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.     

Automated Fluorescent Treponemal Antibody Test: Instrument and Evaluation

Two evaluations of the automated fluorescent treponemal antibody (AFTA) test for the serodiagnosis of syphilis are described. The results of AFTA and manually performed fluorescent treponemal antibody-absorption (FTA-ABS) tests were compared on serum samples from clinically defined donor groups, and the reproducibility of each procedure was studied. Significant improvement of AFTA test results was obtained in the most recent study after developmental modifications of the instrument and test technique. AFTA test agreement with both syphilis and nonsyphilis categories was considered good. With the increasing usage of the FTA-ABS test as an effective tool for the diagnosis of syphilis, successful automation of this procedure is particularly timely and significant.     

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.     

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.     

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.     

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.     

Seroepidemiology of Anaplasma marginale in white-tailed deer (Odocoileus virginianus) from Louisiana

Antibodies to Anaplasma marginale were detected by the indirect fluorescent antibody test (IFA) in six of 331 (2%) serum samples of white-tailed deer (Odocoileus virginianus) from Louisiana. None of the serum samples were positive using the A. marginale modified rapid card agglutination test. Of the six IFA positive sera retested by the complement fixation test four sera gave anticomplementary and two gave seropositive reactions. The low A. marginale reactor rate in this white-tailed deer population was probably a reflection of the lack of cohabitation between cattle and deer and the fact that the primary arthropod vectors in Louisiana are tabanids. The validity of the indirect fluorescent antibody test for A. marginale antibodies in white-tailed deer should be evaluated.     

Positive Fluorescent Treponemal Antibody Reactions in Diabetes

In a survey of 129 diabetic patients and 142 normal individuals, a significantly higher percentage of positive reactions in the fluorescent treponemal antibody-200 (FTA-200) test was found among diabetic patients than in the normal population. Absorption of all FTA-200-reactive sera with an extract of Reiter's treponeme eliminated most of the positive reactions in sera from diabetic patients, and three of the five positive reactions detected in sera from apparently normal subjects. On immunoelectrophoresis, precipitin bands developed most frequently between the Reiter sorbent and sera from diabetic patients positive in the FTA-200 test. Serum components responsible for FTA reactivity and precipitin reactions against the sorbent were resistant to treatment with mercaptoethanol, suggesting antibody of the IgG class. Cross-reacting antibodies produced in response to normal treponemal flora, and perhaps acquiring enhanced reactivity by means of nonspecific interacting substances in sera peculiar to the altered physiological state of diabetes, are suggested as possible causes of positive reactions of unabsorbed sera. No correlation could be made between age of the diabetic patient, treatment or duration of the disease, and FTA or precipitin reactivity of the patient's serum.     

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.     

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.     

Use of the toxin-binding inhibition test for estimation of tetanus antitoxin in serum

The usefulness of the toxin binding inhibition test (ToBI) for the titration of tetanus antibody in human sera was assessed. Sera from 80 healthy people with different vaccination histories that had been previously tested by the in vivo toxin neutralization (TN) test were retested by the ToBI test. The lowest tetanus antibody titre which could be detected by using 0.1 Lf/ml tetanus toxin was 0.01 IU/ml. Comparison between the estimates obtained by the ToBI test and those obtained by the TN test (r = 0.93 and r = 0.7 for titration low titre sera) showed good correlation. No overestimation of antibody content was seen in titrating low titre sera by the ToBI test. It is concluded that the ToBI-test is a reliable alternative to toxin neutralization test in mice.     

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax and its use in a seroepidemiological survey of the Eastern Caribbean Basin

An enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against a South American (New World) strain of Trypanosoma vivax was developed and used for mass screening of cattle from 20 islands in the Eastern Caribbean Basin. The sensitivity and specificity of antigens prepared from a bovine-derived field strain and a murine-adapted laboratory strain of T. vivax, both of New World origin, were compared using an indirect fluorescent antibody (IFA) test, and an antigen prepared from the murine-adapted strain was subsequently used to develop an ELISA test. The results of the ELISA test were then compared with the results of a concurrently run IFA test. There was no cross-reactivity with either test using serum from a Trypanosoma theileri-infected cow. Both tests were weakly cross-reactive with sera from a T. brucei-infected steer, and the IFA test was moderately cross-reactive with several serum samples from a T. evansi-infected steer. For bovine sera collected from herds on islands in the Eastern Caribbean region, only five of 640 tested positive with the ELISA test. Thirty five of 653 sera tested were positive by IFA although the fluorescence elicited was weak as compared to that elicited by sera from known infected animals. Sera collected from 27 cattle in a region known to be free of T. vivax (OH, U.S.A) were negative with the ELISA test, whereas seven of 30 sera from a herd in French Guiana known to be infected with T. vivax were positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme-linked immunosorbent assay for efficient detection of antibody to bluetongue virus in pronghorn (Antilocapra americana)

