Chapter 24: Spirochetes and Other Spiral Microorganisms

The spirochetes are a large, heterogeneous group of spiral, motile bacteria. One family (Spirochaetaceae) of the order Spirochaetales consists of two genera whose members are human pathogens, Borrelia and Treponema. The other family (Leptospiraceae) includes one genus of medical importance: Leptospira.

The spirochetes have many structural characteristics in common, as typified by Treponema pallidum (Figure 24-1). They are long, slender, helically coiled, spiral, or corkscrew-shaped bacilli. T pallidum has an outer sheath or glycosaminoglycan coating. Inside the sheath is the outer membrane, which contains peptidoglycan and maintains the structural integrity of the organisms. Endoflagella (axial filaments) are the flagella-like organelles in the periplasmic space encased by the outer membrane. The endoflagella begin at each end of the organism and wind around it, extending to and overlapping at the midpoint. Inside the endoflagella is the inner membrane (cytoplasmic membrane) that provides osmotic stability and covers the protoplasmic cylinder. A series of cytoplasmic tubules (body fibrils) are inside the cell near the inner membrane. Treponemes reproduce by transverse fission.

Morphology and Identification

A. Typical Organisms

T pallidum are slender spirals measuring about 0.2 μm in width and 5–15 μm in length. The spiral coils are regularly spaced at a distance of 1 μm from one another. The organisms are actively motile, rotating steadily around their endoflagella even after attaching to cells by their tapered ends. The long axis of the spiral is ordinarily straight but may sometimes bend so that the organism forms a complete circle for moments at a time, returning then to its normal straight position.

The spirals are so thin that they are not readily seen unless immunofluorescent stain or dark-field illumination is used. They do not stain well with aniline dyes, but they can be seen in tissues when stained by a silver impregnation method.

B. Culture

Pathogenic T pallidum has never been cultured continuously on artificial media, in fertile eggs, or in tissue culture.

In proper suspending fluids and in the presence of reducing substances, T pallidum may remain motile for 3–6 days at 25°C. In whole blood or plasma stored at 4°C, organisms remain viable for at least 24 hours, which is of potential importance in blood transfusions.

D. Reactions to Physical and Chemical Agents

Drying kills the spirochete rapidly, as does elevation of the temperature to 42°C. Treponemes are rapidly immobilized and killed by trivalent arsenical, mercury, and bismuth (contained in drugs of historical interest in the treatment of syphilis). Penicillin is treponemicidal in minute concentrations, but the rate of killing is slow, presumably because of the metabolic inactivity and slow multiplication rate of T pallidum (estimated division time is 30 hours). Resistance to penicillin has not been demonstrated in syphilis.

E. Genome

The T pallidum genome is a circular chromosome of approximately 1,138,000 base pairs, which is small for bacteria. Most pathogenic bacteria have transposable elements, but T pallidum does not, which suggests that the genome is highly conserved and may explain its continued susceptibility to penicillin. There are few genes involved in energy production and synthesis of nutrients, indicating that T pallidum obtains these from the host.

Antigenic Structure

The fact that T pallidum cannot be cultured in vitro has markedly limited the characterization of its antigens. The outer membrane surrounds the periplasmic space and the peptidoglycan–cytoplasmic membrane complex. Membrane proteins are present that contain covalently bound lipids at their amino terminals. The lipids appear to anchor the proteins to the cytoplasmic or outer membranes and keep the proteins inaccessible to antibodies. The endoflagella are in the periplasmic space. T pallidum has hyaluronidase that breaks down the hyaluronic acid in the ground substance of tissue and presumably enhances the invasiveness of the organism. The endoflagella are composed of three core proteins that are homologous to other bacterial flagellin proteins plus an unrelated sheath protein. Cardiolipin is an important component of the treponemal antigens.

Humans with syphilis develop antibodies capable of staining T pallidum by indirect immunofluorescence, immobilizing and killing live motile T pallidum and fixing complement in the presence of a suspension of T pallidum or related spirochetes. The spirochetes also cause the development of a distinct antibody-like substance, reagin, which gives positive complement fixation (CF) and flocculation test results with aqueous suspensions of cardiolipin extracted from normal mammalian tissues. Both reagin and antitreponemal antibody can be used for the serologic diagnosis of syphilis.

Pathogenesis, Pathology, and Clinical Findings

A. Acquired Syphilis

Natural infection with T pallidum is limited to the human host. Human infection is usually transmitted by sexual contact, and the infectious lesion is on the skin or mucous membranes of genitalia. In 10–20% of cases, however, the primary lesion is intrarectal, perianal, or oral. It may be anywhere on the body. T pallidum can probably penetrate intact mucous membranes, or the organisms may enter through a break in the epidermis. Based on experiments in rabbits, as few as four to eight spirochetes may cause infection.

