Spirochetes, Syphilis, Lyme Disease and Leptospirosis
Spirochetes
Spirochetes are long, slender, helically coiled bacteria with axial filaments (periplasmic flagella) that wind around the cell wall, enclosed by an outer sheath. They are gram-negative, microaerophilic, and exhibit corkscrew-like motility. Spirochete infections often involve a localized skin infection that disseminates to multiple organs, a latent stage, and potential cardiac and neurological complications if untreated.
Key Terms
Borrelia burgdorferi
Chancre
Congenital syphilis
Flocculation
Fluorescent treponemal antibody absorption (FTA-ABS) test
Gummas
Immunoblotting
Leptospira
Leptospirosis
Lyme disease
Microscopic agglutination test (MAT)
Nontreponemal tests
Particle agglutination (PA) tests
Prozone
Rapid plasma reagin (RPR) test
Reagin
Spirochetes
Syphilis
T. pallidum particle agglutination (TP-PA) test
Treponema pallidum
Treponemal test
Venereal Disease Research Laboratory (VDRL) test
Weil’s disease
Syphilis
Syphilis is a spirochete disease primarily transmitted sexually. In the United States, cases rose by 76% from 2013 to 2017, with over 30,600 cases reported in 2017. Homosexual transmission between men accounts for a significant portion of this increase. Early detection and antibiotic treatment are crucial for a cure.
Treponema pallidum Characteristics
The causative agent is Treponema pallidum subspecies pallidum. It lacks a natural environmental reservoir, requiring a living host. Other similar pathogens include:
T. pallidum subspecies pertenue (yaws)
T. pallidum subspecies endemicum (endemic syphilis)
Treponema carateum (pinta)
$T. pallidum$ measures 6-20 µm in length and 0.1-0.2 µm in width, with 6-14 coils. Its outer membrane is a phospholipid bilayer with few exposed proteins (treponemal rare outer membrane proteins or TROMPs), delaying the host immune response.
Transmission of Treponema pallidum
Transmission primarily occurs through direct contact, mainly sexual contact via abraded skin or mucous membranes with active lesions. The risk of acquiring the disease from an infected partner ranges from 30% to 50%. Congenital infections (transplacental) can also occur during pregnancy, often resulting in severe fetal damage.
Congenital Syphilis Manifestations:
Early-onset: Resembles secondary syphilis in adults (cutaneous lesions, snuffles, hepatosplenomegaly, CNS involvement) manifesting before age 2.
Late-stage: Occurs in infants older than 2 years whose mothers have chronic, untreated infections. Manifestations are similar to tertiary syphilis in adults (interstitial keratitis, bone/tooth deformities (Hutchinson’s teeth), eighth-nerve deafness, hard palate perforation).
Other rare transmission routes include parenteral exposure via contaminated needles or blood products. Current American Red Cross guidelines require a 12-month waiting period after syphilis treatment before blood donation.
Stages of Syphilis
Untreated syphilis progresses through primary, secondary, latent, and tertiary stages.
Primary Stage
Endothelial cell thickening and immune cell aggregation occur at the infection site. A chancre, a painless, solitary lesion with raised borders, develops 10-90 days post-infection (average 21 days). Chancres usually occur on the penis in men; they may go undetected in women if located in the vagina or on the cervix. The primary stage lasts 1-6 weeks, with spontaneous lesion healing.
Secondary Stage
Approximately 25% of untreated primary syphilis patients progress to the secondary stage, characterized by systemic dissemination of the organism, usually 1-2 months after chancre disappearance. Symptoms include:
Generalized lymphadenopathy
Malaise
Fever
Pharyngitis
Rash (skin and mucous membranes, including palms and soles)
Neurological signs (visual disturbances, hearing loss, tinnitus, facial weakness) in nearly half of patients, indicating CNS involvement.
Lesions persist for days to weeks, with spontaneous healing.
Latent Stage
This stage follows the resolution of secondary syphilis and is characterized by a lack of clinical symptoms. It is divided into early latent (less than 1 year) and late latent (more than 1 year). Patients are generally non-infectious, except for pregnant women.
Tertiary Stage
Occurs in about one-third of untreated individuals, typically 10-30 years after the secondary stage. Major manifestations include:
Gummatous syphilis: Localized granulomatous inflammation on bones, skin, or subcutaneous tissue, containing lymphocytes, epithelioid cells, and fibroblasts. These may heal with scarring or persist as destructive, chronic inflammation.
Cardiovascular disease: Primarily affects the ascending aorta, causing aortic aneurysm, valve thickening resulting in aortic regurgitation, or ostial narrowing causing angina pectoris.
Neurosyphilis: Can occur at any stage, with early forms presenting as acute meningitis and late forms (after 10+ years) causing degeneration of the lower spinal cord leading to partial paralysis and chronic progressive dementia. Immunodeficient individuals are more susceptible to early neurosyphilis.
Congenital Syphilis
Congenital syphilis occurs when treponemes are transmitted to the fetus during early or early latent syphilis. There was an increase in congenital syphilis cases from 362 in 2013 to 918 in 2017.
Transmission
Can occur at any stage of pregnancy but most severely affects the fetus during the second or third trimester. About 10% of cases result in fetal or perinatal death. Live-born infants may be asymptomatic initially but develop symptoms later if untreated.
Symptoms
Clear or hemorrhagic rhinitis (runny nose)
Skin eruptions (maculopapular rash, especially around the mouth, palms, and soles)
Generalized lymphadenopathy
Hepatomegaly/splenomegaly
Jaundice
Anemia
Painful limbs
Bone abnormalities
Neurosyphilis (in up to 60% of infants)
Immune Response to Treponema pallidum
Intact skin and mucous membranes are the primary defenses. After skin penetration, T cells and macrophages are critical. CD4+ and CD8+ T cells are present in primary lesions. Cytokines from these cells activate macrophages, which phagocytose and heal the chancre. Antibodies' protective role is uncertain: Coating treponemes with antibodies doesn't necessarily destroy them. T. pallidum can coat itself with host proteins, delaying immune recognition. TROMPs trigger complement activation, killing the organism. Chronic disease indicates immune evasion, allowing treponemes to persist for years without antibiotics.
Laboratory Diagnosis of Syphilis
Traditional tests include direct detection of spirochetes, nontreponemal serological tests, and treponemal serological tests.
Direct Detection
Dark-Field Microscopy
Used for primary and secondary syphilis by identifying T. pallidum in skin lesion exudates. A dark-field condenser excludes incidental light, highlighting the organisms. Pathogenic treponemes are identified by corkscrew morphology and flexing motility. Specimens must be examined quickly to observe motility. False negatives can occur due to delays, insufficient specimens, or antibiotic pretreatment. Experienced microscopists are needed to differentiate pathogens from morphologically identical non-pathogens, especially from oral or rectal samples.
Fluorescent Antibody Testing
A sensitive and specific alternative to dark-field microscopy. Uses fluorescent-labeled antibodies to T. pallidum (direct method) or antibody specific for T. pallidum and a second labeled anti-immunoglobulin antibody (indirect method). Live specimens are not required. Monoclonal antibodies enhance sensitivity and specificity but can cross-react with other T. pallidum subspecies.
Serological Tests
Used when active lesions are absent (secondary or tertiary syphilis). Classified as nontreponemal or treponemal based on antibody reactivity.
Nontreponemal Tests
Traditionally used for screening due to high sensitivity and easy performance. False positives are common; positive results require confirmation with treponemal tests. These tests detect antibodies against cardiolipin, a lipid released from damaged cells, referred to as reagin (IgG or IgM). The antigen complex consists of cardiolipin, lecithin, and cholesterol.
Common Nontreponemal Tests:
Venereal Disease Research Laboratory (VDRL) test
Rapid plasma reagin (RPR) test
These tests are based on flocculation reactions (clumping of fine particles). Tests become positive 1-4 weeks after chancre appearance, with titers peaking during secondary or early latent stages. False negatives can occur in secondary syphilis due to the prozone phenomenon (antibody excess). Serial dilutions can resolve the prozone effect.
Cardiolipin antibody titers decline in later stages, even without treatment. About 25% of untreated cases become nonreactive after years. Successful treatment results in a four-fold titer decrease by the third month and an eight-fold decrease by 6-8 months after initial infection. Tests become nonreactive within 1-2 years after successful treatment.
VDRL Test
A qualitative and quantitative slide flocculation test for serum (with modifications for spinal fluid). Antigen must be prepared fresh daily using an alcoholic solution of 0.03% cardiolipin, 0.9% cholesterol, and 0.21% lecithin. Serum specimens are heated at 56°C for 30 minutes to inactivate complement. Results are read microscopically as reactive, weakly reactive, or nonreactive after 4 minutes of rotation at 180 rpm. Tests must be performed at room temperature (23°C to 29°C).
RPR Test
A modified VDRL test involving macroscopic agglutination. The cardiolipin-containing antigen is bound to charcoal particles for easier reading. The suspension is stable for up to 3 months after opening. The antigen contains EDTA, thimerosal, and choline chloride. Serum does not require heat inactivation. Flocculation is read macroscopically after 8 minutes of mechanical rotation at 100 rpm under humid conditions. The RPR test appears to be more sensitive than the VDRL in primary syphilis.
Treponemal Tests
Detect antibodies against the T. pallidum organism or specific treponemal antigens. These tests usually become positive before nontreponemal tests, although patients with early primary syphilis may be nonreactive. Tests are usually 100% reactive in secondary and latent syphilis. Once reactive, individuals remain reactive for life. False positives are fewer compared to reagin tests but can occur with other treponemal diseases (yaws and pinta).
Common Treponemal Tests:
Indirect fluorescent treponemal antibody absorption (FTA-ABS) test
Agglutination tests
These tests are highly specific and used to confirm positive nontreponemal test results. Automated immunoassays for treponemal antibodies have also been developed.
FTA-ABS Test
An early confirmatory test. Heat-inactivated patient serum is incubated with a sorbent (Reiter strain) to remove cross-reacting antibodies. Samples are applied to slides fixed with the Nichols strain of T. pallidum. After incubation and washing, anti-human immunoglobulin conjugated with fluorescein is added. Fluorescence intensity is graded from 0 to 4+. A result of 2+ or above is reactive, 1+ is minimally reactive (repeat test in 1-2 weeks). False positives can occur in patients with SLE or other autoimmune diseases (atypical, beaded fluorescence pattern).
TP-PA (MHA-TP)
Particle agglutination (PA) tests originally used sheep RBCs coated with T. pallidum antigen (MHA-TP). Current PA tests, such as the Serodia T. pallidum particle agglutination (TP-PA) test, use colored gelatin particles coated with treponemal antigens. TP-PA tests are more sensitive in detecting primary syphilis. Agglutination of sensitized gel particles indicates the presence of T. pallidum antibodies. A smooth mat indicates a positive result, while a compact button indicates a negative result.
Automated Immunoassays for T. Pallidum Antibodies
Various automated immunoassays include enzyme immunoassays (EIAs), chemiluminescent immunoassays (CLIAs), and multiplex flow immunoassays (MFIs). EIAs are available in different formats (sandwich assays, competitive assays, and immune capture assays). Capture EIAs are useful in diagnosing congenital syphilis (detect IgM) and monitoring therapy response.
In competitive EIAs, treponemal antibody in the patient sample competes with an enzyme-labeled treponemal antibody conjugate. EIA sensitivities range from 95% to 99%, and specificities are 100%. CLIAs are one-step sandwich techniques using paramagnetic microparticles coated with T. pallidum antigens linked to a chemiluminescent derivative. Relative light units (RLUs) are proportional to the amount of treponemal antibody. CLIAs have higher sensitivity in early syphilis, faster performance, and more stable reagents compared to EIAs.
MFIs involve incubating the patient sample with microspheres coated with recombinant T. pallidum antigens. Bound immune complexes are detected by flow cytometry using a phycoerythrin-labeled reporter antibody. MFIs, EIAs, and CLIAs yield comparable results to the FTA-ABS but have the advantage of automation.
