Syphilis. syphilis. Sera from all stages of syphilis and the. to compare dilution titers with those obtained

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1982, p /82/ $02.00/0 Vol. 15, No. 2 Unheated Serum Reagin Test as a Quantitative Test for Syphilis DEBORAH E. PETTIT, SANDRA A. LARSEN,* VICTORIA POPE, MARY W. PERRYMAN, AND MARY R. ADAMS Bacterial Immunology Branch, Bacteriology Division, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia Received 4 June 1981/Accepted 26 August 1981 The unheated serum reagin (USR) test, a standard qualitative test for syphilis, as evaluated for use as a quantitative test for the serodiagnosis of syphilis and as a means of determining the effectiveness of treatment. Tests ere performed on 664 syphilitic sera from treated and untreated patients in the primary, secondary, and latent stages of the disease. Also included ere 95 false-positive sera and 36 sera from presumably normal donors. The Venereal Disease Research Laboratory slide and rapid plasma reagin 18-mm circle card tests ere used as a basis for comparison ith the USR test. Results of the USR quantitative test paralleled the Venereal Disease Research Laboratory and rapid plasma reagin card quantitative tests ith approximately 84% agreement. The advantages of the USR test make the findings of this study significant in that: (i) less technical manipulation is required to perform the USR test than the Venereal Disease Research Laboratory slide test; (ii) no labor cost is incurred for daily Venereal Disease Research Laboratory antigen preparation; and (iii) the USR antigen is less expensive than the rapid plasma reagin card test antigen. Each year, a great many individuals are tested for syphilis in clinics and various screening programs. Because of the rising costs of nontreponemal tests for syphilis, an evaluation of the unheated serum reagin (USR) test as undertaken. Although the qualitative USR test is a standard test in the 1969 Manual of Testsfor Syphilis (1), an extensive examination of its use as a quantitative test to monitor the treatment of syphilis has not been performed until no. The USR antigen is composed of Venereal Disease Research Laboratory (VDRL) antigen containing choline chloride, hich serves to block interfering substances in the serum (4, 5) and thus eliminates the heating of the serum before testing. The antigen has been stabilized ith EDTA, thereby eliminating the necessity of daily antigen preparation and the attendant variations that could occur in the VDRL test as a result of this variable. The USR test is very similar in appearance to the VDRL test in that reactivity in both tests is gauged by the size of flocculation particles hich are examined microscopically. The USR and VDRL tests are also similar in that the degrees of reactivity are reported in the same manner, i.e., nonreactive, eakly reactive, and reactive. In this study, the tests used for comparison ith the USR test ere the VDRL slide and the rapid plasma reagin (RPR) (1) 18-mm circle card tests, to standard nontreponemal tests for syphilis. Sera from all stages of syphilis and the false-positive sera (FPS) ere tested quantitatively to compare dilution titers ith those obtained ith the VDRL and RPR card tests. The percentage of agreement beteen test titers as calculated by using identical results and titers of ±1 dilution as concordants. MATERIALS AND METHODS Serum sources. The sera for this study ere from three major sources (Table 1). A total of 315 sera from 33 patients (group I) ere used to study the effectiveness of the quantitative USR test to measure the efficacy of antibiotic schedules for the treatment of early syphilis. These sera ere obtained in 1965 as part of an earlier evaluation of various antibiotic schedules by the Venereal Disease Control Division of the Centers for Disease Control (CDC) (6) and had been frozen since that time. To evaluate the use of the USR test on routine specimens, 360 frozen sera ere obtained from the CDC Venereal Disease Serum Bank (group II), and 120 fresh sera ere obtained from a local county sexually transmitted disease clinic (group III). All sera ere categorized by stages of syphilis. The criteria for classifying a serum as false-positive ere: (i) reactivity in at least one of the three nontreponemal tests; and (ii) a nonreactive fluorescent treponemal antibody absorption result. In addition, four sera from the serum bank ere tested 15 times each as a means of establishing the repeatability of the quantitative test. These sera ere coded and randomly entered into testing each 238

