Blood spot screening and confirmatory tests for syphilis antibody.

We developed a blood spot test for syphilis antibody using enzyme-linked immunosorbent assay (ELISA) technology. Dried blood was eluted by buffered saline or, for a supplementary confirmatory test, by treponemal-antibody test diluent. Eluates were diluted in an absorption buffer (Calypte Biomedical, Berkeley, Calif.) and added to plate wells coated with cardiolipin antigen (ADI Diagnostics, Toronto, Ontario, Canada). The wells were washed and treated sequentially with an immunoglobulin G conjugate, buffer washes, and enzyme substrate. Substrate conversion was measured photometrically, and specimen reactivity was determined by reference to nonreactive controls. The optimum test protocol was established by tests of serum and plasma. The serum ELISA specificity with normal specimens was 98.9%. The sensitivity with sera from patients with undefined syphilis was 97.4%, that with sera from patients with documented primary and secondary disease was 100%, and that with sera from patients with early and late latent disease was 95.7%. The specificity of the spot test with donor blood was 94.2%, and its specificity with newborn blood was 94.9%. The sensitivity with 25 spots spiked with reactive sera was 96%. The seroprevalence rates for parturient women in one hospital were 6.01% according to spot tests of sera from 599 newborns and 6.81% according to Rapid Plasma Reagin tests of 499 maternal serum specimens. Seventy percent of infants born to 50 seropositive women were reactive by either the newborn spot or the Rapid Plasma Reagin serum test. The results show that blood spots may be used in seroprevalence or serodiagnostic studies, especially to identify women who are infected or to identify possible cases of congenital infection. The test provides for studies of children and adults when routine venipuncture and serum handling and storage are problematic.     

Hemagglutination tests for syphilis antibody.

In a study of serodiagnosis of syphilis, the authors compared the specificities and sensitivities of two hemagglutination tests, a sheep-erythrocyte test (MHA-TP) and a trukey-erythrocyte test (TPHA), with those of the Fluorescent Treponemal Antibody-Absorption (FTA-ABS) test. In tests of sera from 935 patients without syphilis, the MHA-TP, TPHA, and FTA-ABS tests were reactive for 0.96, 0, and 1.3% respectively. The false-positive results were usually transient and not associated with underlying illness. For the 68 patients with syphilis, the MHA-TP test was as sensitive as the FTA-ABS test in all stages except untreated primary disease. The TPHA test appeared to be undersensitive, and testing of follow-up sera from persons with latent syphilis showed unexplained conversion of false-negative TPHA results to reactive results. Reproducibilities of the two hemagglutination tests were comparable. The MHA-TP test is a valuable confirmatory test for syphilis. Further study is needed before the use of the TPHA test can be recommended.     

Comparison between the automated reagin test and reagin screen test methods of VDRL screening tests for syphilis in use in a routine laboratory.

A comparison is made between the automated reagin test (ART) using Technicon AutoAnalyzer equipment and the reagin screen test (RST) introduced by Lederle using a new antigen formulation. Treponemal haemagglutination tests (TPHA) were done simultaneously as second screen tests. The absorbed fluorescent treponemal antibody test (FTA-ABS) was used in all seropositive cases. Altogether 1154 sera were tested; 1028 were negative with all tests, 66 were positive with all tests, 42 were positive with TPHA and negative with ART and RST, and 16 were positive with TPHA and RST and negative with ART. It was concluded that the use of the RST and TPHA together would be the more sensitive screen test.     

It is time to use treponema-specific antibody screening tests for diagnosis of syphilis

Assays that detect treponema-specific antibodies, which are either automated or can be done as point-of-care tests, have been developed, some of which are FDA approved. These assays have the advantage of being easily performed and demonstrate high sensitivity, both key features of an infectious disease screening test. As a result, many high-volume clinical laboratories have begun to offer a reverse syphilis testing algorithm where a treponema-specific test is used for screening, followed by a nontreponemal test (i.e., rapid plasma reagin [RPR]) to assess disease activity and treatment status. Concerns about physicians being able to understand and apply this new testing algorithm have been expressed (8). In this point-counterpoint, Michael Loeffelholz of the University of Texas Medical Branch at Galveston explains why his laboratory has adopted this reverse algorithmic approach. Matthew Binnicker of the Mayo Clinic, Rochester, MN, explains why the reverse algorithm may not be suitable for all clinical laboratories and every clinical situation.     

Evaluation of a Treponema pallidum enzyme immunoassay as a screening test for syphilis.

