Current problems in syphilis serology

Serodiagnosis

and Immunotherapy

(1987) 1, 393-400

Leading articles

Current

problems

in syphilis serology

Syphilis has been described as a disease that displays “essentially Machiavellian facility in disguise, deceit and malevolence”. Although the incidence of syphilis has decreased significantly since Stokes’ wrote these words, the disease is still a master of disguise that plagues and puzzles modern clinicians. Despite major technical advances in the laboratory diagnosis of other infectious diseases, the tests available for syphilis diagnosis have not changed significantly in the last 25 years. A satisfactory explanation of the events that occur after host-Treponema pallidurn interaction, remains obscure. One of the major factors that contributes to this enigma continues to be the inability to sustain the growth of the organism in vitro. This limitation has severely hampered the isolation and purification of sufficient quantities of constituent antigens of T. paflidum for studies relating to the pathogenesis, immunology, and serodiagnosis of syphilis. Another factor is the existence of antigenic components shared by pathogenic and saprophytic treponemes, which mask the immune response to T. pallidurn. Current methods for syphilis diagnosis Clinicians currently diagnose syphilis by three major criteria: clinical presentation. microscopic identification of Treponema pallidurn, and serodiagnosis. Clinical presentation Syphilis is characterized by periods of clinical activity interrupted by months or decades of latency. In the primary stage, the clinical manifestations can range from an undetected papule to a classical ulcerated chancre. In the secondary stage, from a few barely discernible macules to a florid, pustular body rash. In late syphilis, virtually any organ system may be affected with disease. ranging from subclinical to Iife-threa393

tening. Based upon such transient and variable manifestations, the accuracy of the clinical diagnosis is relatively low. During the latency periods, there are no clinical signs or symptoms to arouse patient or physician suspicions. Thus, diagnosis of latent infection is based solely upon serodiagnosis. Microscopic ident$cution of Treponema pallidum The method for detecting T. pallidum in primary or secondary lesions is darkfield microscopy, in which organisms are identified by their characteristic morphology and motility. The sensitivity of darkfield examination is approximately 75%>. A laboratory alternative is the direct immunofluorescence (DFA-TP) test. which is more sensitive and specific than darkfield microscopy, but not widely used. At present a rapid, simple procedure for the identification of T. pallidurn in lesion exudates or CSF of patients with early syphilis or neurosyphilis, is the use of monoclonal antibodies. This method has high sensitivity, specificity and reproducibility’. Syphilis serology Just 80 years ago, Wasserman introduced the first non-treponemal test for syphilis. Although this test remains a useful clinical diagnostic tool, recognition of its limitations continues to stimulate interest in improved serologic methods. Mammalian infection with Treponema pallidum results in the production of two types of antibody: one is non-specific, reacting with a non-treponemal cardiolipin-lecithincholesterol antigen, and the other is directed at the treponeme itself. Depending on the assay conditions, different tests for non-treponemal antibody (NTA) are possible, the most popular of which are the venereal disease research laboratory test (VDRL), the rapid plasma reagin test (RPR) and the automated reagin test (ART). The various non-treponemal antibody tests (NTA) are similar in sensitivity and specificity. Because the NTA tests are inexpensive. reproducible and relatively easy to perform,

