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Show Journal of Clinical Neuro- ophthalmology 11( 3); 144- 151, 1991. Syphilis Serology in 1991 A. Paris- Hamelin, M. D. Syphilis, the third most common sexually transmitted disease, has numerous clinical manifestations and can remain entirely latent for many years. This article gives an overview of diagnosis, evolution of antibodies, specific cases and types of syphilis, and implications for immunodeficient patients. Key Words: Syphilis- Syphilis antibodies- Neurosyphilis. From the National Center of Reference and the Center of OMS Reference and Research for the Treponematoses, Institute Alfred Fournier, Paris, France. Address correspondence and reprint requests to Dr. A. ParisHamelin at Institut Pasteur, 28, Rue de Dr. Roux, 75724, Paris Cedex 15, France. © 1991 Raven Press, Ltd., New York Syphilis, a bacterial infection caused by Treponema pallidum ( Figs. 1- 3), is an essentially human disease ( although transmissible to laboratory animals). It is primarily transmitted by sexual contact but may also be congenital. Its recrudescence in the past few years has disturbed the entire world. According to the latest statistics that we have in France, it is now the third most common of the sexually transmitted diseases after candida and chlamydia. Syphilis is remarkable in the richness of its clinical manifestations, as well as for its ability to remain completely latent for many years. Recently, in immunodeficient patients, its behavior has taken on unexpected aspects. BIOLOGICAL DIAGNOSIS In the early as well as congenital stages of the disease, bacterial examination of a suspect lesion remains indispensable- but we cannot stop there. It is necessary to study the evolution of the antibodies not only in the initial stages of the disease, but particularly in the course after treatment. Search for the treponemes is conducted by scraping the lesions or by aspiration of a lymph node and looking for the organism under the ultramicroscope or by direct or indirect immunofluorescence. SEROLOGIC DIAGNOSIS Systematic serologic studies are performed in France for various circumstances throughout life: before marriage, during pregnancy, when donating blood, for naturalization certification for immigrants, etc. It is a commonly performed test which is simple and rapid and sufficiently sensitive and specific. Since 1980 French legislation has required biologists performing screening tests to do two examinations: a cardiolipin test, the VORL, which provides evidence of antilipoidal antibodies; and a 144 SYPHILIS SEROLOGY IN 1991 145 f". ~ . .. .. FIG. 1. Treponema pallidum- silver stain ( oil immersion). treponemal test, either the TPHA or the FTA- ABS, which demonstrates specific antibodies to virulent treponemes ( the first showing a passive, simple agglutination reaction which is inexpensive to perform, and the second by an indirect immunofluorescent reaction which is a bit more delicate and costly). A good screening can consequently be performed using two tests- principally the VDRL and the TPHA ( Fig. 4). However, when these tests are positive or disparate ( Le., showing different results), it is recommended that in addition to therapeutic observations to precisely define the diagnosis, one should quantitate these positive reactions as well as obtain the following confirmatory tests: the FTA; the Nelson test ( TPI); and a study of the specific immunoglobulin M ( IgM) response. The enzyme- linked immunosorbent assay ( ELISA) methods are now being proposed for detection of immunoglobulin G ( lgG). It is suggested that they be used as screening tests rather than precise diagnostic tests because they are only semi-quantitative. Many possibilities will present themselves to biologists encountering syphilis serology. If the screening results are negative, the testing will stop there. If the results differ in such a case, interpretation of serodiagnosis will not be completed, in the absence of clinical signs or history, without the confirmatory tests. How does one interpret the serodiagnostic screening data? What needs to be done in such cases? Four responses are possible: TPHA negativelVDRL negative. The two screening reactions are negative. This is evidence against encountering a treponematosis. There is one possible exception: when there has been very recent infection. A chancre mayor may not be visible. The two reactions are in general positive during the third week after contact. The first reactions to become positive are the FTA- ABS and the IgM test. The FTA- ABS is rarely done as a screening test because of its difficult technique, and the same applies to studying the IgM response. TPHA positivelVDRL positive. In this second ex- FIG. 2. Treponema