Further Evaluation of the Automated Fluorescent Treponemal Antibody Test for Syphilis

Improvements in the equipment for the automated fluorescent treponemal antibody (AFTA) test for syphilis prompted this comparative study of the AFTA and its manual counterpart, the fluorescent treponemal antibody-absorption (FTA-ABS) test. The AFTA equipment operated satisfactorily, required only minimal monitoring, and afforded a three-to fourfold increase over the number of sera that could be tested manually by one serologist. The AFTA and FTA-ABS tests agreed well with only 2.1% of the sera yielding conflicting results. The AFTA was less precise than the FTA-ABS on sera retested because of original conflicting results and on sera retested within the same run to determine reproducibility. However, these differences were not large, and AFTA test performance was considered to be within the limits acceptable for a diagnostic serological procedure.     

Evaluation of the Qualitative and Automated Quantitative Microhemagglutination Assay for Antibodies to Treponema pallidum

Qualitative and quantitative microhemagglutination assays for antibodies to Treponema pallidum (MHA-TP) were performed on 314 syphilitic and 597 presumably nonsyphilitic sera, and the results were compared with those of the fluorescent treponemal antibody-absorbed (FTA-ABS), the Treponema pallidum immobilization (TPI), and the Veneral Disease Research Laboratory (VDRL) tests. MHA-TP sensitivity was similar to that of the other tests in all stages of syphilis except primary syphilis, in which MHA-TP reactivity was only 64% compared with 82% in the FTA-ABS test, 73% in the VDRL test, and 67% in the TPI test. MHA-TP specificity was satisfactory and comparable to that of the other treponemal tests. Quantitation of the MHA-TP test was automated by use of Autotiter II equipment. Titers tended to become elevated later in the course of syphilis and to remain elevated longer than did VDRL titers. Reproducibility of the quantitative MHA-TP test was satisfactory, with duplicate tests agreeing within one doubling dilution on 97.5% of 351 reactive sera. Poor reproducibility was obtained with sera giving minimal reactions in the qualitative test, and such sera should be routinely retested. The MHA-TP is less time-consuming and costly than the FTA-ABS test and could be used in conjunction with the VDRL or another reagin test for syphilis to eliminate a large number of the FTA-ABS tests now required.     

Serological diagnosis of syphilis: comparison of the Trep-Chek IgG enzyme immunoassay with other screening and confirmatory tests

The Trep-Chek IgG Enzyme Immunoassay (Trep-Chek IgG EIA) was evaluated with 604 serum specimens submitted for syphilis serology from patients across Canada against a battery of conventional syphilis serology tests, including the Rapid Plasma Reagin (RPR) test, the Venereal Disease Research Laboratory (VDRL) test, the Treponema pallidum passive particle agglutination (TP-PA) test, the fluorescent treponemal antibody absorption (FTA-ABS) test, and the newer confirmatory test, Innogenetics INNO-LIA. On the basis of a consensus result derived from these serologic tests, 34 specimens were found to be syphilis-positive (28 active and six past infections), and 570 were syphilis-negative (including 12 biological false positives). When the test results on this set of samples were compared to those obtained with the conventional tests RPR, VDRL, TP-PA, and FTA-ABS, the sensitivity and specificity of the Trep-Chek IgG EIA were found to be 85.3% and 95.6%, respectively. Without further evaluation, we do not recommend use of the Trep-Chek IgG EIA as a stand-alone test for either screening or confirmatory syphilis serology.     

Current Status of the Treatment of Syphilis

Penicillin remains the treatment of choice for syphilis, with sustained low blood levels curing virtually all patients having early syphilis and halting disease progression in most patients with symptomatic syphilis. Tetracycline, erythromycin or cephalothin yields similar cure rates for patients with early syphilis who are allergic to penicillin. The efficacy of non-penicillin regimens for the treatment of late syphilis is uncertain. Results of Venereal Disease Research Laboratory (VDRL) or other reagin tests should become negative or remain at very low titer following adequate therapy, although results of Treponema pallidum immobilization (TPI) and fluorescent treponemal antibody-absorbed (FTA-ABS) tests often remain positive.     