Molecular Testing by Polymerase Chain Reaction (PCR)
PCR amplifies a specific DNA sequence to detect treponemes in blood, spinal fluid, amniotic fluid, tissues, and swab samples. Quantitative PCR (qPCR) is automated, faster, and more sensitive. PCR can detect as little as one treponeme in some samples. Sensitivity is highest in primary syphilis but reduced in secondary syphilis. PCR may be useful when serological testing is inconclusive and as an alternative to dark-field microscopy. It can also detect treponemes in neonates with congenital syphilis symptoms and in the cerebrospinal fluid (CSF) of patients suspected of having neurosyphilis.
Clinical Applications of Syphilis Tests
Nontreponemal tests are useful as a screening tool and for monitoring disease progress and treatment outcomes (titers decrease with effective treatment). However, they are subject to false positives (e.g., hepatitis, infectious mononucleosis, pregnancy, SLE, leprosy, intravenous drug use). Reactive nontreponemal tests should be confirmed by treponemal tests, especially in pregnancy. Treponemal tests are traditionally used as confirmatory tests and are more sensitive in late latent syphilis or late syphilis.
Testing Algorithms for Syphilis
The traditional algorithm involves screening with a nontreponemal test and confirming positive results with a treponemal test.
The reverse sequence algorithm screens with an automated treponemal immunoassay and confirms positive results with a nontreponemal test. This can detect more early, late, and treated syphilis cases. Discrepant results (positive immunoassay, negative RPR) require reflexive testing with TP-PA. If TP-PA is positive, consider late or latent syphilis or previous syphilis history. If TP-PA is negative, the patient is considered negative for syphilis at the time of testing; reevaluation may be needed.
Testing for Congenital Syphilis
Nontreponemal tests on cord blood or neonatal serum detect IgG (difficult to differentiate from maternal antibodies) and IgM. Late maternal infection may result in nonreactive tests due to low fetal antibody levels. Testing infant spinal fluid often lacks sensitivity. Higher infant titers than maternal titers may indicate congenital disease. IgM capture assays are more sensitive. Western blot assays using four major treponemal antigens have high sensitivity and specificity.
In high-risk populations, nontreponemal tests should be performed on both the mother and infant at birth, regardless of previous maternal tests. Repeat tests within a few weeks if maternal history suggests congenital syphilis. Western blot tests are recommended to confirm congenital syphilis.
Testing of Cerebrospinal Fluid (CSF)
CSF testing determines treponeme invasion of the CNS (more reliable with CNS symptoms). The VDRL test and newer ELISA tests are used. A positive VDRL test on spinal fluid is diagnostic of neurosyphilis (false positives are rare). If the VDRL test is negative, other indicators such as increased lymphocyte count and elevated total protein () are used as signs of active disease. PCR may play an important role in CSF testing in the future.
Treatment for Syphilis
Penicillin is the treatment of choice, administered as a single dose of intramuscular long-acting benzathine penicillin G. Doxycycline and tetracycline are alternatives for penicillin-allergic, non-pregnant patients.
Lyme Disease
Lyme disease was first described in 1975 around Old Lyme, Connecticut. In 1982, the causative agent Borrelia burgdorferi was identified. It is a multisystem illness affecting the skin, nervous system, heart, and joints. Lyme disease is the most common vector-borne disease in the United States, with more than 42,743 confirmed/probable cases reported in 2017.
Borrelia Species Characteristics
Several species cause Lyme disease (B. burgdorferi sensu stricto in North America; B. afzelii, B. garinii, B. burgdorferi sensu stricto in Europe). For simplicity, they are referred to as B. burgdorferi. The organism is a loosely coiled spirochete, 5-25 µm long and 0.2-0.5 µm in diameter. The outer membrane contains lipoproteins (OSPs A to F, encoded by plasmids) that facilitate attachment to mammalian cells. 7-11 endoflagella (periplasmic flagella) run parallel to its long axis, and are composed of 41-kDa subunits eliciting a strong, early antibody response. Flagellin subunit homology with other spirochetes (B. recurrentis, T. pallidum) can cause cross-reactivity, leading to false positives. The organism divides by binary fission approximately every 12 hours. It can be cultured in a complex liquid medium at 33°C, but isolation from patients is difficult. Spirochetemia is short-lived and usually only found early in the illness. Cultures often require 6 weeks or longer to detect growth.
Lyme Disease Transmission
The main reservoir host is the white-footed mouse. Ixodes ticks transmit the disease:
Ixodes scapularis (Northeast and Midwest United States)
Ixodes pacificus (West)
Ixodes ricinus (Europe)
Ixodes persulcatus (Asia
White-tailed deer are the main host for the tick’s adult stage. Nymphs and adult ticks transmit the disease. Peak feeding occurs in late spring, early summer, and fall. Ticks must feed for more than 36 hours to transmit the spirochete (transmission is still low at 72 hours).
Stages of Lyme Disease
Lyme disease progresses through stages, including localized rash, early dissemination, and late dissemination. Lyme disease can be viewed as a progressive infectious disease involving diverse organ systems
Localized Rash Stage
The clinical hallmark of early infection is erythema migrans (EM), appearing 2 days to 2 weeks after a tick bite. EM begins as a small red papule that expands to form a large ring-like erythema, often with a central clearing. Diagnosis relies on recognizing the characteristic rash (at least 5 cm in diameter). Patients may be asymptomatic or have flu-like symptoms. EM usually expands for more than a week and fades within 3-4 weeks if it goes untreated. Approximately 20% of patients do not develop the rash. The antibody response is minimal during this stage, and most serology results are negative.
Early Dissemination Stage
Early dissemination occurs via the bloodstream in the days to weeks following the EM rash. The skin, nervous system, heart, or joints may be affected. Some patients display multiple skin lesions. Migratory pain often occurs in the joints, tendons, muscles, and bones. Without treatment, neurological or cardiac involvement develops in about 15% of patients 4-6 weeks after infection onset. The most prevalent neurological sign is facial palsy. Other symptoms include sleep disturbances, mild chronic confusion, or difficulty with memory and intellectual functioning, as well as aseptic meningitis.
Late Dissemination Stage
Late Lyme disease may develop months to years after infection in untreated patients. Major manifestations include arthritis, peripheral neuropathy, and encephalomyelitis. These symptoms usually respond well to conventional antibiotic treatment, but treatment-resistant arthritis is associated with particular HLA–DRB alleles. Some patients develop chronic fatigue, concentration and short-term memory problems, and musculoskeletal pain that lasts longer than 6 months despite resolution of objective manifestations of Borrelia infection after antibiotic treatment.
Immune Response in Lyme Disease
The immune response is highly variable and complex. Both humoral and cellular responses exist. Spirochete lipoproteins stimulate macrophages to produce cytokines, which further enhance the immune response. The effectiveness of these responses is questionable because late Lyme disease occurs despite high Ab and cellular responses.
Laboratory Diagnosis of Lyme Disease
The diagnosis of Lyme disease is a clinical one, with laboratory testing used as supporting evidence that is often difficult to obtain. If the characteristic rash is present, this can be used as a presumptive finding, but as many as 20% of patients do not develop or do not recognize the rash. Direct isolation of the organism via skin scrapings, spinal fluid, or blood is possible, but the yield of positive cultures is extremely low. For this reason, culture is not used as a routine diagnostic tool. The antibody response is variable and may not be detectable until 3 to 6 weeks after the tick bite. The IgM response occurs first, followed by the IgG response. The IgG response does not peak until the third and fourth weeks of infection. These antibody responses are also not mutually exclusive and can be variable (e.g., an IgM response can occur in late Lyme disease).
In most cases of acute early Lyme disease (first 2 weeks), serological testing is too insensitive to be diagnostically helpful. If patients with symptoms are tested in fewer than 7 days after infection, seropositivity is only about 30%. Therefore, the decision to start treatment for early Lyme disease must be made before seroconversion, similar to many acute infectious diseases. However, untreated seronegative patients having symptoms for 6 to 8 weeks are unlikely to have Lyme disease, and other possible diagnoses should be pursued. Antibiotic therapy begun shortly after the appearance of EM may delay or abrogate the antibody response.
Two-Tiered Approach to Lyme Disease Diagnosis
The CDC recommends a two-tiered approach to providing laboratory support for the diagnosis of Lyme disease. Using the standard testing algorithm, patients with clinical evidence of Lyme disease are screened with a sensitive ELISA or, alternatively, with an IFA. If the first serology test is positive or borderline, a Western blot test is performed on that specimen to confirm the result. Some important limitations to this approach are the cost and complexity of performing and interpreting the Western blot, which must be done in reference laboratories. The standard testing algorithm is highly specific but has low sensitivity in detecting early Lyme disease.
Modified Two-Tiered Algorithm
In this approach, symptomatic patients are first tested with a sensitive ELISA that uses purified Borrelia peptide antigens. Samples with positive or borderline results are retested with a different ELISA method for confirmation. The modified algorithm was approved by the U.S. Food and Drug Administration (FDA). In addition to being easier to perform and interpret, this approach demonstrates comparable specificity to the standard algorithm and increased sensitivity in detecting early Lyme disease.
Lyme testing should not be performed in the absence of supporting clinical evidence. A positive test performed under these circumstances has a low positive predictive value, even when done in an endemic area, whereas it rises to nearly 100% when clinical symptoms and history are present and consistent with Lyme disease.
Common Lyme Disease Testing Procedures
Immunofluorescence Assay (IFA)
The IFA was the first test used to evaluate the antibody response in Lyme disease, followed by various forms of EIAs shortly thereafter. Doubling dilutions of patient serum are incubated with commercially prepared microscope slides coated with antigen from whole or processed Borrelia spirochetes. Following a wash step to remove unbound material, an anti-human globulin with a fluorescent tag attached is added and reacts with any specific antibody bound to the spirochetes on the slide. After a second wash step, the slide is viewed under a fluorescent microscope.
Typically, a test result is only considered positive if a titer of 1:256 or higher is obtained, although this varies between manufacturers. As previously mentioned, specimens obtained in the first few weeks are usually negative because the level of antibody present is below the detection limit of this (and other) assays. As might be expected, other closely related organisms, such as B. recurrentis (relapsing fever), T. denticola and others associated with periodontal disease, and T. pallidum (syphilis), may cross- react and cause biological false-positive results. Autoimmune connective tissue diseases such as rheumatoid arthritis (RA) and SLE can also produce false positives in the IFA for Lyme disease. An astute technologist can recognize a false positive by the beaded fluorescent pattern it produces. Reading of fluorescent patterns tends to be very subjective and requires highly trained individuals. However, if performed correctly by experienced personnel, the test can provide sensitive and accurate results. This test is best suited for low-volume testing.
Enzyme Immunoassay (EIA)
EIA testing has used ELISAs that are relatively inexpensive to perform and yield timely results. The test is reproducible because the results are objective, and the method lends itself well to automation and high-volume testing. For these reasons, EIAs are widely used in the initial evaluation of patients for Lyme disease. In addition, EIAs have recently been recommended by the CDC as an alternative to the Western blot test in the two-tier testing algorithm for Lyme disease, as we discussed previously.
Antigen preparations used in various forms of the assay include crude sonicates of the organism, purified proteins, synthetic proteins, and recombinant proteins such as the VIsE C6 peptide and pepC10 (peptide derived from OSP-C). The manufacturer’s selected antigen is then coated onto 96-well microtiter plates or strips by various proprietary methods. Patient sera is added; during incubation, if antibodies to the B. burgdorferi antigens are present, they will bind to the solid phase. After a washing step, anti-human immunoglobulin conjugated with an enzyme tag such as alkaline phosphatase is added to each well. The conjugate can also be adapted to test for IgM and IgG, IgM only, or IgG only. Adding specific substrate produces a color change. Plates are read in a spectrophotometer, and the antibody is quantitated based on color intensity.