pallidum- electron microscopy. I Gin Neuro- ophthalmol, Vol. 11, No. 3, 1991 146 A. PARIS- HAMELIN FIG. 3. Treponema pallidum- electron microscopy ( higher power). ample one can generally diagnose a treponematosis, but is it recent or remote? Is there a known history of infection or not? Questioning the patient about the disease will resolve this problem. If he knows that he has been infected and treated, one has a positive serologic " cicatrix," and with weak responses- not surprising- this does not necessitate further confirmatory testing. In the case where a history of prior syphilis is unknown, clinical examination and a careful history as well as the serology are required in addition to completing the tests of verification. One can thus resolve this problem. In this circumstance, there are five possibilities ( Fig. 5) The Nelson test ( TPI) is always negative in the primary stage. One exception is in the case of re-infection: the Nelson test is positive ( due to immunologic memory). In cases of recent ( primary and secondary) syphilis, the TPHA always presents a weaker titer than the FTA. The IgM test is always positive in primary and secondary syphilis. The maximum response occurs in secondary syphilis between the third and sixth month. Secondary syphilis evolves over about 2 years, and the IgM decreased after 2 years and disappears. The other reactions decrease also, except the TPI, which is the only rigorously specific test for the treponematoses. The TPI attains its maximum during the tertiary stage. In reality, in the absence of treatment, the TPI never varies- its titer continues to increase regularly. Only TPHA positive. Only the TPHA is positive in R 2 3 FIG. 4: TPHA qualitative r~~ ctions. upper row: negative controls; lower row: ( 1) ( Ieft) negative; ( 2) ( mlddle)- posltlve 2 + ; ( 3) ( right)- positive 4 +. SYPHILIS SEROLOGY IN 1991 147 TPI • NEGATIVE > FTA, TPHA---+ POSITIVE VORL • WEAKLY POSITIVE Primary L IgM • POSITIVE TPI • WEAKLY POSITIVE FTA, TPHA----+ STRONGLY POSITIVE > VORL • MODERATE POSITIVE Early IgM • STRONGLY POSITIVE secondary L TPI • MODERATE POSITIVE ) FTA, TPHA~ STRONGLY POSITIVE VORL • STRONGLY POSITIVE Secondary L IgM • STRONGLY POSITIVE TPI • STRONGLY POSITIVE ) FTA, TPHk----"+ MODERATE POSITIVE VORL • MODERATE POSITIVE Latent L ( untreated patient) IgM • NEGATIVE TPI • STRONGLY POSITIVE > FTA, TPHA~ STRONGLY POSITIVE VORL • MODERATEPOSITIVE Latent L IgM • NEGATIVE FIG. 5. Five serologic possibilities in syphilis. screening. This reaction is specific in about 99.5% ( false positives are rare and in such cases the titer will be very low- 160 units at maximum). One must be certain that there is a question of syphilis on the basis of other reactions ( TPI, FTA- ABS, and IgM). If the TPI and the FTA are positive but with negative IgM, it is then syphilis ( or another treponematosis) which is apparently remote or eradicated. Either the patient knows that he has been infected and treated, or he is unaware of infection. In the first case, it is a serological cicatrix, and one can reassure the patient that he does not need to be treated. In the second case, the patient is ignorant of prior infection, and the findings reflect infection which has been eradicated by antibiotics given for another infection ( as gonorrhea, chlamydia, Vincent's angina, bronchial infection, or the like). Age of the patient, the clinical findings, history, and the patient's temperament, all must be taken into consideration to arrive at a conclusion. If the patient is over 70 years of age, treatment may be superfluous. However, if the patient is young, the infection cannot be very old, and one can propose serologic surveillance and/ or a course of treatment. Table 2 shows the quantification of the various reactions and will aid biologists as well as practitioners in knowing what is considered a high or low titer for each test ( Table 1). The quantification of these tests is performed by diluting the sera by half and half, as is commonly done in immunology. A difference of only one dilution in the same test is of little significance after a few days or weeks. On the other hand, a change of three or four dilutions must be given consideration. Only VORL positive. Only the VORL is positive in screening. It is well known that the cardiolipin ( reagin) reactions are not specific. Therefore, other tests ( TPI and FTA- ABS) are absolutely required in order to assure that the results are indeed negative, as was the TPHA, and one can thus affirm that this is a biologic false positive reaction. If, on the other hand, the TPI or FTA- ABS are found to be positive, one must think that the TPHA is a false negative. This is rare, but can be found in the elderly, in whom the immune defenses decrease. The