Spirochetes multiply locally at the site of entry, and some spread to nearby lymph nodes and then reach the bloodstream. Within 2–10 weeks after infection, a papule develops at the site of infection and breaks down to form an ulcer with a clean, hard base (“hard chancre”). The inflammation is characterized by a predominance of lymphocytes and plasma cells. This “primary lesion” always heals spontaneously, but 2–10 weeks later, the “secondary” lesions appear. These consist of a red maculopapular rash anywhere on the body, including the hands and feet, and moist, pale papules (condylomas) in the anogenital region, axillae, and mouth. The patient may also have syphilitic meningitis, chorioretinitis, hepatitis, nephritis (immune complex type), or periostitis. The secondary lesions also subside spontaneously. Both primary and secondary lesions are rich in spirochetes and are highly infectious. Contagious lesions may recur within 3–5 years after infection, but thereafter the individual is not infectious. Syphilitic infection may remain subclinical, and the patient may pass through the primary or secondary stage (or both) without symptoms or signs yet develop tertiary lesions.

In about 30% of cases, early syphilitic infection progresses spontaneously to complete cure without treatment. In another 30%, the untreated infection remains latent (principally evident by positive serologic test results). In the remainder, the disease progresses to the “tertiary stage” characterized by the development of granulomatous lesions (gummas) in the skin, bones, and liver; degenerative changes in the central nervous system (meningovascular syphilis, paresis, tabes); or cardiovascular lesions (aortitis, aortic aneurysm, aortic valve insufficiency). In all tertiary lesions, treponemes are very rare, and the exaggerated tissue response must be attributed to hypersensitivity to the organisms. However, treponemes can occasionally be found in the eye or central nervous system in late syphilis.

B. Congenital Syphilis

A pregnant woman with syphilis can transmit T pallidum to the fetus through the placenta beginning in the 10th–15th weeks of gestation. Some of the infected fetuses die, and miscarriages result; others are stillborn at term. Others are born live but develop the signs of congenital syphilis in childhood, including interstitial keratitis, Hutchinson’s teeth, saddlenose, periostitis, and a variety of central nervous system anomalies. Adequate treatment of the mother during pregnancy prevents congenital syphilis. The reagin titer in the blood of the child rises with active infection but falls with time if antibody was passively transmitted from the mother. In congenital infection, the child makes IgM antitreponemal antibody.

Diagnostic Laboratory Tests

A. Specimens

Specimens include tissue fluid expressed from early surface lesions for demonstration of spirochetes by either dark-field microscopy or immunofluorescence; such specimens can also be tested by nucleic acid amplification. Blood can be obtained for serologic tests; cerebrospinal fluid (CSF) is useful for Venereal Disease Research Laboratory (VDRL) testing (see later discussion).

B. Dark-Field Examination

A drop of tissue fluid or exudate is placed on a slide, and a coverslip is pressed over it to make a thin layer. The preparation is then examined under oil immersion within 20 minutes of collection with dark-field illumination for typical motile spirochetes. Dark-field microscopy should not be performed on lesions within the oral cavity because it is not possible to differentiate pathogenic from commensal spirochetes.

Treponemes disappear from lesions within a few hours after the beginning of antibiotic treatment.

C. Immunofluorescence

Tissue fluid or exudate is spread on a glass slide, air dried, and sent to the laboratory. It is fixed, stained with a fluorescein-labeled antitreponeme antibody, and examined by means of immunofluorescence microscopy for typical fluorescent spirochetes.

D. Serologic Tests for Syphilis

These tests use either nontreponemal or treponemal antigens.

1. Nontreponemal tests—The nontreponemal tests are universally used as screening tests for syphilis. The tests are widely available, lend themselves to automation with ease of performance in large numbers, and have a low cost. In addition to their function as screening tests, they can be used to follow the efficacy of therapy. The drawbacks to the nontreponemal tests are that they are not very sensitive in early syphilis, and the results may not turn positive until a few weeks after initial infection; false-positive results can occur with many other diseases; and there may be a prozone phenomenon, particularly in secondary syphilis (antibody excess produces a negative result at low serum dilutions but positive results at higher dilutions). The antigens in these tests contain measured amounts of cardiolipin, cholesterol, and purified lecithin in quantities sufficient to yield a standardized amount of reactivity. Historically, the cardiolipin was extracted from beef heart or liver with added lecithin and cholesterol to enhance reaction with syphilitic “reagin” antibodies. Reagin is a mixture of IgM and IgG antibodies reactive with the cardiolipin–cholesterol–lecithin complex. All of the tests are based on the fact that the particles of the lipid antigen remain dispersed in normal serum but flocculate when combining with reagin. The VDRL and unheated serum reagin (USR) tests require microscopic examination to detect flocculation. The rapid plasma reagin (RPR) test and toluidine red unheated serum test (TRUST) have colored particles that become caught in the mesh of the antigen–antibody complex, allowing the tests to be read without microscopic magnification. Results develop within a few minutes, particularly if the suspension is agitated.

