The Spirochetes



The Spirochetes



Objectives



1. Describe the bacterial agents discussed in this chapter in terms of morphology, taxonomy, and growth conditions.


2. Identify the four stages of syphilis (i.e., primary, secondary, latent, and tertiary) according to clinical symptoms, antibody production, transmission, and infectivity.


3. Explain congenital syphilis, including transmission and clinical manifestations.


4. Define reagin, cardiolipin, and biologic false positive.


5. Differentiate reagin and treponemal antibodies, including specificity and association with disease.


6. Identify the various serologic methods that utilize specific treponemal or nonspecific nontreponemal antigens.


7. Describe the basic principles for the RPR, VDRL, FTA-ABS, TP-PA, and MHA-TP assays.


8. Compare Borrelia spp. to the other spirochetes discussed in this chapter, including morphology and growth conditions.


9. Describe the pathogenesis for relapsing fever and Lyme disease, including the routes of transmission, vector, and disease presentation.


10. Explain the methodology and clinical significance for using a two-step diagnostic procedure for Borrelia spp. infections.


11. Describe the pathogenesis associated with leptospirosis, including the two major stages of the disease and the recommended clinical specimens.


12. Describe Brachyspira spp., including potential pathogenesis, appropriate specimen, transmission, and clinical significance.


13. Correlate patient signs and symptoms with laboratory data to identify the most likely etiologic agent.




This chapter addresses the bacteria that belong in the order Spirochaetales. Although there are five genera in this family—the Treponema, Borrelia, Brachyspira, Spirochaeta, and Leptospira—only four are important in clinical diagnostics.


The spirochetes are all long, slender, helically curved, gram-negative bacilli, with the unusual morphologic features of axial fibrils and an outer sheath. These fibrils, or axial filaments, are flagella-like organelles that wrap around the bacteria’s cell walls, are enclosed within the outer sheath, and facilitate motility of the organisms. The fibrils are attached within the cell wall by platelike structures, called insertion disks, located near the ends of the cells. The protoplasmic cylinder gyrates around the fibrils, causing bacterial movement to appear as a corkscrew-like winding. Differentiation of genera within the family Spirochaetaceae is based on the number of axial fibrils, the number of insertion disks present (Table 46-1), and biochemical and metabolic features. The spirochetes also fall into genera based loosely on their morphology (Figure 46-1): Treponema appear as slender with tight coils; Borrelia are somewhat thicker with fewer and looser coils; and Leptospira resemble Borrelia except for their hooked ends. Brachyspira are comma-shaped or helical, with tapered ends with four flagella at each end.





Treponema


General Characteristics


The major pathogens in the genus Treponema—T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, T. pallidum subsp. endemicum, and T. carateum—infect humans and have not been cultivated for more than one passage in vitro. Most species stain poorly with Gram staining or Giemsa’s methods and are best observed with the use of dark-field or phase-contrast microscopy. These organisms are considered to be microaerophilic.


Other treponemes such as T. vincentii, T. denticola, T. refringens, T. socranskii, and T. pectinovorum are normal inhabitants of the oral cavity or the human genital tract. These organisms are cultivable anaerobically on artificial media. Acute necrotizing ulcerative gingivitis, also known as Vincent’s disease, is a destructive lesion of the gums. Methylene blue–stained material from the lesions of patients with Vincent’s disease show certain morphologic types of bacteria. Observed morphologies include spirochetes and fusiforms; oral spirochetes, particularly an unusually large one, may be important in this disease, along with other anaerobes.



Epidemiology and Pathogenesis


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



TABLE 46-2


Epidemiology and Spectrum of Disease of the Treponemes Pathogenic for Humans
















































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


image


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


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



Spectrum of Disease


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


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



Laboratory Diagnosis


Specimen Collection


Samples collected from ulcers and lesions should not be contaminated with blood, microorganisms, or tissue debris. The site should be cleansed with sterile gauze moistened with saline. The sample should be placed on a clean glass slide and cover slipped. Polymerase chain reaction (PCR) samples should be collected on a sterile Dacron or cotton swab and placed in a cryotube containing nucleic acid transport medium or universal transport medium. Tissue or needle aspirates of lymph nodes should be placed in 10% buffered formalin at room temperature. To test for congenital syphilis, a small section of the umbilical cord is collected and fixed in formalin or refrigerated until processed. Serum is the specimen of choice for serology; however, whole blood or plasma may be used in some assays.



Direct Detection


Treponemes can be detected in material taken from skin lesions by dark-field examination or fluorescent antibody staining and microscopic examination. Material for microscopic examination is collected from suspicious lesions. The area around the lesion must first be cleansed with a sterile gauze pad moistened in saline. The surface of the ulcer is then abraded until some blood is expressed. After blotting the lesion until there is no further bleeding, the area is squeezed until serous fluid is expressed. The surface of a clean glass slide is touched to the exudate, allowed to air dry, and transported in a dust-free container for fluorescent antibody staining. A T. pallidum fluorescein-labeled antibody is commercially available for staining (Viro Stat, Portland, Maine). For dark-field examination, the expressed fluid is aspirated using a sterile pipette, dropped onto a clean glass slide, and cover slipped. The slide containing material for dark-field examination must be transported to the laboratory immediately. Because positive lesions may be teeming with viable spirochetes that are highly infectious, all supplies and patient specimens must be handled with extreme caution and carefully discarded as required for contaminated materials. Gloves should always be worn.


Material for dark-field examination is examined immediately under 400× high-dry magnification for the presence of motile spirochetes. Treponemes are long (8 to 10 µm, slightly larger than a red blood cell) and consist of 8 to 14 tightly coiled, even spirals (Figure 46-2). Once seen, characteristic forms should be verified by examination under oil immersion magnification (1000×). Although the darkfield examination depends greatly on technical expertise and the numbers of organisms in the lesion, it can be highly specific when performed on genital lesions.



Lesion exudates or tissue samples may be used for direct fluorescent antibody detection for T. pallidum (DFA-TP). DFA-TP visualizes specimens on slides with fluorescein isothiocyanate (FITC) labeled antibodies. Polyclonal and monoclonal antibodies may be used; however, the Food and Drug Administration (FDA) in the United States has not approved this test.




Serodiagnosis


Serologic tests for treponematosis measure the presence of two types of antibodies: treponemal and nontreponemal. Treponemal antibodies are produced against antigens of the organisms themselves, whereas nontreponemal antibodies, often referred to as reagin antibodies, are produced in infected patients against components of mammalian cells. Reaginic antibodies, although almost always produced in patients with syphilis, are also produced in patients with other infectious diseases such as leprosy, tuberculosis, chancroid, leptospirosis, malaria, rickettsial disease, trypanosomiasis, lymphogranuloma venereum (LGV), measles, chickenpox, hepatitis, and infectious mononucleosis; noninfectious conditions such as drug addiction; autoimmune disorders, including rheumatoid disease and systemic lupus erythematosus; and in conjunction with increasing age, pregnancy, and recent immunization.


The two most widely used nontreponemal serologic tests are the Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin (RPR) tests. Each of these tests is a flocculation (or agglutination) test, in which soluble antigen particles are coalesced to form larger particles that are visible as clumps when they are aggregated in the presence of antibody. The VDRL is used as a quantitative test and may be performed on serum or CSF in suspected cases of neurosyphilis. See Procedures 46-1 and 46-2 on the Evolve site for details and limitations for the VDRL and RPR.



Procedure 46-1   Rapid Plasma Reagin (RPR) Test











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




Principle


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




Method



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Aug 25, 2016 | Posted by in MICROBIOLOGY | Comments Off on The Spirochetes

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