Review of the Standard Tests for Syphilis and Evaluation of a New Commercial ELISA, the Syphilis Bio-EnzaBead Test

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1 Journal of Clinical Laboratory Analysis 1 : (1987) Review of the Standard Tests for Syphilis and Evaluation of a New Commercial ELISA, the Syphilis Bio-EnzaBead Test Sandra A. Larsen, Edith A. Hambie, and Donna D. Cruce Treponema Research Branch, Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia To date, there are eight serologic tests rable to the sensitivity of the microhemagconsidered Standard for the serodiagnosis glutination assay (MHA-TP) of 97.6% in of syphilis; however, the syphilis Bio- untreated syphilis. The specificity of the EnzaBead test is the first commercially syphilis Bio-EnzaBead when read as deavailable enzyme-linked immunosorbent scribed above is 94.7%, compared with assay (ELISA) for the confirmation of a re- the 99.3% of the MHA-TP and the 98.1% active nontreponemal test result in the se- of the fluorescent treponemal antibody-abrodiagnosis of syphilis. The Bio-EnzaBead sorption (FTA-ABS) test. Heating of the has a sensitivity of 95.2% in untreated specimens did not adversely affect the test syphilis when read at 405 nm with 80% of outcome, and it eliminated background the 1 + optical density (OD) used as the readings seen with the negative control breakpoint OD between reactive and non- beads. reactive results. This sensitivity is compa- Key words: FTA-ABS, MHA-TP, nontreponemal syphilis tests, treponemal syphilis tests, Treponema pallidurn INTRODUCTION The first attempt to standardize the tests for syphilis began in 1935 and culminated in the 1939 Manual of Tests for Syphilis (1). Through the subsequent years, the number of tests; their basic technology, e.g., complement fixation, flocculation, etc. ; and their general application have varied. To date, two categories of serodiagnostic tests for syphilis exist: nontreponemal and treponemal. The nontreponemal tests measure antilipid antibodies formed by the host in response to lipoidal material released from damaged host cells early in infection as well as to lipid from the treponeme itself. Although the nontreponemal test antigens have been refined since the inception of the flocculation tests, these tests are still biologically nonspecific. The treponemal tests, in contrast, measure specific antibodies formed by the host in response to immunogenic determinants on the treponemes. Even though these antibodies are relatively specific for the genus Treponema, they are not specific for the various species and subspecies. For example, the current treponemal tests cannot distinguish antibodies produced in response to T. pallidurn subspecies pallidurn from those produced in response to T. pallidurn subspecies pertenue, the causative agent of yaws. Currently, four nontreponemal tests are available commercially in the United States, the veneral disease research Alan R. Liss, Inc. laboratory (VDRL) slide (2), the unheated serum reagin (USR) (2,3), the rapid plasma reagin (RPR) 18 mm circle card (4), and the reagin screen test (RST) (5). All four nontreponemal tests can be used as screening tests and to follow the efficacy of treatment (3,6), yet each test has its own particular advantage. The VDRL slide test, the oldest of the currently used nontreponemal tests, has the fewest advantages. The VDRL is a microscopic test and requires a fresh antigen suspension to be made daily. However, the VDRL slide test antigen is inexpensive, and the test is still the only test recommended for use with cerebrospinal fluid (CSF) for the diagnosis of neurosyphilis (7). The major advantage the USR has over the VDRL is that the USR antigen is stabilized, so the need for daily antigen suspension preparation is eliminated. The USR has not been evaluated as a test for neurosyphilis. A second advantage to the USR, as implied by the name, is that unheated sera can be used in the test. The RPR card test and the RST have the same advantages as the USR along with what Received March 20, 1987; accepted March 22, Address reprint requests to Dr. Sandra A. Larsen, Treponema Research Branch, Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA

