Evaluation of Reagin Screen, a New Serological Test for Syphilis

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1 JOURNAL OF CUNICAL MICROBIOLoGY, Aug. 1976, p Copyright 1976 American Society for Microbiology Vol. 4, No. 2 Printed in U.S.A. Evaluation of Reagin Screen, a New Serological Test for Syphilis JOHN D. DYCKMAN,* REUBEN D. WENDE, DOROTHEA GANTENBEIN, AND ROBERT P. WILLIAMS Department of Microbiology and Immunology, Baylor College of Medicine, and City of Houston Health Department Laboratory,* Houston, Texas Received for publication 6 January 1976 A total of 1,020 serum and plasma specimens were tested using the Venereal Disease Research Laboratory (VDRL), Rapid Plasma Reagin (RPR) card, Reagin Screen (RST) and Fluorescent Treponemal Antibody-Absorption (FTA- ABS) tests. In 257 normal patients, all screening tests were nonreactive; the FTA-ABS test was reactive for one patient. In 588 patients with treated and untreated syphilis, the RST results were 91.7% in agreement with the VDRL and RPR results. In 175 patients with diseases that cause biological false reactions, the RST was 94% in agreement with the other screening tests. The titer of the RST was within one dilution of the corresponding VDRL titer in 91.7% of the 360 specimens tested and within one dilution of the RPR titer in 96.9% of 358 specimens quantitated by both tests. The search for more sensitive and specific laboratory tests to screen for and to confirm syphilis has been constant since the complement fixation test was developed by August von Wassermann, Albert Neisser, and Carl Bruck in 1906 (9). The antigen for this test was prepared from the livers of stillbom, congenital syphilitic infants. The following year Marie and Levaditi demonstrated that saline extracts of nonsyphilitic organs could serve equally well for the antigen (5). Since von Wassermann's discovery, many different serological procedures have been developed to detect the nonspecific substance known as reagin that appears in response to infection with Treponema pallidum. Each test generally has been named for the person who developed the procedure, i.e., the Kline test, the Kolmer test, and the Kahn test, and lipid antigens traditionally have been used in these screening tests. The Venereal Disease Research Laboratory (VDRL) test and the Rapid Plasma Reagin (RPR) card test are currently the most widely used screening tests in the diagnosis of syphilis. The Fluorescent Treponemal Antibody-Absorption (FTA-ABS) Test is commonly used to confirm the reactive results of these less sensitive tests that use nontreponemal antigens. The reactivity ofthese three tests in normal individuals and in patients with various stages of syphilis has been well established in several investigations (1-4, 10). In other reports, the relative reactivity of these tests has been compared without adequate notation of patient categories (7, 8). A new card test using lipid antigens recently has been developed and marketed as a screening test for syphilis. This test, the Reagin Screen Test (RST; Lederle Laboratories, Pearl River, N.Y.), uses lipid antigens stained with a lipid-soluble dye. This dye makes the agglutination of the antigens visible macroscopically. In this respect, the antigen system differs from that of the RPR card test, which uses coagglutination of antigen and charcoal particles that are macroscopically visible when a reactive serum is tested. The purpose of this investigation was to compare the reactivity of these tests (VDRL, RPR, RST, and FTA-ABS). Sera from normal individuals, from patients with various stages of syphilis, and from patients with other diseases and conditions known to cause biological false positive reactions were examined. Qualitative tests were performed on all sera. Quantitative tests were performed on sera that exhibited any degree of reactivity in the qualitative test. MATERIALS AND METHODS Samples. Sera from presumed normal individuals were obtained from persons presenting at the Houston Health Department for a premarital examination. A complete medical history on these patients was not available. Plasma from normal individuals was obtained from children under 10 years of age. Ethylenediaminetetraacetate and oxalate were the anticoagulants used for these specimens. Cord bloods were obtained from Ben Taub Hospital, Houston. A nonreactive VDRL was not a criterion for inclusion or exclusion in this normal group. Sera from patients with mononucleosis, hepatitis, and 145