explanations for interpreting all of the diverse serologic reactions that can be encountered TABLE 1. Quantitative titers of serologic reactions in syphilis Test IgM resultsB TPI FTA- ABS FTA SPHA TPHA VORL Negative < 10 < 200 < 5 < 10 < 80 0 Weakly positive 10- 50 200- 400 10- 20 20- 320 80- 160 1- 2 Moderate positive 100- 300 800- 1,600 40- 80 640- 2,560 320- 1,280 4- 8 Positive 400- 1,000 3,200- 6,400 160- 640 5,120- 10,240 2,560- 5,120 16- 32 Strongly positive 2,000- 10,000 12,800- 819,200 1,280- 5,120 20,480- 40,960 10,240- 1,310,720 64- 4,096 a Test values are given in inverse of dilution. JClin Neuro- ophthalmol, Vol. 11, No. 3, 1991 148 A. PARIS- HAMELIN TABLE 2. Serologic reactions in various stages of syphilis a IgM Infection TPI FTA- ABS FTA SPHA TPHA VORL Very early ~ Negative 200- 400 10- 80 40- 640 Negative Negative Primary ~ 10- 50 800- 3,200 80- 320 1,280- 10,240 80- 320 2- 8 Secondary ~ 100- 5,000 12,800- 819,200 320- 640 10,240- 40,960 10,240- 1,310,120 16- 4,096 Untreated latent ~ 500- 5,000 3,200- 12,800 0- 80 0- 2,560 1,280- 10,240 8- 32 Tertiary evolutive ~ 1,000- 10,000 3,200- 512,000 0- 80 0- 2,560 10,240- 655,360 16- 128 Late- treated ~, residual antibodies 0- 200 400- 3,200 0 0 160- 1,280 D- 4 ~, syphilis. 8 Test values are given in inverse of dilution. in diagnosing the treponematoses is reviewed in Table 2. many years, and the residual antibodies will not protect from reinfection. EVOLUTION OF ANTIBODIES Primary syphilis treated early The first tests to become positive are the FTAABS, the IgM ( i. e. the FTA- ABS- IgM, SPHA [ solid phase hemadsorption assay- IgM] or ELISA) followed by the VORL, and then generally the TPHA. Sometimes the TPHA is positive before the VORL, but this is not generally the rule. The TPI will probably not be positive, and the patient will not retain a serologic cicatrix if the primary infection has been treated immediately and properly. Proper therapy from the onset of chancre will be followed by a negative serology in 4- 6 months ( Fig. 6). Primary- Secondary and secondary syphilis Here all the tests are positive, with TPI weakly positive and IgM strongly positive. The treatment should be for a longer period of time if one desires to obtain a negative serology. This does not occur except late in the treatment course and in only 25% of the cases of secondary syphilis- generally the patients will keep a serologic scar or cicatrix for Latent serologic and late visceral syphilis All reactions are positive, more or less strongly ( Fig. 7). The presence of IgM is the evidence of an infection not very old and still evolving. The absence of IgM signifies, on the other hand, that the infection is quite old and stabilized. Some years afterwards, however, a nontreated patient is susceptible at this phase to presenting with tertiary visceral or nervous system lesions which may be grave and untreatable. Treatment must be instituted but will not completely return the serology to negative. Antibodies will remain for the life of the patient, and one can probably find quiescent treponemes always pathogenic, localized in certain lymph nodes or tissues of the patient, as proven by the work of Collart in France and Dunlop and colleagues in the United States. At this stage one must try to differentiate between a serologic cicatrix and late serologic syphilis. The serologic scar, with low antibody titers, TPI and VORL negative or weakly positive, and absence of IgM should not be disturbing; it corresponds more often to a late- treated syphilis, previously unknown syphilis, syphilis treated insufficiently, and lately, or syphilis treated indirectly FIG. 6. Primary syphilis with early and proper treatment. FIG. 7. Syphilis treated late over years after infection. DAYS 90 120 160 .'"••••••:;;~": Jo.:.::: "..............., .~ " a•• " ..- " "'. , ii ~"."".-.""..... .... II'\." " ::.,.",' · · ' · . · ~ f .....,\ .. , .. ...... \ ANTIBODY ( Iii'") JGin Neuro- ovht/ 4,1," ci. Vol. 1] No. 3, 1991 SYPHILIS SEROLOGY IN 1991 149 during antibiotic therapy instituted for some other infection. Remote, previously unknown serologic syphilis ( showing negative IgM, positive TPI, and average antibody titers to VORL, FTA and TPHA) that has never really been treated can, in a case where spontaneous or acquired immunodeficiency occurs, evolve into a severe tertiary state. In many cases of AIDS, we actually see generalized paresis occurring in patients with serologic findings consistent with a serologic cicatrix or scar. PARTICULAR CASES Differential diagnosis of various treponematoses We employ the term " treponematosis" instead of syphilis when we find a positive serology. In fact, this positive serology exists for four treponematoses: ( a) endemic syphilis or