2 Tests for Syphilis 301 some consider the additional advantage of being macroscopic tests. The RPR card test and the RST differ only in the manner in which a reactive serum is visualized. The RPR card test uses a sized charcoal particle which becomes entrapped in the antigen-antibody lattice formation of a reactive specimen, whereas the antigen of the RST is actually dyed with a lipid-soluble diazo dye, Sudan black B. Although the RST has not been evaluated with CSF, the RPR card test has been found to give false-positive results with CSF from disease cases with possible neurologic involvement other than neurosyphilis at a rate of 14% (139 of 993) (7). All the nontreponemal tests are quite similar in sensitivity and specificity, since all antigens are derived from the VDRL antigen that contains 0.03 % cardiolipin, 0.9% cholesterol, and sufficient lecithin to produce standard reactivity. In a series of studies conducted at the Centers for Disease Control (CDC) with consecutive attendees at the DeKalb County, Georgia, Sexually Transmitted Diseases (STD) Clinic, specificity of nontreponemal tests ranged from 93% to 99% (8). However, the specificity of the nontreponemal tests can vary immensely depending on the patient population examined. Acute false-positive reactions lasting less than 6 months may occur after febrile diseases or during pregnancy and can be associated with IV narcotic use. Chronic false-positive reactions are often associated with the autoimmune diseases and chronic infections such as leprosy. Sensitivity in our studies varied, depending on the stage of syphilis, from a low of 72 % in primary syphilis to a high of 100 % in secondary syphilis (8). The first serologic treponemal test was the T. pallidurn immobilization (TPI) test developed in 1949 (9). Today, the TPI test has been replaced with less cumbersome, less expensive, more sensitive, and equally specific treponemal tests, such as the fluorescent treponemal antibody absorption (FTA-ABS) test (2), the FTA-ABS double staining (DS) test (lo), the microhemagglutination assay for antibodies to T. pallidurn (MHA-TP) (1 l), the hemagglutination treponema1 test for syphilis (HATTS) (12), and an enzyme-linked immunosorbent assay (ELISA) method to be discussed in full below. All the treponemal tests use T. pallidurn subspecies pallidurn as the antigen, all are based on the detection of antibodies directed against cellular components, and all are designed as qualitative confirmatory tests. As with the nontreponemal tests, each of the treponemal tests has its own particular advantage. The most widely used treponemal test is the FTA-ABS, an indirect immunofluorescent antibody test. The test is extremely sensitive, more so than the other treponemal tests, and especially so in early syphilis (8). However, quality control is critical to the reliability and reproducibility of the results. With the change in the reporting system for the FTA- ABS test results (13,14) the specificity of the test increased, as did the positive predictive value. The FTA-ABS DS test is a modification of the FTA-ABS test developed specifically for use with incident illumination microscopes. The use of an antitreponeme conjugate permits visualization of the organisms without the use of a dark field condensor and transmitted light. Location of organism is especially important when a nonreactive specimen is being tested to ensure that antigen is actually present on the slide and that the specimen is nonreactive because of lack of antibodies, not lack of antigen on the slide. The HATTS and MHA-TP tests are similar to each other and differ mainly in the source of erythrocytes used as carriers for the sonicate of T. pallidurn. The hemagglutination tests lend themselves to testing of large numbers of samples since they are simple to set up and read. Packaged as kits, quality control is not as critical for the hemagglutination tests as for the multicomponent FTA-ABS tests. The enzyme immunoassay methods have been heralded as the model test systems for present-day immunologic procedures. For a number of diseases and/or conditions the ELISA offers the sensitivity of the radioimmunoassay without the hazard or expense. The