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they are widely used in the diagnosis of suspected syphilis cases or to screen large numbers of asymptomatic persons. Because a falsepositive NTA may account for up 10% of reactive tests however, an additional treponema1 antibody test is usually necessary to confirm the diagnosis of syphilis. The three important methods for detection of treponemal antibody are the T. pallidum immobilization test (TPI), the fluorescent treponemal antibody absorption test (FTA-ABS) and the T. pallidum haemagglutination assay (TPHA). The TPI test was generally accepted as being the most specific serological test for treponemal infection. The necessity for using living, virulent T. pallidurn as antigen, has restricted the performance of the test to specialized laboratories. It is relatively insensitive in early syphilis and sera can give invalid results because of the presence of treponemicidal drugs. The FTA-ABS test uses as antigen a killed suspension of the organism. It is a standard fluorescence slide test incorporating an absorption step to remove cross-reacting antibodies. The test is sensitive and specific, and is usually the first to be positive following initial infection. False reactivity in the FTA-ABS test has, however, been recorded in collagen-vascular disorders such as systemic lupus erythematosus, rheumatoid arthritis, as well as in several miscellaneous conditions such as genital herpes simplex, alcoholic cirrhosis, diabetes, autoimmune haemolytic anaemia, hypergammaglobuhnaemia, pregnancy, and after smallpox vaccination. Its sensitivity is superior to that of the TPI test in the examination of cerebrospinal fluids. Its disadvantages are an inability to discriminate past from current infection, and inconvenience and unsuitability for large numbers of samples. The Treponema pallidum haemagglutination (TPHA) test, is easier both to perform and to read than either the TPI or the FTA-ABS tests. Its disadvantages are: insensitivity during early primary syphilis, false-positives in some pregnant women, healthy people and diabetics, inability to discriminate past and current infection and it may be poorly reproducible4,5. Syphilis antibodies will persist for years regardless of whether or not the disease is successfully treated. Consequently, an important requirement for serodiagnostic tests is the ability to discriminate between antibodies attributable to a past infection and those associated with active disease. Generally it is those tests that detect primarily IgM antibodies that

are most useful for identifying current infection. The FTA-ABS (IgM) test is an adaptation of the standard FTA-ABS (total antibody) test, whereby fluorescein conjugated anti-human IgM is used as tracer. False-positive and falsenegative can occur: specific IgG will compete for T. pallidum antigenic binding sites and reduce sensitivity for IgM, and the presence of rheumatoid factor can give false-positive results. If the patient is not treated, both classes of specific antibody can be demonstrated during all stages of syphilis and for as long as the disease is active. Following treatment the specific IgM titres decline, eventually to negativity. The rates of decline are, however, variable and it would appear that following successful treatment a patient may require between 6 and 24 months to become IgM negative. The rate of decline varies with the stage of the disease when treatment commenced: patients treated in the primary phase seem to show more rapid IgM decline than patients treated during late syphiW.

Current problems in syphilis serology Two of the major limitations of existing treponemal serologic tests are their inability to measure therapeutic response and their uselessness in patients with a history of syphilis. There is also need for more sensitive and specific tests. The search for these “ideal” tests has been developed in two ways: the application of new serologic techniques to the diagnosis of syphilis, and the isolation and purification of the disease-specific antigens of T. pahdum. Several new serodiagnostic tests based upon enzyme-linked immunoassay (ELISA)“‘” and radioimmunoassay (RIA)” have been described in recent years; these tests, however, all use relatively uncharacterized T. pallidurn antigens or components of the nonpathogenic Reiter treponeme. Although the initial sensitivity and specificity data for several of these tests are good, the tests have inherent theoretical and practical limitations due to the existence of common treponemal antigens. The inability to culture pathogenic treponemes in vitro has prevented the acquisition of Treponema pallidurn in sufficient quantity to permit exploratory immunochemical studies. In efforts to circumvent this difficulty, nonpathogenic cultivable treponemes have been

