Evaluation of a New Rapid Plasma Reagin Card Test as a Screening Test for Syphilis

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1982, p /82/ $02.00/0 Vol. 16, No. 2 Evaluation of a New Rapid Plasma Reagin Card Test as a Screening Test for Syphilis MARY W. PERRYMAN,'* SANDRA A. LARSEN,' EDITH A. HAMBIE,' DEBORAH E. PETTIT,' REBECCA L. MULLALLY,I AND WILLIAM WHITTINGTON2 Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, 1 and Venereal Diseases Control Division, Center for Prevention Services,2 Centers for Disease Control, Atlanta, Georgia Received 22 February 1982/Accepted 6 May 1982 This study evaluates the American Dade (Biokit Laboratories) rapid plasma reagin (Dade RPR) card test, currently used in Spain for the diagnosis of syphilis, which has been recently released to the U.S. market. Used as a basis for comparison with the Dade card test were the 18-mm standard rapid plasma reagin (standard RPR) card test and the Venereal Disease Research Laboratory (VDRL) slide test, using both fresh sera obtained from 505 individuals and paired serumplasma specimens from 174 individuals. Results obtained proved the Dade RPR card test with serum to be very similar to the standard RPR card test; sensitivity was 92.3% and specificity was approximately 99% for both RPR card tests. Although the sensitivity of the VDRL slide test was lower at 88.5%, its specificity was also approximately 99%. Quantitatively, the agreement ± 1 dilution between the two card tests was 93.6%. Agreement ± 1 dilution between the Dade RPR card test and the VDRL slide test was 46.5% for sera, comparable to the standard RPR- VDRL agreement of 50%. In the limited evaluation of the RPR card tests with plasma, the specificity was 99.4% and the sensitivity was 100% for both tests. Quantitative agreement ± 1 dilution between plasma and serum pairs was 100% for the Dade RPR card test. Our results showed that the Dade RPR card test is as sensitive and as specific as the standard RPR card test. Therefore, it was concluded that one card test has no particular advantage over the other. Downloaded from The standard rapid plasma reagin (standard RPR) 18-mm circle card test developed by Portnoy was one of the first modifications of the Venereal Disease Research Laboratory (VDRL) test (3). By addition of EDTA to stabilize the VDRL antigen, choline chloride to eliminate heat inactivation of the serum, and sized charcoal particles to allow the technician to visualize the antigen-antibody reaction, the VDRL test was modified to become the RPR card test (4). Although initial studies evaluated the use of the RPR card antigen with plasma (4), thus suggesting the name, the RPR card test has been accepted as a standard test for syphilis only with sera, but not with plasma. Today in the United States, the RPR card test manufactured by Hynson, Westcott and Dunning is the most commonly used screening test for syphilis. Some of the reasons for the popularity of the RPR card test are as follows. (i) The sensitivity and specificity of the RPR card test are equal to or better than those of the VDRL test (1, 3, 4, 5). (ii) The RPR card test provides serological support for the diagnosis of syphilis at the time the patient is still in the clinic (6). (iii) Materials required for RPR card testing are easy to use, inexpensive, and expendable. Test results are read with the naked eye (1). (iv) The RPR card test can be performed in the clinic or in the field and allows expeditious, large-scale testing (3). Recently, other manufacturers have introduced reagents similar to the product by Hynson, Westcott and Dunning (2). The purpose of this study was to compare a newer RPR (Dade RPR) card test manufactured by Biokit Laboratories, Barcelona, Spain, and distributed by American Dade, Division of American Hospital Supply Corporation, Miami, Fla., with the standard RPR card test, using both plasma and sera for the serodiagnosis of syphilis. For additional comparison, all sera were also tested in the VDRL slide test, and all nontreponemal test reactivity was confirmed with the fluorescent treponemal antibody absorption (FTA-ABS) test. MATERIALS AND METHODS Specimens. Blood was obtained from 505 persons who attended the DeKalb County (Ga.) Sexually Transmitted Disease Clinic. A sample of blood from 174 of these 505 persons was immediately dispensed from a 7-ml Vacutainer tube without anticoagulant on April 9, 2018 by guest 286

