Syphilis & Lyme Disease PDF

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 Prozo...

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 Spirochetes are long, slender, helically coiled bacteria containing axial filaments, or periplasmic flagella, which wind around the bacterial cell wall and are enclosed by an outer sheath. These gram-negative, microaerophilic bacteria exhibit a characteristic corkscrew flexion or motility. Diseases caused by these organisms have many similarities, including a localized skin infection that disseminates to numerous organs as the disease progresses, a latent stage, and cardiac and neurological involvement if the disease remains untreated. This chapter discusses clinical manifestations and laboratory testing for the two major spirochete diseases, syphilis and Lyme disease, and provides an introduction to leptospirosis. Serological testing plays a key role in diagnosis of these diseases because isolation of the organisms themselves is difficult to accomplish in the laboratory and clinical symptoms are not always apparent. Syphilis Syphilis is a commonly acquired spirochete disease in the United States. It is typically spread through sexual transmission. Although the incidence of syphilis in the United States reached an all-time low in 2000, it slowly increased through 2017. According to the Centers for Disease Control and Prevention (CDC) surveillance report for sexually transmitted diseases, more than 30,600 cases of syphilis were reported in the United States in 2017, representing a 76% rise since 2013. Homosexual transmission between men was responsible for much of this increase. Despite the current emphasis on safe sexual practices, syphilis remains a major health problem in many areas of the world; more than 5 million new cases are reported worldwide each year. Early detection of syphilis is of major importance because treatment with antibiotics in the early stages of the disease can usually provide a cure. This section discusses characteristics of the organism that causes syphilis, its clinical manifestations, and laboratory methods essential to its diagnosis. Characteristics of Treponema pallidum The causative agent of syphilis is Treponema pallidum, subspecies pallidum, a member of the family Spirochaetaceae. Organisms in this family have no natural reservoir in the environment and must multiply within a living host. Three other pathogens in this group are so morphologically and antigenically similar to T. pallidum that all but one are classified as subspecies. These other organisms are T. pallidum subspecies pertenue, the agent of yaws; T. pallidum subspecies endemicum, the cause of nonvenereal endemic syphilis; and Treponema carateum, the agent of pinta. Yaws is found in the tropics, pinta is found in Central and South America, and endemic syphilis is found in desert regions. T. pallidum (which will hereafter be used to refer to the subspecies pallidum) varies in length from 6 to 20 µm and in width from 0.1 to 0.2 µm, with 6 to 14 coils (Fig. 21–1). The outer membrane of T. pallidum is a phospholipid bilayer with very few exposed proteins. Several identified membrane proteins, called treponemal rare outer membrane proteins (TROMPs), have been characterized. It appears that the scarcity of such proteins delays the host immune response. Mode of Transmission Pathogenic treponemes are rapidly destroyed by heat, cold, and drying, so they are almost always spread by direct contact. Sexual transmission is the primary mode of dissemination; this occurs through contact of abraded skin or mucous membranes with an open lesion. Approximately 30% to 50% of the individuals who are exposed to a sexual partner with active lesions will acquire the disease. Congenital infections (transplacental) can also occur during pregnancy. In utero syphilis infection is generally severe and mutilating, affecting almost every tissue of the fetus. Infants born to mothers infected with early-stage syphilis tend to have symptoms resembling secondary syphilis in adults, characterized by cutaneous lesions, snuffles, hepatosplenomegaly, and central nervous system (CNS) involvement. The symptoms of early-onset syphilis generally manifest at younger than 2 years of age. Late-stage congenital syphilis occurs in infants older than 2 years when they are conceived by mothers having chronic and untreated infection. This type of manifestation corresponds to tertiary syphilis in adults, and symptoms include interstitial keratitis, bone and tooth deformities (Hutchinson’s teeth), eighth-nerve deafness, and perforation of the hard palate. There may be a variety of manifestations in congenital syphilis, ranging from stillbirth and neonatal death to long-term physical and physiological deformities in the surviving infants. FIGURE 21-1 Treponema pallidum. Electron micrograph showing the coils and periplasmic flagella. (Courtesy of CDC.) Other potential means of transmission include parenteral exposure through contaminated needles or blood, but this is extremely rare. For the past 30 years, the lack of transfusion-transmitted syphilis in the United States has actually called into question the necessity of testing potential donors for the presence of the disease. However, current guidelines by the American Red Cross require that people wait 12 months after treatment for syphilis before donating blood. Because syphilis can only be transmitted by means of fresh blood products, the use of stored blood components has virtually eliminated the possibility of transfusion-associated syphilis. Stages of the Disease Untreated syphilis can progress through four stages: primary, secondary, latent, and tertiary. Primary Stage Once contact has been made with a susceptible skin site, endothelial cell thickening occurs with aggregation of lymphocytes, plasma cells, and macrophages. The initial lesion, called a chancre, develops between 10 and 90 days after infection, with an average of 21 days. A chancre is a painless, solitary lesion characterized by raised and well-defined borders (Fig. 21–2). In men, these usually occur on the outside of the penis, but in women they may appear in the vagina or on the cervix and, thus, may go undetected. This primary stage lasts from 1 to 6 weeks, during which time the lesion heals spontaneously. Secondary Stage About 25% of patients who are untreated in the primary stage progress to the secondary stage, in which systemic dissemination of the organism occurs. This stage is usually observed about 1 to 2 months after the primary chancre disappears; however, in some cases, the primary lesion may still be present. Symptoms of the secondary stage include generalized lymphadenopathy, or enlargement of the lymph nodes; malaise; fever; pharyngitis; and a rash on the skin and mucous membranes. The rash may appear on the palms of the hands and the soles of the feet. Neurological signs, such as visual disturbances, hearing loss, tinnitus, and facial weakness, appear in nearly half of patients with secondary syphilis, indicating involvement of the CNS. Lesions persist from a few days up to 8 weeks, and spontaneous healing occurs, as in the primary stage. FIGURE 21-2 Primary chancre in the early stage of syphilis. (Courtesy of the CDC/Dr. N. J. Fiumara, Public Health Image Library.) Clinical Correlations The Great Imitator Patients with syphilis can be difficult to diagnose