2 146 DYCKMAN ET AL. systemic lupus erythematosis were also obtained from Ben Taub Hospital. Sera from heroin addicts were obtained from the Texas Research Institute of Mental Sciences, Houston. Sera from patients with malaria were obtained from Hermann Hospital, Houston. Sera from patients with yaws were generously donated by William Butler, Department of Microbiology and Immunology, Baylor College of Medicine, Houston. Sera from arthritic individuals were obtained from John Sharp, Department of Medicine, Baylor College of Medicine, Houston. Sera from patients with biological false positive reactions of unknown origin were detected by VDRL or RPR screening. Sera from syphilitic patients were obtained at the Houston Health Department Venereal Disease Clinic. If sera could not be tested within 48 h after collection, they were stored frozen at -20 C. No sera except for yaws specimens were held for more than 2 weeks before being tested. All sera were numbered before testing, and no other identification was indicated for the samples. Serological tests. Qualitative tests using the RPR Test kits (Hynson, Westcott, and Duning, Baltimore, Md.) or the RST kits (Lederle Laboratories. Pearl River, N.Y.) were performed on unheated serum or plasma according to the directions of the manufacturers. Quantitative tests were performed by serial twofold dilution of the serum or plasma with physiological saline in the sample areas on the test card. Capillary pipettes calibrated at 0.05 ml were used to dispense the saline and serum and to perform the dilutions. VDRL and FTA-ABS tests were performed on heated sera by standard, approved methods (8). Reagents for these tests were supplied by the Texas Department of Health Resources, Austin. Tests using lipid antigens were controlled by tests of nonreactive, weakly reactive, and reactive control sera J. CLIN. MICROBIOL. before processing of tests samples. Controls were performed for the FTA-ABS test as described (8). RESULTS Serum from 243 patients with untreated primary, secondary, and latent syphilis and 354 patients previously treated for syphilis were examined by the four tests. For this investigation, the stages of syphilis were defined as follows: (i) primary syphilis -primary, dark-fieldpositive chancre, and no symptoms of secondary syphilis; (ii) secondary syphilis-dark-fieldpositive secondary lesions or multiple symptoms of secondary syphilis such as condyloma lata, alopecia, and lymphadenopathy; and (iii) latent syphilis-no physical signs of syphilis, no history of treatment for syphilis, one reactive nontreponemal test, and a reactive FTA- ABS. The stage of syphilis for each patient was diagnosed by highly trained clinic personnel under the direct supervision of a physician. The reactivity of serum from these patients is presented in Table 1. In serum from patients with primary syphilis, the RPR and VDRL tests were the most sensitive nontreponemal tests and detected some degree of reactivity in 64.9 and 63.1% of these sera. The RST detected some degree of reactivity in 57.7% of the sera. The much greater sensitivity of the FTA-ABS test, which was reactive in 94.6% of these cases, is evident. Serum from all patients with secondary syphilis was reactive by all serological tests. Patients with latent syphilis were initially detected by screening with the VDRL or RPR Patient TABLE 1. Reactivity of the VDRL, RPR, RST, and FTA-ABS in syphilitic patients No. (%) of reactions using: category VDRL RPR RST FTA-ABS (no. tested) N" Wb R' N Md R N W R N Be R Primary (111) (36.9) (10.8) (52.3) (35.1) (9.9) (55.0) (42.3 (8.1) (49.6) (5.4) 0 (94.6) Second ary (0) (0) (100) (0) (0) (100) (0) (0) (100) (0) (0) (100) (56) Latent (76) (1.3) (14.5) (84.2) (7.9) (10.5) (81.6) (9.2) (13.2) (77.6) (0) (0) (100) Treated (345) (15.7) (19.3) (65) (14.7) (23.8) (61.4) (21.6) (24.8) (53.6) (1.6) (0) (97.7) N, Nonreactive. W, Weakly reactive. 'R, Reactive. M, Minimally reactive. B, Borderline. b