EIAs provide objective results, and the titer is based on a continuum range rather than serial dilutions of patient sera. Thus, a more accurate measurement of the specific antibody is possible. Similar to the IFA, EIAs have decreased sensitivity during the early stages of Lyme disease when patients may not have mounted a sufficient antibody response. In addition, as with IFAs, false positives can occur due to syphilis or other treponemal diseases such as yaws and periodontal disease, as well as relapsing fever and leptospirosis. Patients with infectious mononucleosis, Rocky Mountain spotted fever, and other autoimmune diseases may also be positive with an EIA. Lyme disease patients do not test positive with RPR, so this may be helpful if syphilis is in the differential diagnosis.
Western Blot
Immunoblotting, or Western blotting, has been used as a confirmatory test for samples that initially test positive or equivocal by EIA or IFA. It has been employed as the second test in the CDC-recommended two-tier testing scheme for Lyme disease. The CDC does not recommend testing seropositive or borderline patients for IgM antibodies if they have had symptoms for more than 4 weeks. Serological evidence of Lyme disease in these patients is indicated by a positive result in the IgG immunoblot.
The Lyme disease immunoblot is very complex. The technique consists of electrophoresis of Borrelia antigens in an acrylamide gel followed by transfer of the resulting pattern to nitrocellulose paper. This step is performed by the manufacturer, and nitrocellulose antigen strips are provided in the test kit. These strips are reacted with patient serum and developed with an anti-human immunoglobulin (either anti-IgG or anti-IgM) to which an enzyme label is attached. Further incubation with the enzyme’s substrate allows for visualization of any antibody that has bound to a particular antigen. The reactivity is then scored and interpreted.
Ten proteins are used in the CDC-recommended interpretation of this test. For a result to be considered positive for the presence of specific IgM antibody, two of the following bands must be present: 23 (OSP- C), 39, and 41 (flagellin) kDa. An IgG immunoblot is considered positive if any 5 of the 10 bands previously listed are positive. Because of the complexity of the Lyme immunoblots, testing and interpretation of blots should be done only in qualified laboratories that follow CDC-recommended evidence-based guidelines on immunoblot interpretations.
Polymerase Chain Reaction (PCR)
In testing for Lyme disease, the PCR has found a niche in certain scenarios. Although only a few organisms need to be present for detection under optimal conditions, the number of spirochetes in infected tissues and body fluids is low, making specimen collection, transport, and preparation of DNA critical to the accuracy of the test results. Several probes for target DNA that is present only in strains of B. burgdorferi are used in PCR testing. The procedure involves extracting DNA from the patient sample, followed by amplification using specific primers, DNA polymerase, and nucleotides. The patient DNA is combined with a known DNA probe to see if hybridization takes place. The single-stranded Borrelia DNA probe will bind only to an exact complementary strand, thus positively identifying the presence of the organism’s DNA in the patient sample.
This is much more specific than testing for antibody because there is little cross-reactivity. Specificity of PCR ranges from 93% to 100%. However, sensitivity remains problematic. PCR on CSF and synovial fluid is often used in difficult diagnostic neurological and arthritic cases. PCR for Borrelia is typically performed in reference laboratories. Modifications of the PCR, as well as proteomic assays, are being developed and tested for their potential utility in Lyme disease diagnosis as well.
Lyme Disease Treatment
Borrelia is sensitive to several orally administered antibiotics, including penicillins, tetracyclines, and macrolides. Oral doxycycline is the first treatment of choice for patients with early Lyme disease who are not pregnant. Intravenous antibiotic therapy is required for patients with neurological symptoms, cardiac involvement, or arthritis that does not respond to oral therapy.
Prophylaxis, full-course treatment, or serological testing of all patients with tick bites is not recommended. A single dose of doxycycline may be offered to adults and children older than 8 years of age when the tick can be reliably identified, and treatment can begin within 72 hours of tick removal. Currently, there are no effective vaccines for humans. A human vaccine made with the OSP-A surface antigen has had limited usefulness; it has been associated with side effects and has been recalled from the market. There are renewed efforts to create a new vaccine, but as of this writing, no vaccines have been approved for clinical use.
Leptospirosis
Leptospirosis is caused by bacteria of the genus Leptospira. It is more prevalent in temperate zones of the world with a warm climate. The incidence is higher in livestock and dairy farming communities. It is primarily a zoonotic infection commonly associated with occupational and recreational activities. Coming into contact with infected animals, especially rat-infested surroundings, poses increased exposure for veterinarians, dairy farmers, slaughterhouse workers, sewer cleaners, and miners. In the United States, most of the leptospirosis cases are associated with exposure to recreational water activities.
According to the CDC, an estimated 100 to 150 cases are reported annually in the United States; 50% of cases are from Puerto Rico, and Hawaii has the second highest incidence.
Leptospira Species Characteristics
Organisms belonging to the genus Leptospira (“leptospires”) are thin, flexible, and tightly coiled spirochetes. They are 0.1 µm wide and 6 to 20 µm long with pointed ends bent into a typical hook-like shape. Unlike Treponema and Borrelia organisms, the spirals of Leptospira are so close together that they tend to appear similar to a chain of cocci. The genus Leptospira was originally divided into two main species, namely, pathogenic saprophytes, L. interrogans, and environmental saprophytes, L. biflexa. There are more than 200 serovars of L. interrogans and more than 60 for L. biflexa.
Leptospires are obligate aerobes that can grow optimally at a temperature range of 28°C to 30°C. They can be grown in artificial culture media such as Fletcher semisolid medium, Ellinghausen-McCollough-Johnson-Harris (EMJH) semisolid medium, or Stuart liquid medium.
Leptospirosis Mode of Transmission
Leptospires are naturally found inhabiting the renal tubules of infected animals such as rodents, dogs, pigs, horses, and livestock and, thus, are shed in the urine of these animals. The organisms can survive for weeks to months in urine-contaminated water and mud. Humans are exposed through mucous membranes, conjunctiva, skin abrasions, or ingestion when they come into contact with urine-contaminated water of rivers, streams, sewage, or floodwater. The risk of transmission increases by wading, swimming, or boating in floodwater or fresh water that may be contaminated with animal urine.
Exposure to leptospirosis can be avoided by not wading or swimming in potentially contaminated water bodies, especially after heavy rainfall or flooding. Protective clothing such as rubber boots, gloves, and waterproof coveralls should be worn in situations involving occupational exposure.
Leptospirosis Stages of the Disease
The incubation period in leptospirosis varies between 2 to 30 days, but most cases manifest 5 to 14 days after exposure. The clinical presentation of the illness is generally abrupt, starting with a nonspecific febrile episode of headache, myalgia, nausea, vomiting, diarrhea, and a characteristic conjunctival suffusion. Illness may be biphasic, with the patient briefly recovering from mild illness but then developing more severe disease with renal, hepatic, pulmonary, and CNS involvement. Severe systemic disease involving renal failure and jaundice caused by hepatic failure is referred to as Weil’s disease. Patients with severe illness have a fatality rate of 5% to 15%. In pregnant women, leptospirosis can cause fetal abnormalities, death, or abortion.
Laboratory Diagnosis fo Leptospirosis
Leptospires can be demonstrated in blood or serum samples in the first week of illness by dark-field, immunofluorescent, or phase contrast microscopy, but these techniques are not recommended because of lower sensitivity of microscopic examination. Serology is the most common method to diagnose leptospirosis, followed by molecular techniques. During the first week of illness, whole blood and serum are the preferred samples, and afterward, a serum and/or urine sample should be submitted for testing. IgM screening assays are available in the form of ELISA, ImmunoDOT, and lateral flow tests.
Positive screening tests should be confirmed by a microscopic agglutination test (MAT), which is the gold standard for diagnosing leptospirosis. Ideally, acute and convalescent serum samples collected 7 to 14 days apart are tested by MAT. This test is available only at regional or national reference laboratories. PCR assays are also available at the CDC and some commercial laboratories. The key advantage of PCR is quick turnaround time and prompt treatment of positive cases.
Leptospirosis Treatment
Penicillin and doxycycline are the drugs of choice for treatment of leptospirosis. Early treatment leads to reducing the severity and duration of the illness. Studies have shown that prophylaxis with a weekly dose of oral doxycycline is considered effective for people in high-risk environments.
Spirochetes
Spirochetes are distinguished by their unique structure and motility. These bacteria are long, slender, and helically coiled, resembling a corkscrew. The cell's axial filaments, also known as periplasmic flagella, are located within the periplasmic space and wind around the cell wall, contributing to their characteristic motility. Spirochetes are gram-negative but possess an outer sheath surrounding the cell wall, which can complicate Gram staining. They are typically microaerophilic, thriving in low-oxygen environments, and exhibit a distinctive corkscrew-like motility due to the rotation of their axial filaments.
Spirochete infections often follow a complex course. Initially, the infection may manifest as a localized skin infection at the site of entry. However, the bacteria can disseminate rapidly through the bloodstream to multiple organs, leading to systemic involvement. Many spirochetal infections are characterized by a latent stage, during which the bacteria remain dormant and may not cause obvious symptoms. If left untreated, these infections can lead to severe complications, including cardiac and neurological damage.
Key Genera of Spirochetes
Treponema: This genus includes Treponema pallidum, the causative agent of syphilis. Syphilis is a sexually transmitted infection (STI) that can cause long-term complications if not treated.
Borrelia: This genus includes Borrelia burgdorferi, the agent responsible for Lyme disease, which is transmitted through tick bites.
Leptospira: This genus includes various species that cause leptospirosis, a zoonotic disease transmitted through contact with contaminated water or soil.
Key Terms
Borrelia burgdorferi
Chancre
Congenital syphilis
Flocculation
Fluorescent treponemal antibody absorption (FTA-ABS) test
Gummas
Immunoblotting
Leptospira
Leptospirosis
Lyme disease
Microscopic agglutination test (MAT)
Nontreponemal tests
Particle agglutination (PA) tests
Prozone
Rapid plasma reagin (RPR) test
Reagin
Spirochetes
Syphilis
T. pallidum particle agglutination (TP-PA) test
Treponema pallidum
Treponemal test
Venereal Disease Research Laboratory (VDRL) test
Weil’s disease
Syphilis
Syphilis is a systemic infectious disease caused by the spirochete Treponema pallidum. It is primarily transmitted through sexual contact but can also be passed from a pregnant woman to her fetus (congenital syphilis). The disease progresses through several stages if left untreated: primary, secondary, latent, and tertiary.
In the United States, the incidence of syphilis has been increasing. According to the CDC, cases rose by 76% from 2013 to 2017, with over 30,600 cases reported in 2017. A significant portion of this increase is attributed to homosexual transmission between men. Early detection and appropriate antibiotic treatment are essential to cure the infection and prevent long-term complications.
Treponema pallidum Characteristics
The causative agent of syphilis is Treponema pallidum subspecies pallidum. This bacterium lacks a natural environmental reservoir and requires a living host for survival. The genus Treponema includes other pathogens that cause related diseases:
T. pallidum subspecies pertenue (yaws): A tropical infection causing skin lesions.
T. pallidum subspecies endemicum (endemic syphilis or bejel): Primarily affects children in arid regions, causing oral and skin lesions.
Treponema carateum (pinta): Causes skin discoloration and is prevalent in Central and South America.
$T. pallidum$ is a spiral-shaped bacterium, measuring 6-20 µm in length and 0.1-0.2 µm in width, with 6-14 coils. Its outer membrane is a phospholipid bilayer with few exposed proteins, known as treponemal rare outer membrane proteins (TROMPs). This composition delays the host's immune response, allowing the bacteria to disseminate before being effectively targeted by the immune system. The low number of surface proteins also makes it difficult for the immune system to recognize and eliminate the bacteria.
Transmission of Treponema pallidum
Transmission of Treponema pallidum primarily occurs through direct contact with infectious lesions, mainly during sexual activity. The bacteria can penetrate abraded skin or mucous membranes. The risk of acquiring syphilis from an infected partner ranges from 30% to 50% per sexual encounter. Congenital infections can occur when the bacteria cross the placenta during pregnancy, leading to severe fetal damage, including stillbirth, hydrops fetalis, or deformities.