2 VOL. 15, 1982 USR TEST AS A QUANTITATIVE TEST FOR SYPHILIS 239 TABLE 1. Categories of sera tested Group Category No. tested Total I. Frozen sera from treated cases of syphilis Syphilitic 315 Primary treated 165 Secondary treated 150 II. Frozen sera from Venereal Disease Serum Bank Nonsyphilitic 65 False-positive serology 65 Syphilitic 295 Primary treated 20 Primary untreated 79 Secondary treated 46 Secondary untreated 50 Latent treated 38 Latent untreated 62 III. Fresh sera from sexually transmitted disease Nonsyphilitic 66 clinic patients Seronegative 36 False-positive serology 30 Syphilitic 54 Primary 13 Secondary 9 Latent 21 Past syphilis stage unknon 11 day. Repeatability as measured as the ggreatest num- performed in accordance ith the 1969 Manual of ber of times the same result as obtained. Tests for Syphilis (1), except for the quantitative USR All sera ere divided into to parts: ( one for tests test. Each test as performed by a different individual. requiring heated sera and one for tests rrequiring un- The USR test as performed quantitatively by heated sera. diluting the serum on paraffin-ringed glass slides ith Reagents used and test procedures. VDRIL, RPR, and 0.9o saline. A 0.05-ml drop of 0.9% saline as placed USR antigens ere used in this study. All reagents in all but the first ring of the titration ith a microtitra- from the tion dropper. A 0.05-ml drop of serum as placed in used, including the antigens, ere acquijred Biological Products Division of CDC. TIie tests ere the first and second rings, thereby resulting in a 1:2 dilution in the second ring. Tofold dilutions ere performed ith a pipetting device ith disposable plastic tips. The tip of the disposable pipette as od---.c RPR immersed in the saline-serum mixture of the second R128- At --e USR ring hile the contents ere dran up and don five R64- O-OVDRL or six times. When the desired number of dilutions R32- ere made, 1 drop (0.022 ml) of USR antigen as R16- added to the ring ith an 18-gauge needle and syringe. cr R8- ~R4- The slide as rotated for 4 min at 180 rpm and then ~R2 observed for flocculation at x 100 magnification. Rli Quantitative results ere reported as the highest serum dilution producing a reactive (not eakly reac- N- tive) result. For example, a serum reactive at a 1:4,-,--,--,-,.-- dilution, but eakly reactive at a 1:8 dilution, as reported as reactive at four dilutions. Reference may MONTHS be made to the 1969 Manual of Tests for Syphilis (1) FIG. 1. Serological responses after treatment. Ab- for equipment, glassare, and quality control. Agreereactive; R, ments beteen the USR and VDRL, USR and RPR, breviations: N, Nonreactive; W, eakly ions. and RPR and VDRL tests ere evaluated. reactive. Numerals after R indicate diluti

3 240 PETTIT ET AL. J. CLIN. MICROBIOL. Rapid Plasma Reagin Card Test I- 2 R2 8 R4 o CC co St a) _ CD R8 R16 R32 R64 R128 R256 > R512 N W R1 R2 R4 R8 R16 R32 R64 R128 R256 R512 R 1024 R specimen as omitted due to contamination FIG. 2. Agreement beteen the RPR card and the VDRL tests. Concordant results are ithin the heavy lines. RESULTS The titers of the 315 sera in group I, hich consisted of patients hose treatment schedules ere folloed, ere examined in the VDRL, RPR card, and USR quantitative tests. Of the successfully treated cases, i.e., ith no evidence of reinfection or relapse, 19 ere selected to illustrate the arithmetic mean titers in all three N W R1 R2 R4 R8 N W Ri X R Re tests. The mean titers of each test before treatment (point 0) through an 18-month period are illustrated in Fig. 1. The mean titer in the USR test fell beteen that of the RPR card and the VDRL tests. After 12 months of follo-up, sera from five patients (to in the primary and three in the secondary stage of the disease) ere nonreactive as measured by all three serological tests. To patients ith primary syphilis and Rapid Plasrma Reagin Card Test E R R ~ ~ ~ FIG. 3. Agreement beteen the RPR card and the USR tests. Concordant results are ithin the heavy lines.

