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Development and Evaluation of a Simple Latex Agglutination Test for Diagnosis of Tuberculosis' R

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Development and Evaluation of a Simple Latex Agglutination Test for Diagnosis of Tuberculosis' R APPuED MICROBIOLOGY, Oct. 1972, p. 525-534 Vol. 24, No. 4 Copyright 0 1972 American Society for Microbiology Printed in U.S.A. Development and Evaluation of a Simple Latex Agglutination Test for Diagnosis of Tuberculosis' R. V. COLE,2 A. W. LAZARUS, AND H. G. HEDRICK Department of Botany and Bacteriology, College of Life Sciences, Louisiana Tech University, Ruston, Louisiana 71270 Received for publication 1 March 1972 A simple latex agglutination test (SLAT) based on modifications of existing serodiagnostic techniques, in which commercially available reagents are used, was developed for detection of antibodies against Mycobacterium tuberculosis. Tests performed on 553 serum samples from 316 individuals, including 117 bacteriologically confirmed active tuberculosis patients, showed 80% positive titers. Sera from 12 patients with arrested tuberculosis showed 91% positive titers. Nonspecific reactions were noted in 5% of 160 serums from selected normal individuals and patients with diseases other than tuberculosis. The an- tibodies detected by the SLAT method were found to be relatively stable when exposed to low temperatures, whereas high temperatures reduced the antibody titer considerably. Disodium ethylenediaminetetraacetic acid inactivation of serum complement was found to be satisfactory. No variation of tuberculosis antibody titer was noted in tests on multiple specimens from patients whose conditions were stabilized. However, considerable fluctuation was encountered in antibody titers obtained on recently detected individuals. Data obtained in this study indicate that the modified procedures of the SLAT method could replace the tuberculin skin test for simple screening of tuberculosis in adults. The use of tuberculin skin test procedures of serodiagnostic methods for the detection of has been the basis for the detection of new tuberculosis antibodies. Early tests were based cases of tuberculosis in mass testing programs on the standard complement-fixation tech- in recent years. The Heaf test has been used as nique. Emphasis later shifted to simpler agglu- the initial screening procedure because of its tination procedures such as the hemagglutin- simplicity. Positive reactors to this test are ation test and the bentonite and kaolin floccu- usually rechecked by the less sensitive Man- lation tests. Various fractions and metabolic toux intradermal tuberculin test. Individuals products of the tubercle bacillus were used to who have positive reactions with this test are prepare the antigens for these tests. The most followed up with laboratory studies and X successful serodiagnostic procedures for the rays. Skin testing programs have been reason- detection of tuberculosis antibodies have been ably successful in detecting new cases of tuber- the gel double-diffusion test devised by Parlett culosis in children. Mass skin testing programs and Youmans (6) and the latex agglutination in adults have not produced the desired results test of Duboczy and White (3). Although they because many adults have positive reactions to produce satisfactory results, these tests are these tests as a result of acquired subclinical complex and require specially prepared re- infections. agents. The purpose of this study was to de- Recognition of the shortcomings of the tu- velop a method for the detection of tubercu- berculin skin test resulted in the development losis antibodies which would use existing sero- diagnostic procedures and make use of com- 'Presented in part at the 71st Annual Meeting of the mercially available reagents prepared for other American Society for Microbiology, Minneapolis, Minn., May 1971. tests. Emphasis was placed on simplicity of 2Present address: East Texas Chest Hospital, Tyler, Tex. technique, stability of reagents, and reproduci- 75701. bility of results. The simple latex agglutination 525 526 COLE, LAZARUiS. AND HEDRICK APPL. MICROBIOL. test (SLAT) developed is evaluated herein. agglutination was graded according to the following criteria: four plus reactions, indicating maximal ag- glutination, were characterized by the formation of a MATERIALS AND METHODS large, solid mass of latex particles in the bottom of The stock tuberculin reagent was obtained from the tube and a clear supernatant fluid; three plus Difco Laboratories and was prepared from a filter- reactions consisted of large aggregates of latex parti- sterilized, dialyzed flash culture of Mycobacterium cles and a clear supernatant fluid; two plus reactions tuberculosis strain H37Ra which had been grown in consisted of small aggregates of latex particles with a a modified Beck-Proskauer broth according to the clear supernatant fluid; one plus reactions were method of Parlett and Youmans (6). This reagent characterized by fine granulations that were easily was identified by the manufacturer as Bacto-H37Ra distinguishable from the control; and plus-minus antigen and was originally intended for use in a cap- reactions displayed