Congenital Syphilis Manifestations:
Early-onset: Manifests before age 2 and resembles secondary syphilis in adults. Symptoms include cutaneous lesions, snuffles (nasal discharge), hepatosplenomegaly, and CNS involvement.
Late-stage: Occurs in infants older than 2 years whose mothers have chronic, untreated infections. Manifestations are similar to tertiary syphilis in adults, including interstitial keratitis, bone and tooth deformities (Hutchinson’s teeth), eighth-nerve deafness, and hard palate perforation.
Other rare transmission routes include parenteral exposure via contaminated needles or blood products. To minimize the risk of transmission through blood transfusions, the American Red Cross has guidelines requiring a 12-month waiting period after syphilis treatment before blood donation.
Stages of Syphilis
Syphilis progresses through distinct stages if left untreated, each characterized by specific clinical manifestations. These stages include primary, secondary, latent, and tertiary syphilis.
Primary Stage
Following infection, Treponema pallidum multiplies at the site of entry, leading to endothelial cell thickening and immune cell aggregation. The hallmark of primary syphilis is the development of a chancre, a painless, solitary lesion with raised borders. Chancres typically appear 10-90 days post-infection (average 21 days) and most commonly occur on the penis in men. In women, chancres may go undetected if located in the vagina or on the cervix. The primary stage lasts 1-6 weeks, during which the chancre heals spontaneously, even without treatment. However, the infection progresses to subsequent stages if not addressed with antibiotics.
Secondary Stage
Approximately 25% of untreated primary syphilis patients progress to the secondary stage. This stage is characterized by systemic dissemination of the organism, usually 1-2 months after the chancre disappears. Common symptoms include:
Generalized lymphadenopathy: Swollen lymph nodes throughout the body.
Malaise: A general feeling of discomfort, illness, or unease.
Fever: Elevated body temperature.
Pharyngitis: Sore throat.
Rash: Skin and mucous membrane lesions, including on the palms and soles. The rash can vary in appearance, including macules, papules, or pustules.
Neurological signs: Visual disturbances, hearing loss, tinnitus, and facial weakness occur in nearly half of patients, indicating CNS involvement. Syphilis can affect the central nervous system at any stage, but neurological manifestations are more common in the secondary and tertiary stages.
Lesions persist for days to weeks, with spontaneous healing. Even without treatment, the symptoms of secondary syphilis will resolve, but the infection will progress to the latent stage.
Latent Stage
The latent stage follows the resolution of secondary syphilis and is characterized by a lack of clinical symptoms. It is divided into early latent (less than 1 year) and late latent (more than 1 year). During the latent stage, patients are generally non-infectious, except for pregnant women, who can still transmit the infection to their fetus.
Tertiary Stage
Occurs in about one-third of untreated individuals, typically 10-30 years after the secondary stage. Major manifestations include:
Gummatous syphilis: Localized granulomatous inflammation on bones, skin, or subcutaneous tissue, containing lymphocytes, epithelioid cells, and fibroblasts. Gummas can heal with scarring or persist as destructive, chronic inflammation. They may affect any organ but are most common in the skin, bones, and liver.
Cardiovascular disease: Primarily affects the ascending aorta, causing aortic aneurysm, valve thickening resulting in aortic regurgitation, or ostial narrowing causing angina pectoris. Cardiovascular syphilis can lead to significant morbidity and mortality.
Neurosyphilis: Can occur at any stage, with early forms presenting as acute meningitis and late forms (after 10+ years) causing degeneration of the lower spinal cord leading to partial paralysis and chronic progressive dementia. Immunodeficient individuals are more susceptible to early neurosyphilis. Neurosyphilis can manifest as general paresis, tabes dorsalis, or meningovascular syphilis.
Congenital Syphilis
Congenital syphilis occurs when treponemes are transmitted to the fetus during early or early latent syphilis. The rate of congenital syphilis reflects the prevalence of syphilis among women of reproductive age and the adequacy of prenatal care. There was an increase in congenital syphilis cases from 362 in 2013 to 918 in 2017.
Transmission
Congenital syphilis can occur at any stage of pregnancy but most severely affects the fetus during the second or third trimester. About 10% of cases result in fetal or perinatal death. Live-born infants may be asymptomatic initially but develop symptoms later if untreated. Early diagnosis and treatment of pregnant women with syphilis are critical to prevent congenital syphilis.
Symptoms
Clear or hemorrhagic rhinitis (runny nose): Often referred to as "snuffles," this is one of the most common early symptoms.
Skin eruptions (maculopapular rash, especially around the mouth, palms, and soles): The rash can be highly infectious.
Generalized lymphadenopathy: Enlargement of lymph nodes throughout the body.
Hepatomegaly/splenomegaly: Enlargement of the liver and spleen.
Jaundice: Yellowing of the skin and eyes, indicating liver involvement.
Anemia: Reduced red blood cell count.
Painful limbs: Due to bone involvement.
Bone abnormalities: Such as osteochondritis, which can cause painful movement.
Neurosyphilis: Involvement of the central nervous system in up to 60% of infants.
Immune Response to Treponema pallidum
Intact skin and mucous membranes are the primary defenses against Treponema pallidum. Once the bacteria penetrate the skin, T cells and macrophages play a critical role in the immune response. CD4+ and CD8+ T cells are present in primary lesions. Cytokines released from these cells activate macrophages, which phagocytose and facilitate the healing of the chancre. The protective role of antibodies is uncertain; coating treponemes with antibodies does not necessarily lead to their destruction. T. pallidum can coat itself with host proteins, delaying immune recognition. TROMPs trigger complement activation, leading to the killing of the organism. Chronic disease indicates immune evasion, allowing treponemes to persist for years without antibiotics.
Laboratory Diagnosis of Syphilis
Laboratory tests are essential for diagnosing syphilis, especially in the absence of clinical signs or symptoms. Traditional tests include direct detection of spirochetes, nontreponemal serological tests, and treponemal serological tests.
Direct Detection
Dark-Field Microscopy
Used for primary and secondary syphilis by identifying T. pallidum in skin lesion exudates. A dark-field condenser excludes incidental light, highlighting the organisms. Pathogenic treponemes are identified by corkscrew morphology and flexing motility. Specimens must be examined quickly to observe motility. False negatives can occur due to delays, insufficient specimens, or antibiotic pretreatment. Experienced microscopists are needed to differentiate pathogens from morphologically identical non-pathogens, especially from oral or rectal samples.
Fluorescent Antibody Testing
A sensitive and specific alternative to dark-field microscopy. Uses fluorescent-labeled antibodies to T. pallidum (direct method) or antibody specific for T. pallidum and a second labeled anti-immunoglobulin antibody (indirect method). Live specimens are not required. Monoclonal antibodies enhance sensitivity and specificity but can cross-react with other T. pallidum subspecies.
Serological Tests
Serological tests are used when active lesions are absent, such as in secondary or tertiary syphilis. These tests are classified as nontreponemal or treponemal based on antibody reactivity.
Nontreponemal Tests
Traditionally used for screening due to high sensitivity and easy performance. However, false positives are common, and positive results require confirmation with treponemal tests. These tests detect antibodies against cardiolipin, a lipid released from damaged cells, referred to as reagin (IgG or IgM). The antigen complex consists of cardiolipin, lecithin, and cholesterol.
Common Nontreponemal Tests:
Venereal Disease Research Laboratory (VDRL) test
Rapid plasma reagin (RPR) test
These tests are based on flocculation reactions (clumping of fine particles). They become positive 1-4 weeks after chancre appearance, with titers peaking during secondary or early latent stages. False negatives can occur in secondary syphilis due to the prozone phenomenon (antibody excess). Serial dilutions can resolve the prozone effect.
Cardiolipin antibody titers decline in later stages, even without treatment. About 25% of untreated cases become nonreactive after years. Successful treatment results in a four-fold titer decrease by the third month and an eight-fold decrease by 6-8 months after initial infection. Tests become nonreactive within 1-2 years after successful treatment. Monitoring the decline in titers is an important indicator of treatment response.
VDRL Test
A qualitative and quantitative slide flocculation test for serum (with modifications for spinal fluid). Antigen must be prepared fresh daily using an alcoholic solution of 0.03% cardiolipin, 0.9% cholesterol, and 0.21% lecithin. Serum specimens are heated at 56°C for 30 minutes to inactivate complement. Results are read microscopically as reactive, weakly reactive, or nonreactive after 4 minutes of rotation at 180 rpm. Tests must be performed at room temperature (23°C to 29°C).
RPR Test
A modified VDRL test involving macroscopic agglutination. The cardiolipin-containing antigen is bound to charcoal particles for easier reading. The suspension is stable for up to 3 months after opening. The antigen contains EDTA, thimerosal, and choline chloride. Serum does not require heat inactivation. Flocculation is read macroscopically after 8 minutes of mechanical rotation at 100 rpm under humid conditions. The RPR test appears to be more sensitive than the VDRL in primary syphilis.
Treponemal Tests
Detect antibodies against the T. pallidum organism or specific treponemal antigens. These tests usually become positive before nontreponemal tests, although patients with early primary syphilis may be nonreactive. Tests are usually 100% reactive in secondary and latent syphilis. Once reactive, individuals remain reactive for life. False positives are fewer compared to reagin tests but can occur with other treponemal diseases (yaws and pinta).
Common Treponemal Tests:
Indirect fluorescent treponemal antibody absorption (FTA-ABS) test
Agglutination tests
These tests are highly specific and used to confirm positive nontreponemal test results. Automated immunoassays for treponemal antibodies have also been developed. These assays offer high throughput and ease of use.
FTA-ABS Test
An early confirmatory test. Heat-inactivated patient serum is incubated with a sorbent (Reiter strain) to remove cross-reacting antibodies. Samples are applied to slides fixed with the Nichols strain of T. pallidum. After incubation and washing, anti-human immunoglobulin conjugated with fluorescein is added. Fluorescence intensity is graded from 0 to 4+. A result of 2+ or above is reactive, 1+ is minimally reactive (repeat test in 1-2 weeks). False positives can occur in patients with SLE or other autoimmune diseases (atypical, beaded fluorescence pattern).
TP-PA (MHA-TP)
Particle agglutination (PA) tests originally used sheep RBCs coated with T. pallidum antigen (MHA-TP). Current PA tests, such as the Serodia T. pallidum particle agglutination (TP-PA) test, use colored gelatin particles coated with treponemal antigens. TP-PA tests are more sensitive in detecting primary syphilis. Agglutination of sensitized gel particles indicates the presence of T. pallidum antibodies. A smooth mat indicates a positive result, while a compact button indicates a negative result.
Automated Immunoassays for T. Pallidum Antibodies
Various automated immunoassays include enzyme immunoassays (EIAs), chemiluminescent immunoassays (CLIAs), and multiplex flow immunoassays (MFIs). EIAs are available in different formats (sandwich assays, competitive assays, and immune capture assays). Capture EIAs are useful in diagnosing congenital syphilis (detect IgM) and monitoring therapy response.
In competitive EIAs, treponemal antibody in the patient sample competes with an enzyme-labeled treponemal antibody conjugate. EIA sensitivities range from 95% to 99%, and specificities are 100%. CLIAs are one-step sandwich techniques using paramagnetic microparticles coated with T. pallidum antigens linked to a chemiluminescent derivative. Relative light units (RLUs) are proportional to the amount of treponemal antibody. CLIAs have higher sensitivity in early syphilis, faster performance, and more stable reagents compared to EIAs.
MFIs involve incubating the patient sample with microspheres coated with recombinant T. pallidum antigens. Bound immune complexes are detected by flow cytometry using a phycoerythrin-labeled reporter antibody. MFIs, EIAs, and CLIAs yield comparable results to the FTA-ABS but have the advantage of automation.
