Rapid tests for syphilis

Treponema-specific tests

Treponema-specific tests detect antibodies to antigenic components of Treponema pallidum. These tests are used primarily to confirm the diagnosis of syphilis in patients with a reactive non-treponemal test. Treponema-specific tests include, the T. pallidum hemagglutination (TPHA) test, the microhemagglutination test with T. pallidum antigen, the fluorescent treponemal antibody-absorption test (FTA-abs), and the enzyme-linked immunosorbent assay (ELISA) and the related immunochromatographic strip (ICS)tests. All these tests can be used for screening. However, the newer enzyme immunoassay (EIA) and lateral flow tests screen for anti-treponemal IgG, IgM, and IgA also may be more sensitive at various stages of infection.

Treponemal tests have sensitivities and specificities equal to or higher than those for non-treponemal tests. However, treponema-specific tests are more difficult and expensive to perform, which limits their usefulness as screening tests. In addition, false-positive results can occur, especially when the FTA-abs test is used in patients with systemic lupus erythematosus or Lyme disease.

False positives in treponemal assays are commonly due to a related treponemal infection such as yaws (T. pallidum pertenue), bejel (T. pallidum endemicum), or pinta (T. carateum). They may also arise from nonpathological or commensal treponeme exposure such as T. denticola, T. phagedenis, T. refringens, and T. vincentii, or the related spirochetes Borrelia recurrentis and Leptospira interrogans. Newer, more specific monoclonal antibodies and recombinant antigens have minimized this phenomenon, but it should be kept in mind none the less.

Unlike non-treponemal tests, which show a decline in titers or become nonreactive with effective treatment, most treponema-specific tests usually remain reactive for life. Because of the persistence of reactivity, possibly for the life of the patient, treponemal tests are of no value to the clinician in determining relapse, reinfection, or treatment efficacy. Therefore, a reactive treponemal test result only indicates exposure to T. pallidum at some time during a person’s life. It does not indicate that the person currently has an active syphilis infection.

For this reason, a positive treponemal test result should be confirmed with a non-treponemal test. If the non-treponemal test result is also positive and there is no recent history of treatment, it can generally be assumed that the patient has active syphilis. Most persons become seronegative (nonreactive) on non-treponemal tests (Venereal Disease Research Laboratory [VDRL], Rapid Plasma Reagin [RPR] following adequate antibiotic treatment. However, some patients do maintain low antibody titers on non-treponemal tests for life, despite receiving adequate treatment. These patients are described as being serofast.

Treponema-specific test types

Fluorescent Treponemal Antibody Absorption (FTA-abs)

This is sometimes also referred to as a syphilis Immunofluorescent Assay (IFA)
The FTA-abs test detects antibodies to T. pallidum and can be used to detect syphilis infection at any stage except during the first 3 to 4 weeks after exposure (which is about the same time frame that the VDRL/RPR tests become effective) and in tertiary stages of the disease. In the secondary stage of syphilis, the FTA-abs test is most reliable and is reportedly positive in 100 percent of cases. It can be adapted to detect either IgG or IgM antibody. The FTA-abs tests are fairly difficult to do and are relatively expensive, thus they may be better suited as a confirmatory assay for syphilis infection after another method tests positive for the syphilis bacteria.

FTA-abs: test procedure

Before testing the patient’s serum, it is first diluted in an extract of the nonpathogenic T. phagedenis (also known as Reiter’s strain) to remove nonspecific, anti-treponemal antibodies that are produced in some persons in response to nonpathogenic treponemes.

Then the substrate cells are reacted with patient sera. The serum is placed on a microscope slide fixed with nonviable T. pallidum cells. If treponemal antibodies to T. pallidum are present in the patient sera, it will coat the fixed cells.

In the last step, an anti-human immunoglobulin labeled with a fluorescent dye such as fluorescein isothiocyanate (FITC) is added to the slide and combines with any patient antibodies adhering to T. pallidum substrate cells. If the patient has been exposed to syphilis, the spirochetes will stain and be visible when examined by a fluorescence microscope. The intensity of staining is graded on a scale of negative (no fluorescence) through 1+ to 4+. Specimens that are minimally reactive (read as 1+) should be retested.

Atypical staining has been reported in patients with systemic lupus erythematosus and those with other autoimmune diseases.

The FTA-abs test is touchy, and there are numerous factors that can cause a failure. It is a multicomponent test, and each component and reagent must be matched with another. Conjugates and antigen slides must be properly stored and refrigerated. All controls need to be carefully titrated and run with each batch. Nonspecific stain and adsorbent controls must also be run in each batch. Antigen slides must be checked to ensure that the T. pallidum cells are adhering. In addition, the fluorescent microscope must be in good working order with the proper adjustments and filters in place to guarantee the reliability and reproducibility of the test.

The test can be done on blood or spinal fluid.

