Key features of the epidemiology of diseases caused by the pathogenic treponemes are summarized in Table 46-2. In general, these organisms enter the host by either penetrating intact mucous membranes (as is the case for T. pallidum subsp. pallidum—hereafter referred to as T. pallidum) or entering through breaks in the skin. T. pallidum is transmitted by sexual contact and vertically from mother to the unborn fetus. After penetration, T. pallidum subsequently invades the bloodstream and spreads to other body sites. Although the mechanisms by which damage is done to the host are unclear, T. pallidum has a remarkable tropism (attraction) to arterioles; infection ultimately leads to endarteritis (inflammation of the lining of arteries) and subsequent progressive tissue destruction.

TABLE 46-2

Epidemiology and Spectrum of Disease of the Treponemes Pathogenic for Humans

Agent	Transmission	Geographic Location	Disease	Clinical Manifestations*	Age Group
T. pallidum subsp. pallidum	Sexual contact or congenital (mother to fetus)	Worldwide	Venereal syphilis^†	Refer to text in this chapter	All ages
T. pallidum subsp. pertenue	Traumatized skin comes in contact with an infected lesion (person-to-person contact)	Humid, warm climates: Africa, South and Central America, Pacific Islands	Yaws	Skin—papules,^† nodules, ulcers	Children
				Primary lesion (mother yaw), disseminated lesions (frambesia)
				May progress to latent stage and late infection involving destructive lesions to bone and cartilage
T. pallidum subsp. endemicum	Mouth to mouth by utensils, (person-to-person contact)	Arid, warm climates: North Africa, Southeast Asia, Middle East	Endemic nonvenereal syphilis	Skin/mucous patches, papules, macules, ulcers, scars^†	Children or adults; rarely congenital
				May progress to disseminated oropharyngeal with generalized lymphadenopathy
				May demonstrate a latent stage, and late syphilis destructive to skin, bone, and cartilage
T. carateum	Traumatized skin comes in contact with an infected lesion (person-to-person contact)	Semiarid, warm climates: Central and South America, Mexico	Pinta	Skin papules, macules. Hyperkeratotic pigmented may lead to disseminated skin lesions and lymphadenopathy; late stage may result in pigmentary changes in skin (hyper- or hypopigmentation)	All ages but primarily children and adolescents

^*All diseases have a relapsing clinical course and prominent cutaneous manifestations.

^†If untreated, organisms can disseminate to other parts of the body such as bone.

Spectrum of Disease

Treponema pallidum causes venereal (transmitted through sexual contact) syphilis. The clinical presentation of venereal syphilis is varied and complex, often mimicking many other diseases. This disease is divided into stages: incubating, primary, secondary, early latent, latent, and tertiary. Primary syphilis is characterized by the appearance of a chancre (a painless ulcer) usually at the site of inoculation, most commonly the genitalia. Within 3 to 6 weeks, the chancre heals. Dissemination of the organism occurs during this primary stage; once the organism has reached a sufficient number (usually within 2 to 24 weeks), clinical manifestations of secondary syphilis become apparent. During this phase the patient is ill and seeks medical attention. Systemic symptoms such as fever, weight loss, malaise, and loss of appetite are present in about half of the patients. The skin is the organ most commonly affected in secondary syphilis, with patients having a widespread rash with generalized lymphadenopathy. Aseptic meningitis may also occur. After the secondary phase, the disease becomes subclinical but not necessarily dormant (inactive); during this latent period, diagnosis can be made using serologic methods. Relapses are common during early (≤ 1 year) latent syphilis. Late latent syphilis (≥ 1 year) is usually asymptomatic and noninfectious. Tertiary syphilis is the tissue-destructive phase that appears 10 to 25 years after the initial infection in up to 35% of untreated patients. Complications of syphilis at this stage include central nervous disease (neurosyphilis), cardiovascular abnormalities, eye disease, and granuloma-like lesions, called gummas, found in the skin, bones, or visceral organs. Congenital syphilis is transmitted from mother to the unborn fetus during any stage of infection, but is most often associated with early syphilis. The unborn fetus may develop an asymptomatic infection or symptomatic infection with damage to the bone and teeth, deafness, neurosyphilis, or neonatal death.