Molecular Testing by Polymerase Chain Reaction (PCR)
PCR amplifies a specific DNA sequence to detect treponemes in blood, spinal fluid, amniotic fluid, tissues, and swab samples. Quantitative PCR (qPCR) is automated, faster, and more sensitive. PCR can detect as little as one treponeme in some samples. Sensitivity is highest in primary syphilis but reduced in secondary syphilis. PCR may be useful when serological testing is inconclusive and as an alternative to dark-field microscopy. It can also detect treponemes in neonates with congenital syphilis symptoms and in the cerebrospinal fluid (CSF) of patients suspected of having neurosyphilis.
Clinical Applications of Syphilis Tests
Nontreponemal tests are useful as a screening tool and for monitoring disease progress and treatment outcomes (titers decrease with effective treatment). However, they are subject to false positives (e.g., hepatitis, infectious mononucleosis, pregnancy, SLE, leprosy, intravenous drug use). Reactive nontreponemal tests should be confirmed by treponemal tests, especially in pregnancy. Treponemal tests are traditionally used as confirmatory tests and are more sensitive in late latent syphilis or late syphilis.
Testing Algorithms for Syphilis
The traditional algorithm involves screening with a nontreponemal test and confirming positive results with a treponemal test.
The reverse sequence algorithm screens with an automated treponemal immunoassay and confirms positive results with a nontreponemal test. This can detect more early, late, and treated syphilis cases. Discrepant results (positive immunoassay, negative RPR) require reflexive testing with TP-PA. If TP-PA is positive, consider late or latent syphilis or previous syphilis history. If TP-PA is negative, the patient is considered negative for syphilis at the time of testing; reevaluation may be needed.
Testing for Congenital Syphilis
Nontreponemal tests on cord blood or neonatal serum detect IgG (difficult to differentiate from maternal antibodies) and IgM. Late maternal infection may result in nonreactive tests due to low fetal antibody levels. Testing infant spinal fluid often lacks sensitivity. Higher infant titers than maternal titers may indicate congenital disease. IgM capture assays are more sensitive. Western blot assays using four major treponemal antigens have high sensitivity and specificity.
In high-risk populations, nontreponemal tests should be performed on both the mother and infant at birth, regardless of previous maternal tests. Repeat tests within a few weeks if maternal history suggests congenital syphilis. Western blot tests are recommended to confirm congenital syphilis.
Testing of Cerebrospinal Fluid (CSF)
CSF testing determines treponeme invasion of the CNS (more reliable with CNS symptoms). The VDRL test and newer ELISA tests are used. A positive VDRL test on spinal fluid is diagnostic of neurosyphilis (false positives are rare). If the VDRL test is negative, other indicators such as increased lymphocyte count and elevated total protein () are used as signs of active disease. PCR may play an important role in CSF testing in the future.
Treatment for Syphilis
Penicillin is the treatment of choice, administered as a single dose of intramuscular long-acting benzathine penicillin G. The specific dosage and duration of treatment depend on the stage of syphilis. Doxycycline and tetracycline are alternatives for penicillin-allergic, non-pregnant patients. For neurosyphilis, intravenous penicillin is typically required.
Lyme Disease
Lyme disease was first described in 1975 around Old Lyme, Connecticut. In 1982, the causative agent Borrelia burgdorferi was identified. It is a multisystem illness affecting the skin, nervous system, heart, and joints. Lyme disease is the most common vector-borne disease in the United States, with more than 42,743 confirmed/probable cases reported in 2017.
Borrelia Species Characteristics
Several species cause Lyme disease (B. burgdorferi sensu stricto in North America; B. afzelii, B. garinii, B. burgdorferi sensu stricto in Europe). For simplicity, they are referred to as B. burgdorferi. The organism is a loosely coiled spirochete, 5-25 µm long and 0.2-0.5 µm in diameter. The outer membrane contains lipoproteins (OSPs A to F, encoded by plasmids) that facilitate attachment to mammalian cells. 7-11 endoflagella (periplasmic flagella) run parallel to its long axis, and are composed of 41-kDa subunits eliciting a strong, early antibody response. Flagellin subunit homology with other spirochetes (B. recurrentis, T. pallidum) can cause cross-reactivity, leading to false positives. The organism divides by binary fission approximately every 12 hours. It can be cultured in a complex liquid medium at 33°C, but isolation from patients is difficult. Spirochetemia is short-lived and usually only found early in the illness. Cultures often require 6 weeks or longer to detect growth.
Lyme Disease Transmission
The main reservoir host is the white-footed mouse. Ixodes ticks transmit the disease:
Ixodes scapularis (Northeast and Midwest United States)
Ixodes pacificus (West)
Ixodes ricinus (Europe)
Ixodes persulcatus (Asia
White-tailed deer are the main host for the tick’s adult stage. Nymphs and adult ticks transmit the disease. Peak feeding occurs in late spring, early summer, and fall. Ticks must feed for more than 36 hours to transmit the spirochete (transmission is still low at 72 hours).
Stages of Lyme Disease
Lyme disease progresses through stages, including localized rash, early dissemination, and late dissemination. Lyme disease can be viewed as a progressive infectious disease involving diverse organ systems
Localized Rash Stage
The clinical hallmark of early infection is erythema migrans (EM), appearing 2 days to 2 weeks after a tick bite. EM begins as a small red papule that expands to form a large ring-like erythema, often with a central clearing. Diagnosis relies on recognizing the characteristic rash (at least 5 cm in diameter). Patients may be asymptomatic or have flu-like symptoms. EM usually expands for more than a week and fades within 3-4 weeks if it goes untreated. Approximately 20% of patients do not develop the rash. The antibody response is minimal during this stage, and most serology results are negative.
Early Dissemination Stage
Early dissemination occurs via the bloodstream in the days to weeks following the EM rash. The skin, nervous system, heart, or joints may be affected. Some patients display multiple skin lesions. Migratory pain often occurs in the joints, tendons, muscles, and bones. Without treatment, neurological or cardiac involvement develops in about 15% of patients 4-6 weeks after infection onset. The most prevalent neurological sign is facial palsy. Other symptoms include sleep disturbances, mild chronic confusion, or difficulty with memory and intellectual functioning, as well as aseptic meningitis.
Late Dissemination Stage
Late Lyme disease may develop months to years after infection in untreated patients. Major manifestations include arthritis, peripheral neuropathy, and encephalomyelitis. These symptoms usually respond well to conventional antibiotic treatment, but treatment-resistant arthritis is associated with particular HLA–DRB alleles. Some patients develop chronic fatigue, concentration and short-term memory problems, and musculoskeletal pain that lasts longer than 6 months despite resolution of objective manifestations of Borrelia infection after antibiotic treatment.
Immune Response in Lyme Disease
The immune response is highly variable and complex. Both humoral and cellular responses exist. Spirochete lipoproteins stimulate macrophages to produce cytokines, which further enhance the immune response. The effectiveness of these responses is questionable because late Lyme disease occurs despite high Ab and cellular responses.
Laboratory Diagnosis of Lyme Disease
The diagnosis of Lyme disease is a clinical one, with laboratory testing used as supporting evidence that is often difficult to obtain. If the characteristic rash is present, this can be used as a presumptive finding, but as many as 20% of patients do not develop or do not recognize the rash. Direct isolation of the organism via skin scrapings, spinal fluid, or blood is possible, but the yield of positive cultures is extremely low. For this reason, culture is not used as a routine diagnostic tool. The antibody response is variable and may not be detectable until 3 to 6 weeks after the tick bite. The IgM response occurs first, followed by the IgG response. The IgG response does not peak until the third and fourth weeks of infection. These antibody responses are also not mutually exclusive and can be variable (e.g., an IgM response can occur in late Lyme disease).
In most cases of acute early Lyme disease (first 2 weeks), serological testing is too insensitive to be diagnostically helpful. If patients with symptoms are tested in fewer than 7 days after infection, seropositivity is only about 30%. Therefore, the decision to start treatment for early Lyme disease must be made before seroconversion, similar to many acute infectious diseases. However, untreated seronegative patients having symptoms for 6 to 8 weeks are unlikely to have Lyme disease, and other possible diagnoses should be pursued. Antibiotic therapy begun shortly after the appearance of EM may delay or abrogate the antibody response.
Two-Tiered Approach to Lyme Disease Diagnosis
The CDC recommends a two-tiered approach to providing laboratory support for the diagnosis of Lyme disease. Using the standard testing algorithm, patients with clinical evidence of Lyme disease are screened with a sensitive ELISA or, alternatively, with an IFA. If the first serology test is positive or borderline, a Western blot test is performed on that specimen to confirm the result. Some important limitations to this approach are the cost and complexity of performing and interpreting the Western blot, which must be done in reference laboratories. The standard testing algorithm is highly specific but has low sensitivity in detecting early Lyme disease.
Modified Two-Tiered Algorithm
In this approach, symptomatic patients are first tested with a sensitive ELISA that uses purified Borrelia peptide antigens. Samples with positive or borderline results are retested with a different ELISA method for confirmation. The modified algorithm was approved by the U.S. Food and Drug Administration (FDA). In addition to being easier to perform and interpret, this approach demonstrates comparable specificity to the standard algorithm and increased sensitivity in detecting early Lyme disease.
Lyme testing should not be performed in the absence of supporting clinical evidence. A positive test performed under these circumstances has a low positive predictive value, even when done in an endemic area, whereas it rises to nearly 100% when clinical symptoms and history are present and consistent with Lyme disease.
Common Lyme Disease Testing Procedures
Immunofluorescence Assay (IFA)
The IFA was the first test used to evaluate the antibody response in Lyme disease, followed by various forms of EIAs shortly thereafter. Doubling dilutions of patient serum are incubated with commercially prepared microscope slides coated with antigen from whole or processed Borrelia spirochetes. Following a wash step to remove unbound material, an anti-human globulin with a fluorescent tag attached is added and reacts with any specific antibody bound to the spirochetes on the slide. After a second wash step, the slide is viewed under a fluorescent microscope.
Typically, a test result is only considered positive if a titer of 1:256 or higher is obtained, although this varies between manufacturers. As previously mentioned, specimens obtained in the first few weeks are usually negative because the level of antibody present is below the detection limit of this (and other) assays. As might be expected, other closely related organisms, such as B. recurrentis (relapsing fever), T. denticola and others associated with periodontal disease, and T. pallidum (syphilis), may cross- react and cause biological false-positive results. Autoimmune connective tissue diseases such as rheumatoid arthritis (RA) and SLE can also produce false positives in the IFA for Lyme disease. An astute technologist can recognize a false positive by the beaded fluorescent pattern it produces. Reading of fluorescent patterns tends to be very subjective and requires highly trained individuals. However, if performed correctly by experienced personnel, the test can provide sensitive and accurate results. This test is best suited for low-volume testing.
Enzyme Immunoassay (EIA)
EIA testing has used ELISAs that are relatively inexpensive to perform and yield timely results. The test is reproducible because the results are objective, and the method lends itself well to automation and high-volume testing. For these reasons, EIAs are widely used in the initial evaluation of patients for Lyme disease. In addition, EIAs have recently been recommended by the CDC as an alternative to the Western blot test in the two-tier testing algorithm for Lyme disease, as we discussed previously.
Antigen preparations used in various forms of the assay include crude sonicates of the organism, purified proteins, synthetic proteins, and recombinant proteins such as the VIsE C6 peptide and pepC10 (peptide derived from OSP-C). The manufacturer’s selected antigen is then coated onto 96-well microtiter plates or strips by various proprietary methods. Patient sera is added; during incubation, if antibodies to the B. burgdorferi antigens are present, they will bind to the solid phase. After a washing step, anti-human immunoglobulin conjugated with an enzyme tag such as alkaline phosphatase is added to each well. The conjugate can also be adapted to test for IgM and IgG, IgM only, or IgG only. Adding specific substrate produces a color change. Plates are read in a spectrophotometer, and the antibody is quantitated based on color intensity.