3 VOL. 4, 1976 card test at the Health Department or at other clinics. Only 1.3% of sera from patients with latent syphilis was found to be nonreactive by the VDRL test. The RPR test and RST were less sensitive in this group, with 7.9 and 9.2%, respectively, yielding nonreactive results. The FTA-ABS test was 100% reactive. Reagin persisting in the serum of patients after treatment for syphilis was detected more frequently by the RPR and VDRL tests than by the RST. The RST results were nonreactive in 21.6% of these patients. Only 1.6% of these patients had a nonreactive FTA-ABS test. It is not known if any of these nonreactive results represent reversion of a previously reactive FTA-ABS. A total of 257 presumed normal individuals were also tested (Table 2). In this group, the VDRL, RPR, and RST were all nonreactive. Most patients also exhibited a nonreactive NEW SEROLOGICAL TEST FOR SYPHILIS 147 FTA-ABS test. One patient had a borderline FTA-ABS test, and one patient had a reactive FTA-ABS. No history of a previous syphilis infection could be found in these two patients. A total of 175 patients with diseases or conditions known to cause biological false positive reactions also were evaluated (Table 2). Twenty-two patients with hepatitis and 32 patients with mononucleosis were nonreactive by all three nontreponemal tests. Of the two malaria patients who had been treated for the disease and were about to be released from the hospital, one exhibited a minimally reactive RPR card test. The remaining disease categories yielded varying degrees of reactivity to the VDRL, RPR, and RST. No one test was reactive in a significantly greater number of these patients than the other tests. The FTA- ABS test was reactive in all patients with TABLE 2. Reactivity of the VDRL, RPR, RST, and FTA-ABS in nonsyphilitic patients No. (%) of reactions using: Patient cat- VDRL RPR RST FTA-ABS egory Na Wb R' N Md R N W R N Be R Normal (100) (0) (0) (100) (0) (0) (100) (0) (0) (99.2) (0.4) (0.4) Hepatitis (100) (0) (0) (100) (0) (0) (100) (0) (0) (100) (0) (0) Mononu cleosis (100) (0) (0) (100) (0) (0) (100) (0) (0) (96.9) (3.1) (0) Lupus (87) (8.7) (4.3) (78.3) (13) (8.7) (91.3) (4.3) (4.3) (73.9) (0) (26.1) Heroin (84) (4) (12) (88) (8) (4) (84) (12) (4) (96) (4) (0) Malaria (100) (0) (0) (50) (50) (0) (100) (0) (0) (100) (0) (0) Yaws (18.2) (36.4) (45.4) (9.1) (36.4) (54.5) (27.3) (27.3) (45.4) (0) (0) (100) Arthritis " (93) (7) (0) (88.4) (9.3) (2.3) (90.7) (9.3) (0) (93) (0) (7) BFT (5.9) (17.5) (76.5) (29.5) (29.5) (41) (23.5) (47) (29.4) (100) (0) (0) a N, Nonreactive. b W, Weakly reactive. R, Reactive. d M, Minimally reactive. B, Borderline. f The fluorescence seen was an atypical beaded pattern in all cases. This pattern is often seen in patients with lupus. 9 Etiology unknown. i BFP, Biological false positive, cause unknown.

4 148 DYCKMAN ET AL. yaws. Of the patients with systemic lupus erythematosis, 26% yielded the beaded fluorescence characteristic of patients with this disease. The agreement of the qualitative results among the VDRL, RPR, and RST was also analyzed by comparing the individual reactions between any two tests (Table 3). For any given specimen, the tests were taken to be in agreement if the results were both nonreactive, or both reactive to any degree. Thus, if one test was weakly reactive and the other was reactive, the results were said to agree even though the same degree of reactivity was not demonstrated by both tests. Lack of agreement occurred when one test was reactive or weakly reactive and the other tests were nonreactive. There was 100% agreement among all tests compared in the group of normal patients. The agreement in patients with primary syphilis was comparable with all three tests, and 100% agreement was shown in patients with secondary syphilis. The best agreement in patients with latent syphilis was seen between the RPR test and RST. However, the number of patients in this group was relatively small. The agreement in untreated syphilis patients was 94.7% both between the RST and VDRL and between the RPR and VDRL. A 95% agreement between the RST and RPR was seen in the untreated syphilis group. The RPR and VDRL tests had the best agreement in patients whose syphilis infections were treated before this study. In this group, the RST was the most likely to be nonreactive (Table 1), and the biggest discrepancy between test results was seen in this group. In the syphilis group as a whole, the best agreement was seen between the VDRL and RPR tests. Over 90% agreement was seen in all test comparisons. Comparable degrees of agreement were noted among all tests in the group of patients with other diseases. In the total group of 1,020 patients, all test pairs had a 95% + 1% agreement. J. CLIN. MICROBIOL. When a serum had a reactive qualitative end point with a particular serological