2 VOL. 16, 1982 TABLE 1. Qualitative agreement among nontreponemal tests No. of specimens with indicated reaction by following test': Test and reactiona VDRL stide Dade RPR card N R W N R Standard RPR card N R VDRL slide N R 1 39 W 4 6 a N, Nonreactive; R, reactive; W, weakly reactive (considered reactive in tabulations). b Agreement was 99.0% between the standard and Dade RPR card tests, 97.8% between the VDRL slide and Dade RPR card tests, and 97.6% between the standard RPR card and VDRL slide tests. NEW RPR CARD TEST 287 used to originally obtain the blood to a 4-ml Vacutainer tube containing 6.0 mg of EDTA in the potassium form. All blood samples were centrifuged, and the serum or plasma was separated from the cells and tested within 18 h unless otherwise indicated. Samples of sera were dispensed to be heated for testing in the VDRL slide test or FTA-ABS test or both. Histories were obtained on all individuals whose serum or plasma exhibited any degree of reactivity in any of the nontreponemal tests. Reagents. Antigen for the standard RPR card test was supplied by the Biological Products Program, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Ga. A single lot of antigen was used throughout the study. Brewer diagnostic cards for the standard RPR card test, needles, dispensing vials, and stirrers were supplied by Hynson, Westcott and Dunning. The Dade RPR card antigen was supplied as part of a kit containing two 5-ml ampules of antigen; one vial each of reactive, moderate to weakly reactive, and nonreactive control sera; plastic-coated Dade RPR test cards; disposable plastic capillary tubes; rubber bulbs; wooden stirrers; a dispensing vial; and a 20- gauge needle. A single lot of antigen, 2681, was used during the study. The VDRL slide and FTA-ABS test reagents and control sera for these tests, as well as those for the standard RPR card test, were reference reagents furnished by the Biological Products Program. Testing. Each nontreponemal test was performed daily on serum or plasma by a technician who had no knowledge of the results of the other reagin tests. Test assignments were rotated among the technicians to eliminate test preference bias. Quantitative nontreponemal tests were performed on any serum or plasma exhibiting any degree of reactivity in the qualitative test. The FTA-ABS test was performed on any single serum or serum from a plasma pair which was reactive in any of the nontreponemal tests. The VDRL slide, standard RPR card, and FTA-ABS tests were performed according to the 1969 Manual of Tests for Syphilis. The Dade RPR card test was performed according to the directions of the manufacturer. Briefly, for the qualitative test, 0.05 ml of serum or plasma and 1 drop of antigen were placed in a single 18-mm circle of a 10-place, plastic-coated card. The serum and antigen were mixed and spread with a stirrer within the circle. The card, when filled, was placed under a humidifier cover and rotated for 8 min at 100 rpm. Immediately after mechanical rotation, the card was briefly rotated manually and read under a highintensity, incandescent light. The readings were reported as either "reactive" or "nonreactive." Reactive indicated a specimen showing any degree of flocculation from slight to intense with the RPR card antigen. Nonreactive indicated a specimen giving no degree of flocculation or only a slight roughness. The quantitative test was reserved for sera exhibiting any degree of reactivity or roughness. To perform the quantitative test, 0.05 ml of saline was placed in the second through fifth circles of the card, and then 0.05 ml of serum was placed in each of the first two circles. Serial twofold dilutions were prepared in the second through fifth circles. Then, the Dade RPR card antigen was dropped next into the diluted serum or plasma, and the specimen plus antigen was mixed and spread within the circle. The remainder of the quantitative procedure was exactly the same as for the qualitative procedure. Results for the quantitative test were reported as the highest dilution of serum or plasma giving any degree of reactivity. RESULTS The Dade RPR card is similar to the Brewer diagnostic card except for the color of the circles. The Dade RPR card has Kelly-green circles, which may aid in providing a contrast to the gray charcoal color of