EIAs provide objective results, and the titer is based on a continuum range rather than serial dilutions of patient sera. Thus, a more accurate measurement of the specific antibody is possible. Similar to the IFA, EIAs have decreased sensitivity during the early stages of Lyme disease when patients may not have mounted a sufficient antibody response. In addition, as with IFAs, false positives can occur due to syphilis or other treponemal diseases such as yaws and periodontal disease, as well as relapsing fever and leptospirosis. Patients with infectious mononucleosis, Rocky Mountain spotted fever, and other autoimmune diseases may also be positive with an EIA. Lyme disease patients do not test positive with RPR, so this may be helpful if syphilis is in the differential diagnosis.
Western Blot
Immunoblotting, or Western blotting, has been used as a confirmatory test for samples that initially test positive or equivocal by EIA or IFA. It has been employed as the second test in the CDC-recommended two-tier testing scheme for Lyme disease. The CDC does not recommend testing seropositive or borderline patients for IgM antibodies if they have had symptoms for more than 4 weeks. Serological evidence of Lyme disease in these patients is indicated by a positive result in the IgG immunoblot.
The Lyme disease immunoblot is very complex. The technique consists of electrophoresis of Borrelia antigens in an acrylamide gel followed by transfer of the resulting pattern to nitrocellulose paper. This step is performed by the manufacturer, and nitrocellulose antigen strips are provided in the test kit. These strips are reacted with patient serum and developed with an anti-human immunoglobulin (either anti-IgG or anti-IgM) to which an enzyme label is attached. Further incubation with the enzyme’s substrate allows for visualization of any antibody that has bound to a particular antigen. The reactivity is then scored and interpreted.
Ten proteins are used in the CDC-recommended interpretation of this test. For a result to be considered positive for the presence of specific IgM antibody, two of the following bands must be present: 23 (OSP- C), 39, and 41 (flagellin) kDa. An IgG immunoblot is considered positive if any 5 of the 10 bands previously listed are positive. Because of the complexity of the Lyme immunoblots, testing and interpretation of blots should be done only in qualified laboratories that follow CDC-recommended evidence-based guidelines on immunoblot interpretations.
Polymerase Chain Reaction (PCR)
In testing for Lyme disease, the PCR has found a niche in certain scenarios. Although only a few organisms need to be present for detection under optimal conditions, the number of spirochetes in infected tissues and body fluids is low, making specimen collection, transport, and preparation of DNA critical to the accuracy of the test results. Several probes for target DNA that is present only in strains of B. burgdorferi are used in PCR testing. The procedure involves extracting DNA from the patient sample, followed by amplification using specific primers, DNA polymerase, and nucleotides. The patient DNA is combined with a known DNA probe to see if hybridization takes place. The single-stranded Borrelia DNA probe will bind only to an exact complementary strand, thus positively identifying the presence of the organism’s DNA in the patient sample.
This is much more specific than testing for antibody because there is little cross-reactivity. Specificity of PCR ranges from 93% to 100%. However, sensitivity remains problematic. PCR on CSF and synovial fluid is often used in difficult diagnostic neurological and arthritic cases. PCR for Borrelia is typically performed in reference laboratories. Modifications of the PCR, as well as proteomic assays, are being developed and tested for their potential utility in Lyme disease diagnosis as well.
Lyme Disease Treatment
Borrelia is sensitive to several orally administered antibiotics, including penicillins, tetracyclines, and macrolides. Oral doxycycline is the first treatment of choice for patients with early Lyme disease who are not pregnant. Intravenous antibiotic therapy is required for patients with neurological symptoms, cardiac involvement, or arthritis that does not respond to oral therapy.
Prophylaxis, full-course treatment, or serological testing of all patients with tick bites is not recommended. A single dose of doxycycline may be offered to adults and children older than 8 years of age when the tick can be reliably identified, and treatment can begin within 72 hours of tick removal. Currently, there are no effective vaccines for humans. A human vaccine made with the OSP-A surface antigen has had limited usefulness; it has been associated with side effects and has been recalled from the market. There are renewed efforts to create a new vaccine, but as of this writing, no vaccines have been approved for clinical use.
Leptospirosis
Leptospirosis is caused by bacteria of the genus *Lept
Spirochetes
Spirochetes are distinguished by their unique structure and motility. These bacteria are long, slender, and helically coiled, resembling a corkscrew. The cell's axial filaments, also known as periplasmic flagella, are located within the periplasmic space and wind around the cell wall, contributing to their characteristic motility. Spirochetes are gram-negative but possess an outer sheath surrounding the cell wall, which can complicate Gram staining. They are typically microaerophilic, thriving in low-oxygen environments, and exhibit a distinctive corkscrew-like motility due to the rotation of their axial filaments.
Spirochete infections often follow a complex course. Initially, the infection may manifest as a localized skin infection at the site of entry. However, the bacteria can disseminate rapidly through the bloodstream to multiple organs, leading to systemic involvement. Many spirochetal infections are characterized by a latent stage, during which the bacteria remain dormant and may not cause obvious symptoms. If left untreated, these infections can lead to severe complications, including cardiac and neurological damage.
Key Genera of Spirochetes
Treponema: This genus includes Treponema pallidum, the causative agent of syphilis. Syphilis is a sexually transmitted infection (STI) that can cause long-term complications if not treated.
Borrelia: This genus includes Borrelia burgdorferi, the agent responsible for Lyme disease, which is transmitted through tick bites.
Leptospira: This genus includes various species that cause leptospirosis, a zoonotic disease transmitted through contact with contaminated water or soil.
Key Terms
Borrelia burgdorferi
Chancre
Congenital syphilis
Flocculation
Fluorescent treponemal antibody absorption (FTA-ABS) test
Gummas
Immunoblotting
Leptospira
Leptospirosis
Lyme disease
Microscopic agglutination test (MAT)
Nontreponemal tests
Particle agglutination (PA) tests
Prozone
Rapid plasma reagin (RPR) test
Reagin
Spirochetes
Syphilis
T. pallidum particle agglutination (TP-PA) test
Treponema pallidum
Treponemal test
Venereal Disease Research Laboratory (VDRL) test
Weil’s disease
Syphilis
Syphilis is a systemic infectious disease caused by the spirochete Treponema pallidum. It is primarily transmitted through sexual contact but can also be passed from a pregnant woman to her fetus (congenital syphilis). The disease progresses through several stages if left untreated: primary, secondary, latent, and tertiary.
In the United States, the incidence of syphilis has been increasing. According to the CDC, cases rose by 76% from 2013 to 2017, with over 30,600 cases reported in 2017. A significant portion of this increase is attributed to homosexual transmission between men. Early detection and appropriate antibiotic treatment are essential to cure the infection and prevent long-term complications.
Treponema pallidum Characteristics
The causative agent of syphilis is Treponema pallidum subspecies pallidum. This bacterium lacks a natural environmental reservoir and requires a living host for survival. The genus Treponema includes other pathogens that cause related diseases:
T. pallidum subspecies pertenue (yaws): A tropical infection causing skin lesions.
T. pallidum subspecies endemicum (endemic syphilis or bejel): Primarily affects children in arid regions, causing oral and skin lesions.
Treponema carateum (pinta): Causes skin discoloration and is prevalent in Central and South America.
$T. pallidum$ is a spiral-shaped bacterium, measuring 6-20 µm in length and 0.1-0.2 µm in width, with 6-14 coils. Its outer membrane is a phospholipid bilayer with few exposed proteins, known as treponemal rare outer membrane proteins (TROMPs). This composition delays the host's immune response, allowing the bacteria to disseminate before being effectively targeted by the immune system. The low number of surface proteins also makes it difficult for the immune system to recognize and eliminate the bacteria.
Transmission of Treponema pallidum
Transmission of Treponema pallidum primarily occurs through direct contact with infectious lesions, mainly during sexual activity. The bacteria can penetrate abraded skin or mucous membranes. The risk of acquiring syphilis from an infected partner ranges from 30% to 50% per sexual encounter. Congenital infections can occur when the bacteria cross the placenta during pregnancy, leading to severe fetal damage, including stillbirth, hydrops fetalis, or deformities.
Congenital Syphilis Manifestations:
Early-onset: Manifests before age 2 and resembles secondary syphilis in adults. Symptoms include cutaneous lesions, snuffles (nasal discharge), hepatosplenomegaly, and CNS involvement.
Late-stage: Occurs in infants older than 2 years whose mothers have chronic, untreated infections. Manifestations are similar to tertiary syphilis in adults, including interstitial keratitis, bone and tooth deformities (Hutchinson’s teeth), eighth-nerve deafness, and hard palate perforation.
Other rare transmission routes include parenteral exposure via contaminated needles or blood products. To minimize the risk of transmission through blood transfusions, the American Red Cross has guidelines requiring a 12-month waiting period after syphilis treatment before blood donation.
Stages of Syphilis
Syphilis progresses through distinct stages if left untreated, each characterized by specific clinical manifestations. These stages include primary, secondary, latent, and tertiary syphilis.
Primary Stage
Following infection, Treponema pallidum multiplies at the site of entry, leading to endothelial cell thickening and immune cell aggregation. The hallmark of primary syphilis is the development of a chancre, a painless, solitary lesion with raised borders. Chancres typically appear 10-90 days post-infection (average 21 days) and most commonly occur on the penis in men. In women, chancres may go undetected if located in the vagina or on the cervix. The primary stage lasts 1-6 weeks, during which the chancre heals spontaneously, even without treatment. However, the infection progresses to subsequent stages if not addressed with antibiotics.
Secondary Stage
Approximately 25% of untreated primary syphilis patients progress to the secondary stage. This stage is characterized by systemic dissemination of the organism, usually 1-2 months after the chancre disappears. Common symptoms include:
Generalized lymphadenopathy: Swollen lymph nodes throughout the body.
Malaise: A general feeling of discomfort, illness, or unease.
Fever: Elevated body temperature.
Pharyngitis: Sore throat.
Rash: Skin and mucous membrane lesions, including on the palms and soles. The rash can vary in appearance, including macules, papules, or pustules.
Neurological signs: Visual disturbances, hearing loss, tinnitus, and facial weakness occur in nearly half of patients, indicating CNS involvement. Syphilis can affect the central nervous system at any stage, but neurological manifestations are more common in the secondary and tertiary stages.
Lesions persist for days to weeks, with spontaneous healing. Even without treatment, the symptoms of secondary syphilis will resolve, but the infection will progress to the latent stage.
Latent Stage
The latent stage follows the resolution of secondary syphilis and is characterized by a lack of clinical symptoms. It is divided into early latent (less than 1 year) and late latent (more than 1 year). During the latent stage, patients are generally non-infectious, except for pregnant women, who can still transmit the infection to their fetus.
Tertiary Stage
Occurs in about one-third of untreated individuals, typically 10-30 years after the secondary stage. Major manifestations include:
Gummatous syphilis: Localized granulomatous inflammation on bones, skin, or subcutaneous tissue, containing lymphocytes, epithelioid cells, and fibroblasts. Gummas can heal with scarring or persist as destructive, chronic inflammation. They may affect any organ but are most common in the skin, bones, and liver.
Cardiovascular disease: Primarily affects the ascending aorta, causing aortic aneurysm, valve thickening resulting in aortic regurgitation, or ostial narrowing causing angina pectoris. Cardiovascular syphilis can lead to significant morbidity and mortality.
Neurosyphilis: Can occur at any stage, with early forms presenting as acute meningitis and late forms (after 10+ years) causing degeneration of the lower spinal cord leading to partial paralysis and chronic progressive dementia. Immunodeficient individuals are more susceptible to early neurosyphilis. Neurosyphilis can manifest as general paresis, tabes dorsalis, or meningovascular syphilis.
Congenital Syphilis
Congenital syphilis occurs when treponemes are transmitted to the fetus during early or early latent syphilis. The rate of congenital syphilis reflects the prevalence of syphilis among women of reproductive age and the adequacy of prenatal care. There was an increase in congenital syphilis cases from 362 in 2013 to 918 in 2017.