Bejel, ( b) yaws, ( c) pinta, and ( d) venereal or sporadic syphilis. The first three treponematoses are not venereal but differ in their clinical manifestations. They have been recognized for years, even before the causative agent was identified. Endemic syphilis or Bejel is found in hot, dry regions; it affects children between the ages of 1 and 15 years. Transmission is from infant to infant by direct contact and by exchanging toys or clothes. It is an infection of the skin and mucous membranes; late complications are possible, especially cardiovascular. Yaws is found in hot and humid regions. The lesions, often florid, profoundly affect the skin and bones and are followed by mutilating sequelae. On the other hand, the viscera and nervous system are rarely involved. It is not transmitted congenitally. Pinta ( or carate) is a very superficial treponematosis, touching just the epidermis. It is benign and is still encountered in South America and Mexico. Are these treponematoses due to one or several distinct treponemes? Different theories have addressed this question. In reality we cannot differentiate between them. The organisms manufacture antibody that the usual serologic tests cannot differentiate. Therefore, clinicians encountering African patients with a positive serology and without apparent clinical signs must determine the etiology of the seropositivity. Is this venereal syphilis, or is it a scar of pinta or Bejel? First, one can question the patient about his ethnic origin, history of previous blood tests, or previous treatment for infection during his youth. Generally, if it is a question of a nonvenereal treponematoses in a young patient ( 20- 30 years), the serology will show weak titers for all the tests. On the other hand, if the titers are average or high, this is probably evidence of venereal syphilis. It is known that there is no immunity between the different treponematoses. For example, a patient who is seropositive due to pinta could be infected with venereal syphilis as easily as a healthy individual. The treatment is identical for all of these treponemes, i. e., antibiotic therapy ( penicillin as first choice), and it is prudent to treat pregnant women, very young patients, and those with a serologic cicatrix after 40 years, when it is unknown whether this is an infectious " scar" of venereal or acquired syphilis that has been more or less eradicated or treated very late. False positive VORL reactions One must be aware of the late false positive reactions which occur with antigens of a lipid nature. One must distinguish between acute false positive reactions and chronic false positive reactions. Febrile illnesses can produce falsely positive VORL reactions which are transitory. This generally accompanies viral or parasitic infections ( infectious mononucleosis, cirrhosis, hepatitis, toxoplasmosis, tuberculosis, and the like) and clears easily with or without appropriate therapy. After a few months, febrile false positive reactions disappear during the period of convalescence. Chronic false positive VORL reactions that remain positive can show increasing titers over the course of months to years. These may indicate severe problems, such as autoimmune disease, dysproteinemias ( myeloma, Waldenstrom's macroglobulinemia), scleroderma, and the like. The antibodies recognize simultaneously the phospholipid micelle showing the constitution of prothrombinase and the phospholipid antigen present in the VORL reactions. Therefore, one can look for anticardiolipid antibodies by the ELISA reaction and for the antithrombinases by the presence of cephalinkaoline. It is possible to have false positive reactions appear after vaccinations. Remarks about the FTA- ABS and the Nelson- Mayer ( TPI) test Some authors view the FTA- ABS as the test of reference, considering it to be as specific as the TPHA and yielding results just as early; appearance these authors do not give sufficient interest to the TPI test of Nelson and Mayer. I disagree with this emphasis for the following reasons: The FTA- ABS, commonly done as an indirect I Clin Neuro- ophthalmol, Vol. 11, No. 3, 1991 150 A. PARIS- HAMELIN immunofluorescent reaction, is positive in a patient whose serum contains rheumatoid factor or antinuclear antibodies. The rheumatoid factor is not specific for rheumatoid arthritis, but is a no~ specific inflammatory reaction. The F~ A- ABS IS generally found with anti- Ig total conjugate, as well as IgM with rheumatoid factor. This error occurs very rarely with TPHA. The FTA- ABS is positive earlier than the TPHA, but it is less specific. The FTA- ABS shows cross reactions with other spirochetes, in particular with the antibody of Lyme borreliosis. We have found false positives also with leukemias