The nontreponemal tests can give quantitative results using serial twofold dilutions. An estimate of the amount of reagin present in serum can be expressed as the titer or as the highest dilution giving a positive result. Quantitative results are valuable in establishing a diagnosis and in evaluating the effect of treatment. Positive nontreponemal test results develop after 2–3 weeks of untreated syphilis and are positive in high titer in secondary syphilis. Positive nontreponemal test results typically revert to negative, often in 6–18 months and generally by 3 years after effective treatment of syphilis. A positive nontreponemal test result late after treatment for syphilis suggests ineffective treatment or reinfection.

The VDRL test is standardized for use on CSF, and the result becomes positive in patients with neurosyphilis. Reagin antibodies generally do not reach the CSF from the bloodstream but are probably formed in the central nervous system in response to syphilitic infection. The serologic diagnosis of neurosyphilis is complex.

2. Treponemal antibody tests—The treponemal tests measure antibodies against T pallidum antigens. The tests are used to determine if a positive result from a nontreponemal test is truly positive or falsely positive. A positive result of a treponemal test on a serum specimen that is also positive on a nontreponemal test is a strong indication of T pallidum infection. The traditional treponemal tests are less useful as screening tests because once positive after initial syphilitic infection the tests remain positive for life independent of therapy for syphilis. Serial dilutions of serum are not done in the treponemal tests, and results are reported as reactive or nonreactive (or occasionally inconclusive). The treponemal antibody tests tend to be more costly than the nontreponemal test, which is important when large groups of people (eg, blood donors) are being screened.

The T pallidum–particle agglutination (TP-PA) test is perhaps the most widely used treponemal test in the United States. Gelatin particles sensitized with T pallidum subspecies pallidum antigens are added to a standard dilution of serum. When anti-T pallidum antibodies (IgG, IgM, or both) react with the sensitized particles, a mat of agglutinated particles forms in the well of the microdilution tray. Gelatin particles that are not sensitized are tested with diluted serum to exclude nonspecific agglutination.

The T pallidum hemagglutination (TPHA) and the microhemagglutination T pallidum (MHA-TP) are based on the same principles as the TP-PA but use sheep erythrocytes rather than gelatin particles and may be more prone to nonspecific agglutination.

The fluorescent treponemal antibody absorbed (FTA-ABS) test is the treponemal antibody test used for many years. Because it is difficult to perform, the test is used only in selected circumstances. The test uses indirect immunofluorescence to detect reactive antibodies, including killed T pallidum and the patient’s serum absorbed with sonicated saprophytic Reiter spirochetes plus antihuman γ-globulin labeled with a fluorescent compound. The presence of IgM FTA in the blood of newborns is a good evidence of in utero infection (ie, congenital syphilis). A negative FTA-ABS result on CSF tends to exclude neurosyphilis, but a positive FTA-ABS result on CSF can occur by transfer of antibodies from serum and is not helpful in the diagnosis of neurosyphilis.

Multiple relatively similar treponemal antibody tests using enzyme immunoassay (EIA) or chemiluminescence (CIA) formats for T pallidum are available. These tests use antigens obtained by sonication of T pallidum or recombinant antigens. An aliquot of serum at a standard dilution is added to a sensitized well of a microdilution plate. After washing, addition of an enzyme-labeled conjugate, and further washing, a precursor substrate is added. A color change or CIA indicates a reactive serum. Because some of these assays are available as high-throughput automated tests, many laboratories have now reversed the traditional algorithm for screening. Instead of screening with the nontreponemal test and verifying with a treponemal assay, the high throughput allows screening with a more sensitive treponemal test. The advantage to this approach is that patients with early disease or untreated latent disease are more likely to be detected (see earlier discussion).

There are some concerns about variability in assay performance among these tests that result in more false positives when testing low-prevalence populations. Because of this, the Centers for Disease Control and Prevention (CDC) has recommended an algorithm for confirming a positive EIA or CIA test result with a quantitative RPR or other nontreponemal test. If the RPR result is positive, a current or past infection with syphilis is likely. If the RPR result is negative, then additional testing with a traditional treponemal test such as the TP-PA is recommended. If the TP-PA result is positive, syphilis is likely; if it is negative, syphilis is unlikely.

Immunity

A person with active or latent syphilis appears to be resistant to superinfection with T pallidum. However, if early syphilis is treated adequately and the infection is eradicated, the individual again becomes fully susceptible. The various immune responses usually fail to eradicate the infection or arrest its progression.