Transmission
Congenital syphilis can occur at any stage of pregnancy but most severely affects the fetus during the second or third trimester. About 10% of cases result in fetal or perinatal death. Live-born infants may be asymptomatic initially but develop symptoms later if untreated. Early diagnosis and treatment of pregnant women with syphilis are critical to prevent congenital syphilis.
Symptoms
Clear or hemorrhagic rhinitis (runny nose): Often referred to as "snuffles," this is one of the most common early symptoms.
Skin eruptions (maculopapular rash, especially around the mouth, palms, and soles): The rash can be highly infectious.
Generalized lymphadenopathy: Enlargement of lymph nodes throughout the body.
Hepatomegaly/splenomegaly: Enlargement of the liver and spleen.
Jaundice: Yellowing of the skin and eyes, indicating liver involvement.
Anemia: Reduced red blood cell count.
Painful limbs: Due to bone involvement.
Bone abnormalities: Such as osteochondritis, which can cause painful movement.
Neurosyphilis: Involvement of the central nervous system in up to 60% of infants.
Immune Response to Treponema pallidum
Intact skin and mucous membranes are the primary defenses against Treponema pallidum. Once the bacteria penetrate the skin, T cells and macrophages play a critical role in the immune response. CD4+ and CD8+ T cells are present in primary lesions. Cytokines released from these cells activate macrophages, which phagocytose and facilitate the healing of the chancre. The protective role of antibodies is uncertain; coating treponemes with antibodies does not necessarily lead to their destruction. T. pallidum can coat itself with host proteins, delaying immune recognition. TROMPs trigger complement activation, leading to the killing of the organism. Chronic disease indicates immune evasion, allowing treponemes to persist for years without antibiotics.
Laboratory Diagnosis of Syphilis
Laboratory tests are essential for diagnosing syphilis, especially in the absence of clinical signs or symptoms. Traditional tests include direct detection of spirochetes, nontreponemal serological tests, and treponemal serological tests.
Direct Detection
Dark-Field Microscopy
Used for primary and secondary syphilis by identifying T. pallidum in skin lesion exudates. A dark-field condenser excludes incidental light, highlighting the organisms. Pathogenic treponemes are identified by corkscrew morphology and flexing motility. Specimens must be examined quickly to observe motility. False negatives can occur due to delays, insufficient specimens, or antibiotic pretreatment. Experienced microscopists are needed to differentiate pathogens from morphologically identical non-pathogens, especially from oral or rectal samples.
Fluorescent Antibody Testing
A sensitive and specific alternative to dark-field microscopy. Uses fluorescent-labeled antibodies to T. pallidum (direct method) or antibody specific for T. pallidum and a second labeled anti-immunoglobulin antibody (indirect method). Live specimens are not required. Monoclonal antibodies enhance sensitivity and specificity but can cross-react with other T. pallidum subspecies.
Serological Tests
Serological tests are used when active lesions are absent, such as in secondary or tertiary syphilis. These tests are classified as nontreponemal or treponemal based on antibody reactivity.
Nontreponemal Tests
Traditionally used for screening due to high sensitivity and easy performance. However, false positives are common, and positive results require confirmation with treponemal tests. These tests detect antibodies against cardiolipin, a lipid released from damaged cells, referred to as reagin (IgG or IgM). The antigen complex consists of cardiolipin, lecithin, and cholesterol.
Common Nontreponemal Tests:
Venereal Disease Research Laboratory (VDRL) test
Rapid plasma reagin (RPR) test
These tests are based on flocculation reactions (clumping of fine particles). They become positive 1-4 weeks after chancre appearance, with titers peaking during secondary or early latent stages. False negatives can occur in secondary syphilis due to the prozone phenomenon (antibody excess). Serial dilutions can resolve the prozone effect.
Cardiolipin antibody titers decline in later stages, even without treatment. About 25% of untreated cases become nonreactive after years. Successful treatment results in a four-fold titer decrease by the third month and an eight-fold decrease by 6-8 months after initial infection. Tests become nonreactive within 1-2 years after successful treatment. Monitoring the decline in titers is an important indicator of treatment response.
VDRL Test
A qualitative and quantitative slide flocculation test for serum (with modifications for spinal fluid). Antigen must be prepared fresh daily using an alcoholic solution of 0.03% cardiolipin, 0.9% cholesterol, and 0.21% lecithin. Serum specimens are heated at 56°C for 30 minutes to inactivate complement. Results are read microscopically as reactive, weakly reactive, or nonreactive after 4 minutes of rotation at 180 rpm. Tests must be performed at room temperature (23°C to 29°C).
RPR Test
A modified VDRL test involving macroscopic agglutination. The cardiolipin-containing antigen is bound to charcoal particles for easier reading. The suspension is stable for up to 3 months after opening. The antigen contains EDTA, thimerosal, and choline chloride. Serum does not require heat inactivation. Flocculation is read macroscopically after 8 minutes of mechanical rotation at 100 rpm under humid conditions. The RPR test appears to be more sensitive than the VDRL in primary syphilis.
Treponemal Tests
Detect antibodies against the T. pallidum organism or specific treponemal antigens. These tests usually become positive before nontreponemal tests, although patients with early primary syphilis may be nonreactive. Tests are usually 100% reactive in secondary and latent syphilis. Once reactive, individuals remain reactive for life. False positives are fewer compared to reagin tests but can occur with other treponemal diseases (yaws and pinta).
Common Treponemal Tests:
Indirect fluorescent treponemal antibody absorption (FTA-ABS) test
Agglutination tests
These tests are highly specific and used to confirm positive nontreponemal test results. Automated immunoassays for treponemal antibodies have also been developed. These assays offer high throughput and ease of use.
FTA-ABS Test
An early confirmatory test. Heat-inactivated patient serum is incubated with a sorbent (Reiter strain) to remove cross-reacting antibodies. Samples are applied to slides fixed with the Nichols strain of T. pallidum. After incubation and washing, anti-human immunoglobulin conjugated with fluorescein is added. Fluorescence intensity is graded from 0 to 4+. A result of 2+ or above is reactive, 1+ is minimally reactive (repeat test in 1-2 weeks). False positives can occur in patients with SLE or other autoimmune diseases (atypical, beaded fluorescence pattern).
TP-PA (MHA-TP)
Particle agglutination (PA) tests originally used sheep RBCs coated with T. pallidum antigen (MHA-TP). Current PA tests, such as the Serodia T. pallidum particle agglutination (TP-PA) test, use colored gelatin particles coated with treponemal antigens. TP-PA tests are more sensitive in detecting primary syphilis. Agglutination of sensitized gel particles indicates the presence of T. pallidum antibodies. A smooth mat indicates a positive result, while a compact button indicates a negative result.
Automated Immunoassays for T. Pallidum Antibodies
Various automated immunoassays include enzyme immunoassays (EIAs), chemiluminescent immunoassays (CLIAs), and multiplex flow immunoassays (MFIs). EIAs are available in different formats (sandwich assays, competitive assays, and immune capture assays). Capture EIAs are useful in diagnosing congenital syphilis (detect IgM) and monitoring therapy response.
In competitive EIAs, treponemal antibody in the patient sample competes with an enzyme-labeled treponemal antibody conjugate. EIA sensitivities range from 95% to 99%, and specificities are 100%. CLIAs are one-step sandwich techniques using paramagnetic microparticles coated with T. pallidum antigens linked to a chemiluminescent derivative. Relative light units (RLUs) are proportional to the amount of treponemal antibody. CLIAs have higher sensitivity in early syphilis, faster performance, and more stable reagents compared to EIAs.
MFIs involve incubating the patient sample with microspheres coated with recombinant T. pallidum antigens. Bound immune complexes are detected by flow cytometry using a phycoerythrin-labeled reporter antibody. MFIs, EIAs, and CLIAs yield comparable results to the FTA-ABS but have the advantage of automation.
Molecular Testing by Polymerase Chain Reaction (PCR)
PCR amplifies a specific DNA sequence to detect treponemes in blood, spinal fluid, amniotic fluid, tissues, and swab samples. Quantitative PCR (qPCR) is automated, faster, and more sensitive. PCR can detect as little as one treponeme in some samples. Sensitivity is highest in primary syphilis but reduced in secondary syphilis. PCR may be useful when serological testing is inconclusive and as an alternative to dark-field microscopy. It can also detect treponemes in neonates with congenital syphilis symptoms and in the cerebrospinal fluid (CSF) of patients suspected of having neurosyphilis.
Clinical Applications of Syphilis Tests
Nontreponemal tests are useful as a screening tool and for monitoring disease progress and treatment outcomes (titers decrease with effective treatment). However, they are subject to false positives (e.g., hepatitis, infectious mononucleosis, pregnancy, SLE, leprosy, intravenous drug use). Reactive nontreponemal tests should be confirmed by treponemal tests, especially in pregnancy. Treponemal tests are traditionally used as confirmatory tests and are more sensitive in late latent syphilis or late syphilis.
Testing Algorithms for Syphilis
The traditional algorithm involves screening with a nontreponemal test and confirming positive results with a treponemal test.
The reverse sequence algorithm screens with an automated treponemal immunoassay and confirms positive results with a nontreponemal test. This can detect more early, late, and treated syphilis cases. Discrepant results (positive immunoassay, negative RPR) require reflexive testing with TP-PA. If TP-PA is positive, consider late or latent syphilis or previous syphilis history. If TP-PA is negative, the patient is considered negative for syphilis at the time of testing; reevaluation may be needed.
Testing for Congenital Syphilis
Nontreponemal tests on cord blood or neonatal serum detect IgG (difficult to differentiate from maternal antibodies) and IgM. Late maternal infection may result in nonreactive tests due to low fetal antibody levels. Testing infant spinal fluid often lacks sensitivity. Higher infant titers than maternal titers may indicate congenital disease. IgM capture assays are more sensitive. Western blot assays using four major treponemal antigens have high sensitivity and specificity.
In high-risk populations, nontreponemal tests should be performed on both the mother and infant at birth, regardless of previous maternal tests. Repeat tests within a few weeks if maternal history suggests congenital syphilis. Western blot tests are recommended to confirm congenital syphilis.
Testing of Cerebrospinal Fluid (CSF)
CSF testing determines treponeme invasion of the CNS (more reliable with CNS symptoms). The VDRL test and newer ELISA tests are used. A positive VDRL test on spinal fluid is diagnostic of neurosyphilis (false positives are rare). If the VDRL test is negative, other indicators such as increased lymphocyte count and elevated total protein () are used as signs of active disease. PCR may play an important role in CSF testing in the future.
Treatment for Syphilis
Penicillin is the treatment of choice, administered as a single dose of intramuscular long-acting benzathine penicillin G. The specific dosage and duration of treatment depend on the stage of syphilis. Doxycycline and tetracycline are alternatives for penicillin-allergic, non-pregnant patients. For neurosyphilis, intravenous penicillin is typically required.
Lyme Disease
Lyme disease was first described in 1975 around Old Lyme, Connecticut. In 1982, the causative agent Borrelia burgdorferi was identified. It is a multisystem illness affecting the skin, nervous system, heart, and joints. Lyme disease is the most common vector-borne disease in the United States, with more than 42,743 confirmed/probable cases reported in 2017.
Borrelia Species Characteristics
Several species cause Lyme disease (B. burgdorferi sensu stricto in North America; B. afzelii, B. garinii, B. burgdorferi sensu stricto in Europe). For simplicity, they are referred to as B. burgdorferi. The organism is a loosely coiled spirochete, 5-25 µm long and 0.2-0.5 µm in diameter. The outer membrane contains lipoproteins (OSPs A to F, encoded by plasmids) that facilitate attachment to mammalian cells. 7-11 endoflagella (periplasmic flagella) run parallel to its long axis, and are composed of 41-kDa subunits eliciting a strong, early antibody response. Flagellin subunit homology with other spirochetes (B. recurrentis, T. pallidum) can cause cross-reactivity, leading to false positives. The organism divides by binary fission approximately every 12 hours. It can be cultured in a complex liquid medium at 33°C, but isolation from patients is difficult. Spirochetemia is short-lived and usually only found early in the illness. Cultures often require 6 weeks or longer to detect growth.