because their clinical presentations can vary widely. Because the symptoms of syphilis can mimic those of many other diseases or conditions, the disease has often been referred to as “The Great Imitator.” Latent Stage The latent stage follows the disappearance of secondary syphilis. This stage is characterized by a lack of clinical symptoms. It is arbitrarily divided into early latent (fewer than 1 year’s duration) and late latent, in which the primary infection has occurred more than 1 year previously. Patients are not infectious at this time, with the exception of pregnant women, who can pass the disease on to the fetus even if they exhibit no symptoms. Tertiary Stage About one-third of the individuals who remain untreated develop tertiary syphilis. This stage occurs most often between 10 and 30 years following the secondary stage. Tertiary syphilis has three major manifestations: gummatous syphilis, cardiovascular disease, and neurosyphilis. Gummas are localized areas of granulomatous inflammation that are most often found on bones, skin, or subcutaneous tissue. Such lesions contain lymphocytes, epithelioid cells, and fibroblasts. They may heal spontaneously with scarring, or they may remain destructive areas of chronic inflammation. It is likely that they represent the host response to infection. Cardiovascular complications usually involve the ascending aorta, and symptoms are caused by destruction of elastic tissue in the aortic arch. The destruction may result in aortic aneurysm, thickening of the valve leaflets causing aortic regurgitation, or narrowing of the ostia, producing angina pectoris. Neurosyphilis is the complication most often associated with the tertiary stage, but it actually can occur any time after the primary stage and can span all stages of the disease. Immunodeficient individuals such as HIV patients are susceptible to early neurosyphilis. During the first 2 years following infection, CNS involvement often takes the form of acute meningitis. Late manifestations of neurosyphilis include degeneration of the lower spinal cord with partial paralysis and chronic progressive dementia. It usually takes more than 10 years for these to occur; both are the result of structural CNS damage that cannot be reversed. Fortunately, symptoms of tertiary syphilis are now very rare because of early detection and effective treatment with antibiotics such as penicillin. Connections Gummas A gumma is a form of granuloma characteristic of tertiary syphilis. As discussed in Chapter 14, granulomas are organized clusters of white blood cells (WBCs) and epithelial cells that are formed because of a type IV hypersensitivity reaction. This cell- mediated mechanism develops in response to chronic persistence of the antigen.  Granulomas can form in patients with various infectious diseases, including leprosy, tuberculosis, cutaneous leishmaniasis, yaws, and syphilis. Congenital Syphilis Congenital syphilis occurs when a woman who has early syphilis or early latent syphilis transmits treponemes to the fetus. The occurrence of congenital syphilis rose from 362 cases in the year 2013 to 918 cases in 2017. Although the disease can be transmitted at any stage of pregnancy, typically the fetus is most affected during the second or third trimester. Fetal or perinatal death occurs in approximately 10% of the cases. Infants who are born live often have no clinical signs of disease during the first few weeks of life. Some may remain asymptomatic, but the majority of these infants develop later symptoms if not treated at birth. Such infants may exhibit clear or hemorrhagic rhinitis (runny nose). Skin eruptions, in the form of a maculopapular rash that is especially prominent around the mouth, the palms of the hands, and the soles of the feet, are also common. Other symptoms include generalized lymphadenopathy hepatosplenomegaly jaundice, anemia, painful limbs, and bone abnormalities. Neurosyphilis may occur in up to 60% of infants with congenital disease. Nature of the Immune Response The primary body defenses against treponemal invasion are intact skin and mucous membranes. Once the skin is penetrated, T cells and macrophages play a key role in the immune response. Primary lesions show the presence of both CD4+ and CD8+ T cells. Cytokines produced by these cells activate macrophages, and it is ultimately macrophage phagocytosis that heals the primary chancre. The protective role of antibodies is uncertain, however, as coating the treponemes with antibodies does not necessarily bring about their destruction. T. pallidum is also capable of coating itself with host proteins, which delays the immune system’s recognition of the pathogen. The rare treponemal proteins, or TROMPs, are important in triggering the activation of complement, which ultimately kills the organism. However, the chronic nature of the disease is an indicator that the organisms are able to evade the immune response. Treponemes may persist in the host for years if antibiotic therapy is not obtained. Laboratory Diagnosis Traditional laboratory tests for syphilis can be classified into three main types: direct detection of spirochetes, nontreponemal serological tests, and treponemal serological tests. These vary in their ability to detect syphilis at different stages of the disease. Principles and procedures of each type of testing are discussed in the text that follows. Special considerations in laboratory testing for neurosyphilis and congenital syphilis are also introduced. Direct Detection Direct detection of spirochetes can be accomplished by dark-field microscopy or fluorescent antibody testing. The performance of either test requires that the patient have active lesions. Dark-Field Microscopy Primary and secondary syphilis can be diagnosed by demonstrating the presence of T. pallidum in exudates from skin lesions. In dark-field microscopy, a dark-field condenser is used to keep all incidental light out of the field except for that captured by the organisms themselves. It is essential to have a good specimen in the form of serous fluid from a lesion. The serous fluid is usually obtained by cleaning the lesion with sterile saline and rubbing it with clean gauze. Pathogenic treponemes are identified on the basis of their characteristic corkscrew morphology and flexing motility. Because observation of motility is the key to identification, specimens must be examined as quickly as possible before they dry out. False-negative results can occur when there is a delay in evaluating the slides, an insufficient specimen is obtained, or the patient is pretreated with antibiotics. Thus, a negative test does not exclude a diagnosis of syphilis. In addition, an experienced microscopist should perform testing. If a specimen is obtained from the mouth or the rectal area, morphologically identical nonpathogenic microbes can be found that must be differentiated from the true pathogens. Fluorescent Antibody Testing The use of a fluorescent-labeled antibody is a sensitive and highly specific alternative to dark-field microscopy. Testing can be performed by either a direct method, which uses a fluorescent-labeled antibody conjugate to T. pallidum, or an indirect method using antibody specific for T. pallidum and a second labeled anti-immunoglobulin antibody. An advantage of these methods is that live specimens are not required. A specimen