Use of Tunable, Pulsed Dye Laser for Quantitative Fluorescence in Syphilis Serology (FTA-ABS Test)

A pulsed dye laser was used as an excitation source in a fluorescent treponemal antibody absorption (FTA-ABS) test. A high precision in quantitative fluorescence was obtained with this high-power excitation source coupled to an electronic detection system and a storage oscilloscope by standardization of fluorescence evaluation and through elimination of human error. One 0.4-mus pulse exposure was sufficient to record fluorescence intensity data on the oscilloscope. Absence of fading of fluorescence after repeated excitation permitted multiple readings of the same microscope field. Almost 100% reproducible results were obtained for the FTA-ABS test with 40 samples. Electronic detection of fluorescence and the high sensitivity obtained with laser excitation raise doubts about the relative value of quantitative immunofluorescence in the FTA-ABS test.     

A Cost-Benefit Analysis of California's Mandatory Premarital Screening Program for Syphilis

As do most states, California requires premarital serologic tests for syphilis. The Venereal Disease Research Laboratory (VDRL) test and a fluorescent treponemal antibody-absorbed (FTA-ABS) are often used in series for this purpose. In 1979 in California, there were approximately 300,000 persons tested premaritally, but only 35 were found to have asymptomatic infectious syphilis (incidence=0.012%). Including all the direct costs of this screening program, the yearly costs of premarital screening is $8.5 million or almost $240,000 per case found. If one takes into account the sensitivities and specificities of the tests, one still has 6 false-negative and 90 false-positive tests using the 1979 figures. The benefits of the program are the number of cases of congenital syphilis that are prevented. Using a worse-case method, no more than 1.5% of the cases of syphilis detected would result in a case of congenital syphilis. The estimated benefits would result in a savings of approximately $161,000. The economic costs of the premarital screening program far outweigh the benefits.     

Reduction of Nonspecific Background Staining in the Fluorescent Treponemal Antibody-Absorption Test

The nonspecific background fluorescence which occurs with the fluorescent treponemal antibody-absorption test for syphilis was found to result from a reaction between serum-treated Treponema pallidum organisms and the conjugated antihuman gamma-globulin. It was also shown that beta-lipoprotein and albumin were the important contributing factors in human serum. Various dilutions of 2.5% trypsin in phosphate-buffered saline specifically reduced background fluorescence under proper test conditions. By employing a trypsin digestion method, a semiautomated procedure utilizing a visual readout has been postulated as feasible.     

Routine Serologic Testing for Syphilis in a Community Medical Practice

A retrospective review of 8,100 serologic tests for syphilis ordered during a 42-month period yielded positive rapid plasma reagin test results in 127 patients (1.6 percent) and a positive fluorescent treponemal antibody absorption reaction in 91 patients (1.1 percent). Of the 36 cases of biologic false-positive reactions, most were in prenatal patients. Forty-six cases of syphilis were previously undiagnosed but antibiotic therapy was given in only 26 of the patients. Some 24 percent of syphilitic patients were not treated because the positive serologic findings were overlooked. Cerebrospinal fluid determinations were analyzed and cost-effectiveness of finding a single case of previously undiagnosed syphilis was calculated. We found that routine serologic tests and cerebrospinal fluid studies for syphilis in asymptomatic patients had low rates of positivity in our community hospital and outpatient practice.     

Depressed CD4/CD8 ratio in TPHA-negative patients with syphilis.

The Treponema pallidum hemagglutination assay (TPHA) is a widely used method for screening syphilis. We report our experience with six elderly (age 72.4 +/- 8.3 years) patients with syphilis, whose TPHA was negative. Their cardiolipin (RPR) and absorbed fluorescence treponemal tests (FTA-ABS) were positive. TPHA-negative patients with syphilis were compared with TPHA-positive syphilitics by immunological analysis. We found that both the numbers and the percentage of CD4 cells in TPHA-negative syphilitics were significantly lower than those in TPHA-positive syphilitics (722 +/- 142 vs. 1,064 +/- 141/mm3, P less than 0.01: 35.1 +/- 6.9 vs. 48.4 +/- 6.4%, P less than 0.01) and that the ratio of CD4 to CD8 was also lower in TPHA-negative syphilitics compared with TPHA-positive syphilitics (1.08 +/- 0.46 vs. 2.24 +/- 1.07, P less than 0.05). These data suggest that the TPHA is insufficient for excluding elderly syphilitics because of immunological impairment seen in aged patients.     