The additional pathogenic treponemes are major health concerns in developing countries. Although morphologically and antigenically similar, these agents differ epidemiologically and with respect to their clinical presentation from T. pallidum. The diseases caused by these treponemes are summarized in Table 46-2.

Laboratory Diagnosis

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 dark-field 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 46-2). 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.

Figure 46-2 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 antigens 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 46-1 and 46-2 on the Evolve site for details and limitations for the VDRL and RPR.

Procedure 46-1 Rapid Plasma Reagin (RPR) Test

Purpose

The rapid plasma reagin (RPR) test is a presumptive serologic screening test for syphilis.

Principle

The test is based on the presence of reagin, a nonspecific antilipid antibody found in the serum of patients infected with T. pallidum. Syphilis infection results in the breakdown of human tissue releasing fatty substances that combine with T. pallidum protein to form an antigen, resulting in the formation of nonspecific and specific T. pallidum antibodies. The RPR antigen consists of cardiolipin, lecithin, and cholesterol bound to charcoal particles. The nonspecific antibody reacts with the test antigen resulting in flocculation. The flocculation is visible on a white test card by incorporating charcoal particles in the antigen preparation.

Specimen

Patient serum or plasma is tested at room temperature, 20° to 25° C.

Method

1. Place a 50 µL sample of patient serum or plasma, positive and negative controls into three separate circles on the test card.

2. Mix the RPR-carbon reagent gently prior to applying to each circle.

3. Add exactly one drop (20 µL) of reagent to each sample.

4. Mix the specimen and reagent with a stirrer carefully spreading each sample over the entire surface of the circle. Use a clean stirrer for each sample.

5. Place the test card on a mechanical rotator or shaker at 80 to 100 rpm for 8 minutes.

6. Read the results prior to drying.

Expected Results

Reactive: samples that demonstrate small to large clumps; a weak positive may show fine granulation or partial clumping.

Nonreactive: samples that demonstrate no clumping or very slight roughness.

Limitations

1. Slide flocculation tests are affected by room temperature. All test reagents and specimens should be warmed to room temperature prior to testing.

2. Failure to read results within 8 minutes may lead to drying and the appearance of false-positive results.

3. Patient samples that are hemolyzed, lipemic, or contain a high concentration of bilirubin may interfere with test results.

Quality Control

Positive controls should demonstrate readily visible flocculation

Negative controls will appear uniformly turbid.

Procedure 46-2 Venereal Disease Research Laboratory (VDRL) Test

Purpose

The Venereal Disease Research Laboratory Test (VDRL) is a slide flocculation test used for screening patients for infection with T. pallidum.

Principle

Patients infected with T. pallidum produce nonspecific antibodies capable of reacting with the cardiolipin test antigen. Cardiolipin is a lipid antigen extracted from beef heart that contains cardiolipin, lecithin, and cholesterol. The VDRL test is typically positive within 1 to 2 weeks following the appearance of the primary lesion. The test becomes reactive in late phase primary syphilis and highly reactive in secondary syphilis. The results will slowly decrease and become less reactive in late or tertiary syphilis. The VDRL test is also useful in the diagnosis for congenital syphilis. Maternal antibodies are capable of crossing the placenta; a positive VDRL immediately following birth may be solely a result of the presence of maternal antibodies. A quantitative titer is therefore required at birth, followed by a second titer approximately 1 month following birth. No increase in titer will assist in ruling out the possibility of congenital syphilis.

Specimen

Patient serum or CSF samples. The serum must be inactivated prior to testing. The sample is heated at 56° C for 30 minutes. This inactivates serum proteins such as complement within the patient specimen. CSF samples should not be heated prior to testing.

Method

1. Place 50 µl of patient sample, a positive reactive, and a negative nonreactive control into separate circles on the test card.

2. Place 20 µl of reagent next to the sample and stir each, making sure to completely cover the circle on the test card. Use a separate stirrer for each sample to avoid contamination.

3. Place the test card on a rotator or shaker at 180 rpm for 4 minutes.

Expected Results

Strongly reactive results will demonstrate large visible clumps.

Weakly reactive results will appear as small clumps.

Nonreactive results will appear as a diffuse, evenly turbid reaction.