Treatment

Penicillin in concentrations of 0.003 U/mL has definite treponemicidal activity, and penicillin is the treatment of choice. Syphilis of less than 1 year’s duration is treated by a single injection of benzathine penicillin G 2.4 million units intramuscularly. In older or latent syphilis, benzathine penicillin G intramuscularly is given three times at weekly intervals. In neurosyphilis, the same therapy is acceptable, but larger amounts of intravenous penicillin are sometimes recommended. Other antibiotics (eg, tetracyclines or erythromycin) can occasionally be substituted. Treatment of gonorrhea is thought to cure incubating syphilis. Prolonged follow-up is essential. In neurosyphilis, treponemes occasionally survive such treatment. Severe neurologic relapses of treated syphilis have occurred in patients with AIDS who are infected with both HIV and T pallidum. A typical Jarisch-Herxheimer reaction may occur within hours after treatment is begun. It is caused by the release of toxic products from dying or killed spirochetes.

Epidemiology, Prevention, and Control

With the exceptions of congenital syphilis and the rare occupational exposure of medical personnel, syphilis is acquired through sexual exposure. Reinfection in treated persons is common. An infected person may remain contagious for 3–5 years during “early” syphilis. “Late” syphilis, of more than 5 years’ duration, is usually not contagious. Consequently, control measures depend on (1) prompt and adequate treatment of all discovered cases, (2) follow-up on sources of infection and contacts so they can be treated, and (3) safe sex with condoms. Several sexually transmitted diseases can be transmitted simultaneously. Therefore, it is important to consider the possibility of syphilis when any one sexually transmitted disease has been found.

Concept Checks

• T pallidum cannot be cultivated on artificial media; therefore, they are detected directly in tissues or exudates using dark-field microscopy, immunofluorescence, or molecular tests.

• Infections with T pallidum are limited to humans and are acquired by direct sexual contact and less commonly transplacentally (congenital disease) or through occupational exposure.

• The primary lesion at the site of inoculation is the painless “hard chancre,” a type of genital ulcer.

• Untreated primary infection can lead to secondary disease with spread of the spirochetes systemically; latent disease is characterized by the absence of symptoms but with a positive serologic test result. The tertiary stage involves serious disease with central nervous system or cardiac manifestations.

• In addition to direct detection in clinical specimens, most often the diagnosis is made by serologic testing. The traditional testing algorithm involves screening using a nontreponemal test followed by confirmation with a treponemal test such as the TP-PA.

• The availability of high-throughput EIA or CIA tests has resulted in the reversal of this sequence of testing with many laboratories offering screening with one of these treponemal tests followed by confirmation with the nontreponemal test. Concern for false-positive test results has prompted the CDC to recommend an algorithm that confirms a negative nontreponemal test result with one of the traditional treponemal tests.

• Penicillin is the drug of choice for treatment of all stages of syphilis.

BORRELIA SPECIES AND RELAPSING FEVER

Relapsing fever in epidemic form is caused by Borrelia recurrentis, which is transmitted by the human body louse; it does not occur in the United States. Endemic relapsing fever is caused by borreliae transmitted by ticks of the genus Ornithodoros. The species name of the Borrelia genus is often the same as that of the tick. Borrelia hermsii, the cause of relapsing fever in the western United States, is transmitted by Ornithodoros hermsii.

Morphology and Identification

A. Typical Organisms

The borreliae form irregular spirals 10–30 μm long and 0.3 μm wide. The distance between turns varies from 2 to 4 μm. The organisms are highly flexible and move both by rotation and by twisting. Borreliae stain readily with bacteriologic dyes as well as with blood stains such as Giemsa stain or Wright stain (Figure 24-2).

B. Culture

The organism can be cultured in fluid media containing blood, serum, or tissue, but it rapidly loses its pathogenicity for animals when transferred repeatedly in vitro. Multiplication is rapid in chick embryos when blood from patients is inoculated onto the chorioallantoic membrane.

C. Growth Characteristics

Little is known of the metabolic requirements or activity of borreliae. At 4°C, the organisms survive for several months in infected blood or in culture. In some ticks (but not in lice), spirochetes are passed from generation to generation.

D. Variation

The only significant variation of Borrelia species is with respect to its antigenic structure.

Antigenic Structure

Antibodies develop in high titer after infection with borreliae. The antigenic structure of the organisms changes in the course of a single infection. The antibodies produced initially act as a selective factor that permits the survival only of antigenically distinct variants. The relapsing course of the disease appears to be caused by the multiplication of such antigenic variants, against which the host must then develop new antibodies. Ultimate recovery (after 3–10 relapses) is associated with the presence of antibodies against several antigenic variants.

Pathology

Fatal cases show spirochetes in great numbers in the spleen and liver, necrotic foci in other parenchymatous organs, and hemorrhagic lesions in the kidneys and the gastrointestinal tract. Spirochetes have occasionally been demonstrated in the spinal fluid and brain of persons who have had meningitis. In experimental animals (guinea pigs, rats), the brain may serve as a reservoir of borreliae after they have disappeared from the blood.