test, the serum titer was also determined by using that test. A serum yielding a reactive qualitative result by two of the tests and a nonreactive result by the third would only be titered by the two tests yielding a reactive qualitative result. The titer for the VDRL test was the highest dilution yielding a reactive result. The titer for the RPR and RST was the highest dilution yielding any degree of reactivity. A comparison of the titers of these tests is shown in Table 4. Two tests were compared only when a specimen was reactive and a titer was established with both tests. In the 360 samples quantitated by the VDRL and RST, the RST titers ranged from three dilutions lower than the VDRL titer on the same sample to two dilutions higher than the corresponding VDRL titer. Most RST titers were equal to or one dilution lower than the VDRL titer. The agreement between quantitative results of these two tests within one dilution was 91.7%. In the 393 samples quantitated by the VDRL and RPR tests, the RPR titer ranged from two dilutions lower than the VDRL to two dilutions higher than the VDRL. Most RPR titers were equal to or one dilution higher than the corresponding VDRL titer. The agreement within one dilution was 93.9%. Of the 358 samples titered by the RST and RPR, the RST titer was always within two dilutions of the RPR titer. Most RST titers were equal to or one dilution lower than the RPR. The agreement between these two tests within one dilution was 96.9%. Although several sera tested in this study had titers as high as 1:128 and 1:256, no prozone reactions were encountered in the qualitative tests. All the high-titer sera were read as reactive in the VDRL, RPR, and RST. DISCUSSION In past studies, the reported VDRL reactivity of patients with dark-field-positive primary TABLE 3. Agreement of qualitative results among VDRL, RPR, and RST Patient category (no. RST-VDRL agreement RPR-VDRL agreement RST-RPR agreement tested) No. % No. % No. % Normal (257) Syphilis Primary (111) Secondary (56) Latent (76) Treated (345) Total (588) Other diseases (175) Total (1,020)

5 VOL. 4, 1976 TABLE 4. NEW SEROLOGICAL TEST FOR SYPHILIS 149 Comparison of quantitative results among VDRL, RPR, and RST Titer comparison Tests compared % Agreement + 1 dii - 3 dilsa - 2 dil - 1 dil Equal + 1 dil + 2 dil RST titer versus /360 = 91.7 VDRL RPR titer versus /393 = 93.9 VDRL RST titer versus /358 = 96.9 RPR a dil, Dilutions. syphilis at the Houston Health Department was 73% (1, 11). This reactivity is in agreement with other published results (2, 6, 10). In the present study, only 63.1% of patients with darkfield-positive, primary syphilis yielded a weakly reactive or reactive VDRL test. The results of the RPR and RST qualitative tests were similar with 64.9% and 57.7%, respectively, yielding some degree of reactivity. The FTA-ABS test was reactive in 94.6% of these patients. This figure reemphasizes the value of the FTA-ABS test, which can aid in the diagnosis of syphilis in patients who have a visible chancre that cannot be examined by dark-field microscopy and who have a nonreactive VDRL (1). However, the FTA-ABS test should not be used as a screening test in an attempt to detect primary syphilis. The reason for the lower percentage of reactive VDRLs in patients with primary syphilis in this study is probably due to a change in patient population in this study compared with past studies. We have noted a 143% increase in the number of white males with syphilis attending the clinic over the past year. The number of black males with syphilis declined 36% over the same time period. White males, who were 30% of the population with primary syphilis in this study, exhibited a 53% incidence of nonreactive VDRL tests. Only 30% of black males with primary syphilis were nonreactive by the VDRL test. The great majority of the results of the qualitative RST were in agreement with the results of the VDRL and RPR tests, although the sensitivity of the RST was slightly lower than that of the VDRL and RPR. The RST failed to detect six cases of primary syphilis that were weakly reactive by the VDRL test. However, since these patients all had diagnostic symptoms (dark-field-positive primary chancres), this deficiency would not seem to be a basis for severe criticism of the RST, especially in facilities capable of performing dark-field examinations. The RST also failed to detect reactivity in six cases of latent syphilis