Leading articles investigated, for the purpose of identifying and extracting antigenic components shared with pathogens. It was shown that purified T. Reiter filaments are composed of two protein subunits, at least one of which is shared with the pathogenic treponemes.‘Z.‘3. Forty antigens have been demonstrated in Treponema Reiter by means crossed immunoelectrophoresis (CIE). Five of these 40 antigens reacted with antibodies in sera from patients with secondary syphilis. Enzyme-immunosorbent assays for the detection of both IgG and IgM antibodies, against this T. pallidurn antigen were developed with high sensitivity and specificity’““. Early studies on T. pallidurn antigen structure demonstrated the existence of SIX major surface proteins named P, to P,. Among them, P? and P, were used as the antigens for detecting antibody to T. pallidurn in an ELISA test with very promising results’*,19. The recent application of technical advances including immunoblotting, hybridoma production, and gene cloning has resulted in an impressive amount of information concerning the antigens of T. pallidurn. At least 32 antigenie molecules have been identified using immunoblotting and radioimmunoprecipitation. The molecular weight of these molecules varies between 13 and 200 kDa2+lZ. Among them, 23 polypeptides cross-reacting with T. phagedenis biotype Reiter. 21 with T. refringens, and 26 with T. vincentii have been identified. The T. pallidurn-immune sera reacted with 21 of its own polypeptides. Thus it is evident that the species T. pallidurn is an antigenic mosaic, consisting of more crossreacting than specific proteins”. The ability to obtain large quantities of purified or E. coli recombinant pathogen-specific antigens has important implications in the development of new specific serologic tests for syphilis. Different authors have evaluated the use of some of these antigens such as 17, 14,89, 37 and 32 kDa molecules as well as the first T. pallidurn cloned antigen for serodiagnosis: a purified recombinant 90 kDa surface antigen. These studies are in progress now2h27. Western blott analysis showed that, in plasma from persons with primary syphilis as early as 3 days after appearance of the lesion, both IgG and IgM were present, with the most strongly apparent reactivity against a 47-kDa molecule. IgM reactitiity against six antigens then diminished as IgG antibodies against additional molecules (most notably the 14- and I2-kDa polypeptides) developed. IgM reacti-

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vity declines rapidly within 6 months after therapy of primary syphilis. IgG also diminishes but appears to persist in proportion to the duration of symptoms: the shorter the duration of disease, the more transient the IgG antibody’*. Plasma from patients with untreated secondary and early latent syphilis contain IgM with faint-to-moderate reactivity against a subset of 32 treponemal antigens, including the 12- and 14-kDa molecules. Strong IgG reactivity was present against the 32 treponemal antigens. Following treatment, IgM reactivity against all specificities declined. In both secondary and early latent infection, IgG intensity declined but some reactivity persisted against all molecules detected initiallyZv. Some of these T. pallidum antigens are common to T. Reiter but in spite of this, healthy people develop antibodies against only two of these antigens: the 30 and 33 kDa molecules, those corresponding to the axial filament of both treponemes. It seems possible that by studying the sequence of appearance of the M or G antibodies against the above-mentioned antigens, it would be possible to make an accurate diagnosis on the evolution of the disease in one given patient. Studies on the structure of T. pallidurn could lead to a greater knowledge of surface antigens that bind to eukaryotic cells. The antibodies directed against these antigens could have a protective role. These studies indicate that there is great potential for improved serodiagnostic tests for syphilis using well-characterized T. pallidum antigens. Two of the major limitations of existing treponemal serologic tests, their inability to measure therapeutic response and their lack of utility in patients with a history of syphilis have not yet been addressed with the newer tests. Another important consideration in developing new serodiagnostic tests is cost: a new more expensive test would not be acceptable as a screening tool unless it had very significant technical and diagnostic advantages over the existing, inexpensive cardiolipin tests. Such a new test may, however, have application as a confirmatory test. In summary, the recent rapid advances in our knowledge of the pathogen-specific antigens of T. pallidurn and the applicatioriof new technologies including hybridoma production and gene cloning, have resulted in the development of new diagnostic tools. These methods have the theoretical and actual advantages of

396

Leading articles

increased specificity and predictive value, thus aiding the clinician in the diagnosis of syphilis, “the great imitator”.