first application of the ELISA to the area of syphilis serology was in 1975 (15). Since that time, several ELISA methods for syphilis have been described ( 16-22). In 1984, the first commercially available ELISA technique for the serodiagnosis of syphilis received Provisional Status from the CDC. This test, the syphilis Bio-Enzabead, developed by Litton Bionetics, now a part of Organon Teknika Corporation, uses T. pallidurn subspecies pallidurn (Nichols strain) as the antigen fixed to ferrous metal beads (23). Control beads coated with normal rabbit tissue extract are used to determine nonspecific reactions. The detection system consists of horseradish peroxidase-conjugated antihuman IgG as the second antibody and 2,2'-azino- di(3-ethyl-2,3dihydro-6-benzthiaoline-sulfonate) (ABTS) as the enzyme substrate. In an initial evaluation by Stevens and Schmitt (24), the syphilis Bio-EnzaBead when read visually was found to be comparable to the FTA-ABS test. A major feature of the ELISA techniques is their adaptability to automated spectrophotometric read-out. With spectrophotometric readings, the subjectivity of the technician, often a part of the current immunofluorescent and hemagglutination tests for syphilis, is eliminated. Recently, Moyer et al. (25) evaluated the Bio-EnzaBead test using the spectrophotometer available from Organon Teknika Corporation and a wavelength of 690 nm. These investigators found the results with the Bio-EnzaBead test comparable to those obtained with the FTA-ABS test. The study presented here compares the syphilis Bio- EnzaBead test results read both visually and with the Dynatech Microelisa Reader (MR580) at a wavelength of 405 nm with results obtained in the RPR 18 mm circle card test, the FTA-ABS test, and the MHA-TP test. The results of these tests are also compared with patient history and treatment status to determine the sensitivity and specificity of the tests.

3 302 Larsen, Hambie, and Cruce TABLE 1. Determination of Wavelength for Reading Syphilis Bio-EnzaBead Test Results on Dvnatech MR580 Wavelength (nm) and breakooint ODs I + 8O%l+ I+ 80%1+ I+ 8O%l+ Nonsyphilis sera No. reactive No. nonreactive I Syphilis sera No. reactive I No. nonreactive % Specificity % Sensitivity TABLE 2. Reproducibility of Reading Methods Reading method Visible OD 1+ OD 80% I + Pair Ra N R N R N Cateeorv Treated early latent Nonsyphilis Treated late latent Treated secondary Nonsyphilis Treated secondary Treated secondary Nonsyphilis Nonsyphilis Treated primary Nonsyphilis Untreated secondary Treated secondary Nonsyphilis Treated primary Treated secondary I0 Nonsyphilis Treated secondary Nonsyphilis Treated early latent R = reactive; N = nonreactive. Since the Bio-EnzaBead test is designed to be confirmatory for syphilis and sera may be heated for the screening tests, the effect on results of the use of heated and unheated sera was determined. As with most clinical tests, the Bio- EnzaBead will be included in proficiency testing programs; therefore, the effect of pooled specimens as used in the programs on the outcome of results was investigated. MATERIALS AND METHODS Sera A total of 680 sera were used to compare the syphilis Bio- EnzaBead test with the FTA-ABS, MHA-TP, and RPR card tests. One hundred twenty-one specimens were frozen specimens from the Sexually Transmitted Diseases Laboratory Program s (CDC) serum bank; the remaining 559 speci- mens were obtained from individuals attending the DeKalb County, Georgia, Sexually Transmitted Disease Clinic. Of the 680 sera tested, 430 were from persons without a history of syphilis, 46 were from persons with primary syphilis, 83 were from those with secondary syphilis, 87 were from those with early latent syphilis of less than 1 year s duration, and 34 were from those with late latent syphilis. In addition, 35 individual sera not included in the above 680 were used to evaluate the effect of heating on test outcome, and another 74 specimens prepared from pooled sera were used to assess the effect of these specimens on test outcome. Testing The MHA-TP test (11) was performed with Sera-Tek kits (Fujirebio, Inc., Tokyo, Japan; distributed by Ames Divi-

