Chapter 18 Syphilis

The 2 types of antibody tests for syphilis are:

(1) those detecting a nonspecific antibody (2) those detecting a specific treponemal antibody Both types will be positive during this secondary stage

The laboratory assays most often used for syphilis diagnosis are based on the detection of 2 different types of antibody in the infected patient.

-nonspecific nontreponemal reaginic antibody, utilized as a screening test only, not diagnostic -specific treponemal antibody, used as a confirmatory test for a positive nonspecific test

Diagnostic Tests and Serology Evaluation based on three factors:

1. Clinical findings. 2. Demonstration of spirochetes in clinical specimen. 3. Present of antibodies in blood or cerebrospinal fluid.

Daily quality control:

20 gauge needle checked for delivery of 60 drops/mL Rotator checked for 100 rpms Room temperature must be 23-29 C. Three levels of control must be run and give appropriate results.

Percentage (stage 1)

30% of patients become serologically positive one week after appearance of chancre. 90% positive after 3 weeks. If left untreated, about 25% of patients progress to Stage 2 (secondary).

Syphulus test procedure

50 uL of serum added to circle on ceramic slide and spread. Add one calibrated drop of antigen to each circle. Rotate at 180 rpms for 4 minutes. Read microscopically at 100x and grade reaction if positive. Perform titer on positive samples, report out titer. A modified version is used for CSF. VDRL used primarily to screen cerebral spinal fluid.

Syphilis stage 1 symptoms (2)

A lesion does not always develop, or it may go unnoticed in the tissue folds of the vagina or anorectum. Multiple chancres can also occur, especially in persons co- infected with HIV. The chancre will heal spontaneously in 2 to 8 weeks with or without treatment. However, the serous fluid of this lesion is filled with T pallidum, and demonstration of these organisms forms the basis of the first available laboratory test (see below).

Treponema pallidum hemagglutination (TPHA)

Adapted for use in a microtiter plate (MHA-TP) Sheep or turkey RBCs are coated with T. pallidum antigen from Nichol's strain (a pathogenic strain). If antibodies to T. pallidum are present, agglutination occurs. Agglutination of the RBCs is a positive result. Appears as a "halo-like" distribution of RBCs in well. If no antibodies are present, the RBCs sink to bottom of the well forming a well-defined, small button.

Stage 2: Secondary Syphilis

At this point syphilis becomes a systemic disease. The symptoms are body rash, fever, malaise, headache, meningitis, alopecia, and mucous patch lesions. Some or all of these symptoms may be present, and this is when the greatest number of treponemes is in the body, particularly in the bloodstream. Highly infectious

Problem Areas with Testing

Biologic False Positives(BFP), reactive screening tests when patient does not have syphilis. Collagen diseases such as arthritis, LE, etc., sometimes result in increased amount of reagin. Certain infections: infectious mononucleosis, malaria, leprosy. Other treponemal infections.

Rapid Plasma Reagin test - RPR

CANNOT be performed on CSF. The VDRL cardiolipin antigen is modified with EDTA, thimerosal, choline chloride to make it more stable and inactivate complement. It is attached to charcoal particles to allow macroscopic reading, the antigen comes prepared and is very stable. Serum or plasma may be used for testing, serum is not heated.

Syphilis - Stage 1 (Primary) (2)

Clinically this incubation period is about 3 weeks but may range from 3 to 90 days. The incubation period is indirectly proportional to inoculum size and is called the "window phase," a time during which an infectious agent is undetectable by all available laboratory tests.

Treatment

Drug of choice penicillin, if allergic, doxycycline. Correlation of treatment with test results. Treatment at the primary stage, serology tests become non-reactive after 6 months. Treatment at secondary stage, tests usually non- reactive after 12-18 months. If treatment is not initiated until 10 or more years, the reagin tests probably positive for life.

ElISA

ELISA Tubes coated with T. pallidum antigen. Antibody in serum attaches to antigen. Following washing, add an anti-antibody tagged with enzyme alkaline phosphatase. Detectable color change occurs. Due to increasing availability and decreasing costs, may become screening test.

Western Blot

FDA is currently evaluating several Western blot assays against specific treponemal antigens

Venereal Disease Research Laboratory - VDRL

Flocculation test, antigen consists of very fine particles that precipitate out (flakes) in the presence of reagin. Utilizes an antigen which consists of cardiolipin, cholesterol and lecithin. 1) Antigen very technique dependent. 2) Must be made up fresh daily

Molecular Assays (primary and secondary)

Lab developed assays only-nothing FDA approved. Not in general use, but some assays target the DNA polymerase I gene (Pol A) which is specific to T. palladium sub. pallidum and is not present in other T. pallidum subspecies.

Syphilis congenital (2)

Live-born infants show no signs during first few weeks. -60 to 90 % develop clear or hemorrhagic rhinitis. skin eruptions (rash) especially around mouth, palms of hands and soles of feet. -Other signs: general lymphadenopathy, hepatosplenomegaly, jaundice, anemia, painful limbs, and bone abnormalities.

nonspecific nontreponemal tests

Non treponemal tests become positive 1 to 4 weeks after appearance of primary chancre

Congenital syphilis

Non treponemal tests on cord blood or baby serum detect IgG antibody, may be of maternal origin. Detection of IgM (ELISA) lacks sensitivity (about 80%). If it negative, does not rule out congenital syphilis Western Blot using four major treponemal antigens has demonstrated high sensitivity and specificity. All mothers undergoing prenatal care in the US are tested.

