unit is made of a stainless It has been mentioned earlier that a new steel disc from which project four tines, 2 mm. standard tuberculin, RT 23, has been produced long and 4 mm. apart. This disc is attached in Copenhagen, in quantity large enough that to a plastic handle. The tines are dipped into it is hoped that improvements in antigens will a standard solution of OT and allowed to make tuberculin testing much more specific dry, redipped, and dried again repeatedly. The by the time the present supply of standard is amount of OT deposited in the skin by each exhausted. Here are some of the comparisons3 of the tines is equivalent to the intermediate between RT 23 and PPD-S. Tuberculin RT strength dose in the Mantoux test (5 TU). 23, once diluent is added, can be used for about 6 months, provided that it is protected Application of the tine test. There is more to from direct sunlight or strong daylight and the application of a tine test to a patient than stored at temperatures not exceeding 20 C cleansing the arm with alcohol and pressing except for short periods. the tines into the skin. The thickness of the In 1960 the WHO expert committee on skin apparently does not have a great effect biologic standardization, at its fourteenth on the accuracy of the test procedure. How- meeting, decided that the existing definition ever, the proper depth of administration is of the international unit of PPD of mammalian important, as is the period of contact of the tuberculin in high dilutions was highly un- tines with the skin. The tines should be predictable because of adsorption of the tu- pressed into skin that is held taut. The pres- berculin onto the glassware and containers sure should be sufficient to cause a ring-shaped after dilution. Tweens°, a nonionic detergent, mark to be imprinted in the skin, but not prevents this and is added to RT 23. deeply. Excessive pressure is associated with The standard dose of RT 23 is 0.2 micro- bleeding from the puncture sites. The tines gram (1 TU) in 0.1 ml. of diluent. This dose should be left in place at least 1 second (about produces reactions of about the same size as the time it takes to say "one thousand") and 0.06 microgram (3 TU) of PPD-S in 0.1 ml. then removed. It has been shown that the same of diluent without the nonionic detergent results were obtained when the tine test was Tween80. The standard dose of 0.2 microgram applied for 5 seconds as for 1 second, but of RT 23 with Tweens° produced reactions that a large percentage of false negative reac-

361/72 tions occurred when the tines were left in of the comparison. The Mantoux tests were place for less than a second. done by nurse technicians highly trained in It has been suggested7 that the skin be dry the technique. prior to the administration of a tine test be- Considering the utilization of the tine and cause of the possibility of denaturing OT with the Mantoux test, the American Thoracic So- alcohol or washing it away so that it is not ciety committee on diagnostic skin testing placed intradermally. made the following recommendations: Palpable induration of 2 or 3 mm. around (1) For both screening and diagnostic purposes the any one of the four tines indicates a reaction Mantoux with a standard dose of 5 TU (0.0001 mg.) of of about the same significance as a 5 to 9 mm. PPD is the most accurate test. Skillful administration reaction to the Mantoux test. Palpable indura- and careful reading are essential. (2) For clinical screening use when tuberculosis is tion of from 4 to 10 mm. with confluence is not suspected, a multiple-puncture test is acceptable. comparable to induration of from 10 to 19 If an equivocal reaction is obtained, a repeat test with mm. after the Mantoux test. Induration of the intermediate-strength of 5 TU (0.0001 mg.) of PPD Mantoux is indicated. more than 10 mm. with or without vesicula- (3) For differential diagnostic purposes when tuber- tion is equivalent to from 20 to 39 mm. of culosis is suspected, the multiple-puncture test is ac- induration after the Mantoux test.? ceptable as an initial test. If a negative or equivocal reaction is obtained, a repeat test should be given with Badger and associates8 found a slightly bet- intermediate-strength PPD. If still negative, a stronger ter statistical concordance of the 6 mm. re- dose should be used in an effort to rule out tuberculous action to PPD-S with the 3-mm. reaction than infection. (4) For large-scale screening programs, multiple- with the 2-mm. reaction to the tine test. They puncture tests are acceptable for purposes of case stated: finding and determination of a rough index of tuber- culous infection. Unless the number is prohibitive, These two reactions of 3 mm.+ or 2 would those with smaller or questionable reactions should be appear to be a judiciously liberal concept of a positive retested with the standard Mantoux test. TTT . . . quite analogous to the concept of a positive PPD reaction at 6 mm. . . . Therefore, a minimal posi- tive TTT should be recognized when any one of the Another multiple-puncture method is the four tine reactions in the skin is equivalent to 3 mm. Sterneedle using PPD and a spring-loaded de- (or 2 mm.) of palpable induration. vice which quickly propels six tines into the Maximum reactions to the tine test occur dermis is available. This test utilizes a steril- between 48 and 72 hours. The fact that in some izable, reusable tip containing six tines approx- subjects the maximum reactions occur at 72 imately 2 mm. long, which retract into a pro- hours indicates that fewer reactions will be tective housing. The tip is attached to a device missed at 72 hours than at 48 hours if only a which propels the tines from the housing. The single reading is to be done.8 PPD is removed by dropper from its bottle, and When both the tine test and the Mantoux a drop or two is placed in the inverted cap. test were done on both forearms of 93 sub- The sterile tip attached to the spring-loaded jects, the results of the Mantoux test on the device is dipped into the solution to moisten two arms disagreed in 6 percent of subjects it with PPD. Then it is placed on the selected and those of the tine test in 10 percent. In- site for testing and rotated through a 90- durations of 6 mm. or more to the Mantoux degree angle to spread the PPD onto the skin test and of 2 or 3 mm. to the tine test were evenly, and the tines are quickly injected in- considered positive reactions for the purposes to the skin. One drop of PPD should be suf-