4 ~~ Tests for Syphilis 303 TABLE 3. Test Results for Sera From 430 Patients Without Syphilis RPR Card FTA-ABS MHA-TP Bio-EnzaBead Visual OD 1+ OD 80% I f Category R N R N R N R N R N R N DOTS Biologic false positives Nonsyphilis Soecificitv Diseases other than syphilis. sion, Miles Laboratories, Inc., Elkhart, IN) according to the manufacturer s directions. The FTA-ABS and RPR card tests were performed according to the 1969 Manual of Tests for Syphilis (2) with reagents produced at the CDC. The syphilis Bio-EnzaBead test was performed with kits supplied by the manufacturer (Organon Teknika Corporation, Durham, NC) and according to the directions provided. Briefly, sera to be tested were diluted 1 : 100 in test diluent, and 0.2 ml was placed into microtitration tray wells in duplicate. Reconstituted control sera (low-positive (1 +), high-positive (4 +), and negative) were also placed into designated wells of the microtiter plate. Positive and negative control beads were placed into the wells of an empty plate and then transferred with a magnetic transfer device into each well of the plate containing test sera and controls. The plate was covered with a plastic cover sheet and incubated for 90 min at 37 C. After serum incubation, the beads were thoroughly washed to remove unbound serum proteins and transferred with a magnetic device to a plate containing 0.2 ml conjugated goat antihuman IgG labeled with horseradish peroxidase (Enzabody). The plate containing Enzabody and beads was covered and incubated for 90 min at 37 C. After incubation of beads in Enzabody, the beads were washed and transferred with a magnetic device to a plate containing 0.2 ml ABTS substrate and incubated at room temperature (20-25 C) for 10 min. Plates were not disturbed during the incubation of ABTS substrate and beads. Twenty-five microliters of Stop solution, which contains 1.25 % sodium fluoride, was added to wells of each plate after incubation to stop the color reaction, the plates were gently swirled, and the beads were removed. Tests were read within 2 hr. Visual readings were reported as either reactive or nonreactive. A positive reaction was indicated by a green coloration that developed after the substrate incubation period. Degrees of green color intensities ranged from 1 + to 4 +. A negative reaction was indicated by no color or only slight coloration. The Dynatech MR580 Microelisa reader was used for spectrophotometric readings. Initially, 126 sera from persons without syphilis and 134 sera from patients in various stages of syphilis were used to determine the proper wavelength of 405 nm. Other wavelengths evaluated were 490 and 570 nm. After selecting 405 nm as the wavelength of choice, 20 serum pairs (40 specimens) were tested five times per specimen or 10 times per pair to determine the reproducibility of the reading methods. These tests were performed over a 5 day testing period. An analysis of variance of the optical density (OD) of the low positive 1 + control was computed to determine the control limits. To determine the OD breakpoint between a nonreactive and reactive result, we placed the 1 + control in the run at least four times, and the ODs of the 1 + control sera minus the control bead ODs for the 1+ control were averaged. Any serum with an OD greater or equal to that of the 1 + control was recorded as reactive. Additionally, test results were read based on 80% of the mean OD of the 1 + control serum tested four times. Again, any test serum with an OD of greater than or equal to 80% of the l+ control serum mean was considered as reactive. RESULTS For two of the three lots of syphilis Bio-EnzaBead used in this study, the mean OD of the 1 + control serum at 405 nm was f Ninety-five percent confidence levels were set at ODs of and For an individual run to be considered as satisfactory or in control, the mean OD of the 1 + control serum had to fall between ODs of and For the third lot, the mean OD of the 1 + control serum was 0.120, with 95% confidence levels of and Twice during the testing period of 2 years, the Bio- EnzaBead 1+ control mean fell outside the satisfactory range. Both times, the working dilutions of the reagents were at or near the indicated stability expiration time. The sensitivities and specificities of the test read at wavelengths of 405,490, and 570 nm were basically comparable (Table 1). We selected the 405 nm wavelength, since unsensitized control beads ODs were higher at the higher wavelengths, i.e., for 405 nm, for 490 nm, and for 570 nm. Results of the reproducibility testing indicated that use of 80% of the 1 + control OD as the breakpoint between reactive and nonreactive results gave the most consistent results of the three reading methods. These results also agreed more closely with the original diagnosis of the patient (Table 2). For the visible reading method, agreement with diagnosis was 88% (176/200), whereas agreement with diagnosis was 85% (170/200) for the OD 1 + method and

5 304 Larsen, Hambie, and Cruce TABLE 4. Test Results for Sera From 250 Patients With Treated or Untreated Syphilis Bio-EnzaBead - RPR Card FTA-ABS MHA-TP Visual OD I+ OD 80% I+ ~~ Category R N R N R N R N R N R N Primary (n =46) Untreated Treated Secondary (n = 83) Untrcatcd Treated Early latent (n=87) Untreated I Treated I Late latent (n=34) Untrcated I1 2 Treated n = total number tested in each category. TABLE 5. Serologic Results for 74 Pooled Samples Tested Unheated and Heated With the Bio-EnzaBead Test Unheated Bio-EnzaBead Hcated RPR ~- FTA-ABS Visual OD I+ OD 80% If Visual OD I+ OD 80% I+ R N R N R N R N R N R N R N R N Number % ( ) for the method using 80% of the OD of the I+ control. Results obtained with sera from patients without syphilis are shown in Table 3. Those sera classified as biologic false-positives (BFPs) were classified originally on the basis of past reactivity in the VDRL slide test (2). The specificities of the MHA-TP and FTA-ABS tests were 99.3% and 98.1 %, respectively, for the 430 sera examined, whereas, of the three methods of reading the Bio-EnzaBead, a bredkpoint of 80% of the 1 + OD proved to be the least specific at 94.7% specificity. Decreasing the breakpoint OD to 80% of the 1 + control OD increased the nonspecificity in the category of nonsyphilis in particular. With the 1 + control OD as the breakpoint between reactive and nonreactive, specificity was 97.1 % for this category in contrast to the 95 % specificity with 80% of the 1 + control OD as the breakpoint OD. For sera in the category of diseases other than syphilis (DOTS), decreasing the OD breakpoint did not affect the specificity (73.3 %); however, the Bio-EnzaBead specificity was significantly lower in this category than either the MHA- TP (93.3%) or the FTA-ABS test (100%). Of the 250 sera from patients with syphilis (Table 4), 83 were from patients who had no history of prior treatment. Of these 83, the MHA-TP test failed to detect two cases, for a sensitivity of 97.6% in untreated syphilis. In comparison, both the Bio-EnzaBead read visually and with the 1 + con- trol serum OD as the breakpoint failed to detect four cases of untreated syphilis, for a sensitivity of 95.2%. Three of the four cases were from the late latent category and were nonreactive on the RPR card test as well. The sensitivity increased to 96.4% when 80% of the 1 + control OD was used as the breakpoint. The FTA-ABS test was the most sensitive of the tests in untreated syphilis (98.8%). The sensitivities of the Bio-EnzaBead test in treated syphilis (167 cases) more closely resembled the sensitivity of the RPR card test (86.2%) than either the MHA-TP (98.2%) or the FTA-ABS test (99.4 %). The sensitivities in treated syphilis, based on the reading method of the Bio-EnzaBead test, were visually 90.4%, read with the OD of the 1 + control as the breakpoint 88.6%, and read at 80% of the 1 + control OD 92.2%. Of the 74 pooled specimens tested both heated and unheated, 39 were reactive in the FTA-ABS test regardless of whether the samples were tested before or after heating, whereas the remaining 35 specimens were consistently nonreactive regardless of heat treatment. A comparison of results obtained with heated and unheated samples in the Bio- EnzaBead test is shown in Table 5. Heating these specimens did not adversely affect the outcome of the tests. In fact, results with heated specimens more closely resembled the results obtained with the FTA-ABS test than did the Bio- EnzaBead results with unheated specimens. The percentage of agreement between tests and reading methods with heated