Fluorescent treponemal antibody absorption test (FTA- ABS)

One of the most used confirmatory tests. Diluted, heat inactivated serum added to Reiter's strain of T. pallidum to remove cross reactivity due to other Treponemes. Slides are coated with Nichol's strain of T. pallidum and add absorbed patient serum.

Stage 3: Tertiary Syphilis

One third to one half of untreated syphilis patients will go on to the last stage, called tertiary or late syphilis. Can occur in as little as a year after initial exposure, but typically is decades later.

nonspecific nontreponemal reaginic antibody

Reagin is an antibody formed against cardiolipin, a lipid released from treponemes and generated from damaged cells. -Found in sera of patients with syphilis as well as other diseases such as hepatitis, malaria, Chagas, endocarditis, measles, varicella, and various autoimmune disorders. -Also IV drug use, sample contamination, and recent vaccination can cause false positives.

Nonspecific Nontreponemal Serological Tests

Reagin test

Syphilis QC

Run three levels of control: Non-reactive, weakly reactive and reactive. Glass syringe with 18g delivery needle must be checked daily to ensure delivery of 60±2 drops/mL. Rotator rpms must be checked to ensure 180 rpms. Room temperature must be 23-29 C.

Spirochete Diseases

Serologic testing plays a key role in diagnosis due to the difficulty in isolation of the organisms. Clinical symptoms are not always present. Syphilis (Treponema pallidum sub. pallidum) Lyme Disease (Borrelia burgdorferi)

Syphulus serum

Serum must be heated to 56 C for 30 minutes to inactivate complement which may cause false positive. If serum is not tested within 4 hours must be reheated for 10 minutes. Calibrated syringe utilized to dispense antigen must deliver 60 drops/mL +/- 2 drops.

Test Procedure syphilis

Serum or plasma added to circle on card and spread. One drop of antigen from a needle capable of delivering 60±2 drops/mL is added. Rotate at 100 rpms for 8 minutes. Results are read macroscopically.

Test Procedure:

Serum or plasma added to circle on card and spread. One drop of antigen from a needle capable of delivering 60±2 drops/mL is added. Rotate at 100 rpms for 8 minutes. Results are read macroscopically.

Syphilis Fluorescence

Slides are washed, and incubated with antibody bound to a fluorescent tag. After washing again the slides are examined for fluorescence. Requires experienced personnel to read. Need a fluorescent microscope to read. Highly sensitive and specific, but time consuming to perform.

Syphilis fluorescense

T pallidum is identified in the serous transudate of lesions in primary and early secondary syphilis by either darkfield microscopy or fluorescent staining. They cannot be cultured or gram stained

Syphilis stage 1 symptoms

The first recognizable symptom of syphilis occurs in the primary stage. This is characterized by the usual formation of a single, painless, indurated ulcer called a chancre that forms at the site of inoculation

Syphilis 2nd stage detection

The immune response becomes quite intense as a result and is responsible for the inflammatory symptoms of this stage. It is during this time that antibody detection is the most robust.

Stage 2 (secondary) length

The secondary or disseminated stage follows 2 to 12 weeks after infectious contact, although there may be no sharp demarcation between the primary and secondary stage (still may have chancre).

Congenital Syphilis

Transmitted from mother to fetus. Mom typically has secondary syphilis or early latent syphilis. Fetus affected during second or third trimester. Forty percent result in syphilitic stillbirth-fetal death that occurs after a 20 week gestation.

Stage 3 of inflammation

Typical of this stage is the formation of gummas (granulomas), which are festering pockets of inflammatory cells and chemicals trying to clear the organism. Can occur anywhere (bones, tissues, skin) and can cause grotesque abnormalities.

Syphilis stage 2

Undiagnosed or untreated syphilis will progress to the latent period where the secondary symptoms subside. Still infected, the patient appears to get better without treatment. Patients can remain in this stage for the rest of their lives and are thought to be noninfectious to others. During this latent stage, syphilis can be diagnosed only with laboratory antibody tests.

Cerebrospinal Fluid tests

Used to determine if Treponemes have invaded CNS. VDRL utilized to confirm neurosyphilis. Highly specific. Lacks sensitivity.

Treponema pallidum particle agglutination (TP-PA)

Uses gel particles sensitized with T. pallidum of the Nichol's strain. Agglutination appears as a uniform halo in the well, and negative results are indicated by a well-defined button at the bottom of the well. TP-PA and TPHA/MHA-TP can cross react with other treponemes

False negatives

Very early in disease or latent, inactive stage Immunosuppressed patients Consumption of alcohol prior to testing (temporary)

Syphilis

Very fragile, destroyed rapidly by heat, cold, and drying. Predominately sexually transmitted, but can be transmitted congenitally, and very rarely needle stick and blood transfusion. Syphilis is treated with antibiotics, but contracting the disease offers no long-term immunity. People can be reinfected whenever exposed to the causative organism, Treponema pallidum sub pallidum (will call just T. pallidum in rest of notes).

This end stage of syphilis is a slowly progressive inflammatory disease that can involve any organ of the body but most:

cardiovascular: enlarging of the heart, aneurysms, etc. central nervous system: changes in behavior and personality, changes in emotional reactions, and psychotic symptoms

Syphilis - Stage 1 (Primary)

open ulcerated lesions small, hard chancre Stage 1(primary) The first stage of any disease begins at the point of infection. T pallidum penetrates mucosal membranes or broken skin, dividing every 30 to 33 hours, and when the concentration is approximately 107 organisms per milligram of tissue, a lesion will appear.

nonspecific nontreponemal tests (2)

secondary stage may have false negative due to prozone tertiary 25% are negative after successful treatment will become nonreactive after 1 to 2 years