can be brought to the laboratory in a capillary tube, and fixed slides can be prepared for later viewing. Treponemes can be washed off the slide even after fixing; therefore, each slide must be handled individually, and rinsing must be carefully performed. The use of monoclonal antibodies has made fluorescent antibody testing very sensitive and specific. However, monoclonal antibodies can still cross-react with other subspecies of T. pallidum, and this must be taken into account when making a diagnosis. Serological Tests If a patient does not have active lesions, as may be the case in secondary or tertiary syphilis, then serological testing for antibodies is the key to diagnosis. Serological tests can be classified as nontreponemal or treponemal, depending on the reactivity of the antibody that is detected. Nontreponemal tests have traditionally been used to screen for syphilis because of their high sensitivity and ease of performance. However, false- positive results are common because of the nonspecific nature of the antigen. Therefore, any positive results must be confirmed by a more specific treponemal test, which detects antibodies to T. pallidum. Connections Complement Fixation The first nontreponemal serological test for syphilis was developed in 1906 by the bacteriologist August Paul von Wassermann. This test used a crude liver extract from a fetus that was infected with syphilis as the source of the lipid antigen. The Wasserman test was based on the principle of complement fixation. Patient serum was incubated with cardiolipin antigen in the presence of rabbit serum as the source of complement; this was followed by a detection system consisting of antibody-coated sheep red blood cells (RBCs). If the patient serum contained cardiolipin antibody, complexes were formed that bound the reagent complement, and the indicator RBCs were not lysed. In contrast, if cardiolipin antibody was not present in the patient serum, the reagent complement was free to react with the antibody-sensitized sheep RBCs to cause hemolysis. Nontreponemal Tests Nontreponemal tests determine the presence of an antibody that forms against cardiolipin, a lipid material released from damaged host cells. This antibody has sometimes been referred to as reagin. It is found in the sera of patients with syphilis and several other disease states. An antigen complex consisting of cardiolipin, lecithin, and cholesterol is used in the reaction to detect the nontreponemal reagin antibodies, which are either of the immunoglobulin G (IgG) or immunoglobulin M (IgM) class. Connections Reagin The term reagin as it applies to syphilis should not be confused with the same word that was originally used to describe immunoglobulin E (IgE). They are not the same. Fortunately, the term reagin in reference to IgE is rarely used today. The most widely used nontreponemal tests are the Venereal Disease Research Laboratory (VDRL) test and the rapid plasma reagin (RPR) test. These tests are based on flocculation reactions in which patient antibody complexes with the cardiolipin antigen. Flocculation is a specific type of precipitation that occurs over a narrow range of antigen concentrations. The antigen consists of very fine particles that clump together in a positive reaction. Typical serological results for nontreponemal tests during the course of untreated and treated syphilis are shown in Figure 21–3. In general, nontreponemal tests become positive within 1 to 4 weeks after the appearance of the primary chancre. Titers usually peak during the secondary or early latent stages. In primary disease, up to about 40% of individuals appear nonreactive; however, by the secondary stage, almost all patients have reactive test results. However, testing of sera from patients in the secondary stage is subject to false negatives because of the prozone phenomenon (antibody excess). In this case, a nonreactive pattern that is typically granular or rough in appearance is seen. If a prozone is suspected, serial two-fold dilutions of the patient’s serum should be made to obtain a titer.  Cardiolipin antibody titers tend to decline in the later stages of the disease, even if the patient remains untreated. After several years, about 25% of untreated syphilis cases become nonreactive for reagin. This decline occurs more rapidly in individuals who have received treatment. A first- time infection, if in the primary or secondary stage, should show a four-fold decrease in titer by the third month following treatment and an eight-fold decrease by 6 to 8 months. Following successful treatment, tests typically become completely nonreactive within 1 to 2 years. VDRL Test The VDRL test, which was designed by the Venereal Disease Research Laboratories, is both a qualitative and quantitative slide flocculation test for serum that includes a modification for use on spinal fluid. Antigen for all tests must be prepared fresh daily and in a highly regulated fashion. The antigen is an alcoholic solution of 0.03% cardiolipin, 0.9% cholesterol, and 0.21% lecithin. The antigen suspension is prepared by adding the VDRL antigen with a dropper to a buffered saline solution while continuously rotating the mixture on a flat surface; attention must be paid to required rotation speed and timing. A daily calibrated Hamilton syringe is used to deliver one drop of antigen for the slide test. If the delivery is off by more than 2 drops out of 60, the syringe must be cleaned with alcohol and recalibrated. FIGURE 21-3 Typical nontreponemal and treponemal antibody patterns in syphilis. (Adapted from Peeling RW, Ye H. Diagnostic tools for preventing and managing maternal and congenital syphilis: an overview. Bull World Health Organ 2004;82(6):439–446.)  The serum specimens to be tested are heated at 56°C for 30 minutes to inactivate complement, after which 0.05 mL is pipetted into a ceramic ring of a glass slide. Three control sera—nonreactive, minimally reactive, and reactive—are pipetted into separate rings on the glass slide in the same manner. Control sera and patient samples are spread out to fill the entire ring. One drop (1/60 mL) of the VDRL antigen is then added to each ring. The slide is rotated for 4 minutes on a rotator at 180 rpm. It is read microscopically to determine the presence of flocculation, or small clumps. The results are recorded as reactive (medium to large clumps), weakly reactive (small clumps), or nonreactive (no clumps or slight roughness). Tests must be performed at room temperature within the range of 23°C to 29°C (73°F to 85°F) because results may be affected by temperature changes. All sera with reactive or weakly reactive results must be tested using the quantitative slide test, in which two-fold dilutions of serum ranging from 1:2 to 1:32 are initially used. Sera yielding positive results at the 1:32 dilution are titered further. RPR Test The RPR test is a modified VDRL test involving macroscopic agglutination. The cardiolipin-containing antigen suspension is bound to charcoal particles, making the test easier to read. The suspension is contained in small glass vials, which are stable for up to 3 months after opening. The antigen is similar to the VDRL antigen, with the addition of ethylenediaminetetraacetic acid (EDTA), thimerosal, and choline chloride, which stabilize the antigen and inactivate complement so that serum does not have to be