questionable reactivities difficult illary gel double-diffusion test for the detection of to differentiate from the negative. control. The titer tuberculosis antibodies. was determined to be the reciprocal of the highest Polystyrene latex particles were obtained from dilution showing a minimum of a one plus reaction. Difco in a stock 3% aqueous suspension. The particle Several diluents were evaluated with variable re- diameter was standardized by the manufacturer at sults. Phosphate and borate buffers at pH 8.2, as 0.81 um. This reagent was originally intended for use recommended by Singer and Plotz (7) for use in the in the Bacto-RA test for the rheumatoid factor asso- RA test, appeared to inhibit agglutination. A glycine ciated with rheumatoid arthritis (Singer et al. [8]). buffer (4, 5) was also tested and gave negative re- To determine the optimal concentration of the sults. Physiological saline, as suggested by Carlisle antigen needed to sensitize the polystyrene latex and Saslaw (1) for a diluent in the latex agglutina- particles, it was necessary to perform a series of tion test for histoplasmosis, was found to work well cross-titrations with positive serum and a standard- at pH 6.2. The serum diluent was prepared in 0.0015 ized concentration of latex particles sensitized with M disodium EDTA in physiological saline at pH 5.0. various dilutions of the stock tuberculin reagent. The individuals furnishing the sera for this study Serial dilutions of the antigen were prepared with were grouped according to their clinical diagnosis: physiological saline as the diluent. The initial dilu- group 1 included patients with minimal tuberculosis; tion was 1:10, with a maximal dilution of 1:320. group 2, patients with moderately advanced pulmo- Final volume of each dilution was 10 ml. A 0.1-ml nary tuberculosis; group 3, patients with far ad- amount of the stock latex reagent was added to each vanced pulmonary tuberculosis; group 4, patients tube of the diluted antigen. A latex control was also with mycobacterioses due to organisms other than prepared by adding 0.1 ml of the stock latex reagent M. tuberculosis; group 5, patients with extrapul- to 10 ml of saline. The antigen-latex suspensions and monary tuberculosis; group 6, patients with pulmo- the latex control suspension was then incubated in a nary tuberculosis, classification and activity unde- water bath at 37 C for 2 hr. termined; group 7, patients with inactive or arrested The antigen titration procedure was performed in tuberculosis; group 8, patients admitted to the hos- triplicate with new, scratch-free serological test pital for the purpose of ruling out tuberculosis; group tubes (10 by 75 mm). Seven titrations of eight tubes 9, patients with nontuberculous diseases which each were set up. To assure that the seven titrations might result in abnormal globulin fractions and indi- would be identical, the serum dilutions were pre- viduals with high antibody titers resulting from non- pared in 5-ml amounts and distributed to appropri- tuberculous diseases or immunization; and group 10, ately marked tubes for each titration. An initial dilu- selected normal controls. tion of 1: 2 was used. The diluent was 0.0015 M diso- Blood specimens from individuals in test groups 1 dium ethylenediaminetetraacetic acid (EDTA) in through 8 were collected in the laboratory at the physiological saline. Each tube of the first six titra- Harlingen State Tuberculosis Hospital, Harlingen, tions received 0.2 ml of the appropriate antigen- Tex. These specimens included patients confined to latex suspension. The tubes in the seventh series the hospital as well as those receiving treatment or served as latex controls for the detection of nonspe- evaluation in the out-patient and follow-up clinics. cific agglutination and received 0.2 ml of the saline- Bacteriological data were obtained from the hospital latex suspension. laboratory records. Information pertaining to com- The optimal incubation conditions were deter- plement-fixation and latex agglutination tests for mined by incubating each of the triplicate sets of ti- deep systemic mycoses was obtained from the same trations under three different conditions: (i) incu- source. Specimens from patients in group 9 were col- bated in a water bath overnight at 37 C (approxi- lected at the laboratory of a local hospital in San mately 15 hr); (ii) incubated at room temperature Benito, Tex., and at the venereal disease clinics held (22 to 25 C) for the same length of time; and (iii) al- by the Cameron County Health Department in San lowed to stand at room temperature for 4 hr, and Benito. Specimens from individuals in group 10 were then refrigerated at 4 to 6 C overnight and allowed also collected at the Cameron County Health De- to warm to room temperature the following morning. partment. The collected serum was stored at 4 to 6 Tubes of all three sets of titrations were centri- C prior to testing, with the exception of those speci- fuged for 5 min at 2,000 rev/min in a size 1 model mens which were to be used to determine the effects SBV International centrifuge and examined macro- of freezing on the antibodies detected by the SLAT scopically for evidence of agglutination.
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