Run time: 1 to 2 hours

Lab equipment required:

  • Water bath adjusted to 56°C
  • 35°C to 37°C incubator
  • Serological pipette, and preferably a pipetting device for delivery of 0.01 mL
  • Timer
  • Test tubes and test tube racks
  • Volumetric flasks
  • Staining dish
  • Moist chamber
  • Coverslips, 24 x 60 mm, thickness No. 1
  • Distilled water
  • Bibulous paper
  • Properly equipped fluorescence microscope

Microhemagglutination assay (MHA-TP)and TPHA (T. pallidum hemagglutination assay)

For all intents and purposes, these tests are equivalent, but the MHA-TP was the earlier iteration of the TPHA. Occasionally, these tests are simply referred to as an indirect hemagglutination assay (IHA). The hemagglutination tests generally are simpler to perform than the fluorescent antibody tests and lend themselves to automation. The MHA-TP and TPHA tests are very rarely used currently. Both tests are quickly being replaced by newer and easier TP-PA and EIA-based tests (see below), including lateral flow strip tests.

Hemagglutination tests using treponemal antigen for T. pallidum slowly gained acceptance since their emergence in the mid-1960s as a confirmatory procedure following a reactive RPR or VDRL assay.

The MHA-TP and TPHA are used to confirm a syphilis infection after another method tests positive for the syphilis bacteria. The MHA-TP and TPHA tests detect antibodies to the bacteria that cause syphilis and can be used to detect syphilis in all stages, except during the first 3 to 4 weeks when antibody levels are too low. These tests are also suitable for use as a screening procedure. Neither of these tests is suitable for use on cerebrospinal fluid (CSF).

The MHA-TP/TPHA tests may be less sensitive than the FTA-abs test for identifying patients with primary syphilis.

TPHA: test procedure

The TPHA test is an indirect hemagglutination test for the detection and titration of specific anti-T. pallidum antibodies. In the assay, avian or ovine erythrocytes are coated with antigens from T. pallidum. In the presence of syphilitic positive serum, the erythrocytes aggregate to form characteristic patterns on the surface of the microplate wells. Antibodies directed against other nonpathogenic treponemas are absorbed out of the patient sample with an extract of Reiter’s treponeme, T. phagedenis, as is done in the FTA-abs test. This step greatly reduces false positives. Nonspecific reactions are detected using the control cells, which are erythrocytes that have not been coated with T. pallidum antigens. Nonspecific reactions may also be absorbed out using these control cells. The positive cell agglutination patterns are both easily read and long lasting.

This test is ideally suited for routine syphilis screening, or for confirming the results of tests performed using less specific methods. The TPHA will remain positive after treatment and thus is not a viable option for post-treatment follow up. False positives are most commonly seen in patients infected with leprosy.

Run time: 60 minutes to 12 hours (overnight incubation).

Lab equipment required:

  • Centrifuge to spin blood tubes for serum
  • Accurate pipettes for quantitative/titer testing
  • Microwell plates

T. pallidum particle agglutination assay (TP-PA)

The TP-PA test uses the same treponemal antigen as the MHA-TP/TPHA, but in this assay they are bound to gelatin particles. This modification eliminates virtually all nonspecific reactions with the patient sample.

The TP-PA test is primarily used as a confirmatory test for syphilis infection after another method gives a positive result for the syphilis treponeme. This test detects antibodies to T. pallidum. It can be used to detect syphilis in all stages except during the first 3 to 4 weeks, as with the MHA-TP/TPHA. Also like the MHA-TP/TPHA, the TP-PA test is not a suitable assay for use with CSF. Many studies have shown that TP-PA is more sensitive than TPHA and thus is a widely used confirmatory assay. It has also widely replaced both the FTA-abs and TPHA as the confirmatory test of choice.

The TP-PA assay is a treponema-specific test, and many clinicians recommend against its use for screening as it will be positive with treponemal infections other than syphilis (i.e., bejel, pinta, yaws). It is as sensitive and as specific as FTA-abs in all stages of syphilis, except the primary stage, in which it is less sensitive (but more sensitive than VDRL). Like FTA-abs and MHA-TP/TPHA, once the TP-PA is positive, it will remain so. Because of this, it cannot be used to evaluate the effectiveness of treatment.

TP-PA: test procedure

The TP-PA shares many similarities with the TPHA since it was developed from that platform. The TP-PA test is based on the agglutination of color-enhanced gelatin particles. Like the erythrocytes in the TPHA the gelatin particles are coated with T. pallidum antigens. It is used almost exclusively in microtiter volumes and wells.

In this test, the patient serum or plasma samples are mixed in a diluent containing an extract of Reiter’s treponeme T. phagedenis. This is typically done in microplate wells, and may be done as a serial dilution to assess titration levels. Then the antigen-coated gelatin particles are added to the appropriate wells and the plate is mixed by hand or on a tray mixer. The tray is then incubated for 2 hours at room temperature. During this step, the plate should not be moved or agitated. If the patient sample contains treponeme-specific antibodies, they will react with the antigen-coated gelatin particles to form a smooth mat of agglutinated particles within the well. If the patent sample is negative, there will be no agglutination of the particles, and a compact bead will form at the bottom of the well.