The CAPTIA Syphilis-G enzyme immunoassay for the detection of antibodies to Treponema pallidum was evaluated as a screening test for syphilis in comparison with the standard rapid plasma reagin (RPR) test. One thousand samples were tested, and the standard fluorescent treponemal antibody absorption test and the standard microhemmaglutination test were used to confirm the presence of treponemal antibodies. Diagnosis of syphilis was based on traditional standard serology results. Clinical data used in the diagnosis of patients whose samples yielded conflicting results were provided by physicians. Initially, 7 patients whose samples were reactive in the RPR test and 14 patients whose samples yielded positive or equivocal results in the CAPTIA Syphilis-G test were diagnosed as not being infected. After discrepancies due to technical problems were reconciled, samples from six patients remained reactive in the RPR test and that from one patient remained positive in the CAPTIA Syphilis-G test. In addition, seven patients whose samples were nonreactive in the RPR test and two patients whose samples were negative in the CAPTIA Syphilis-G test were diagnosed as having untreated syphilis. After discrepancies were reconciled, samples from five patients remained nonreactive in the RPR test and none remained negative in the CAPTIA Syphilis-G test. Final results indicate that the specificities are 99.4 and 99.9%, respectively. In addition to the improved sensitivity and specificity of the CAPTIA Syphilis-G screen, other potential benefits of this assay lead us to believe that this method could serve as a better screening tool than the RPR test.     

Evaluation of NCS rapid plasma reagin card test, a new screening kit for syphilis.

Agreements in qualitative and quantitative test results between the recently marketed NCS rapid plasma reagin card test (NCS Diagnostics Corp.) and the Macro-Vue rapid plasma reagin 18-mm circle card test (Hynson, Westcott and Dunning) were 99.2 and 99.1%, respectively, indicating the NCS RPR card test to be an acceptable alternative procedure for the serodiagnosis of syphilis.     

An automated sample preparation system for large-scale DNA sequencing.

Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.     

Evaluation of three commercial screening tests for AIDS virus antibodies

Three commercial enzyme-linked immunosorbent assays (ELISA) for acquired immune deficiency syndrome (AIDS) virus antibodies were evaluated using serum that had been characterized by an ELISA and western blot procedure developed at the University of California at Davis (UCD). Each of the commercial tests was more specific than the UCD ELISA, but the UCD ELISA was more sensitive in the detection of sera that lacked reactivity by western blot to the envelope glycoprotein (gp-41). The HTLV-III Bio-EnzaBead (Litton Bionetics, Charleston, SC) was less sensitive and specific than the Abbott HTLV-III EIA (Abbott Laboratories, North Chicago, IL) or the Virgo HTLV-III ELISA (Electro-Nucleonics Inc., Columbia, MD). Overall, 22.9% (57 of 250) and 51.0% of sera that were repeatedly (X2) positive by commercial screening kits tested at blood donor centers and clinical laboratories, respectively, were confirmed by western blot. These results indicate that the screening assays for AIDS virus antibodies are not equal in performance and that positive screening test results must be confirmed by a more specific test like western blot before results are released.     

Evaluation of an automated system in bacteriology. Application to bacteriological susceptibility tests

Susceptibility tests have been compared between a new automated system and the reference method by agar diffusion. The COBAS-Micro apparatus for bacterial susceptibility tests is intended for rapid determinations with, normally, an incubation time of 5 h at 37 degrees C. Its is compatible with strains requiring greater than 18 h incubation. With this technique a large range of antibacterial substances (greater than 60) can be studied, in groups of fifteen. With the help of a computerized soft-ware, one growth-index:EPR (End Point Ratio) is calculated, in comparison with a standard, for each antibacterial agent tested and expressed in three categories: SIR. The findings are optimized according to the limits prescribed by the NCCLS. The reference method, by agar-diffusion, is as described by the Comité de l'Antibiogramme de la Société Fran?aise de Microbiologie (CA-SFM). A total of 1,048 strains were tested by the two techniques: 518 Gram negative rods, 530 Gram positive cocci, with four common antibiotics. The percentages of agreement between the two groups were: 96% full agreement and minor discrepancy, 4% major and very major discrepancy. This automated system seems to be perfectly suitable for susceptibility testing in a routine laboratory, especially with strains isolated from ambulatory patients.     

Evaluation of the Bio-EnzaBead test for syphilis.

The sensitivity and specificity of the Bio-EnzaBead test for syphilis and the fluorescent treponemal antibody-absorption (FTA-ABS) test were determined by examining 262 serum samples, including 202 serum samples from patients with confirmed syphilis in various stages. Overall correlation with patient history was 95.8% with both tests. False-negative Bio-EnzaBead tests occurred in 9 of 86 (10.5%) cases of late-latent syphilis (greater than 2 years) and in 1 of 38 (2.6%) cases for which the stage of disease could not be determined. False-negative FTA-ABS tests occurred in 5 of 86 (5.8%) cases of late-latent syphilis (greater than 2 years) and in 2 of 38 (5.3%) cases for which the stage of disease could not be determined. One false-positive test occurred with Bio-EnzaBead, and the cause could not be determined. The reproducibility of the Bio-EnzaBead test was excellent when spectrophotometric readings were calibrated against either air or substrate blanks. The Bio-EnzaBead test for syphilis is a suitable alternative to the FTA-ABS test.