Pathogenesis and Clinical Findings

The incubation period is 3–10 days. The onset is sudden, with chills and an abrupt rise of temperature. During this time, spirochetes abound in the blood. The fever persists for 3–5 days and then declines, leaving the patient weak but not ill. The afebrile period lasts 4–10 days and is followed by a second attack of chills, fever, intense headache, and malaise. There are 3–10 such recurrences, generally of diminishing severity. During the febrile stages (especially when the temperature is rising), organisms are present in the blood; during the afebrile periods, they are absent.

Antibodies against the spirochetes appear during the febrile stage, and the attack is probably terminated by their agglutinating and lytic effects. These antibodies may select out antigenically distinct variants that multiply and cause a relapse. Several distinct antigenic varieties of borreliae may be isolated from a single patient’s sequential relapses even after experimental inoculation with a single organism.

Diagnostic Laboratory Tests

A. Specimens

Blood specimens are obtained during the rise in fever for smears and animal inoculation.

B. Smears

Thin or thick blood smears stained with Wright or Giemsa stain reveal large, loosely coiled spirochetes among the red cells.

C. Animal Inoculation

White mice or young rats are inoculated intraperitoneally with blood. Stained films of tail blood are examined for spirochetes 2–4 days later.

D. Serology

Spirochetes grown in culture can serve as antigens for CF tests, but the preparation of satisfactory antigens is difficult. Patients with epidemic (louse-borne) relapsing fever may develop a positive VDRL test result.

Immunity

Immunity following infection is usually of short duration.

Treatment

The great variability of the spontaneous remissions of relapsing fever makes evaluation of chemotherapeutic effectiveness difficult. Tetracyclines, erythromycin, and penicillin are all believed to be effective. Treatment for a single day may be sufficient to terminate an individual attack.

Epidemiology, Prevention, and Control

Relapsing fever is endemic in many parts of the world. Its main reservoir is the rodent population, which serves as a source of infection for ticks of the genus Ornithodoros. The distribution of endemic foci and the seasonal incidence of the disease are largely determined by the ecology of the ticks in different areas. In the United States, infected ticks are found throughout the West, especially in mountainous areas, but clinical cases are rare. In the tick, Borrelia species may be transmitted transovarially from generation to generation.

Spirochetes are present in all tissues of the tick and may be transmitted by the bite or by crushing the tick. The tickborne disease is not epidemic. However, when an infected individual harbors lice, the lice become infected by sucking blood; 4–5 days later, they may serve as a source of infection for other individuals. The infection of the lice is not transmitted to the next generation, and the disease is the result of rubbing crushed lice into bite wounds. Severe epidemics may occur in louse-infected populations, and transmission is favored by crowding, malnutrition, and cold climate.

In endemic areas, human infection may occasionally result from contact with the blood and tissues of infected rodents. The mortality rate of the endemic disease is low, but in epidemics, it may reach 30%.

Prevention is based on avoidance of exposure to ticks and lice and on delousing (cleanliness, insecticides).

BORRELIA BURGDORFERI AND LYME DISEASE

Lyme disease is named after the town of Lyme, Connecticut, where clusters of cases in children were identified. Since 1992, three species of Borrelia have been associated with Lyme disease, Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. All three species cause disease in Europe, but only B burgdorferi is responsible for disease in North America. The spirochete B burgdorferi is transmitted to humans by the bite of a small Ixodes tick. The disease has early manifestations with a characteristic skin lesion, erythema migrans, along with flulike symptoms, and late manifestations often with arthralgia and arthritis.

Morphology and Identification

A. Typical Organisms

B burgdorferi is a spiral organism 20–30 μm long and 0.2–0.3 μm wide. The distance between turns varies from 2 to 4 μm. The organisms have variable numbers (7–11) of endoflagella and are highly motile. B burgdorferi stains readily with acid and aniline dyes and by silver impregnation techniques.

B. Culture and Growth Characteristics

B burgdorferi grows most readily in a complex liquid medium, Barbour-Stoenner-Kelly medium (BSK II). Rifampin, fosfomycin (phosphonomycin), and amphotericin B can be added to BSK II to reduce the rate of culture contamination by other bacteria and fungi. B burgdorferi has been most easily isolated from erythema migrans skin lesions; isolation of the organism from other sites has been difficult. The organism can also be cultured from ticks. Because culture of the organism is a complex and specialized procedure with a low diagnostic yield, it is seldom used.

Antigenic Structure and Variation

B burgdorferi has a morphologic appearance similar to that of other spirochetes. The entire genome of B burgdorferi has been sequenced, allowing prediction of many antigenic structures. There is an unusual linear chromosome of about 950 kb and multiple circular and linear plasmids. There are a large number of sequences for lipoproteins, including outer surface proteins OspA to F. Differential expression of these proteins is thought to help B burgdorferi live in the very different tick and mammalian hosts. OspA and OspB along with lipoprotein 6.6 are expressed primarily in ticks. Other outer surface proteins are upregulated during tick feeding when the organisms migrate from the tick’s midgut to the salivary gland. This may explain why the tick must feed for 24–48 hours before transmission of B burgdorferi occurs.