heat-inactivated before use. Patient serum (approximately 0.05 mL) is placed in an 18-mm circle on a plastic-coated disposable card using a micropipette or Dispenstir device. Cardiolipin antigen is dispensed from a small plastic dispensing bottle with a calibrated 20-gauge needle. One free-falling drop of antigen is placed onto each test area, and the card is mechanically rotated at 100 rpm for 8 minutes under humid conditions. Cards are read under a high-intensity light source; if flocculation is evident, the test is positive (Fig. 21–4). All reactive tests should be confirmed by retesting using doubling dilutions in a quantitative procedure. The RPR test appears to be more sensitive than the VDRL in primary syphilis. FIGURE 21-4 RPR test results. Well 1: reactive control, showing large clumps. Well 2: weakly reactive control, showing small clumps. Well 3: nonreactive control, showing slight roughness and a “tail” upon swirling. Wells 6 to 10 show results of a serially diluted sample of patient serum with a titer of 8. (Courtesy of Linda Miller.) Treponemal Tests Treponemal tests detect antibody directed against the T. pallidum organism or against specific treponemal antigens. Typical treponemal antibody results during various stages of syphilis are shown in Figure 21–3. Treponemal tests usually become positive before nontreponemal tests, although patients with early primary syphilis may be nonreactive. In secondary and latent syphilis, tests are usually 100% reactive. Once a patient is reactive, that individual remains reactive for life. Although there are fewer false positives compared with reagin tests, reactivity is seen with other treponemal diseases, notably yaws and pinta. Connections Antibody Response Recall from Chapter 5 that IgG antibody produced In the secondary Immune response Is of high titer and persists for a long period of time. This is the reason that treponemal tests, which mainly detect IgG, remain positive for life after they have become reactive. Two main types of manual treponemal tests are the indirect fluorescent treponemal antibody absorption (FTA-ABS) test and agglutination tests. Because these tests are highly specific for syphilis, they have been used to confirm positive nontreponemal test results. More recently, automated immunoassays for treponemal antibodies have been developed. Their applications will be discussed later. FTA-ABS test The FTA-ABS test is one of the earliest confirmatory tests developed. In this test, a dilution of heat-inactivated patient serum is incubated with a sorbent consisting of an extract of nonpathogenic treponemes (Reiter strain), which removes antibodies that cross-react with treponemes other than T. pallidum. Diluted patient samples and controls are applied to individual wells on a test slide fixed with the Nichols strain of T. pallidum. Following a 30-minute incubation at 37°C, the slides are washed, and air- dried and antibody conjugate (anti-human immunoglobulin conjugated with fluorescein) is added to each well. Slides are re-incubated as before and washed to remove excess conjugate. Mounting medium is applied, and coverslips are placed on the slides, which are then examined under a fluorescence microscope. If specific patient antibody is present, it will bind to the T. pallidum antigens. The antibody conjugate will, in turn, only bind where patient immunoglobulin is present and bound to the spirochetes. When slides are read under a fluorescence microscope, the intensity of the green color is reported on a scale of 0 to 4 +. No fluorescence indicates a negative test, whereas a result of 2+ or above is considered reactive. A result of 1 + means that the specimen was minimally reactive, and the test must be repeated with a second specimen drawn in 1 to 2 weeks. Experienced personnel are needed to read and interpret fluorescent test results. False- positive results may occur in patients with systemic lupus erythematosus (SLE) or other autoimmune diseases and can appear as an atypical, beaded pattern of fluorescence. The FTA-ABS is highly sensitive and specific, but it is time consuming to perform and has been replaced in many laboratories with particle agglutination methods. TP-PA (MHA-TP) The particle agglutination (PA) tests originally used sheep RBCs coated with T. pallidum antigen and were referred to as MHA-TP (microhemagglutination assay for T. pallidum antibody). Current PA tests for T. pallidum, such as the Serodia T. pallidum particle agglutination (TP-PA) test, use colored gelatin particles coated with treponemal antigens and are more sensitive in detecting primary syphilis. In the Serodia TP-PA test, patient serum or plasma is diluted in microtiter plates and incubated with either T. pallidum–sensitized gel particles or unsensitized gel particles as a control. The presence of T. pallidum antibodies is indicated by agglutination of the sensitized gel particles, which bind to the antibodies to form a lattice-like structure that spreads to produce a smooth mat covering the surface of the well. If a sample is negative for the antibody, the gel particles settle to the bottom of the well and form a compact button (Fig. 21–5). FIGURE 21-5 TP-PA test results. Row A: positive control. Row B: negative control. Row C: serum from a positive patient. T. pallidum– sensitized gel particles were placed in column 3 of each row, and unsensitized gel particles were pipetted into column 4 of each row. Positive wells (A3 and C3) are indicated by a diffuse mat of particles that spread over the surface of the well, whereas negative results are indicated by a compact button. (Courtesy of Linda Miller.) Automated Immunoassays for T. Pallidum Antibodies A variety of automated immunoassays have been developed for the detection of antibodies to T. pallidum. These include enzyme immunoassays (EIAs), chemiluminescent immunoassays (CLIAs), and multiplex flow immunoassays (MFIs). EIAs have been manufactured in a variety of formats, including one- or two-step sandwich assays, one-step competitive assays, and immune capture assays. In the sandwich assays, antibodies in the patient sample bind to recombinant T. pallidum antigens coated onto microtiter plate wells. An enzyme-labeled antibody or antigen conjugate and substrate are added to detect binding. In the immune capture format, microtiter wells are coated with antibody to IgM or IgG and are reacted with patient serum. Antigens that are labeled with an enzyme are then added (Fig. 21–6). The capture EIA tests are especially useful in diagnosing congenital syphilis in infants because they look for the presence of IgM, which cannot cross the placenta. They can also be used to monitor response to therapy in the early stages of syphilis because many patients are negative for IgM treponemal antibodies 6 to 12 months after treatment. In competitive EIAs, treponemal antibody in the patient sample competes with an enzyme-labeled treponemal antibody conjugate for T. pallidum antigens bound to microtiter plate wells. Test sensitivities of the various EIAs range from about 95% to 99%, and test specificities are 100%. FIGURE 21-6 Antibody capture enzyme-linked immunosorbent assay (ELISA) test. Only specific anti-treponemal antibody will react with enzyme-labeled antigen.  