4 VOL. 15, 1982 USR TEST AS A QUANTITATIVE TEST FOR SYPHILIS 241 Unhated Serum Regin Test i; R1 R2 t.8 R4 U a9. 8 R3 a. R12 e= R325 it R51R 048 COR > R ER N W R1 R2 R4 R8 R16 R32 R64 R128 R256 R512 R1024 R i FIG. 4. Agreement beteen the USR and the VDRL tests. Concordant results are ithin the heavy lines. one ith secondary syphilis had nonreactive serological results ith only the RPR card test, hereas the serum from one patient as nonreactive in both the USR and VDRL test, yet remained reactive in the RPR card test. Of the 10 patients ith remaining reactive serological results, all ere at least one dilution higher in titer in the RPR card test than in either the USR or VDRL test. Excluding those sera hich ere either nonreactive in one of the to tests compared or hich gave equivocal eak reactions in the USR or VDRL test, the overall agreement ± 1 dilution of the 795 sera tested as 71.3% (357/501 reactive by both tests) beteen the VDRL and RPR tests (Fig. 2), 84.4% (421/499 reactive by both tests) beteen the USR and RPR tests (Fig. 3), and 87.3% (420/481 reactive by both tests) beteen the USR and VDRL tests (Fig. 4). With all results from group I sera, the agreement as 93% beteen the USR and VDRL tests; 73.0% beteen the VDRL and RPR tests; and 79.0% beteen the RPR and USR tests. With group II sera, hich ere obtained from the CDC serum bank, the agreement as 87.5% beteen the USR and VDRL tests; 86.6% beteen the RPR and USR tests; and 78.6% beteen the VDRL and RPR tests. With group III sera, hich ere obtained from a local health department, the agreement as 92% beteen the USR and VDRL tests; 72.5% beteen the VDRL and RPR tests; and 89.9% beteen the RPR and USR tests. When the 95 FPS ere considered as a separate category, 71.6% ere positive in the RPR card test, 64.2% ere positive in the USR test, and 85.7% ere positive in the VDRL test. Of the 65 frozen sera, no differences ere seen in the percentage of FPS reactives among the three tests. Hoever, ith the 30 FPS in fresh sera, more than tice as many of these sera ere reactive in the VDRL as in either the USR or RPR card test. Repeat testing of these sera after freezing reduced the FPS reactivity in the VDRL test to a level comparable ith that of the RPR card and USR tests. In the repeatability study (Table 2), 100% repeatability ±1 dilution as achieved. The USR test exhibited more repeatability than the VDRL or RPR test at the same dilutions; i.e., more often having identical titers on repeat testing. DISCUSSION In the past fe years, the rising cost of performing nontreponemal screening tests on a routine basis has become a matter of increasing concern. Our study indicates that the rather inexpensive USR antigen may be used successfully in the quantitative USR test procedure for the serodiagnosis of syphilis and to follo the serological responses of patients to treatment. When used as an indicator of treatment responses, more sera remained reactive, regardless of the stage of syphilis, ith the USR and VDRL

5 242 PETTIT ET AL. J. CLIN. MICROBIOL. TABLE 2. Repeatability study Serum and reactivity" Test FC44 FC48 FC52 N WR R1 N WR RI R, R128 R256 RsI2 R1024 R208 R16 R32 USR RPR VDRL 4b a N, Nonreactive; WR, eakly reactive; R, reactive. Subscript numerals indicate dilutions. b One specimen could not be tested. slide tests than ith the RPR card test. Those sera hich remained reactive in the RPR card test retained reactivity usually one dilution, or more, higher than that in the USR or VDRL test. These findings are similar to those reported by others (2, 3, 6). Based upon the results of our study, e caution against changing test methods hen monitoring the efficacy of therapy. In all three groups of sera tested, the agreement ±1 dilution beteen the USR and VDRL slide tests as consistently better than that beteen the VDRL slide and RPR card tests or the USR and RPR card tests. This greater degree of concordance beteen the USR and VDRL slide test titers can be partially accounted for by the method of reporting the quantitative test. The USR and VDRL slide tests both include a eakly reactive reading not included in the RPR card test. Both of the quantitative microscopic tests are reported as the last dilution giving a reactive result, not a eakly reactive result. Therefore, a serum reactive at 1:4 but eakly reactive at 1:8 ould be reported in both the USR and VDRL slide tests as reactive 4 dilutions. In contrast, the same serum tested in the RPR card test and giving a minimally reactive reading at 1:8 ould be reported as reactive 8 dilutions. Thus, a +1 dilution difference beteen the USR or VDRL slide test and the RPR card test is inherent in the RPR card test reporting system. With the fresh sera categorized as false-positive, the USR and RPR card tests appeared to be more specific than the VDRL test. This difference apparently disappeared once the sera ere frozen and retested, ith all tests having comparable specificity. Besides possible antigen cost reduction, the USR test offers to those ho prefer a microscopic test the advantages of: (i) the use of unheated serum; (ii) a stable antigen suspension; (iii) the elimination of variation in antigen suspensions prepared each day; and (iv) the use of leftover antigen, hich need not be discarded unless the expiration date has been exceeded or the appearance of the antigen suggests bacterial contamination. LITERATURE CITED 1. Centers for Disease Control Manual of tests for syphilis, publication 411. U.S. Department of Health, Education, and Welfare, Washington, D.C. 2. Dyckman, J. D., R. D. Wende, D. Gantenbein, and R. P. Williams Evaluation of Reagin Screen, a ne serological test for syphilis. J. Clin. Microbiol. 4: Fiumara, N. J Serologic responses to treatment of 128 patients ith late latent syphilis. Sex. Transm. Dis. 6: Lantz, M. A., and V. H. Falcone The necessity for heating sera for the VDRL slide test. Am. J. Med. Technol. 34: Portnoy, J., W. Garson, and C. A. Smith Rapid plasma reagin test for syphilis. Public Health Rep. 72: Schroeter, A. L., J. B. Lucas, E. V. Price, and V. H. Falcone Treatment for early syphilis and reactivity of serologic tests. J. Am. Med. Assoc. 221: