Percoll-purified Treponema pallidum was evaluated as a fluorescent treponemal antibody-absorbed antigen. Borderline and false-positive reactions were essentially eliminated, resulting in sensitivity and specificity of 100 and 95.5%, respectively. The lack of background debris improved the ease and speed of reading the test

Fernandez, Conchita

Folds, James D.

Hanff, Philip A.

Carolina Digital Repository

Vol. 23, No. 5JOURNAL OF CLINICAL MICROBIOLOGY, May 1986, p. 980-9820095-1137/86/050980-03$02.00/0Copyright X) 1986, American Society for MicrobiologyPercoll-Purified Treponema pallidum, an Improved FluorescentTreponemal Antibody-Absorbed AntigenPHILIP A. HANFF,lt* CONCHITA FERNANDEZ,'t AND JAMES D. FOLDS' 2Clinical Microbiology and Immunology Laboratories, Division of Laboratory Medicine, North Carolina MemorialHospital,' and Department of Microbiology and Immunology, University of North Carolina,2 Chapel Hill,North Carolina 27514Received 19 September 1985/Accepted 13 January 1986Percoll-purified Treponema pallidum was evaluated as a fluorescent treponemal antibody-absorbed antigen.Borderline and false-positive reactions were essentially eliminated, resulting in sensitivity and specificity of 100and 95.5%, respectively. The lack of background debris improved the ease and speed of reading the test.Since the introduction of the fluorescent treponemal anti-body (FTA) test for the serodiagnosis of syphilis (2), thepreparation of Treponema pallidum antigen from infectedrabbit testicular tissue has not substantially changed. This isdespite the development of the hemagglutination (15, 17, 18,22) and enzyme immunoassay (10, 14, 20) tests and modifi-cations to the FTA test (7, 9, 11). The laclk of change hasbeen due, in part, to the difficulties associated with obtaininglarge quantities of purified antigen that are free of contami-nating host testicular tissue and retain morphological char-acteristics and immunological reactivity.Although Hunter et al. (7) standardized methodology forpreparing FTA-absorbed (ABS) antigen that was free ofrabbit globulin, sensitive techniques revealed that these T.pallidum preparations were still contaminated with rabbittesticular material (5). The development of the continuousparticle electrophoresis method for separation of T. pallidumfrom testicular material (16) resulted in the availability of apurified antigen for use in the FTA-ABS test. This continu-ous particle electrophoresis antigen is no longer available,and its preparation was technically difficult and expensive.Furthermore, it was a low-yield procedure that resulted innonmotile, avirulent, and ultrastructurally damaged orga-nisms (5, 16). The possibility exists, therefore, that antigenicchanges accompanying cell death were responsible for lot-to-lot variability in FTA-ABS reactivity.A new, rapid purification technique utilizing Percoll den-sity gradient centrifugation has been described recently (5).The organisms were shown to be ultrastructurally intact,motile, virulent, and free of detectable soluble and insolublehost testicular material. In this report we evaluate the use ofPercoll-purified T. pallidum as an FTA-ABS antigen. Theevidence presented suggests that the purity and quality ofthe T. pallidum antigen preparation may have a direct effecton the occurrence of equivocal and borderline reactions.T. pallidum, Nichols strain, was maintained withoutcorticosteroids and harvested in FTA-ABS buffer (CSI, Inc.,Whippany, N.J.) as previously described (5). To increasebacterial yields for preparation of FTA-ABS antigen, rabbitswere given cortisone acetate from days 3 to 10 postinfection(19). Soluble and insoluble host testicular tissue was re-* Corresponding author.t Present address: Clinical Microbiology, Beth Israel Hospital,Boston, MA 02215.t Present address: Cutter Biological, P. 0. Box 1986, Berkeley,CA 94701.moved from treponemal suspensions by low-speed differen-tial centrifugation followed by density gradient centrifuga-tion through Percoll (Pharmacia Fine Chemicals,Piscataway, N.J.) as described elsewhere (5). Percoll wasremoved from the purified treponemes by centrifugation at100,000 x g for 1 h at 4°C (5), and the organisms were diluted1:20 in lyophilization buffer (8) and maintained at 4°C for 10days. Samples (0.5 ml) were lyophilized and stored at 4°Cuntil used (8). Purified T. pallidum antigen was reconstitutedin 0.5 ml of double-distilled water and adjusted to a workingconcentration in FTA-ABS buffer. Before use, 10-well slides(Carlson Scientific, Peotone, Ill.) were washed in 7x deter-gent, rinsed in water, soaked in 95% ethanol, and allowed toair dry. Ten microliters of the treponemal suspension wasplaced in each well and allowed to air dry in a laminar-flowhood. Antigen-coated slides were fixed in acetone for 10 minand stored at -20°C (21). Antigen testing and titration-of thefluorescein-conjugated anti-human globulih (BBL Microbiol-ogy Systems, Cockeysville, Md.) were performed as de-scribed (21). The reactive control serum and a total of 122diagnostically and serologically well-characterized sera frompatients with syphilis and from patients with false-positive ornonspecific fluoresence in the FTA-ABS test were gener-ously provided by Sandra Larsen (Treponemal ResearchBranch, Sexually Transmitted Disease Laboratory Program,Center for Infectious Disease, Centers for Disease Control[CDC]). The sera were selected for use on the basis of theirFTA-ABS reactivity. Approximately 20 sera each werecategorized as nonreactive, borderline, or 1-, 2-, 3-, and4-plus reactive. The FTA-ABS test was performed as de-scribed (21) and interpreted in a double-blind fashion by twoexperienced readers. A positive test was considered to be aresult of 1 to 4+. A typical 3+ reaction is shown in Fig. 1.Borderline reactions were noted but interpreted as negative(1).The FTA-ABS test with the purified antigen (FTA-P) andthe test performed at the CDC (FTA-CDC) detected 77 truepositives (Table 1). False-positives occurred with both tests,but it is important to note that the false-positive results wereobtained with different sera. It also appears that the ability ofeach test to correctly identify true negatives (normals andindividuals with diseases other than syphilis) was similar.A significant difference in the number of borderline reac-tions was observed, however. A total of 21 borderlinereactions occurred with the FTA-CDC test (Table 1). Thisincluded 3 patients with treated latent syphilis, 1 withcongenital syphilis, 3 false-positives, and 14 normal individ-980TABLE 1. FTA-ABS testing with Percoll-purified T. pallidumNo. of sera by testa:Diagnostic FTA-CDC FTA-PcategoryReactiveb Borderline Nonreactive Reactiveb Borderline NonreactiveSyphilisc 77 4 0 77 1 3Other diseases 0 0 20 0 0 20Normal 0 14 0 2 1 11False-positive 4 3 0 0 0 7a FTA-ABS testing performed at North Carolina Memorial Hospital with Percoll-purified T. pallidum (FTA-P) or by CDC with nonpurified antigen (FTA-CDC).b Reactive, >1 + reaction.c The diagnosis of syphilis was based on detailed historical, clinical, and serologic information provided to or obtained by the CDC. There were 21 patients withuntreated and treated early syphilis, 52 with untreated and treated latent disease, 7 with congenital syphilis, and 1 with late cardiovascular syphilis.uals. When these sera were tested by the FTA-P 17 werenonreactive, 2 remained borderline, and 2 became reactive.Similarly, seven sera that were diagnostically categorized