assayfor syphilis.J Clin Microbial 1982; 16: 483-6. 11. Baughn RE, Musher DM. Radioimmunoassaysfor the detectionof antibodiesto treponemalpolypeptide antigensin serum. M. V. BOROBIO J Clin Microbial 1985;21: 922-9. Department of Microbiology, 12. Hardy PH, FredericksWR, Nell E. IsolaSchool of Medicine, University of SeviIla, tion and antigenic characteristicsof axial Apdo. 914, 41009-Sevilla. Spain filamentsfrom the Reiter treponeme.Infect Immun 1975;11: 380-6. 13. Nell E, Hardy PH. Counterimmunoelectrophoresisof Reiter treponemeaxial filaments as a diagnostic test for syphilis. J Clin Microbial 1978;8: 148-52. References 14. StrandbergPedersenN, Axelsen NH, JorgensenBB, SandPetersenC. Antibodies in 1. Stokes JH, Beerman H, Ingraham NR. secondary syphilis against five of forty Modern clinical syphilology. In: PhiladelReiter treponemeantigens.Stand J Immuno11980;11: 629-33. phia: WB SaundersCompany, 1944,p. 1. 2. EdwardsEA. Detecting T. pallidurn in prim- 15. StrandbergPedersenN, Sand PetersenC. ary lesionsby the fluorescentantibody techAxelsen NH. Ribonucleic acid antigen nique. Pub Health Rep 1962;77: 427-30. from the Reiter treponeme used in an 3. Hook III HW, Roddy RE, Lukerhart SA, ELISA for antibodiesin syphilis.Stand J Horn J, HolmesKK, Tam MR. Detectionof Immunol 1982;16: 431-6. Treponemapallidum in lesionexudatewith a 16. PedersenNS, Sand PetersenC, Vejtorp M, Axelsen NH. Serodiagnosisof syphilisby pathogen-specificmonoclonal antibody. J Clin Microbial 1985;22: 241-4. an enzyme-linked immunosorbent assay for IgG antibodies against Reiter trepo4. Borobio MV, Martin E, PereaEJ. Specificity of three serologicaltests for syphilis: nemeflagellum.Stand J Immunol 1982;15: 341-8. VDRL, FTA-abs and TPHA in healthy people,pregnantwomenand diabetics.Eur 17. PedersenNS, Sand PetersenC, Axelsen J SexTrans Dis 1984;1: 155-8. NH. Enzyme-linkedimmunosorbentassay 5. PereaEJ, Dwyer RStC, Borobio MV. Enferfor detection of immunoglobulinM antibody againstthe Reiter treponemeflagelmedadesde TransmisidnSexual. In: Perea lum in syphilis.J Clin Microbial 1982;16: EJ, ed. EnfermedadesInfecciosas:Patoge604-14. nesisy Diagnbstico.Barcelona: Salvat Ed, 18. Morrison-Plummer J, Alderete JF, Base1983:478-510. man JB. Enzyme-linked immunosorbent 6. StevensRW, Schmitt ME. Evaluation of an enzyme-linked immunosorbent assay for assayfor the detection of serumantibody to outer membraneproteins of Treponema treponemal antibody. J Clin Microbial pallidurn. Br J Vener Dis 1983;59: 75-9. 1985;21: 399402. Verldkam J, VisserAM. Application of the 19. PetersonKM, BasemanJB, Alderete JF. Treponema pallidum receptor proteins enzyme-linked immunosorbent assay interact with fibronectin. J Exp Med 1983; (ELISA) in the serodiagnosis of syphilis.Br 157: 1958-70. J Vener Dis 1975;51: 227-31. 20. Lukehart SA, Baker-Zander SA, Gubish Borobio MV, Gallardo RM, Alvarez-DarER. Identification of Treponema pallidurn det C. Evaluation of a solid-phaseenzyme antigens:Comparisonwith a nonpathogeimmunoassayin the diagnosisof syphilis. nit treponeme.J Immunol 1982;129:833Eur J SexTrans Dis 1985;2: 231-33. 8. PopeV, Hunter EH, FeeleyJC. Evaluation of the microenzyme-linkedimmunosorbent 21. Hanff PA, Miller JA, Lovett MA. Molecular characterization of common treponeassaywith Treponema pallidurn antigen. J ma1antigens.Infect Immun 1983;40: 825Clin Microbial 1982;15: 630-4. 8. 10. Hunter EF, Farshy CE, Liska SL, Cruce DD, Crawford JA, Feeley JC. Sodium 22. HenselV, WelensiekHJ, Bhakdi S.Sodium dodecyl sulfate-polyacrylamidegel electrodesoxycholate-extractedtreponemal antiphoresisimmunoblotting as a serological gen in an enzyme-linked immunosorbent