CLIAs are available as a one-step sandwich technique in which the patient sample is incubated with paramagnetic microparticles that have been coated with T. pallidum antigens linked to a chemiluminescent derivative. After a wash step to remove unbound material, a catalyst is added and a chemical reaction occurs, producing emissions of light if the test sample is positive. The number of relative light units (RLUs) is proportional to the amount of treponemal antibody in the sample. CLIAs have many advantages as compared with EIAs, including a higher sensitivity in the early stages of syphilis, faster performance, and more stable reagents. MFIs involve incubation of the patient sample with microspheres coated with recombinant T. pallidum antigens. Microspheres that have bound immune complexes are detected after addition of a phycoerythrin-labeled reporter antibody and are analyzed by flow cytometry. This method can simultaneously detect antibodies to multiple T. pallidum antigens in a small volume of sample and has a rapid turnaround time. Treponemal MFI, EIA, and CLIA yield comparable results to the FTA-ABS but have the advantage of automation. Molecular Testing by Polymerase Chain Reaction (PCR) PCR technology, which involves isolating and amplifying a specific sequence of DNA, has been used to test for the presence of treponemes in whole blood, spinal fluid, amniotic fluid, various tissues, and swab samples from syphilis lesions. Although there are many variations of this procedure, basically DNA is extracted from the sample and then replicated using a DNA polymerase enzyme and a primer pair to start the reaction (see Chapter 12). One variation is (real-time) quantitative PCR (qPCR), which is automated, faster, and more sensitive than traditional PCR. PCR is an extremely sensitive technique capable of detecting as little as one treponeme in some clinical samples. Sensitivity is highest in patients with primary syphilis but is greatly reduced in detecting disease in secondary syphilis. The clinical availability of PCR is currently limited; however, in the future, PCR could be a useful tool for diagnosis when serological testing is inconclusive and may provide a viable alternative to dark-field microscopy in directly detecting the organism in ulcers from patients with primary disease. PCR may also be helpful in detecting treponemes in the blood of neonates with symptoms of congenital syphilis and in the cerebrospinal fluid (CSF) of patients suspected of having neurosyphilis. Better standardization of PCR may help the method to gain more widespread use in testing for syphilis in the future. Clinical Applications Nontreponemal tests are sensitive, inexpensive, and simple to perform. Thus, they have been very useful as a screening tool for syphilis. In addition, because antibody titers can be determined by testing serial dilutions of the patient sample, these methods have also been useful in monitoring the progress of the disease and in determining the outcome of treatment. As noted previously, nontreponemal titers will decrease if treatment is effective. Their main disadvantage is that they are subject to false positives. Transient false positives occur in diseases such as hepatitis, infectious mononucleosis, varicella, herpes, measles, malaria, and tuberculosis, as well as during pregnancy. Chronic conditions causing sustained false-positive results include SLE, leprosy, intravenous drug use, autoimmune arthritis, advanced age, and advanced malignancy. A reactive nontreponemal test should be confirmed by a more specific treponemal test. In pregnancy, this is especially important because nontreponemal titers from a previous syphilis infection may increase nonspecifically. Titers can be considered to be nonspecifically increased if lesions are absent, the increase in titer is less than four-fold, and documentation of previous treatment is available. Although treponemal tests are usually reactive before nontreponemal tests in primary syphilis, they suffer from a lack of sensitivity in congenital syphilis and neurosyphilis. Nontreponemal tests should be used for these purposes. Treponemal tests have been traditionally used as confirmatory tests to distinguish false-positive from true-positive nontreponemal results. They also help establish a diagnosis in late latent syphilis or late syphilis because they are more sensitive than nontreponemal tests in these stages. Testing Algorithms The traditional testing algorithm for syphilis involves screening the sample with a nontreponemal test and confirming any positive results with a more specific treponemal test (Fig. 21–7). This testing strategy was used in most clinical laboratories for many years. Because of the development of sensitive, automated methods for treponemal antibodies that can be easily performed in the clinical laboratory, a change in the testing strategy for syphilis has become increasingly popular, especially among large reference laboratories. Under this “reverse sequence algorithm,” the testing order is reversed from the traditional algorithm, in that patient samples are screened by an automated treponemal immunoassay, and positive results are confirmed by a nontreponemal test. The reverse algorithm has several advantages over the traditional algorithm. The first advantage is cost. Automated testing can be performed on LIS-interfaced high-throughput analyzers as opposed to labor-intensive manual methods, saving time and reducing errors. Secondly, this algorithm can potentially detect more early, late, and treated syphilis cases because of the higher sensitivity of the specific treponemal tests. In the traditional algorithm, these cases may be missed because testing stops with a negative nontreponemal test result and the treponemal test is not run. In the reverse algorithm, if the initial assay is negative, no further testing is done unless early syphilis is suspected (i.e., before seroconversion). If the automated assay result is positive and the subsequent RPR is positive, the results are considered positive for syphilis. However, discrepant results can be obtained in some cases, with the initial automated test result being positive and the RPR that follows giving a negative result. This combination of results can be problematic because it could be caused by a false-positive treponemal antibody test result, a past syphilis infection, or early primary syphilis, in which patients have not yet produced nontreponemal antibodies. To help distinguish between these possibilities, the CDC recommends that if laboratories choose to use the reverse algorithm, all discrepant results should be tested reflexively using the TP- PA test as a secondary confirmatory treponemal test. If the TP-PA is positive, then late or latent syphilis or previous history of syphilis would be considered. If the TP-PA is negative, the patient would be considered negative for syphilis at the time of testing. Careful evaluation of the patient’s history should be considered, and the patient may be reevaluated for syphilis at a later date (Fig. 21–8). Testing for Congenital Syphilis Nontreponemal tests for congenital syphilis performed on cord blood or neonatal serum detect the IgG class of antibody in addition to IgM. It is difficult to differentiate passively transferred IgG maternal antibodies from those produced by the neonate, so there are problems in establishing a definitive diagnosis. Late maternal infection may result in a nonreactive test because of low levels of fetal