asfalse-positives were nonreactive in the FTA-P. In contrast,one 2+, three 1+, and three borderline reactions occurredwith the FTA-CDC. The sensitivity and specificity of theFTA-P were 100 and 95.5%, and those of the FTA-CDCwere 98.7 and 90.9%, respectively.All treponemal tests for syphilis utilize treponemal anti-gens that are contaminated with rabbit testicular material.The inability to obtain sufficient quantities of purified antigenresults from the fact that only limited in vitro multiplicationof the organism has been reported (3, 4, 12, 13). Further-more, an efficient method for purification of T. pallidum fromhost tissue has not been available. Recently, however, theseparation of soluble and insoluble host material from T.pallidum suspensions by Percoll density gradient centrifuga-tion was described (5). The resultant organisms were biolog-ically, morphologically, and immunologically intact. Prelim-inary evidence suggested that the use of Percoll-purified T.pallidum as an FTA-ABS antigen reduced the number ofFIG. 1. Percoll-purified T. pallidum as an FTA-ABS antigen.equivocal reactions and enhanced the ease and speed ofreading (P. A. Hanff, C. Fernandez, J. Waters, and J. D.Folds, Progr. Abstr. 83rd Intersci. Conf. Antimicrob. AgentsChemother., abstr. no. 669, 1983). This appeared to be due,in part, to the purity of the antigen and the resultant lack ofbackground debris (5). Additionally, Percoll-purified antigenremained firmly fixed to the slides throughout the procedure.In contrast, some loss of the standard nonpurified antigen isexpected (6, 21).In an attempt to demonstrate more accurately that Percoll-purified T. pallidum could be effectively utilized as anFTA-ABS antigen, we tested diagnostically and serologi-cally well-characterized sera, using lyophilized purified an-tigen. The sensitivity and specificity of the FTA-P and theFTA-CDC were essentially similar: 100 and 95.5% versus98.7 and 90.9%, respectively. There was a marked differ-ence, however, in the number of borderline reactions ob-served with the two tests. Of the 21 borderline resultsmeasured by the CDC, 19 (95%) were nonreactive by theFTA-P, and 2 became positive. The CDC currently recom-mends that borderline reactions be interpreted as negative(1). Thus, the differences observed between the FTA-P andFTA-CDC may not be clinically significant.In contrast, the fact that all patients classified as false-positive were nonreactive with the FTA-P may have impor-tant clinical and serologic implications. We have shown thatthe quality and purity of the T. pallidum antigen have aneffect on borderline and false-positive FTA-ABS results.Similar results have been obtained by comparing the reac-tivity of a commercial FTA-ABS antigen with nonpurifiedand Percoll-purified T. pallidum prepared from the samesuspension (G. Tringali, P. A. Hanff, G. Scuderi, and G.Fragale, submitted for publication). If these results areconfirmed in larger prospective evaluations utilizing FTA-ABS, hemagglutination, or enzyme-linked immunosorbentassays, it is conceivable that many of the interpretiveproblems encountered with the treponemal tests for syphilisand the resultant diagnostic dilemmas will be resolved.We thank Elizabeth Hunter for invaluable conversations andguidance, Jennifer Waters for technical assistance, and LydiaMoretti for excellent secretarial assistance.LITERATURE CITED1. Centers for Disease Control, Biological Products Program. 1984.Current status of FTA-ABS tests for syphilis. Supplement no.27. Centers for Disease Control, Atlanta, Ga.2. Deacon, W. E., V. H. Falcone, and A. Harris. 1957. A fluores-cent test for treponemal antibodies. Proc. Soc. Exp. Biol. Med.96:477-490.NOTES 981VOL. 23, 1986J. CLIN. MICROBIOL.3. Fieldsteel, A. H., D. L. Cox, and R. A. Moeckli. 