Pathogenesis and Clinical Findings

The transmission of B burgdorferi to humans is by injection of the organism in tick saliva or by regurgitation of the tick’s midgut contents. The organism adheres to proteoglycans on host cells; this is mediated by a borrelial glycosaminoglycan receptor. After injection by the tick, the organism migrates out from the site, producing the characteristic skin lesion. Dissemination occurs by lymphatics or blood to other skin and musculoskeletal sites and to many other organs.

Lyme disease, similar to other spirochetal diseases, occurs in stages with early and late manifestations. A unique skin lesion that begins 3 days to 4 weeks after a tick bite often marks stage 1. The lesion, erythema migrans, begins as a flat reddened area near the tick bite and slowly expands, with central clearing. With the skin lesion, there is often a flulike illness with fever, chills, myalgia, and headache. Stage 2 occurs weeks to months later and includes arthralgia and arthritis; neurologic manifestations with meningitis, facial nerve palsy, and painful radiculopathy; and cardiac disease with conduction defects and myopericarditis. Stage 3 begins months to years later with chronic skin, nervous system, or joint involvement. Spirochetes have been isolated from all of these sites, and it is likely that some of the late manifestations are caused by deposition of antigen–antibody complexes.

Diagnostic Laboratory Tests

In some symptomatic patients, the diagnosis of early Lyme disease can be established clinically by observing the unique skin lesion. When this skin lesion is not present and at later stages of the disease, which must be differentiated from many other diseases, it is necessary to perform diagnostic laboratory tests. There is, however, no one test that is both sensitive and specific.

A. Specimens

Blood is obtained for serologic tests. CSF or joint fluid can be obtained, but culture usually is not recommended. These specimens and others can be used to detect B burgdorferi DNA by PCR.

B. Smears

B burgdorferi has been found in sections of biopsy specimens, but examination of stained smears is an insensitive method for diagnosis of Lyme disease. B burgdorferi in tissue sections can sometimes be identified using antibodies and immunohistochemical methods.

C. Culture

Culture is generally not performed because it takes 6–8 weeks to complete and lacks sensitivity.

D. Nucleic Acid Amplification Methods

The PCR assay has been applied to detection of B burgdorferi DNA in many body fluids. It is rapid, sensitive, and specific, but it does not differentiate between DNA from live B burgdorferi in active disease and DNA from dead B burgdorferi in treated or inactive disease. It has about 85% sensitivity when applied to synovial fluid samples, but the sensitivity is much lower when it is applied to CSF samples from patients with neuroborreliosis.

E. Serology

Serology has been the mainstay for the diagnosis of Lyme disease, but 3–5% of normal people and persons with other diseases (eg, rheumatoid arthritis, many infectious diseases) may be seropositive by initial EIA or indirect fluorescent antibody (IFA) assay. When the prevalence of Lyme disease is low as it is in many geographic areas, there is a much greater likelihood that a positive test result is from a person who does not have Lyme disease than from a person who does have the disease (a positive predictive value of <10%). Thus, serology for Lyme disease should only be done when there are highly suggestive clinical findings. A diagnosis of Lyme disease should not be based on a positive EIA or IFA test result in the absence of suggestive clinical findings. A two-stage approach to the serodiagnosis is strongly recommended that includes EIA or IFA followed by an immunoblot assay for reactivity with specific B burgdorferi antigens.

The EIA and IFA are the most widely used initial tests for Lyme disease. Multiple variations of these assays using different antigen preparations, techniques, and end points have been marketed. Results of the initial tests are generally reported as positive, negative, or indeterminate.

The immunoblot assay is generally performed to confirm results obtained by the EIA tests. Recombinant B burgdorferi antigens or antigens from whole-cell lysates are electrophoretically separated, transferred to a nitrocellulose membrane, and reacted with a patient’s serum. Interpretation of the immunoblot is based on the number and molecular size of antibody reactions with the B burgdorferi proteins. Blots can be analyzed for IgG or IgM. The antigen–antibody band patterns on the immunoblots should be interpreted with knowledge of known results from patients at various stages of Lyme borreliosis, and caution should be used to avoid overinterpretation of minimally reactive blots.

Immunity

The immunologic response to B burgdorferi develops slowly. Sera obtained in stage 1 are positive in 20–50% of patients. Sera obtained during stage 2 are positive in 70–90% with reactive IgG and IgM; IgG predominates in longstanding infection. In stage 3, nearly 100% of patients have IgG reactive with B burgdorferi. The antibody response can expand from months to years and appears to be directed sequentially against a series of B burgdorferi proteins. Early antimicrobial treatment decreases the antibody response. Antibody titers fall slowly after treatment, but most patients with later manifestation of Lyme disease remain seropositive for years.