the antigen. The Dade antigen is similar in color and texture to the standard RPR card antigen. The qualitative comparisons of the results from the 505 sera tested in the Dade and standard RPR card tests and the VDRL slide test are shown in Table 1. Qualitative agreement between the Dade and standard RPR card tests was 99.0%. If the VDRL slide test results that were weakly reactive were considered as reactive, then agreement between the Dade RPR card test and the VDRL slide test was 97.8%, whereas agreement between the standard RPR card test and the VDRL slide test was 97.6%. Fifty-seven sera were reactive in at least one of the nontreponemal tests. The distribution of the reactive sera among the three nontreponemal tests and the FTA-ABS test is shown in Table 2. The agreement of the FTA-ABS test results with the standard RPR card test, the Dade RPR card test, and the VDRL slide test was 87.0% (47 of 54 results in agreement) for both RPR card tests and 77.8% (42 of 54 results in agreement) for the VDRL slide test. The three sera giving borderline FTA-ABS test results were not entered into tabulation. The diagnoses

3 288 PERRYMAN ET AL. RPR card test TABLE 2. Distribution of reactive sera among the three nontreponemal tests and the FTA-ABS test No. of sera Nontreponemal test with following pattern of reactivity No. of result by FTAsera with pattem ABS test': Dade Stan- slide R B N dard R R R N R R 1 1 N N R N R N 1 1 R R N 5 5 R N N 1 1 R N R a R, Reactive; B, borderline; N, nonreactive. associated with reactive nontreponemal tests are shown in Table 3. The sensitivity of all three nontreponemal tests, based on the 21 untreated cases of syphilis, was 95.2% (20 of 21 results in agreement). With the 31 sera from individuals with treated syphilis, both the Dade and standard RPR card tests were 90.3% reactive (28 of 31 results in agreement). On the the other hand, the sensitivity of the VDRL slide test with treated cases was 83.9% (26 of 31 results in agreement). Therefore, overall sensitivity, i.e., treated and untreated, was 92.3% for both RPR card tests and 88.5% for the VDRL slide test. Comparative specificities of the three nontreponemal tests based on the 453 sera drawn from individuals presumed not to have syphilis were 99.3% for the Dade RPR card test, 99.6% for the standard RPR card test, and 98.9% for the VDRL slide test. Quantitative agreement among the three nontreponemal tests is shown in Table 4. The results of only those sera which were reactive in both of the tests compared are tabulated. Agreement + 1 dilution (dil) between the two RPR card tests was 93.6%. Again, assuming weakly reactive VDRL slide test readings as equivalent to reactive 1 dil, then agreement ± 1 dil between the standard RPR card test and the VDRL slide test was 50%, and agreement between the Dade RPR card test and the VDRL slide test was 46.5%. Of the 174 plasma and serum pairs tested, qualitative agreement between the results with plasma and serum was 98.9% for the standard RPR card test and 99.4% for the Dade RPR card test. Agreement between the two RPR card tests when plasmas were tested was 100%, whereas agreement between the two tests for the 174 sera was 99.4%. In the standard RPR card test, 14 specimens were reactive and 158 specimens were nonreactive when either plasma or serum was used; 2 specimens were nonreactive for serum and reactive for plasma. In the Dade RPR card test, 15 specimens were reactive and 158 specimens were nonreactive when either plasma or serum was used; 1 specimen, drawn from an individual diagnosed as having treated secondary syphilis, was nonreactive for serum and reactive for plasma. A total of 16 specimens were reactive and 158 specimens were nonreactive for plasma by either test, whereas 15 specimens were reactive and 158 specimens were nonreactive for serum by either RPR card test. One specimen was reactive for serum by the TABLE 3. Diagnoses associated with sera reactive in nontreponemal tests No. of sera with indicated reaction by following testa: RPR card test Stage of syphilis VDRL slide FTA-ABS Dade Standard R N R N R W N R B N Primary Untreated Treated Secondary Untreated Treated Latent Untreated Treated Past history of syphilis J. CLIN. MICROBIOL. Other sexually transmitted diseases a R, Reactive; N, nonreactive; W, weakly reactive (considered reactive in tabulations); B, borderline.