1981. Cultiva-tion of virulent Treponema pallidum in tissue culture. Infect.Immun. 32:908-915.4. Fieldsteel, A. H., D. L. Cox, and R. A. Moeckli. 1982. Furtherstudies on replication of virulent Treponema pallidum in tissuecultures of SflEP cells. Infect. Immun. 35:449-455.5. Hanff, P. A., S. J. Norris, M. A. Lovett, and J. N. Miller. 1984.Purification of Treponema pallidum, Nichols strain, by Percolldensity gradient centrifugation. Sex. Transm. Dis. 11:275-286.6. Hunter, E. F., M. R. Adams, L. H. Orrison, B. J. Pender, andS. A. Larsen. 1979. Problems affecting performance of thefluorescent treponemal antibody-absorption test for syphilis. J.Clin. Microbiol. 9:163-169.7. Hunter, E. F., E. T. Creighton, and J. S. Lewis. 1970. Animproved antigen for the FTA-ABS test. Health Lab. Sci.7:237-241.8. Hunter, E. F., E. T. Creighton, and J. S. Lewis. 1971. Alyophilization medium for FTA-ABS test antigen.' Health Lab.Sci. 8:35-39.9. Hunter, E. F., W. E. Deacon, and P. E. Meyer. 1964. Animproved FTA test for syphilis, the absorption procedure (FTA-ABS). Public Health Rep. 79:410-412.10. Hunter, E. F., C. E. Farshy, S. L. Liska, D. D. Cruce, J. A.Crawford, and J. C. Feeley. 1982. Sodium desoxycholate-extracted treponemal antigen in an enzyme-linked immunosor-bent assay for syphilis. J. Clin. Microbiol. 16:483-486.11. Hunter, E. F., R. M. McKinney, S. E. Maddison, and D. D.Cruce. 1979. Double-staining procedure for the fluorescenttreponemal antibody absorption (FTA-ABS) test. Br. J. Vener.Dis. 55:105-108.12. Levy, J. A. 1984. Confirmation of the successful cultivation ofTreponema pallidum in tissue culture. Microbiologica7:367-370.13. Norris, S. J. 1982. In vitro cultivation of Treponema pallidum:independent confirmation. Infect. Immun. 36:437-439.14. Pope, V., E. F. Hunter, and J. C. Feeley. 1982. Evaluation of themicroenzyme-linked immunosorbent assay with Treponemapallidum antigen. J. Clin. Microbiol. 15:630-634.15. Rathlev, T. 1967. Hemagglutination test utilizing pathogenicTreponema pallidum for the sero-diagnosis of syphilis. Br. J.Vener. Dis. 43:181-185.16. Schmale, J. D., D. S. Kellogg, Jr., C. E. Miller, P. Schammel,and J. D. Thayer. 1970. Separation of Treponema pallidum fromtissue debris through continuous-particle electrophoresis. Appi.Microbiol. 19:287-289.17. Tomizawa, T., and S. Kasamatsu. 1966. Hemagglutination testsfor the diagnosis of syphilis. A preliminary report. Jpn. J. Med.Sci. Biol. t9:305-308.18. Tomizawa, T., S. Kasamatsu, and S. Yamaya. 1969. Usefulnessof the hemagglutination test using Treponema pallidum antigen(TPHA) for the serodiagnosis of syphilis.;Jpn. J. Med. Sci. Biol.22:341-350.19. Turner, T. B., and D. H. Hollander. 1954. Studies on themechanism of action of cortisone in experimental syphilis. J.Syph. Gonorrhea Vener. Dis. 38:371-387.20. Veldkamp, J., and A. M. Visser. 1975. Application of theenzyme-linked immunosorbent assay (ELISA) in the sero-diagnosis of syphilis. Br. J. Vener. Dis. 51:227-231.21. Venereal Disease Program, National Communicable Disease Cen-ter. 1969. Manual of tests for syphilis. U. S. GovernmentPrinting Office, Washington, D.C.22. Wentworth, B. B., M. A. Thompson, C. R. Peter, R. E. Bawson,and D. L. Wilson. 1978. Comparison of a hemagglutinationtreponemal test for syphilis (HATTS) with other serologicmethods for the diagnosis of syphilis. Sex. Transm. Dis.5:103-111.982 NOTES

Percoll-purified Treponema pallidum, an improved fluorescent treponemal antibody-absorbed antigen.

https://cdr.lib.unc.edu/downloads/nv935948m

Percoll-purified Treponema pallidum, an improved fluorescent treponemal antibody-absorbed antigen.

Abstract

Similar works

Full text

Available Versions

Carolina Digital Repository