accompanying VORL ( in which TPI and TPHA are both negative). The study of specific IgM in the FTA- ABS also has another possible error- negative reactions occur when the total IgG is elevated, ( phenomenon of competition for antigenic sites). For this reason, the research of IgM must be done using another technique, the modified SPHA which is an immunocaptive method. This technique is as sensitive and specific as the FTA- ABS- IgM. The ELISA reactions begin to give good results. However, this method shows that the IgM persists for a long time in a patient and can result in a mixed interpretation, and one cannot differentiate between reinfection or a therapeutic relapse. Finally, the Nelson test is the test of reference, rigorously specific and positive ( above 200 units) where there is persistence of quiescent treponemes in the organism. It is considered in Europe to be the reaction with the greatest specificity. The TPI is performed only in very special laboratories, and it is of great diagnostic and prognostic value. It is found to be regularly positive in the course of untreated syphilis. NEUROSYPHILIS During the secondary period one can encounter certain cases of specific meningitis. Spinal fluid is very positive in evolving neurosyphilis, with lymphocytosis and increased CSF protein showing the beginning of the involvement of the nervous system. The serum treponemal reactions are positive ( TPI, FTA, TPHA), but the lipoidal reactions may be negative or doubtful. On the other hand, there does not exist an example of positive treponemal test results in the spinal fluid when the blood test results are entirely negative. When considering the diagnosis of neurosyphilis it is important to differentiate between the phenomenon of transudation of antibodies by crossing the meningeal barrier ~ ... ~ ; J'IT.'~ hecal productions produced by Iympho-cytes in the cerebrospinal fluid. One can establish this by calculating different parameters of blood and cerebrospinal fluid ( dosages of albumin, IgG) and establishing an index which permits one to differentiate between intrathecal production or antibodies and simple transudation. SEROLOGY IN THE IMMUNOCOMPROMISED The humoral response after treponemal infection is the base of diagnostic serology. With the loss of antibodies found with severe HIV infection, one can find negative or weakly positive syphilis serologies. The clinical manifestations of the secondary state can evolve rapidly, frequently, and severely into neurosyphilis- and in particular into generalized paresis. In HIV- positive subjects, more or less late syphilis can be frequently observed with either nearly normal humoral responses or with an elevated VORL and quantitative treponemal reactions. In a recent study, antiphospholipid antibodies detected by an ELISA method showing the presence of circulating anticoagulants occurred with false positive VORL parameters. HIV- seropositive subjects infected with syphilis may show treponemal antibodies and anticardiolipid antibodies, specific and nonspecific, which may be elevated or negative. HIV infection causes difficult interpretations in serology. PREVENTION There does not exist at this time a vaccination against syphilis. If the primary infection is well treated and does not progress to secondary syphilis, the patient will be initially seropositive, but after reinfection a second time can again show the same serological responses as occurred in primary infection. CONCLUSION The serology of syphilis is complex in that different reactions can be found throughout the course of the disease. It is advantageous that we have test with differences of sensitivity, specificity, and timing of appearance that permit one to date the infection even when the onset is inapparent and unknown to the patient. SYPHILIS SEROLOGY IN 1991 151 BIBLIOGRAPHY 1. Collart P. Etat actuel des recherches sur la persistance des treponerries au caurs de la syphilis tardivement traitee. Bull Soc France Derm SYl'h 1965; 72: 562. 2. Deacon WA, Falcone VH, Harris A. A fluorescent test for treponemal antibodies. Proc Soc Exl' Bioi Med 1957; 96: 479. 3. Dunlop EMC. Problems in syphilis. Persistence of treponemes after treatment. Br Med / 1972; 2: 577. 4. Nelson R, Mayer MM. Immobilization of Treponema pallidum in vitro by antibody produced in syphilitic infection. J Exl' Med 1969; 89: 369. 5. Paris- Hamelin A, Dreux P, Vuthien H. Treponematoses aspects cliniques et biologiques. Feuil Bioi 1991; 32: 73. 6. Vaisman A, Paris- Hamelin A, Fustec S. La reaction d'hemagglutination passive pas Ie serodiagnostic de la syphilis ( TPHA). I micromethode en plaque. Techniques utilisee a l'Institut Alfred Faurives. Med Molin! 1976; 4: 159. , C/ iI, Neuro- ophthalmol, Vol. 11, No. 3, 1991 |
