Obligate Intracellular and Nonculturable Bacterial Agents



Obligate Intracellular and Nonculturable Bacterial Agents



Objectives



1. Define the following: bubo, proctitis, bartholinitis, salpingitis, elementary body, reticulate body, Whipple’s disease, morulae, and Donovan body.


2. Describe the general characteristics for the organisms included in this chapter including gram stain characteristics, cultivation methods (media and growth conditions), transmission and clinical significance.


3. Explain the mechanism and location for the replication of Chlamydia spp.


4. Compare the clinical manifestations and diagnosis of trachoma and other oculogenital infections associated with Chlamydia spp.


5. List the appropriate specimens used for the isolation of the organisms included in this chapter.


6. Describe the correct collection method for a specimen to be submitted for C. trachomatis screening from the female genital tract.


7. Explain the three stages associated with lymphogranuloma venereum, and compare the disease with other genital infections.


8. Describe the laboratory methods used for the diagnosis of Chlamydia infections, including sensitivity, limitations and appropriate use for culture, cytology, antigen (DFA), and nucleic acid testing (NAAT).


9. Compare hybridization and amplification nucleic acid–based testing for chlamydia.


10. Describe the triad of symptoms associated with Rickettsia spp.


11. Compare human monocytic ehrlichiosis (HME) and granulocytic anaplasmosis (HGA).


12. Distinguish and describe the three groups of Rickettsia based on mode of transmission, clinical manifestations, and intracellular growth characteristics.


13. Describe the Weil-Felix reaction, including chemical principle and limitations.


14. Describe the clinical significance for Coxiella burnetii phase I and phase II forms, including laboratory diagnosis.


15. Explain the limitations of the laboratory tests used to diagnose disease caused by the obligate intracellular and nonculturable bacteria.


16. Correlate signs, symptoms, and laboratory data for the identification of the organisms included in this chapter.



Genera and Species to Be Considered
















































Current Name Previous Name
Chlamydia trachomatis  
Chlamydia psittaci Chlamydophila psittaci
Chlamydia pneumoniae Chlamydophila pneumoniae
Rickettsia rickettsii  
Rickettsia prowazekii  
Rickettsia typhi  
Orientia tsutsugamushi  
Ehrlichia chaffeensis  
Anaplasma phagocytophilum Ehrlichia phagocytophila, Ehrlichia equi, and human granulocytic ehrlichiosis agent
Neorickettsia sennetsu Ehrlichia sennetsu
Coxiella burnetii  
Tropheryma whipplei T. whippelii
Klebsiella granulomatis Calymmatobacterium granulomatis


The organisms addressed in this chapter are obligate intracellular bacteria or are considered either extremely difficult to culture or unable to be cultured. Organisms of the genera Chlamydia, Rickettsia, Orientia, Anaplasma, and Ehrlichia are prokaryotes that differ from most other bacteria with respect to their very small size and obligate intracellular parasitism. Three other organisms, Coxiella, Calymmatobacterium granulomatis, and Tropheryma whipplei, are discussed in this chapter because they are also difficult to cultivate or are noncultivable.



Chlamydia


The Chlamydia spp. are members of the order Chlamydiales and the family Chlamydiaceae. The members of the family Chlamydiaceae had been regrouped in 1999 from one genus, Chlamydia, into two genera, Chlamydia and Chlamydophila, based on differences in phenotype, 16S rRNA, and 23S rRNA. This nomenclature change was controversial, however, and additional research led to the rejection of Chlamydophila as a separate genus in the family, thereby returning all species to the genus Chlamydia.


Members of the order Chlamydiales are obligate intracellular bacteria that were once regarded as viruses because, like viruses, the chlamydiae require the biochemical resources of the eukaryotic host cell to fuel their metabolism for growth and replication by providing high-energy compounds such as adenosine triphosphate. Chlamydia spp. are similar to the gram-negative bacilli in that they have lipopolysaccharide (LPS) as a component of the cell wall. The chlamydial LPS, however, has little endotoxic activity. The chlamydiae have a major outer membrane protein (MOMP) that is very diverse. The variation in MOMP in C. trachomatis is used to separate the species into 18 distinct serovars, yet highly conserved in C. pneumoniae.


Chlamydiae have a unique developmental life cycle reminiscent of parasites, with an intracellular, replicative form, the reticulate body (RB), and an extracellular, metabolically inert, infective form, the elementary body (EB). The EB cannot survive outside of a host cell for an extended period. Following infection of a host cell, the EB differentiates into a RB. The RB divides by binary fission within vacuoles. As the numbers of RB increase, the vacuole expands forming an intracytoplasmic inclusion. The RB then revert to EB, and 48 to 72 hours postinfection, the EB are released from the host cell (Figure 44-1). In addition to the replicative cycle associated with acute chlamydial infections, there is evidence that Chlamydia can persist in an aberrant form in vitro depending on the amount of interferon-gamma (IFN-γ) and tryptophan in the host cell as well as the function of the tryptophan synthase encoded by the organism. Removal of the IFN-γ or increase in tryptophan will result in the differentiation of the chlamydiae into an active EB infection. The therapeutic implications of this persistence in vivo has not yet been completely defined; however, evidence suggests that the activity of the tryptophan synthase gene in C. trachomatis differs between isolates recovered from the eye versus the genital tract.


image
Figure 44-1 The life cycle of chlamydiae. The entire cycle takes approximately 48 to 72 hours.