Lyme Disease Transmission
The main reservoir host is the white-footed mouse. Ixodes ticks transmit the disease:
Ixodes scapularis (Northeast and Midwest United States)
Ixodes pacificus (West)
Ixodes ricinus (Europe)
Ixodes persulcatus (Asia
White-tailed deer are the main host for the tick’s adult stage. Nymphs and adult ticks transmit the disease. Peak feeding occurs in late spring, early summer, and fall. Ticks must feed for more than 36 hours to transmit the spirochete (transmission is still low at 72 hours).
Stages of Lyme Disease
Lyme disease progresses through stages, including localized rash, early dissemination, and late dissemination. Lyme disease can be viewed as a progressive infectious disease involving diverse organ systems
Localized Rash Stage
The clinical hallmark of early infection is erythema migrans (EM), appearing 2 days to 2 weeks after a tick bite. EM begins as a small red papule that expands to form a large ring-like erythema, often with a central clearing. Diagnosis relies on recognizing the characteristic rash (at least 5 cm in diameter). Patients may be asymptomatic or have flu-like symptoms. EM usually expands for more than a week and fades within 3-4 weeks if it goes untreated. Approximately 20% of patients do not develop the rash. The antibody response is minimal during this stage, and most serology results are negative.
Early Dissemination Stage
Early dissemination occurs via the bloodstream in the days to weeks following the EM rash. The skin, nervous system, heart, or joints may be affected. Some patients display multiple skin lesions. Migratory pain often occurs in the joints, tendons, muscles, and bones. Without treatment, neurological or cardiac involvement develops in about 15% of patients 4-6 weeks after infection onset. The most prevalent neurological sign is facial palsy. Other symptoms include sleep disturbances, mild chronic confusion, or difficulty with memory and intellectual functioning, as well as aseptic meningitis.
Late Dissemination Stage
Late Lyme disease may develop months to years after infection in untreated patients. Major manifestations include arthritis, peripheral neuropathy, and encephalomyelitis. These symptoms usually respond well to conventional antibiotic treatment, but treatment-resistant arthritis is associated with particular HLA–DRB alleles. Some patients develop chronic fatigue, concentration and short-term memory problems, and musculoskeletal pain that lasts longer than 6 months despite resolution of objective manifestations of Borrelia infection after antibiotic treatment.
Immune Response in Lyme Disease
The immune response is highly variable and complex. Both humoral and cellular responses exist. Spirochete lipoproteins stimulate macrophages to produce cytokines, which further enhance the immune response. The effectiveness of these responses is questionable because late Lyme disease occurs despite high Ab and cellular responses.
Laboratory Diagnosis of Lyme Disease
The diagnosis of Lyme disease is a clinical one, with laboratory testing used as supporting evidence that is often difficult to obtain. If the characteristic rash is present, this can be used as a presumptive finding, but as many as 20% of patients do not develop or do not recognize the rash. Direct isolation of the organism via skin scrapings, spinal fluid, or blood is possible, but the yield of positive cultures is extremely low. For this reason, culture is not used as a routine diagnostic tool. The antibody response is variable and may not be detectable until 3 to 6 weeks after the tick bite. The IgM response occurs first, followed by the IgG response. The IgG response does not peak until the third and fourth weeks of infection. These antibody responses are also not mutually exclusive and can be variable (e.g., an IgM response can occur in late Lyme disease).
In most cases of acute early Lyme disease (first 2 weeks), serological testing is too insensitive to be diagnostically helpful. If patients with symptoms are tested in fewer than 7 days after infection, seropositivity is only about 30%. Therefore, the decision to start treatment for early Lyme disease must be made before seroconversion, similar to many acute infectious diseases. However, untreated seronegative patients having symptoms for 6 to 8 weeks are unlikely to have Lyme disease, and other possible diagnoses should be pursued. Antibiotic therapy begun shortly after the appearance of EM may delay or abrogate the antibody response.
Two-Tiered Approach to Lyme Disease Diagnosis
The CDC recommends a two-tiered approach to providing laboratory support for the diagnosis of Lyme disease. Using the standard testing algorithm, patients with clinical evidence of Lyme disease are screened with a sensitive ELISA or, alternatively, with an IFA. If the first serology test is positive or borderline, a Western blot test is performed on that specimen to confirm the result. Some important limitations to this approach are the cost and complexity of performing and interpreting the Western blot, which must be done in reference laboratories. The standard testing algorithm is highly specific but has low sensitivity in detecting early Lyme disease.
Modified Two-Tiered Algorithm
In this approach, symptomatic patients are first tested with a sensitive ELISA that uses purified Borrelia peptide antigens. Samples with positive or borderline results are retested with a different ELISA method for confirmation. The modified algorithm was approved by the U.S. Food and Drug Administration (FDA). In addition to being easier to perform and interpret, this approach demonstrates comparable specificity to the standard algorithm and increased sensitivity in detecting early Lyme disease.
Lyme testing should not be performed in the absence of supporting clinical evidence. A positive test performed under these circumstances has a low positive predictive value, even when done in an endemic area, whereas it rises to nearly 100% when clinical symptoms and history are present and consistent with Lyme disease.
Common Lyme Disease Testing Procedures
Immunofluorescence Assay (IFA)
The IFA was the first test used to evaluate the antibody response in Lyme disease, followed by various forms of EIAs shortly thereafter. Doubling dilutions of patient serum are incubated with commercially prepared microscope slides coated with antigen from whole or processed Borrelia spirochetes. Following a wash step to remove unbound material, an anti-human globulin with a fluorescent tag attached is added and reacts with any specific antibody bound to the spirochetes on the slide. After a second wash step, the slide is viewed under a fluorescent microscope.
Typically, a test result is only considered positive if a titer of 1:256 or higher is obtained, although this varies between manufacturers. As previously mentioned, specimens obtained in the first few weeks are usually negative because the level of antibody present is below the detection limit of this (and other) assays. As might be expected, other closely related organisms, such as B. recurrentis (relapsing fever), T. denticola and others associated with periodontal disease, and T. pallidum (syphilis), may cross- react and cause biological false-positive results. Autoimmune connective tissue diseases such as rheumatoid arthritis (RA) and SLE can also produce false positives in the IFA for Lyme disease. An astute technologist can recognize a false positive by the beaded fluorescent pattern it produces. Reading of fluorescent patterns tends to be very subjective and requires highly trained individuals. However, if performed correctly by experienced personnel, the test can provide sensitive and accurate results. This test is best suited for low-volume testing.
Enzyme Immunoassay (EIA)
EIA testing has used ELISAs that are relatively inexpensive to perform and yield timely results. The test is reproducible because the results are objective, and the method lends itself well to automation and high-volume testing. For these reasons, EIAs are widely used in the initial evaluation of patients for Lyme disease. In addition, EIAs have recently been recommended by the CDC as an alternative to the Western blot test in the two-tier testing algorithm for Lyme disease, as we discussed previously.
Antigen preparations used in various forms of the assay include crude sonicates of the organism, purified proteins, synthetic proteins, and recombinant proteins such as the VIsE C6 peptide and pepC10 (peptide derived from OSP-C). The manufacturer’s selected antigen is then coated onto 96-well microtiter plates or strips by various proprietary methods. Patient sera is added; during incubation, if antibodies to the B. burgdorferi antigens are present, they will bind to the solid phase. After a washing step, anti-human immunoglobulin conjugated with an enzyme tag such as alkaline phosphatase is added to each well. The conjugate can also be adapted to test for IgM and IgG, IgM only, or IgG only. Adding specific substrate produces a color change. Plates are read in a spectrophotometer, and the antibody is quantitated based on color intensity.
EIAs provide objective results, and the titer is based on a continuum range rather than serial dilutions of patient sera. Thus, a more accurate measurement of the specific antibody is possible. Similar to the IFA, EIAs have decreased sensitivity during the early stages of Lyme disease when patients may not have mounted a sufficient antibody response. In addition, as with IFAs, false positives can occur due to syphilis or other treponemal diseases such as yaws and periodontal disease, as well as relapsing fever and leptospirosis. Patients with infectious mononucleosis, Rocky Mountain spotted fever, and other autoimmune diseases may also be positive with an EIA. Lyme disease patients do not test positive with RPR, so this may be helpful if syphilis is in the differential diagnosis.
Western Blot
Immunoblotting, or Western blotting, has been used as a confirmatory test for samples that initially test positive or equivocal by EIA or IFA. It has been employed as the second test in the CDC-recommended two-tier testing scheme for Lyme disease. The CDC does not recommend testing seropositive or borderline patients for IgM antibodies if they have had symptoms for more than 4 weeks. Serological evidence of Lyme disease in these patients is indicated by a positive result in the IgG immunoblot.
The Lyme disease immunoblot is very complex. The technique consists of electrophoresis of Borrelia antigens in an acrylamide gel followed by transfer of the resulting pattern to nitrocellulose paper. This step is performed by the manufacturer, and nitrocellulose antigen strips are provided in the test kit. These strips are reacted with patient serum and developed with an anti-human immunoglobulin (either anti-IgG or anti-IgM) to which an enzyme label is attached. Further incubation with the enzyme’s substrate allows for visualization of any antibody that has bound to a particular antigen. The reactivity is then scored and interpreted.
Ten proteins are used in the CDC-recommended interpretation of this test. For a result to be considered positive for the presence of specific IgM antibody, two of the following bands must be present: 23 (OSP- C), 39, and 41 (flagellin) kDa. An IgG immunoblot is considered positive if any 5 of the 10 bands previously listed are positive. Because of the complexity of the Lyme immunoblots, testing and interpretation of blots should be done only in qualified laboratories that follow CDC-recommended evidence-based guidelines on immunoblot interpretations.
Polymerase Chain Reaction (PCR)
In testing for Lyme disease, the PCR has found a niche in certain scenarios. Although only a few organisms need to be present for detection under optimal conditions, the number of spirochetes in infected tissues and body fluids is low, making specimen collection, transport, and preparation of DNA critical to the accuracy of the test results. Several probes for target DNA that is present only in strains of B. burgdorferi are used in PCR testing. The procedure involves extracting DNA from the patient sample, followed by amplification using specific primers, DNA polymerase, and nucleotides. The patient DNA is combined with a known DNA probe to see if hybridization takes place. The single-stranded Borrelia DNA probe will bind only to an exact complementary strand, thus positively identifying the presence of the organism’s DNA in the patient sample.
This is much more specific than testing for antibody because there is little cross-reactivity. Specificity of PCR ranges from 93% to 100%. However, sensitivity remains problematic. PCR on CSF and synovial fluid is often used in difficult diagnostic neurological and arthritic cases. PCR for Borrelia is typically performed in reference laboratories. Modifications of the PCR, as well as proteomic assays, are being developed and tested for their potential utility in Lyme disease diagnosis as well.
Lyme Disease Treatment
Borrelia is sensitive to several orally administered antibiotics, including penicillins, tetracyclines, and macrolides. Oral doxycycline is the first treatment of choice for patients with early Lyme disease who are not pregnant. Intravenous antibiotic therapy is required for patients with neurological symptoms, cardiac involvement, or arthritis that does not respond to oral therapy.
Prophylaxis, full-course treatment, or serological testing of all patients with tick bites is not recommended. A single dose of doxycycline may be offered to adults and children older than 8 years of age when the tick can be reliably identified, and treatment can begin within 72 hours of tick removal. Currently, there are no effective vaccines for humans. A human vaccine made with the OSP-A surface antigen has had limited usefulness; it has been associated with side effects and has been recalled from the market. There are renewed efforts to create a new vaccine, but as of this writing, no vaccines have been approved for clinical use.
Leptospirosis
Leptospirosis is caused by bacteria of the genus *Lept