Specimen Collection
Samples collected from ulcers and lesions should not be contaminated with blood, microorganisms, or tissue debris. The site should be cleansed with sterile gauze moistened with saline. The sample should be placed on a clean glass slide and cover slipped. Polymerase chain reaction (PCR) samples should be collected on a sterile Dacron or cotton swab and placed in a cryotube containing nucleic acid transport medium or universal transport medium. Tissue or needle aspirates of lymph nodes should be placed in 10% buffered formalin at room temperature. To test for congenital syphilis, a small section of the umbilical cord is collected and fixed in formalin or refrigerated until processed. Serum is the specimen of choice for serology; however, whole blood or plasma may be used in some assays.
Direct Detection
Treponemes can be detected in material taken from skin lesions by dark-field examination or fluorescent antibody staining and microscopic examination. Material for microscopic examination is collected from suspicious lesions. The area around the lesion must first be cleansed with a sterile gauze pad moistened in saline. The surface of the ulcer is then abraded until some blood is expressed. After blotting the lesion until there is no further bleeding, the area is squeezed until serous fluid is expressed. The surface of a clean glass slide is touched to the exudate, allowed to air dry, and transported in a dust-free container for fluorescent antibody staining. A T. pallidum fluorescein-labeled antibody is commercially available for staining (Viro Stat, Portland, Maine). For dark-field examination, the expressed fluid is aspirated using a sterile pipette, dropped onto a clean glass slide, and cover slipped. The slide containing material for darkfield examination must be transported to the laboratory immediately. Because positive lesions may be teeming with viable spirochetes that are highly infectious, all supplies and patient specimens must be handled with extreme caution and carefully discarded as required for contaminated materials. Gloves should always be worn.
Material for dark-field examination is examined immediately under 400× high-dry magnification for the presence of motile spirochetes. Treponemes are long (8 to 10 µm, slightly larger than a red blood cell) and consist of 8 to 14 tightly coiled, even spirals (Figure 1). Once seen, characteristic forms should be verified by examination under oil immersion magnification (1000×). Although the darkfield examination depends greatly on technical expertise and the numbers of organisms in the lesion, it can be highly specific when performed on genital lesions.
Fig1. Appearance of Treponema pallidum in dark-field preparation.
Lesion exudates or tissue samples may be used for direct fluorescent antibody detection for T. pallidum (DFA-TP). DFA-TP visualizes specimens on slides with fluorescein isothiocyanate (FITC) labeled antibodies. Polyclonal and monoclonal antibodies may be used; however, the Food and Drug Administration (FDA) in the United States has not approved this test.
Molecular Diagnostics
Although molecular diagnostic assays are not currently available within many clinical laboratories, several methods have been developed using PCR for the detection of T. pallidum. These methods are primarily useful in the identification of organisms within exudate or lesions.
Serodiagnosis
Serologic tests for treponematosis measure the presence of two types of antibodies: treponemal and nontreponemal. Treponemal antibodies are produced against anti gens of the organisms themselves, whereas nontreponemal antibodies, often referred to as reagin antibodies, are produced in infected patients against components of mammalian cells. Reaginic antibodies, although almost always produced in patients with syphilis, are also produced in patients with other infectious diseases such as leprosy, tuberculosis, chancroid, leptospirosis, malaria, rickettsial disease, trypanosomiasis, lymphogranuloma venereum (LGV), measles, chickenpox, hepatitis, and infectious mononucleosis; noninfectious conditions such as drug addiction; autoimmune disorders, including rheumatoid disease and systemic lupus erythematosus; and in conjunction with increasing age, pregnancy, and recent immunization.
The two most widely used nontreponemal serologic tests are the Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin (RPR) tests. Each of these tests is a flocculation (or agglutination) test, in which soluble antigen particles are coalesced to form larger particles that are visible as clumps when they are aggregated in the presence of antibody. The VDRL is used as a quantitative test and may be performed on serum or CSF in suspected cases of neurosyphilis. See Procedures 1 and 2 on the Evolve site for details and limitations for the VDRL and RPR.