An indirect enzyme-linked immunosorbent assay (ELISA), using cell-associated viral antigen, was developed for detection of antibody to bluetongue virus (BTV) in field-collected pronghorn (Antilocapra americana) sera. To test the applicability of the ELISA to seroepizootiologic studies, pronghorn serum samples from three Wyoming counties (USA) were tested. Bluetongue virus ELISA results were compared to those of the bluetongue immunodiffusion assay. Discrepant serum samples were retested for reaction to either BTV or epizootic hemorrhagic disease virus. The pronghorn BTV ELISA gave rapid, quantitative, objective results and should facilitate testing large numbers of sera for BT diagnostic and seroepizootiologic studies.     

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.     

Comparative evaluation of antibody positive titer by ELISA and IFA in Theileria annulata vaccinated cattle in Iran

An enzyme linked immunosorbent assay (ELISA) was used to evaluate antibody positive titer in vaccinated and non-vaccinated cattle using schizont infected myeloid cells as an antigen. The result was compared with indirect fluorescent antibody level in the same animals. For this study 116 milking cows, 95 vaccinated and 21 non-vaccinated, were bleeded in order to prepare sera. They were tested with both ELISA and IFA tests. 94 sera had positive antibody titer and 22 sera were negative through ELISA test but, with IFA test, only 89 sera showed positive antibody titer and 27 were negative. Thereby, it was concluded that the sensitivity and specificity of ELISA test in comparison with IFA test was 95.5% and 66.6% respectively. This study generally indicated that ELISA could be an effective test for sero-epidemiological investigations of bovine tropical theileriosis, and it is considered to be valid as an additional test to distinguish the vaccinated from the non vaccinated cattle in order to schedule vaccination programs.     

Evaluation of ELISA for the Detection of Toxoplasma Antibodies in Swine Sera

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from > 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving < 10 EIU were negative in the other tests, and all those with > 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10–70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.     

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.     

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.     

Validation of Serological Tests for the Detection of Antibodies Against Treponema pallidum in Nonhuman Primates

There is evidence to suggest that the yaws bacterium (Treponema pallidum ssp. pertenue) may exist in non-human primate populations residing in regions where yaws is endemic in humans. Especially in light of the fact that the World Health Organizaiton (WHO) recently launched its second yaws eradication campaign, there is a considerable need for reliable tools to identify treponemal infection in our closest relatives, African monkeys and great apes. It was hypothesized that commercially available serological tests detect simian anti-T. pallidum antibody in serum samples of baboons, with comparable sensitivity and specificity to their results on human sera. Test performances of five different treponemal tests (TTs) and two non-treponemal tests (NTTs) were evaluated using serum samples of 57 naturally T. pallidum-infected olive baboons (Papio anubis) from Lake Manyara National Park in Tanzania. The T. pallidum particle agglutination assay (TP-PA) was used as a gold standard for comparison. In addition, the overall infection status of the animals was used to further validate test performances. For most accurate results, only samples that originated from baboons of known infection status, as verified in a previous study by clinical inspection, PCR and immunohistochemistry, were included. All tests, TTs and NTTs, used in this study were able to reliably detect antibodies against T. pallidum in serum samples of infected baboons. The sensitivity of TTs ranged from 97.7-100%, while specificity was between 88.0-100.0%. The two NTTs detected anti-lipoidal antibodies in serum samples of infected baboons with a sensitivity of 83.3% whereas specificity was 100%. For screening purposes, the TT Espline TP provided the highest sensitivity and specificity and at the same time provided the most suitable format for use in the field. The enzyme immune assay Mastblot TP (IgG), however, could be considered as a confirmatory test.