6 ~ Visual ~~ ~ and unheated sera is shown in Table 6. The highest percentage of agreement was within the Bio-EnzaBead test between heated samples read at the 1 + control OD and unheated samples read at 80% of the 1 + control OD. The 35 specimens nonreactive in the FTA-ABS test were also nonreactive in the Bio-EnzaBead test regardless of heat treatment. Thus, when evaluating the effect of heating on fresh specimens, only specimens that were reactive in at least one serologic test were used. In Table 7 the serologic results of the 35 individual sera used to study the effect of heating on test outcome are shown. As in the major portion of the study, the Bio-EnzaBead test was less sensitive than the FTA-ABS test in treated syphilis. In this portion of the study, the FTA-ABS test was 100% sensitive for both heated and unheated sera, whereas the Bio-EnzaBead test detected only ( X) of the 27 treated cases depending on the method used to read the test results. Heating the sera had no significant effect on test outcome. DISCUSSION Although we found a slight problem with reagent stability, Burdash et a!. (26), who did a systematic reagent stability study as a part of their evaluation, found the reagents to be stable when reconstituted and stored as directed by Organon TABLE 6. Percentage of Agreement Among Tests and Reading Methods Between Heated and Unheated Pooled Samples Unheated samples Bio-EnzaBead Results with FTA-ABS Visual OD 1 + OD 80% 1 + Heated samples FTA-ABS Bio-EnzaBead (visual) OD I OD 80% Tests for Syphilis 305 Teknika. Our problem may have been related to the actual age of the kit, since the kits were near their expiration date and the reagents had been reconstituted for at least 21 days. Since the diagnosis of latent syphilis is based solely on serologic results and patient history, the sensitivity of 96.4% seen in the Bio-EnzaBead test and the MHA-TP test in untreated early latent syphilis may not represent a case missed by these tests but rather false-positive results in the RPR card and FTA-ABS tests. Conversely, the one case missed in untreated late latent syphilis by the FTA-ABS test and reported reactive solely by the MHA-TP test may represent a false positive in the MHA-TP test. If this is actually the case, then the sensitivity of the Bio-EnzaBead in this category is increased to 83.3% for the visual and OD 1 + readings and to 91.7% for the reading based on 80% of the 1 + control OD. A sensitivity of the Bio-EnzaBead ELISA of 91.7% in latent syphilis is comparable to the sensitivities in that category seen in ELISA procedures to detect T. pallidurn antibodies developed in our laboratory, which ranged from 94% (21) to 95 % (19). The lack of sensitivity of the Bio-EnzaBead test in treated syphilis should not be considered a disadvantage to the use of the test at this point in its evaluation. Theoretically, one would like both nontreponemal and treponemal antibodies to disappear after the patient is adequately treated. If this should occur, then diagnosis of reinfection would be clear-cut, as would the need for retreatment. The length of time between treatment and a nonreactive Bio-EnzaBead test result has not been established. The decrease in specificity to 94.5% seen when the breakpoint OD was lowered to 80% of the OD of the 1 + control is of concern for those cases when the nontreponemal test result may also be false-positive. In our study, this did not occur. Therefore, if the syphilis Bio-EnzaBead test is used as the CDC recommends for other treponemal tests, i.e., when the nontreponemal test is reactive, unless late latent syphilis is suspected, then the decrease in specificity should not present a problem. TABLE 7. Serologic Results for 35 Individual Sera Tested Both Unheated and Heated Bio-EnzaBead FTA- Unheated Heated RPR card ABS OD I+ OD 80% I + Visual _ OD _ I + OD _ 80% I f Category R N R N R N R N R N R N R N R N Primary untreated I Primary treated Secondary untreated I 1 I I 1 Secondary treated Early latent untreated Early latent treatcd I I Late latent untreated Late latent treated I Biologic false Biologic false positives