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tool in the diagnosis of syphilitic infections. J Clin Microbial 1985; 21: 82-7. Lukehart SA, Baker-Zander SA, Gubish ER Jr. Identification of Treponema pallidum antigens: Comparison with a nonpathogenic Treponema. J Immunol 1982; 129: 833-8. Stamm LV, Folds JD, Bassford PJ. Expression of Treponema pallidum antigens in Escherichia coli K-12. Infect Immun 1982; 36: 123841. Norgard MV, Miller JN. Cloning and expression of Treponema pallidurn (Nichols) antigen genes in Escherichia coli. Infect Immun 1983; 42: 43545. Fehniger TE, Walfield AM, Cunningham TM, Radolf JD, Miller JN, Lovett MA. Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen. Infect Immun 1984; 46: 5988607. Radolf JD, Lernhardt EB, Fehniger TE, Lovett MA. Serodiagnosis of syphilis by enzyme-linked immunosorbent assay with purified recombinant Treponema pallidurn antigen 4D. J Infect Dis 1986; 153: 1023-7. Moskophidis M, Mtiller F. Molecular analysis of immunoglobulins M and G immune response to protein antigens of Treponema pallidum in human syphilis. Infect Immun 1984; 43: 127-132. Baker-Zander SA, Hook EW III, Bonin P et al. Antigens of T. pallidurn recognized by IgG and IgM antibodies during syphilis in humans. J Infect Dis 1985; 151: 26478.

*Casoli C, Tremolada F, Lori F et al. & Tremolada F, Casoli C, Loreggian M et al. Reverse transcriptase activity in post-transfusion non-A, non-B hepatitis. I & 11. Serodiag Immunother Infect Dis 1987; t(5): 339-346, 347-352.

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articles

Hepatitis non-A, non-B-is retrovirus involved?

a

Researchers in the field of hepatitis may feel that they have come to a dead end as regards hepatitis non-A, non-B (HNANB) but nevertheless there have been a gradual progress in our knowledge about these diseases. The characterization of a hepatitis A-like agent causing epidemic water-borne non-A, non-B hepatitis’ and the clarification of the different forms of HNANB2 are examples of this progress. A few years ago it was suggested that the bloodtransmitted type of HNANB may be caused by a retroviruslike agent3,4 and in the previous issue of Serodiagnosis and Immunotherap? further evidence of that was presented*. Different epidemiological

types of HNANB

Three epidemiological types on non-A, non-B hepatitis are usually recognised: a water-borne or epidemic type; a blood- or needle-associated type: and a sporadically occurring type. The water-borne hepatitis A-like form of non-A. non-B hepatitis, often named epidemic non-A hepatitis, is transmitted by the faecaloral routes.h. This variety of non-A, non-B hepatitis has occurred in epidemics in developing countries in Africa and Asia. An association with consumption of shellfish has been reported in the U.S.A. and Europe’.*. The epidemiology of this type thus resembles that of hepatitis A, but the virus is serologically unrelated to hepatitis A virus. Sporadically occurring cases of the epidemic type of non-A. bon-B hepatitis may also occur. Such cases are sometimes referred to as “community-acquired”. Recently Caredda et a/. described 20 patients with non-A, non-B hepatitis associated with shellfish who showed the same clinical characteristics as cases from a waterborne outbreak of non-A, non-B hepatitis. Sporadic cases of HNANB may thus very well represent sporadic spread of the epidemic water-borne type of non-A, non-B hepatitis. It seems probable that the communityacquired type of HNANB also include parenterally transmitted cases for instance through sexual contacts or via unapparent blood transmission. If so, there may be only two epidemiological forms of HNANB-one hepatitis A-like which is transmitted orally and one hepatitis Blike form which is transmitted parenterally by blood or blood products.