4 VOL. 16, 1982 NEW RPR CARD TEST 289 TABLE 4. Quantitative comparisons of the three nontreponemal tests No. of indicated type of reaction (dil) obtained by following RPR card test': Comparison Dade Standard Equal Equal Dade and standard RPR card tests Dade RPR card and VDRL slide tests Standard RPR card and VDRL slide tests a Agreement ± 1 dil was 44 of 47 results in agreement (93.6%) between the Dade and standard RPR card tests, 20 of 43 results in agreement (46.5%) between the Dade RPR card and VDRL slide tests, and 22 of 44 results in agreement (50%o) between the standard RPR card and VDRL slide tests. Dade test and nonreactive for serum by the standard test. The diagnoses for the 16 reactive plasmas are shown in Table 5. Both RPR card tests were 100% sensitive with plasma. The specificity of the card tests with plasma was 99.4%, based on the 159 specimens from individuals without syphilis. Quantitative agreement + 1 dil (Table 6) between the plasma and serum pairs within the same test was 92.9% for the standard RPR card test and 100% for the Dade RPR card test. The between-test quantitative agreement with plasma was 93.8% ± 1 dil. All of the above plasma results are based on results of tests performed on plasma which had been separated from the cellular components of the blood by centrifugation. In addition to these tests, 33 of the plasmas were tested after 18 h at 25 C but before centrifugation and separation from the cellular material. The plasma portion of the unspun blood contained fibrous material that was avoided when transferring the plasma to the card. Of these plasmas, 32 gave results that were identical, even to titer, to those obtained on the plasma after centrifugation, for a repeatability of 97%. One plasma was reactive 1 dil in the Dade RPR card test before centrifugation but nonreactive in both RPR card tests after centrifugation. The serum pair of the plasma was nonreactive in all three nontreponemal tests and the FTA-ABS test. Two groups of centrifuged separated plasmas were frozen after initial testing. One group of 39 specimens was frozen for 1 week. Upon thawing, these plasmas were clear in appearance. Although retesting of the specimens gave 100% repeatable results + 1 dil, some increased negative roughness was noted in both card tests. Five plasmas were read as negative rough with the standard RPR card test, whereas three were read with this result in the Dade RPR card test. The second group of 33 specimens was retested after 3 weeks of storage at -20 C. A fibrous precipitate was seen in these plasmas. The amount of fibrous material transferred to the card was proportional to the amount of disturbance of the fibrous clot. Negative roughness was especially increased with the Dade RPR TABLE 5. Diagnosis associated with plasmas reactive in the RPR card tests No. of specimens with indicated reaction by following test': RPR Card Test Stage of syphilis FTA-ABS Dade Standard R N R N R B N Primary Untreated Treated Secondary Untreated Treated Latent Untreated Treated Other sexually transmitted diseases 1 1 a R, Reactive; N, nonreactive; B, borderline.

5 290 PERRYMAN ET AL. J. CLIN. MICROBIOL. TABLE 6. Quantitative comparison No. of indicated type of reaction (dil)a Comparison Serum Dade RPR card test -2-1 Equal Equal +1 Serum and plasma in standard RPR card test Serum and plasma in Dade RPR card test Plasma in Dade and in standard RPR card tests a Agreement ± 1 dil was 13 of 14 results in agreement (92.9%) between serum and plasma in the standard RPR card test, 15 of 15 results in agreement (100%) between serum and plasma in the Dade RPR card test, and 15 of 16 results in agreement (93.8%) between plasma in the Dade and in the standard RPR card test. card test. Repeatability for the Dade card test was 100%, whereas that for the standard RPR card test was 97%. One sample, which had been reactive 1 dil in both RPR card tests with the fresh plasma, was reported as nonreactive in the standard test on repeat after freezing. This was the same plasma whose serum pair had been nonreactive in both the standard RPR card and the Dade RPR card tests. DISCUSSION Our evaluation indicates the Dade RPR card test to be as sensitive and as specific as the standard RPR card test for the serodiagnosis of syphilis. Since one card test has no particular advantage over the other, the choice of product will be a matter of personal preference. In a previous evaluation of the use of EDTA plasma in the standard RPR card test, we found that the plasma may be stored at either 25 or 4 C for up to 24 h without an increase in negative roughness. Results from the current study with plasma indicated that (i) when testing plasma which has not been centrifuged or separated or both from the cellular constitutents, care must be taken to avoid the fibrous material in the plasma portion; and (ii) separated plasma may be frozen for up to 1 week without the formation of an additional fibrous clot. Only plasma from blood drawn with EDTA as the anticoagulant has been evaluated in the RPR card tests; therefore, we caution against the testing of plasma when the anticoagulant used is other than EDTA or is unknown. The use of plasma to follow the efficacy of treatment or in the treponemal tests has not been evaluated. If a plasma is reactive in the RPR card test and confirmation is necessary, then we suggest that a serum be drawn, the quantitative RPR card test be repeated with the serum, and a treponemal test be performed. LITERATURE CITED 1. Falcone, V. H., G. W. Stout, and M. B. Moore, Jr Evaluation of rapid plasma reagin (circle) card test. Public Health Rep. 79: March, R., and G. E. Stiles The reagin screen test: a new reagin card test for syphilis. Sex. Transm. Dis. 7: Portnoy, J Modification of the rapid plasma reagin (RPR) card test for syphilis for use in large scale testing. Am. J. Clin. Pathol. 40: Portnoy, J., W. Garson, and C. A. Smith Rapid plasma reagin test for syphilis. Public Health Rep. 72: Reed, E. L The rapid plasma reagin (circle) card test for syphilis as a routine screening procedure. Public Health Lab. 23: Tiedemann, J. H., and J. Mullins The RPR card test as a diagnostic aid in a venereal disease clinic. Public Health Lab. 24:12-15.