Treatment

Early infection, either local or disseminated, should be treated with doxycycline, amoxicillin, or cefuroxime axetil for 14–21 days. Treatment relieves early symptoms and promotes resolution of skin lesions. Doxycycline may be more effective than amoxicillin in preventing late manifestations. Established arthritis may respond to prolonged therapy with doxycycline or amoxicillin orally or penicillin G or ceftriaxone intravenously. In refractory cases, ceftriaxone has been effective. Nearly 50% of patients treated with doxycycline or amoxicillin early in the course of Lyme disease develop minor late complications (eg, headache, joint pains).

Epidemiology, Prevention, and Control

B burgdorferi is transmitted by a small tick of the genus Ixodes. The vector is Ixodes scapularis (also called Ixodes dammini) in the Northeast and Midwest and Ixodes pacificus on the West Coast of the United States. In Europe, the vector is Ixodes ricinus, and other tick vectors appear to be important in other areas of the world. The Ixodes ticks are quite small and often are not noticed when feeding on the skin. The larvae are about 1 mm; the nymphs about the size of a poppy seed or piece of cracked pepper (~2 mm), and the adult female 3–4 mm. All stages are smaller by one-half or more than comparable stages of the dog tick Dermacentor variabilis. Depending on the developmental stage and the Ixodes species, the ticks must feed for 2–4 days to obtain a blood meal. Transmission of B burgdorferi occurs late in the feeding process. Mice and deer constitute the main animal reservoirs of B burgdorferi, but other rodents and birds may also be infected. In the eastern part of the United States, 10–50% of ticks are infected; in the western states, the infection rate in ticks is much lower, about 2%.

Most exposures are in May through July, when the nymphal stage of the ticks is most active; however, the larval stage (August and September) and adult stage (spring and fall) also feed on humans and can transmit B burgdorferi.

Prevention is based on avoidance of exposure to ticks. Wearing long sleeves and long pants tucked into socks is recommended. Careful examination of the skin for ticks after being outdoors can locate ticks for removal before they transmit B burgdorferi.

Environmental control of ticks using application of insecticides has provided modest success in reducing the number of nymphal ticks for a season.

Concept Checks

• A variety of Borrelia species cause disease, usually after the bite of an arthropod or other vector.

• B recurrentis, transmitted by the human body louse, causes epidemic relapsing fever; endemic disease is usually transmitted by ticks of the genus Ornithodoros. B hermsii is the cause of relapsing fever in the western United States.

• Relapsing fever is characterized by abrupt rise in temperature that persists for 3–5 days. After a brief afebrile hiatus, a second attack occurs, usually related to antigenic variants.

• Diagnosis of relapsing fever is best done by obtaining thick and thin blood smears and staining them with Wright or Giemsa stain.

• Treatment requires penicillin, tetracycline, or erythromycin.

• B burgdorferi is responsible for Lyme disease and is most often transmitted by the nymphal stage of the Ixodes tick.

• Lyme disease occurs in stages. The hallmark of stage 1 is erythema migrans at the site of the tick bite. Stages 2 and 3 are characterized by arthritis and cardiac and neurologic manifestations.

• Diagnosis rests on a two-stage serologic approach beginning with an EIA or IFA test followed by an immunoblot assay for reactivity to specific antigens if the screening test result is positive.

• Treatment depends on the stage of the disease; penicillin, doxycycline, cefuroxime, and parenteral ceftriaxone have all been effective.

Leptospirosis is a zoonosis of worldwide distribution. It is caused by spirochetes of the genus Leptospira. There is one pathogenic species, Leptospira interrogans, but more than 200 serovars of L interrogans. These serovars are further organized into over two dozen serogroups. The serogroups are based on shared antigenicity and are primarily for laboratory use.

Morphology and Identification

A. Typical Organisms

Leptospirae are tightly coiled, thin, flexible spirochetes 5–15 μm long, with very fine spirals 0.1–0.2 μm wide; one end is often bent, forming a hook. They are actively motile, which is best seen using a dark-field microscope. Electron micrographs show a thin axial filament and a delicate membrane. The spirochete is so delicate that in the dark-field view, it may appear only as a chain of minute cocci. It does not stain readily but can be impregnated with silver.

B. Culture

Leptospires grow best under aerobic conditions at 28–30°C in semisolid medium (eg, Ellinghausen-McCullough-Johnson-Harris, EMJH) in 10 mL test tubes with 0.1% agar and 5-fluorouracil (see also Diagnostic Laboratory Tests). After 1–2 weeks, the leptospires produce a diffuse zone of growth near the top of the tube and later a ring of growth at a level in the tube corresponding to the level of the optimal oxygen tension for the organisms.

C. Growth Requirements

Leptospirae derive energy from oxidation of long-chain fatty acids and cannot use amino acids or carbohydrates as major energy sources. Ammonium salts are a main source of nitrogen. Leptospirae can survive for weeks in water, particularly at alkaline pH.