Limitations

1. Results should be read immediately to avoid drying of sample and the appearance of clumping caused by evaporation.

2. Biologic false positives occur as a result of other physiologic or pathologic conditions. Various conditions including pregnancy, menstruation, vaccination, trauma, and a variety of infectious diseases and immunologic disorders may cause biologic false positives.

Quality Control

A reactive and nonreactive control should be included in each assay, demonstrating results as indicated in expected outcomes.

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 antigens (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 antitreponemal 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 antibody 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 46-3 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 46-3).

TABLE 46-3

Sensitivity of Commonly Used Serologic Tests for Syphilis

METHOD	STAGE
	Primary	Secondary	Late
Nontreponemal (Reaginic Tests)—Screening
Venereal Disease Research Laboratory (reaginic) test (VDRL)	70%	99%	60%-98%
Rapid plasma-reagin (RPR) card test and automated reagin test (ART)	80%	99%	60%-98%
Specific Treponemal Tests—Confirmatory
Fluorescent treponemal antibody absorption test (FTA-ABS, TP-PA, TPHA, MHA-TP, EIA, PaGIA)	85%	100%	98%

Figure 46-3 Traditional testing versus reverse testing.

Antimicrobial Susceptibility Testing and Therapy

Because the treponemes cannot be cultivated, susceptibility testing is not performed. For all treponemal infections, penicillin G is the drug of choice. Ceftriaxone is also highly active in most cases of syphilis other than early syphilis. Tetracycline or doxycycline is often the treatment of choice when patients are allergic to penicillin. Treatment varies depending on the stage of disease and the host (e.g., children or adults, HIV-infected or infected with congenital syphilis).

Borreliosis is considered a relapsing fever that is transmitted by a human-specific body louse or a tick. Organisms belonging to the genus Borrelia are composed of 3 to 10 loose coils (see Figure 46-1) and are actively motile. They contain endoflagella located beneath the outer membrane. The cells contain a protoplasmic cylinder that is composed of a peptidoglycan layer and an inner membrane. In contrast to the treponemes, Borrelia spp. stain well with Giemsa’s stain. Species that have been grown in vitro are microaerophilic or anaerobic.

Spectrum of Disease

Relapsing Fever

Two to 15 days following infection, patients have an abrupt onset of fever, headache, and myalgia that lasts for 4 to 10 days. Physical findings often include petechiae, diffuse abdominal tenderness, and conjunctival effusion. As the host produces specific antibody in response to the agent, organisms disappear from the bloodstream, becoming sequestered (hidden) in different organs during the afebrile period. Subsequently, organisms reemerge with newly modified antigens and multiply, resulting in another febrile period. Subsequent relapses are usually milder and of shorter duration. Generally, more relapses are associated with cases of untreated tickborne relapsing fever, but louseborne relapsing fevers tend to be more severe.

Treatment of relapsing fever with antibiotics may result in the formation of the Jarisch-Herxheimer reaction. This reaction is associated with the clearance of the organisms from the bloodstream and release of cytokines within hours of antibiotic treatment. The patient experiences tachycardia, chills, rigors, hypotension, fever, and diaphoresis. Death may be associated with the reaction. An acute respiratory distress syndrome has also been recognized in cases associated with tickborne relapsing fever.

Lyme Disease

Lyme disease is characterized by three stages, not all of which occur in any given patient. The first stage, erythema migrans (EM), is the characteristic red, ring-shaped skin lesion with a central clearing that first appears at the site of the tick bite but may develop at distant sites as well (Figure 46-4). Patients may experience headache, fever, muscle and joint pain, and malaise during this stage. The second stage, beginning weeks to months after infection, may include arthritis, but the most important features are neurologic disorders (i.e., meningitis, neurologic deficits) and carditis. This is a result of the hematogenous spread of spirochetes to organs and tissues. In addition, neurologic symptoms and infection may occur in the meninges, spinal cord, peripheral nerves, and brain. The third stage is usually characterized by chronic arthritis or acrodermatitis chronica atrophicans (ACA), a diffuse skin rash, and may continue for years. There is an association between Borrelia species and distinct clinical manifestations. For example, B. garinii has been associated with up to 72% of European cases of neuroborreliosis.