antibody. Additionally, testing the infant’s spinal fluid for the presence of treponemes often lacks sensitivity. Nontreponemal titers in the infant that are higher than those in the mother may be a good indicator of congenital disease, but this does not always occur. Several approaches have focused on detecting IgM antibodies in the infant. An FTA-ABS test for IgM alone lacks sensitivity, and the test is subject to interference because of the presence of rheumatoid factor. However, an IgM capture assay is more sensitive, and a Western blot assay (see Chapter 24 for details) using four major treponemal antigens has demonstrated high sensitivity and specificity. FIGURE 21-7 Traditional testing algorithm for syphilis. EIA = enzyme immunoassay; CIA = chemiluminescence immunoassay; TP-PA = Treponema pallidum particle agglutination. FIGURE 21-8 The algorithm for reverse sequence syphilis screening involves treponemal test screening followed by nontreponemal test confirmation. The CDC recommends that discrepant results be followed by performance of the TP-PA test. (Adapted from Centers for Disease Control and Prevention. Discordant results from reverse sequence syphilis screening: five laboratories, United States, 2006–2010. MMWR. 20U;60:133–137). Currently, it is recommended that in high-risk populations, nontreponemal tests be performed on both the mother and infant at birth, regardless of previously negative maternal tests. Because symptoms are not always present at birth, if congenital syphilis is suspected because of maternal history, tests should be repeated on infant serum within a few weeks. If infection is present in the infant, the titer will remain the same or will increase. The Western blot test is recommended to confirm congenital syphilis. Clinical Applications Testing of Cerebrospinal Fluid CSF is typically tested to determine whether treponemes have invaded the CNS. Such testing is usually more reliable if CNS symptoms are present. The VDRL test and some of the newer enzyme-linked immunosorbent assay (ELISA) tests are the only ones routinely used for the testing of spinal fluid. For VDRL spinal fluid testing, the antigen volume used is less than the serum test and is at a different concentration. In addition, different slides are used (Boerner agglutination slides). The test is read microscopically as in the VDRL serum test. If a test is reactive, two-fold dilutions are made and retested following the same protocol to determine the titer. A positive VDRL test on spinal fluid is diagnostic of neurosyphilis because false positives are extremely rare. However, sensitivity is lacking because samples from fewer than 70% of patients with active neurosyphilis give positive results. If a negative test is obtained, other indicators such as increased lymphocyte count and elevated total protein (45 mg/dL) are used as signs of active disease. PCR has been advocated in diagnosing neurosyphilis and may play an important role in CSF testing in the future. Treatment Penicillin has been the treatment of choice for syphilis since its availability in the late 1940s. Infection can be easily cured by administering a single dose of intramuscular long-acting benzathine penicillin G. Doxycycline and tetracycline are other alternatives for patients who are allergic to penicillin and are not pregnant. Lyme Disease Lyme disease was first described in the United States in 1975 when an unusually large number of cases of juvenile arthritis appeared in a geographically clustered rural area around Old Lyme, Connecticut (hence the disease name), in the summer and fall. Two mothers recognized this and brought it to the attention of health officials. Because of the epidemiological features of this newly described “Lyme arthritis,” transmission by an arthropod vector was suggested. In 1982, the agent was isolated and identified as a new spirochete. It was given the name Borrelia burgdorferi after Willy Burgdorfer (first author in the original description). The clinical features of Lyme disease were soon recognized to extend beyond arthritis; it is now known to be a multisystem illness involving the skin, nervous system, heart, and joints. Lyme disease is the most common vector-borne disease in the United States; more than 42,743 confirmed and probable cases were reported in 2017, almost 9% more than in 2016. According to the CDC, the number of cases in high-incidence states has remained stable but has increased in neighboring states with previously low incidence. Characteristics of Borrelia Species Several species of Borrelia are known to be the causative agents of Lyme disease. In North America, it is exclusively B. burgdorferi sensu stricto, whereas in Europe, several species are known to cause Lyme disease (Borrelia afzelii, Borrelia garinii, Borrelia sensu stricto, and occasionally other Borrelia species). All share similar characteristics; for simplicity, they will be referred to as B. burgdorferi. The organism is a loosely coiled spirochete, 5 to 25 µm long and 0.2 to 0.5 µm in diameter. The outer membrane, which consists of glycolipid and protein, is extremely fluid and only loosely associated with the organism. Several important lipoprotein antigens, outer surface proteins (OSPs) OSP-A through OSP-F, are located within this structure and are actually encoded by plasmids. Surface proteins allow the spirochetes to attach to mammalian cells. Just underneath the outer envelope are 7 to 11 endoflagella or periplasmic flagella. These run parallel to the long axis of the organism and are made up of 41-kDa subunits that elicit a strong antibody response. This immunodominant characteristic is of diagnostic importance because the response is not only strong but is also very early. Unfortunately, the flagellin subunit has homology to that of other nonpathogenic and pathogenic spirochetes, notably B. recurrentis and T. pallidum, causing cross-reactivity in serological testing. Because of this, a large number of uninfected people have low levels of antibodies to the 41-kDa protein. Although this is usually not a problem in the current diagnostic scheme of testing, it can become an issue when these individuals become ill with certain viruses that are known polyclonal B-cell activators (such as Epstein- Barr virus). In these cases, this normally low-level antibody becomes high enough to cause biological false positivity.  