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ficient for approximately six tests. (3) Concentrated tuberculin produces fre- The reaction to the test is read in from 3 quent false positive reactions. to 7 days. Positive reactions are graded from (4) Quantitative measures or reactions can 1 to 4. Palpable induration of at least four be only approximate guides to degrees of sensi- puncture points is considered to be a grade 1 tivity in view of variables affecting the dose. reaction. Coalescence of the indurated points Whereas the Mantoux test is considered to to form an edematous ring is considered to be be the most accurate intradermal test for a grade 2 reaction. A coin pattern reaction 1 tuberculosis available today because of the cm. in diameter is considered to be a grade 3 specificity of the dose administered, skill is reaction, and extensive induration with pos- required to deliver proper dose intradermally. sible sloughing of the center is considered to The tuberculin tine test is highly accurate and be a grade 4 reaction. convenient to use, and requires a minimum of skill to perform. In some circumstances either Comparison of tests the Mantoux and the multiple-puncture test The Mantoux test has several advantages: The procedure will be the more appropriate or test dose can be measured accurately; ma- practical, depending on the desires of the phy- terials and equipment are easily available, and sician responsible for the tests. reactions can be read and recorded quanti- tatively. It has the following disadvantages, however: (1) Skill in administering the test is re- 1. Jawetz, E., Melnick, J. L., and Adelberg, E. A.: Review of medical microbiology. Ed. 2. Lange Medical Publications. Los quired for best results. Altos, Calif., 1956 (2) There are psychologic objections to a 2. National Tuberculosis Association: Diagnostic standards and classification of tuberculosis. Ed. 11. National Tuberculosis Asso- hypodermic needle. ciation, New York, 1961 (3) For individual use, the procedure is 3. The WHO standard tuberculin test. WHO/TB technical guide/3, 22 Feb 68, Tuberculin Laboratory, Copenhagen, 1968 cumbersome. 4. American Thoracic Society, Committee on Diagnostic Skin Test- ing: Tuberculin skin-testing techniques. Current status. Amer Rev The multiple-pressure tests have the follow- Reap Die 87:607-10, Apr 63 ing advantages: 5. A child-centered program to prevent tuberculosis. Public Health Service Publication No. 1280. U.S. Government Printing Office. (1) Minimal technical skill is required for Washington, 1966 administration. 6. American Trudeau Society, Committee on Diagnostic Skin Test- ing: Homologous serum jaundice. Amer Rev Rasp Dis 81:126, Jan (2) The concentrated tuberculin punched 60 into the skin is essentially a strong dose, which 7. Lederle Laboratories: Testing for tuberculosis. A community approach. A manual of organization and procedures. Lederle Lab- ensures the rarity of false negative reactions. oratories, Pearl River, New York (3) Some types of test are convenient for 8. Badger, T. L., Breitwieser, E. R., and Muench, H.: Tuberculin tine test. Multiple-puncture intradermal technique compared with individual use. PPD-S, intermediate strength (5 TU). Amer Rev Reap Dis 87:888- (4) Materials are stable at room temper- 53, Mar 63 ature. It has the following disadvantages, however: (1) The tuberculins are not well standard- Dr. Hagen, who is an associate professor in preventive medicine and chairman, Department of Preventive Medicine, Kirksville Col- ized. lege of Osteopathy and Surgery, Kirksville, Mo., was the re- cipient of a 1969-70 National Osteopathic Foundation fellowship (2) The dose of tuberculin retained in the grant through the cooperation of the Merck Company Foundation. skin is unknown. Dr. Hagen, 17 Scott Street Terrace, Kirksville, Mo. 68501.

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