that gave some degree of reactivity by the VDRL test. Five of these cases also were nonreactive by the RPR card test. Most of the differences in reactivity between the RST and VDRL were seen in the population that had been treated previously for syphilis. In this group the RST yielded a nonreactive result more frequently than the VDRL or RPR tests. These nonreactive results with the RST could be an advantage of this test in following treated cases of syphilis. Reinfection or relapse might be more readily detected upon repeat qualitative testing as the results turn from nonreactive to weakly reactive or reactive. However, the slight insensitivity of the RST in the treated-patient category might prevent this early detection. Certainly further studies of responses of treated patients are warranted to determine possible advantages of the RST. Excellent agreement was seen in the population that had untreated syphilis. Thus, the RST exhibited a sensitivity similar to that of the VDRL and RPR tests, although where discrepancies occurred the RST was usually less sensitive than the other two. Excellent specificity of the RST was noted in the group of normal patients who were uniformly nonreactive. In patients with diseases or conditions that sometimes yield a biological false positive reaction, the specificity of the RST was slightly superior to that of the VDRL and RPR tests. Agreement also was noted in comparison of the titers of the RST with the VDRL and RPR titers. A 91.7% agreement within one dilution was demonstrated between the RST and VDRL, and a 96.9% agreement was seen between the RST and RPR. The closer agreement between the two card tests probably is due to the similarity of methods involved in dilution of patient's sera and in the degree of reactivity used to determine the titer end point. These two factors differ between the card tests and the

6 150 DYCKMAN ET AL. VDRL quantitative procedure. Despite the agreement in titer, the most meaningful information in terms of rise in titer before therapy or decline after therapy would be provided if a patient was consistently evaluated by the same test. The RST does offer several advantages over the VDRL and RPR tests. The antigen used in the test is stable for 1 year according to the manufacturers' instructions and is packaged in the dispensing bottle. Thus, transfer of the antigen from an ampoule to the dispensing bottle is eliminated. A set of control sera is included with each kit. These control sera are stable over 1 year, yielding reproducible results. The antigen is nonparticulate, yielding a smooth, even background with nonreactive sera. The RPR test has a particulate (charcoal-associated) antigen which yields a slightly coarse background. Since the RST antigen is not particulate, it is especially important to read the results carefully under high-intensity lighting while rocking the card slightly. The slight agglutination of some weakly reactive sera could be missed by a less careful examination. LITERATURE CITED 1. Duncan, W. C., J. M. Knox, and R. D. Wende J. CLIN. MICROBIOL. The FTA-ABS Test in darkfield-positive primary syphilis. J. Am. Med. Assoc. 288: Falcone, V. H., G. W. Stout, and M. B. Moore Evaluation of rapid plasma reagin (Circle) card test. Public Heath Rep. 79: Hunter, E. F., L. C. Norins, V. H. Falcone, and G. W. Stout The fluorescent treponemal antibody absorption (FTA-ABS) test. W.H.O. Bull. 39: Mackey, D. M., E. V. Price, J. M. Knox, and A. Scotti Specificity of the FTA-ABS test for syphilis. J. Am. Med. Assoc. 207: Marie, A., and C. Levaditi Wasserman antigen from normal tissue extracts. Ann. Inst. Pasteur Paris 21: Sparling, P. F Diagnosis and treatment of syphilis. N. Engl. J. Med. 284: Reed, E. L Rapid Reagin tests in the public health laboratory RPR card test. J. Conf. Public Heath Lab. 27: U.S. Public Health Service Manual of tests for syphilis. Public Health Service Publication no U.S. Government Printing Office, Washington, D.C. 9. von Wassermann, A., A. Neisser, and C. Bruck Eine serodiagnostische Reaction bei Syphilis. Dtsch. Med. Wochenschr. 32: Wallace, A. L., and L. C. Norins Syphilis serology today, p In Progress in clinical pathology, vol. II. Grune and Stratton, Inc., New York. 11. Wende, R. D., R. L. Mudd, J. M. Knox, and W. R. Holden The VDRL slide test in 322 cases of darkfield positive primary syphilis. South. Med. J. 64:

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