The organism divides by binary fission approximately every 12 hours. It can be cultured in the laboratory in a complex liquid medium (Barbour- Stoenner-Kelly) at 33°C, but it is difficult to isolate from patients. The spirochetemia (presence of spirochetes in the blood) is short-lived and is generally only found early in the illness. Cultures often must be incubated for 6 weeks or longer to detect growth and are therefore of little diagnostic utility. Mode of Transmission The main reservoir host is the white-footed mouse (Peromyscus leucopus), although in California and Oregon the spirochete is also harbored by the dusky-footed woodrat. Several types of Ixodes ticks serve as vectors of transmission: Ixodes scapularis in the Northeast and Midwest United States (Fig. 21–9), Ixodes pacificus in the West, Ixodes ricinus in Europe, and Ixodes persulcatus in Asia. White-tailed deer are the main host for the tick’s adult stage. Nymphs and adult stages of the tick can transmit the disease. The peak feeding is in the late spring, early summer, and fall, which corresponds to the peak biphasic occurrence of Lyme disease. The tick must feed for a period of time before the spirochete can be transmitted, typically more than 36 hours; one study found that transmission is still low at 72 hours. Stages of the Disease Lyme disease resembles syphilis in that manifestations occur in several stages. These have been characterized as (1) localized rash, (2) early dissemination to multiple organ systems, and (3) a late disseminated stage that often includes arthritic symptoms. These stages are not always sharply delineated; therefore, it may be easier to view Lyme disease as a progressive infectious disease that involves diverse organ systems. FIGURE 21-9 Adult tick I. scapularis, which transmits Lyme disease. (Courtesy of the CDC/Michael L. Levin, PhD, and Jim Gathany, Public Health Image Library.) Localized Rash Stage The clinical hallmark of early infection is the rash known as erythema migrans (EM), which appears between 2 days and 2 weeks after a tick bite. EM begins as a small red papule where the bite occurred, then rapidly expands to form a large ring-like erythema and often a central area that exhibits partial clearing (Fig. 21–10). The clinical diagnosis of early Lyme disease relies on the recognition of this characteristic rash, which should be at least 5 cm in diameter. At this stage, the patient may be asymptomatic or have nonspecific flu-like symptoms. The EM usually continues to expand for more than a week; even if untreated, it gradually fades within 3 to 4 weeks. Unfortunately, approximately 20% of patients do not develop the rash. At this early stage, the antibody response is minimal, 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. If treatment is not obtained, neurological or cardiac involvement develops in about 15% of patients within 4 to 6 weeks after the onset of infection. The most prevalent neurological sign is facial palsy, a peripheral neuritis that usually involves one side of the face. Pain and weakness can occur in the limb that was bitten. Some patients develop sleep disturbances, mild chronic confusion, or difficulty with memory and intellectual functioning. An aseptic meningitis can also be seen. Late Dissemination Stage Late Lyme disease may develop in some untreated patients months to years after acquiring the infection. The major manifestations of late Lyme disease are arthritis, peripheral neuropathy, and encephalomyelitis. These symptoms usually respond well to conventional antibiotic treatment, but treatment-resistant arthritis has been associated with particular HLA–DRB alleles. Despite resolution of objective manifestations of Borrelia infection after antibiotic treatment, a small percentage of patients develop chronic fatigue, concentration and short-term memory problems, and musculoskeletal pain. These symptoms can last longer than 6 months in some cases. FIGURE 21-10 Erythema chronicum migrans rash, which appears after a tick bite in Lyme disease. (Courtesy of the CDC/Jim Gathany, Public Health Image Library.) Nature of the Immune Response The immune response in Lyme disease is highly variable and complex. A well-documented humoral and cellular response is known to exist. Spirochete lipoproteins also trigger the production of macrophage-derived cytokines, which further enhance the immune response. However, the clinical effectiveness of these responses is questionable and not necessarily protective because late Lyme disease occurs despite high levels of circulating antibody and cellular responses. Laboratory Diagnosis The diagnosis of Lyme disease is a clinical one, with laboratory testing used as supporting evidence. Unfortunately, the clinical diagnosis is often difficult. 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 from skin scrapings, spinal fluid, or blood is possible, but the yield of positive cultures is extremely low. Therefore, 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. The CDC recommends a two-tiered approach to providing laboratory support for the diagnosis of Lyme disease (Fig. 21–11). The standard approach has been in use since 1995. 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.  The limitations of the standard algorithm have led to the development of alternative testing strategies, referred to as a modified two-tiered algorithm (see Fig. 21–11). 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. Some of the current testing procedures are discussed next. 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. Basically, 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. FIGURE 21-11 Two-tiered testing for Lyme disease (Adapted from Centers for Disease Control and Prevention [https://www.cdc.gov/lyme/healthcare/clinician_twotier.html] and Branda JA, Strle K, Nigrovic LE, Lantos PM, Lepore TJ, et al. Evaluation of modified 2- tiered serodiagnostic testing algorithms for early Lyme disease. Clin Infec Dis. 2017;64:1074–1080.) 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 (Fig. 21–12). 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. They are designated by their molecular weights: 18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kDa. 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. FIGURE 21-12 Immunoblot for Lyme disease. 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. The specificity of PCR ranges from 93% to 100%. However, sensitivity remains problematic. In a series of studies, the median sensitivity of PCR on skin biopsies was 69%; of blood components, 14%; of CSF, 38%; and of synovial fluid, 78%. However, the range of sensitivities in any one type of specimen is quite large, suggesting that testing remains to be standardized. Furthermore, it would be hard to clinically justify a skin biopsy for PCR as a diagnostic method for an EM rash in most cases. However, 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. 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. Leptospirosis was a nationally reportable disease up until 1995, at which time it was dropped from the list of notifiable diseases. However, it was reinstated as a reportable infection in 2013. 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. Characteristics of Leptospira Species 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 (Fig. 21–13). The genus Leptospira was originally divided into two main species, namely, pathogenic saprophytes, L. interrogans, and environmental saprophytes, L. biflexa. However, more characteristic distinction can be achieved by serotyping. 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- McCullough-Johnson-Harris (EMJH) semisolid medium, or Stuart liquid medium. FIGURE 21-13 Scanning electron microscope (SEM) image shows many corkscrew-shaped Leptospira spirochetes atop a 0.1-µm polycarbonate filter. (Courtesy of the CDC/Rob Weyant and Janice H. Carr, Public Health Image Library.) 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. 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 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. 