株纯化轴丝在梅毒血清学中初步应用

本试验以纯化的Reiter株轴丝为抗原,建立了AF—ELISA试验方法,用该方法检测了35份自家免疫病人血清、285份非梅毒病人血清及78份各期梅毒病人血清共398份血清,并同时与FTA-ABS等进行比较,结果AF-ELISA特异性为99.3%,FTA—ABS为99.7%,两者敏感性为100%.     

Treponema paraluis-cuniculi infection in a commercial rabbitry: epidemiology and serodiagnosis

The epidemiology of Treponema paraluis-cuniculi infection in a commercial rabbit breeding facility was described using several serologic tests. The Venereal Disease Research Laboratory, rapid plasma reagin, microhemagglutination and fluorescent treponemal antibody-absorption tests were used to detect antibodies to T paraluis-cuniculi. Young adult New Zealand white rabbits, tested prior to entry into the breeding program, were nearly always free of T paraluis-cuniculi infection. In adult females, the prevalence of T paraluis-cuniculi infection increased with parity; females para 6 or greater were usually seropositive. Most adult males seroconverted within 6 months of entering the breeding program; all males were seropositive after 12 months in the breeding program. This suggested that T paraluis-cuniculi spreads mainly by horizontal transmission during breeding in adult rabbits. Of the two nontreponemal antigen tests used, the Venereal Disease Research Laboratory test was more sensitive, whereas the rapid plasma reagin test was more specific in detecting T paraluis-cuniculi infection; the fluorescent treponemal antibody-absorption test was used as the confirmatory treponemal antigen test. However, neither nontreponemal antigen test was completely satisfactory. On the other hand, the sensitivity and specificity of the microhemagglutination test compared favorably with the fluorescent treponemal antibody-absorption test. Since the microhemagglutination test combines desirable features of both a screening and verification procedure, it should be the test of choice for detection of T paraluis-cuniculi infection.     

Quantitative profile of cardiolipin and group treponemal IgD antibodies in syphilis estimated by single radial immunodiffusion technique (SRID)

150 serum samples (reactive in VDRL, Reiter-ELISA, FTA-Abs tests), from male patients 25-45 years old, in various stages of syphilis whether treated or untreated, were tested for IgD by SRID. On 154 sera from healthy males 25-45 years old, the reference normal values for IgD levels were established, as: 0-131.2 IU/ml with a mean of 29.92 +/- 29.61 IU/ml. Cardiolipin and group treponemal fraction values for IgD class were obtained by assessing the difference between the immunodiffusion diameter values produced by sera before and after complete absorption with VDRL antigen or delipidated T. reiteri suspension. The individual, mean +/- SD values (expressed in IU/ml) and the percentage of cardiolipin and treponemal IgD of the total IgD class were calculated for each stage. The mean value of the total IgD class, excepting secondary syphilis (sigma 2) 52.53 +/- 26.66 IU/ml), did not overstep the normal levels but all minimal individual values from syphilitic patients (7.09-14.89 IU/ml) surpassed significantly the normal minimal values which were less than or equal to 3.54 IU/ml. The total lack of cardiolipin (IgD and the presence of group treponemal IgD in all sera of the syphilis stages studied were manifest. The group treponemal IgD mean values ranged between 7-9 IU/ml, with a maximum of 19.32 +/- 10.58 IU/ml in sigma 2 followed by latent syphilis (sigma lat) with a mean value of 9.37 +/- 4.9 IU/ml. A significant percentage of treponemal IgD vs total IgD was recorded: primary syphilis (sigma 1) 32.01%, primary-secondary syphilis (sigma 1-2) 28.76%, sigma 2 36.77%, sigma lat and treated persistent seroreactive syphilis (sigma t+) 29.61%. The high proportion of treponemal IgD in latent and treated persistent reactive syphilis suggests a steady activation of B lymphocytes by treponemal antigens and presumably is an expression of an active infectious process. The absence of cardiolipin IgD and the presence of only the treponemal IgD, in all sera from all stages, might confer to their detection an extremely specific diagnostic value in syphilis.     