Procedures 1
Procedures 2
Specific treponemal serologic tests include automated enzyme immunoassays (EIAs) and agglutination tests, such as the T. pallidum particle agglutination (TP-PA) test, the microhemagglutination assay (MHA-TP), T. pallidum indirect hemagglutination (TPHA), particle gel immunoassay (PaGIA), and the fluorescent treponemal antibody absorption (FTA-ABS) test. Once positive, their usefulness is limited because these tests tend to yield positive results throughout the patient’s life. The FTA-ABS test is performed by overlaying whole treponemes fixed to a slide with serum from patients suspected of having syphilis. This test is typically performed following a positive VDRL or RPR screening test. The patient’s serum is first absorbed with non–T. pallidum treponemal anti gens (sorbent) to reduce nonspecific cross-reactivity. Fluorescein-conjugated antihuman antibody reagent is then applied as a marker for specific antitreponemal antibodies in the patient’s serum. This test should not be used as a primary screening procedure. TP-PA (Fujirebio America, Fairfield, New Jersey) tests utilize gelatin particles sensitized with T. pallidum subsp. pallidum antigens. Serum samples are diluted in a microtiter plate and sensitized gelatin particles are added. The presence of specific antibody causes the gelatin particles to agglutinate and form a flat mat across the bottom of the microdilution well in which the test is performed. The MHA-TP is a passive hemagglutination assay of sensitized erythrocytes that are tested against the patient’s serum. Agglutination indicates the presence of IgG or IgM anti treponemal antibodies in the patient’s serum. TPHA is an indirect hemagglutination assay that uses sensitized red blood cells that aggregate when exposed to positive patient serum. This test is similar to the MHA-TP. PaGIA test, which uses gel immunoassay technology, an established method in blood group serology. The assay contains recombinant antigens for the detection of T. pallidum antibodies in the patient’s serum or plasma. The results are available in approximately 15 minutes. Several EIAs are available that utilize the direct, indirect sandwich, or competitive assay methodology. EIAs use recombinant antigens to detect IgM, IgG, or both. To date, no evidence indicates that these assays are more sensitive than the traditional treponeme tests. The Centers for Disease Control and Prevention (CDC) is currently evaluating rapid testing formats for syphilis that use lateral flow or flow through cassette methodology.
Several automated systems currently exist that use bead-capture technology. These assays use a capture anti body attached to a suspension of small micro polystyrene beads. The beads are dyed with fluorophores of differing intensity, giving each a unique fingerprint. The sandwich immunoassay uses a flow cytometry dual-laser system for detection. There are currently three Luminex commercial platforms that utilize this technology; Abbott Architect (Abbott Laboratories, Abbott Park, Illinois), Bio-Rad Bioplex (Bio-Rad Laboratories, Hercules, California), and Zeus AtheNA (Zeus Scientific, Branchburg, New Jersey). A fourth system, the DiaSorin Liaison (DiaSorin S.p.A., Vercelli, Italy) uses magnetic beads to capture patient antibodies with an isoluminol-antigen conjugate. Positive samples are then detected using a flash chemiluminescent signal.
The nontreponemal serologic tests for syphilis can be used to determine antibody quantitative titers, which are useful to follow the patient’s response to therapy. The relative sensitivity of each test is shown in Table 1 to confirm that a positive nontreponemal test result is due to syphilis rather than to one of the other infections or biologic false-positive conditions previously mentioned. Traditional diagnosis for syphilis is useful in active infections. However, early or treated infections may be incorrectly diagnosed. In addition, primary testing using RPR or VDRL may result in a high rate of false-positives. The Centers for Disease Control has recommended a reverse algorithm to detect early primary or treated infections that may be missed using traditional nonspecific screening methods. Reverse testing suggests the use of specific antibody testing for syphilis, using enzyme-linked immunoassay (EIA) for IgM and IgG or a similar technique. T. pallidum antibodies persist for many years following infection. Specific tests may then be followed by nonspecific screening tests, which become less reactive over time. However, reverse testing is not currently widely accepted, and more data are needed to resolve clinical diagnostic discrepancies (Figure 2).
Table1. Sensitivity of Commonly Used Serologic Tests for Syphilis
Fig2. Traditional testing versus reverse testing.