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. SUMMARY Syphilis and Lyme disease are two major diseases caused by spirochetes. Spirochetes are distinguished by the presence of axial filaments that wrap around the cell wall inside a sheath and give the organisms their characteristic motility. Syphilis is caused by the organism Treponema pallidum, subspecies pallidum. The disease is acquired by direct contact, usually through sexual transmission. Syphilis can be separated into four main clinical stages: The primary stage is characterized by the presence of a painless ulcer called a chancre at the site of initial contact. An untreated patient may progress from the primary stage to the secondary stage, in which systemic dissemination of the organism occurs and symptoms such as generalized lymphadenopathy, malaise, sore throat, and skin rash appear.  Disappearance of the secondary stage is followed by a lengthy latent stage in which patients are usually free of clinical symptoms. About one-third of the individuals who remain untreated develop tertiary syphilis. This late-stage disease is characterized by three major clinical manifestations: granulomatous inflammation (gummas), cardiovascular disease, and neurosyphilis. Early diagnosis and treatment help to prevent later complications. Direct laboratory diagnosis of syphilis involves detecting the organism from a lesion and using dark-field microscopy, fluorescence microscopy, or PCR. If an active lesion is not present, diagnosis must be made on the basis of serological tests. Nontreponemal serological tests (i.e., the VDRL and RPR) determine the presence of antibody to cardiolipin, also known as reagin. These tests are fairly sensitive and simple to perform; however, they lack specificity, so specimens with positive results must be confirmed with a more specific treponemal antibody test. Traditional treponemal antibody tests include the FTA-ABS and particle agglutination (TP-PA). These tests detect antibody formed against T. pallidum itself. Treponemal tests are more specific and sensitive in early stages of the disease. Titers of treponemal antibodies remain detectable for life, whereas nontreponemal titers decline after successful treatment. More recent developments in testing include EIA and CLIA technology and PCR. EIA and CLIA tests for antibody to specific treponemal antigens, and separation of antibodies by class is possible. For large- volume testing, an EIA or CLIA is commonly used as a screening test, followed by confirmation with an RPR test. This testing sequence is known as the reverse screening algorithm. Lyme disease is the most common vector-borne infection in the United States. The organism responsible is the spirochete Borrelia burgdorferi, which is transmitted by the deer tick. Although an expanding red rash is often the first symptom noted in Lyme disease, the disease can be characterized as a progressive infectious syndrome involving diverse organ systems. Despite antibiotic treatment, a small percentage of patients continue to have fatigue; concentration and short-term memory problems; and  musculoskeletal pain, which may last 6 months or longer. These symptoms may be caused by persistence of the infection. The presence of IgM and IgG to B. burgdorferi cannot usually be detected until 3 to 6 weeks after symptoms initially appear. A two-tier testing protocol is used that involves screening with an IFA or EIA and follow-up of equivocal or positive tests with immunoblotting or a different EIA. All serological findings must be interpreted carefully and in conjunction with clinical findings. Leptospirosis is a zoonosis associated with exposure to water contaminated with animal urine. It is caused by spirochetes of the genus Leptospira and has its highest incidence in tropical and subtropical regions of the world. Symptoms of severe Leptospira infection include fever, headache, myalgia, conjunctivitis, jaundice, and combined liver and renal failure, a condition usually referred to as Weil’s disease. The microscopic agglutination test (MAT) is the goldstandard confirmatory method in the laboratory diagnosis of leptospirosis. Study Guide: Comparison of Tests Used for the Diagnosis of Syphilis TEST ANTIGEN ANTIBODY COMMENTS Direct Microscopic Dark-field T. pallidum from None Requires active lesion; patient must have good specimen, experienced technologist; inexpensive Fluorescent T. pallidum from Anti-treponemal Requires active lesion; antibody patient antibody with more specific than dark- fluorescent tag field; specimen does not have to contain live organisms Nontreponemal VDRL Cardiolipin Anti-cardiolipin Microscopic flocculation; (Reagin) may be used for screening, treatment monitoring, spinal fluid testing; false positives are common RPR Cardiolipin Anti-cardiolipin Macroscopic flocculation (Reagin) with charcoal particles; more sensitive than VDRL in primary syphilis; false positives are common Treponemal FTA-ABS Nichols strain of T. Anti-treponemal Confirmatory; specific, pallidum sensitive; may be negative in primary stage Serodia TP-PA Gel particles Anti-treponemal Not as sensitive as FTA- (formerly, sensitized with T. ABS MHA-TP) pallidum sonicate (formerly sensitized sheep RBCs) EIA, CLIA, MFI Treponemal antigen Anti-treponemal Sensitive, automated testing provides objective results; used to screen for syphilis in many large laboratories; EIAs have been  developed as competitive, sandwich, or capture immunoassays that can detect IgM or IgG antibodies PCR T. pallidum DNA in None Highest sensitivity is in patient sample is primary-stage syphilis; amplified availability is limited CLIA = chemiluminescent immunoassay; EIA = enzyme immunoassay; FTA-ABS = fluorescent treponemal antibody absorption; MFI = multiplex flow immunoassay; MHA-TP = microhemagglutination assay for antibodies to Treponema pallidum; PCR = polymerase chain reaction; RPR = rapid plasma reagin; TP-PA = T. pallidum particle agglutination; VDRL = Venereal Disease Research Laboratory. Study Guide: Comparison of Tests for the Diagnosis of Lyme Disease TEST ANTIGEN ANTIBODY COMMENTS IFA Whole or processed Anti-Borrelia antibody Initial test for Lyme B. burgdorferi from patient, anti- disease; labor intensive human globulin to perform; false with fluorescent tag positives; subjective EIA Sonicated B. Anti-Borrelia antibody Initial test for Lyme burgdorferi from patient, anti- disease; easy to human globulin perform; false positives; with enzyme tag more sensitive than IFA Purified flagellin Anti-flagellin antibody Initial test for Lyme protein from patient, anti- disease; easy to human globulin perform; highly specific; with enzyme tag sensitive in early Lyme disease C6 peptide Conserved region of Easy to perform; highly surface lipoprotein specific; sensitive in (VlsE) early and late Lyme disease; may be used as a confirmatory test for Lyme disease Western blot or Antigens of B. Detects antibodies Technically difficult to immunoblot burgdorferi (IgG or IgM) to perform; scoring the blot separated by individual B. can be challenging; molecular weight burgdorferi used as a confirmatory antigens test for Lyme disease PCR None. B. burgdorferi None Available in reference DNA in patient laboratories sample is amplified EIA = enzyme immunoassay; IFA = immunofluorescence assay; PCR = polymerase chain reaction. CASE STUDIES 1. A 30-year-old woman saw her physician to complain about repeated episodes of arthritis-like pain in the knees and hip joints. She recalled having seen a very small tick on her arm about 6 months before the development of symptoms. However, no rash was ever seen. Laboratory tests for rheumatoid arthritis and SLE were negative. An