Shelf Life of Fluorescent Treponemal Antibody-Absorption Test Reagents

Properly prepared, standarized, and stored fluorescent treponemal antibody-absorption (FTA-ABS) reagents have been shown to have stabilities equal to other biological reagents. A liquid antigen over 10 years old has been shown to give a satisfactory reaction. Newer preparations have now been shown to be stable for over 5 years, and the tests on each are being continued. The very new liquid antigens which were originally standardized by the FTA-ABS method have shown no decrease in potency over a 20-month period. Stability studies on antigens dried on slides are now in their eighth month, with no apparent loss in potency. The stability of the conjugate is constant when stored frozen at -20 C or lyophilized. When stored as a liquid at 4 C, the stability is governed by the pH and the molarity of the buffer. The standardized and lyophilized sorbent has been shown to be stable for over 1 year.     

Treponemal serologic tests; experiences of the Bacteriology Laboratory,California State Department of Public Health

In a study of the relationship of clinical impression regarding syphilis and age, sex and pregnancy status to treponemal serologic test reactivity, it was noted that in diagnostic "problem cases" the standard lipid serologic test titers did not differentiate between syphilitic and biologic false positive reactors. Preliminary data indicated that heroin addiction may be a source of biologic false reactions and that pregnant women with standard serologic test reactivity have a lower treponemal reactivity rate than other women with lipid serologic reactivity.     

Fluorescent Treponemal Antibody Tests—A Summary and Comparison

A comparison of current serologic tests for syphilis shows that treponemal tests are preferable to reagin tests in detecting specific antibodies, but that reagin tests are best for determining the response to treatment. The newly developed fta-absorption technique is suggested as a reliable, inexpensive test for treponemal antibodies.     

TREPONEMAL SEROLOGIC TESTS—Experiences of the Bacteriology Laboratory,California State Department of Public Health

In a study of the relationship of clinical impression regarding syphilis and age, sex and pregnancy status to treponemal serologic test reactivity, it was noted that in diagnostic “problem cases” the standard lipid serologic test titers did not differentiate between syphilitic and biologic false positive reactors. Preliminary data indicated that heroin addiction may be a source of biologic false reactions and that pregnant women with standard serologic test reactivity have a lower treponemal reactivity rate than other women with lipid serologic reactivity.     

Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.     

Syphilis 2001 a palaeopathological reappraisal

The origin and subsequent spread of the treponematoses, especially that of venereal syphilis, has been the subject of considerable scientific attention. Various theories were put forth and palaeopathological specimens were used for their validation in recent times. One influential contribution was the paper by Baker & Armelagos in 1988. Numerous new findings and results on both sides of the Atlantic call for a new evaluation of the available osseous material. A review of the recent literature leads to the suggestion of a worldwide distribution of non-venereal treponemal disease since the emergence of Homo and to a first epidemic outbreak of venereal syphilis in Europe of the late 15th and the early 16th century, which was a time of change and enormous sexual liberty. Old World specimens with pathological alterations attributed to venereal syphilis and dated to precolumbian times seem to invalidate the Columbian theory and call for a more differentiated analysis of the phenomenon of syphilis than a theory based on a single factor can provide. With the help of molecular methods which now allow a positive identification of Treponema pallidum pallidum, causative agent of venereal syphilis, in palaeopathological material, it seems possible to elucidate the matter of origin and spread of syphilis further and to evaluate previous diagnoses of treponemal disease.     

FACTS AND FALLACIES ABOUT GONORRHEA AND SYPHILIS

The limitations and special usefulness of clinical and laboratory diagnostic techniques in the diagnosis of gonorrhea are poorly understood and utilized by the average practitioner today. Most physicians and clinics, lulled by complacency or lack of ancillary aid in the area of diagnosis, proceed by measures based in many instances upon past fallacy rather than upon the facts recently developed by research in this disease. The same circumstances apply concerning treatment and management of this disease, particularly in females.All physicians are potentially capable of giving excellent treatment for syphilis today. The problem is to properly diagnose the disease, manage the patient and deal with the source. Looming large in the area of diagnosis is the interpretation of serologic tests for syphilis. No serologic test diagnoses syphilis, but rather gives information as to the immunologic status of the the patient in relation to reagin and treponemal antibodies. None of the antibodies measured in these tests are absolutely specific for syphilis alone.There is no substitute for a well-informed physician, who knows his patient, to relate and interpret even the best of treponemal serologic tests.