Antigenic Structure

The main strains (“serovars”) of L interrogans are all serologically related and exhibit cross-reactivity in serologic tests. This indicates considerable overlapping in antigenic structure, and quantitative tests and antibody absorption studies are necessary for a specific serologic diagnosis. The outer envelope contains large amounts of lipopolysaccharide of antigenic structure that is variable from one strain to another. This variation forms the basis for the serologic classification of the Leptospira species. It also determines the specificity of the human immune response to leptospirae.

Pathogenesis and Clinical Findings

Human infection usually results from leptospires, often in bodies of water, entering the body through breaks in the skin (cuts and abrasions) and mucous membranes (mouth, nose, conjunctivae). Ingestion is considered to be less important. After an incubation period of 1–2 weeks, there is a variable febrile onset during which spirochetes are present in the bloodstream. They then establish themselves in the parenchymatous organs (particularly liver and kidneys), producing hemorrhage and necrosis of tissue and resulting in dysfunction of those organs (jaundice, hemorrhage, nitrogen retention). The illness is often biphasic. After initial improvement, the second phase develops when the IgM antibody titer rises. It manifests itself often as “aseptic meningitis” with an intense headache, stiff neck, and pleocytosis of the CSF. Nephritis and hepatitis may also recur, and there may be skin, muscle, and eye lesions. The degree and distribution of organ involvement vary in the different diseases produced by different leptospirae in various parts of the world. Many infections are mild or subclinical. Hepatitis is frequent in patients with leptospirosis.

Kidney involvement in many animal species is chronic and results in the shedding of large numbers of leptospirae in the urine; this is probably the main source of environmental contamination resulting in infection of humans. Human urine also may contain spirochetes in the second and third weeks of disease.

Agglutinating, complement-fixing, and lytic antibodies develop during the infection. Serum from convalescent patients protects experimental animals against an otherwise fatal infection. The immunity resulting from infection in humans and animals appears to be serovar specific.

Diagnostic Laboratory Tests

A. Specimens

Specimens consist of aseptically collected blood in a heparin tube, CSF, or tissues for microscopic examination and culture. Urine should be collected using great care to avoid contamination. Serum is collected for agglutination tests.

B. Microscopic Examination

Dark-field examination or thick smears stained by the Giemsa technique occasionally show leptospirae in fresh blood from early infections. Results of dark-field examination of centrifuged urine may also be positive. Fluorescein-conjugated antibodies or other immunohistochemical techniques can be used also.

C. Culture

Whole fresh blood or urine can be cultured in a semisolid medium. Because of inhibitory substances in blood, only one or two drops should be placed in each of five tubes containing 5 or 10 mL of medium. Up to 0.5 mL of CSF can be used. One drop of undiluted urine can be used followed by one drop each of 10-fold serially diluted urine for a total of four tubes. Tissue approximately 5 mm in diameter should be crushed and used as the inoculum. Growth is slow, and cultures should be kept for at least 8 weeks.

D. Serology

The diagnosis of leptospirosis in most cases is confirmed serologically. Agglutinating antibodies first appear 5–7 days after infection and develop slowly, reaching a peak at 5–8 weeks. Very high titers may be attained (>1:10,000). The reference laboratory standard for detection of leptospiral antibody uses microscopic agglutination of live organisms, which can be hazardous. The test is highly sensitive, but it is difficult to standardize; the end point is 50% agglutination, which is difficult to determine. Agglutination of the live suspensions is most specific for the serovar of the infecting leptospires. Agglutination tests are generally performed only in reference laboratories. Paired sera that show a significant change in titer or a single serum with high-titer agglutinins plus a compatible clinical illness can be diagnostic. Because of the difficulty in performing the definitive agglutination tests, a variety of other tests have been developed for use primarily as screening tests.

Immunity

Serovar-specific immunity follows infection, but reinfection with different serovars may occur.

Treatment

Treatment of mild leptospirosis should be with oral doxycycline, ampicillin, or amoxicillin. Treatment of moderate or severe disease should be with intravenous penicillin or ampicillin.

Epidemiology, Prevention, and Control

The leptospiroses are essentially animal infections; human infection is only accidental, occurring after contact with water or other materials contaminated with the excreta of animal hosts. Rats, mice, wild rodents, dogs, swine, and cattle are the principal sources of human infection. They excrete leptospirae in urine both during the active illness and during the asymptomatic carrier state. Leptospirae remain viable in stagnant water for several weeks; drinking, swimming, bathing, or food contamination may lead to human infection. Persons most likely to come in contact with water contaminated by rats (eg, miners, sewer workers, farmers, and fishermen) run the greatest risk of infection. Children acquire the infection from dogs more frequently than adults do. Control consists of preventing exposure to potentially contaminated water and reducing contamination by rodent control. Doxycycline, 200 mg orally once weekly during heavy exposure, is effective prophylaxis. Dogs can receive distemper–hepatitis–leptospirosis vaccinations.