7 306 Larsen, Hambie, and Cruce TABLE 8. Percentage of Sensitivities and Specificities of the Bio-EnzaBead ELISA as Reported by Moyer et al. (25), Burdash et al. (26), and Larsen et al. (this study) Moyer et al. Burdash et al." Larsen et al. 405 nm Bio-EnzaBead RXD 405 nm' Category 690 nm Visual FTA-ABS 690 nm 405 nm Visual OD1 + OD 80% 1 + Visual MHA-TP FTA-ABS Primary Secondary Early latent Late latent Late Suecificities 'Based on specimens reactive in both RPR card and FTA-AES tests. bmean OD of the nonreactive control run three times to Although the instructions provided by the manufacturer state that serum inactivation is not required, heating of test specimens, whether individual or pooled, had no adverse effect on test outcome. Background/control bead levels were reduced in some cases with the heated specimens, thereby increasing the OD of specimen minus OD of control bead reading and thus leading to a reactive result rather than the nonreactive result reported with the unheated specimen. Additionally, heating of the specimen offers one more assurance against the possible exposure to human immunodeficiency virus. The Bio-EnzaBead study presented here was undertaken in part to establish the syphilis Bio-EnzaBead test as a Standard test for syphilis as designated by the CDC. A test that has achieved Standard Status has a specific written technique and has undergone independent, large-scale evaluations sufficient to prove that the test is as useful for medical management decisions as other Standard Status tests. This implies that the test must be at least as specific and as sensitive as the Standard tests to which it has been compared. Evaluations of the Bio-EnzaBead test have been conducted by Stevens and Schmitt (24), Moyer et al. (25), and Burdash et al. (26) in addition to our study. Each of these studies has approached the evaluations of the Bio-EnzaBead in a slightly different manner. The Stevens and Schmitt study (24) and an early evaluation in our laboratory were the basis on which the syphilis Bio-EnzaBead test achieved the Provisional Status (Reagents Evaluation Program Supplement No 28, 1984 CDC, Atlanta, GA) as a visually read test. The Moyer et al. study (25) compared results obtained with the Bio-EnzaBead test read visually with those obtained using the spectrophotometric reading method suggested by Organon Teknika. The Burdash et al. study (26) compared the results obtained with the Bio-EnzaBead test read visually with those obtained spectrophotometrically at wavelenghs of 405 and 690 nm; in this study, various methods for determining the OD to be used to separate reactive results from nonreactive results were also reported. In Table 8, the results of the Moyer et al. (25) and Burdash et al. (26) studies are compared with the results we obtained with sera from untreated cases of syph- ilis. Neither the Moyer et al. nor the Burdash et al. study separated results on the basis of treatment. As is indicated in Table 8, the sensitivity of the Bio-EnzaBead test read spectrophotometrically approaches the sensitivity of the FTA- ABS test. However, before the Organon Teknika syphilis Bio-EnzaBead ELISA can attain Standard Status, the method of reading the test must be finalized. As the test now stands, it may be used for medical management decisions, but it must be shown to give results comparable to those of the Standard Status treponemal tests that it replaces in a particular laboratory. REFERENCES 1. Public Health Service: Manual of Tests for syphilis J Vener Dis Inform (Suppl9) National Communicable Disease Center: Manual of Tests for syphilis. Atlanta; DHEW Publ. No. PHS 411, Pettit DE, Larsen SA, Pope V, Perryman MW, Adams MR: Unheated serum reagin test as a quantitative test for syphilis. J Clin Microbiol 15:238, Portnoy J, Brewer JH, Harris A: Rapid plasma reagin card test for syphilis and other treponematosis. Pub Health Reg 77545, March RW, Stiles GE: The reagin screen test: A new reagin card test for syphilis. Sex Transm Dis 7:66, Brown ST, Zaidi A, Larsen SA, Reynolds GH: Serological response to syphilis treatment a new analysis of old data. 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