The agglutination patterns and interpretation of the test are clear cut and easy to read visually or with the aid of a tray viewer. Specimens giving inconclusive results in the assay should be retested.

False positives and inconclusive results are most common in patients with HIV, leprosy, toxoplasmosis, Helicobacter pylori, a history of drug abuse, and nonsyphilitic treponemal infections (i.e., bejel, pinta, yaws). The TP-PA assay will also give a positive result in a small percentage (less than 1%) of normal, healthy persons. These false-positive results are often transient, and their cause is unknown.

Run time: approximately 3 to 4 hours (overnight incubation is possible).

Lab equipment required:

  • Centrifuge to spin blood tubes for serum
  • Plate shaker
  • Accurate pipettes for quantitative/titer testing
  • Microwell plates

There are a number of  treponemal assays which are no longer performed but may be of interest for further reading such as the following.

Treponema pallidum immobilization (TPI) and surface immunofluorescence assay (SIFA). These have fallen out of favor because of their high complexity and use of live spirochetes which pose the risk of accidental infection. They also require animals in which to culture the treponemes. The advent of effective and sensitive EIAs and lateral flow platforms has helped to propagate the trend away from these tests.

EIA

Enzyme immunoassay (EIA), also known as an enzyme linked immunosorbent assay (ELISA), for syphilis is a relatively new invention first appearing on the market in the mid-1990s. There are numerous benefits to the EIA platform over earlier technologies. Firstly, the majority of diseases that are considered to be of clinical and public health importance already exist in an EIA format, which is highly standardized even across international boundaries. This familiarity allows new EIAs to be readily accepted by clinicians and technicians with minimal difficulty. It also limits the need to purchase new capital equipment since most labs will already be equipped to handle EIAs.

This standardization and conformity of procedure, even between assays for highly disparate diseases, lends itself to automation of EIA-based platforms and facilitates the addition of more assays once an EIA format test is established in a laboratory.

EIAs are relatively fast, taking on average about 2 hours total per batch, and allow for the screening of vast numbers of patients in a short period of time. Along with this, new syphilis EIA tests use a cocktail of recombinant antigens to detect T. pallidum specific IgG, IgM, and IgA antibodies in serum or plasma samples. This makes the assay an effective diagnostic in all stages of infection except those that are very early primary syphilis. It should be kept in mind that the EIA and ICS assays are still treponemal tests and will likely remain positive for the lifespan of those who have had syphilis, even with adequate treatment.

EIA: test procedure

This is a general protocol for an ELISA. Most diagnostic EIAs/ELISAs are based on a qualitative sandwich immunoassay. Most of these assays come in a prepackaged kit. A kit typically includes a number of 96-well microtiter plates. This kit is precoated with highly specific recombinant T. pallidum antigens. The more common ones currently available are TpN15, TpN16, TpN17, and TpN47, with the number reflecting the antigen’s weight in kiloDaltons (kD). Some manufactures do not disclose their antigens nor how many types are included in the test. Controls and samples are added to the plate wells and incubated, usually between 30 and 60 minutes. If anti-T. pallidum antibodies are present, they will bind to and become immobilized by the antigens coated in the wells. The microtiter plate wells are thoroughly washed to remove unbound components of the samples to help eliminate background caused by nonspecific reactions. After the wash step, a preparation of an enzyme, most commonly a peroxidase, which has been conjugated to an anti-human antibody, is then added to each test and control well. After adding the peroxidase bound antibody, a second incubation of another 30 to 60 minutes is done. After the incubation, the microtitre plate is thoroughly washed to remove all the unbound peroxidase antibody conjugate and a substrate that is reactive with it is added to each well. The most common is TMB (3,3’, 5,5’ tetramethyl benzidine) which turns a bright blue in the presence of a peroxidase. The peroxidase and substrate are allowed to incubate for 10 to 30 minutes. The wells that contain T. pallidum-specific antibodies, either in the patient sample or the positive controls added earlier, bound the peroxidase-antibody conjugate will turn from clear to blue in color. The intensity of this color will be based on numerous factors including temperature and length of reaction or incubation, amount of antigen coated on to the wells, and the amount of the antibodies in the patient/control sample. This enzyme-substrate reaction can be stopped by the addition of the “stop solution,” a .1-M sulphuric acid solution. This solution will change the original blue color to a bright yellow. This color can measured spectrophotometerically as an optical density (OD) value, when an EIA plate reader is available, at a wavelength of 450 nm. If the background is low enough, the wells can be read visually for a simple yes or no answer. If the sample has an OD values greater than or equal to the predetermined cutoff value, it is considered positive for syphilis.

Run time: approximately 2 to 4 hours

Lab equipment required:

  • Centrifuge to spin blood tubes for serum
  • Plate washer*
  • Plate reader*
  • Incubator*
  • Refrigerator
  • Accurate pipettes for quantitative/titer testing
  • Microwell plates

* These may be optional depending on the test protocol or format.