C. trachomatis, C. pneumoniae, and C. psittaci are important causes of human infection; C. psittaci and C. pecorum are common pathogens among animals. The three species that infect humans differ with respect to their antigens, host cell preference, antibiotic susceptibility, EB morphology, and inclusion morphology (Table 44-1).




Chlamydia Trachomatis


Over the past few decades, the importance of both acute and chronic infections caused by Chlamydia trachomatis has been recognized. Not only are C. trachomatis infections associated with infertility and ectopic pregnancy, but oftentimes C. trachomatis infections are asymptomatic, resulting in inadvertent transmission and high prevalence rates.



General Characteristics


C. trachomatis infects humans almost exclusively and is responsible for various clinical syndromes. Based on MOMP antigenic differences, C. trachomatis is divided into 18 different serovars that are associated with different primary clinical syndromes (Table 44-2).




Epidemiology and Pathogenesis


C. trachomatis causes significant infection and disease worldwide. In the United States, C. trachomatis is the most common sexually transmitted bacterial pathogen and a major cause of pelvic inflammatory disease (PID), ectopic pregnancy, and infertility (see Chapter 74 for more information on PID). An estimated 3 million cases of C. trachomatis infection occur annually in the United States. In 2010, more than 1.3 million cases of C. trachomatis infection were reported to the Centers for Disease Control and Prevention (CDC), corresponding to a rate of infection of 426 per 100,000 population and a 5.1% increase over the cases reported in 2009. In fact, of all the organisms causing sexually transmitted disease reported to the CDC, only C. trachomatis cases have increased every year. Genital tract infections caused by C. trachomatis were identified most frequently in women between the ages of 15 and 24 years. It is important to note, however, that data reported to the CDC, especially with regards to Chlamydia, come as a result of screening programs that primarily target women between the ages of 15 and 24 years.


Ocular trachoma, on the other hand, is a much more prevalent disease, affecting 84 million individuals worldwide, with 7 to 9 million infections resulting in blindness. Remote rural areas of Africa, Asia, Central and South America, Australia, and the Middle East are hyperendemic for trachoma, where the prevalence rate of C. trachomatis is 60% to 90% in preschool children. Trachoma is the cause for 3% of the cases of blindness in individuals around the world, with adult women more likely to be affected as a result of their exposure to children who serve as the major reservoir of the organism.


C. trachomatis infections are primarily transmitted from human to human by direct contact with infected secretions. Some infections, such as neonatal pneumonia or inclusion conjunctivitis, are transmitted from mother to infant during birth. The various routes of transmission for C. trachomatis infection are summarized in Table 44-2.


The natural habitat of C. trachomatis is humans. The mechanisms by which C. trachomatis cause inflammation and tissue destruction are not completely understood. The chlamydiae can infect a variety of different cells, including epithelial cells of the mucosa as well as blood vessels, smooth muscle cells, and monocytes. The chlamydial EB is phagocytosed into a host cell and resides in a vacuole that fails to fuse with a lysosome, leading to the intracellular persistence of the organism and escape from the host immune response. Chlamydiae are able to either turn on or turn off apoptosis (programmed cell death pathways) in infected host cells. By inducing host cell death, the organism facilitates its transmission to neighboring host cells and down-regulating inflammation in the acute disease process, whereas, by inhibiting apoptosis, the organism keeps the host cell alive, allowing for sustained survival in chronic infections.


The host’s immune response accounts for the majority of the tissue destruction following infection with C. trachomatis. Infected epithelial cells secrete pro-inflammatory cytokines including Interleukin-1α (IL-1α), tumor necrosis factor (TNF) and IL-6. Quickly upon infection, neutrophils and monocytes migrate to the mucosa and eliminate exposed EB. Later CD4 T helper cells migrate to the site of infection. Responding neutrophils and T helper cells release cytokines, resulting in the influx of additional immune cells. The importance of multiple, recurrent infection with C. trachomatis is associated with the development of ocular trachoma. Immunity provides little protection from reinfection and appears to be short lived following infection with C. trachomatis.



Spectrum of Disease


As previously mentioned, infection with different C. trachomatis serovars can lead to several clinical syndromes. These infections are summarized in Table 44-2.



Trachoma.

Trachoma is manifested by a chronic inflammation of the conjunctiva and remains a major cause of preventable blindness worldwide. The organism is acquired as a result of contact with infected secretions on towels or fingers or by flies. Early symptoms of infection include mild irritation and itching of the eyes and eyelids. There may also be some discharge from the infected eye. The infection progresses slowly with increasing eye pain, blurred vision, and photophobia. Repeated infections result in scarring of the inner eyelid that may then turn the eyelid in toward the eye (entropion). As the inner eyelid continues to turn in, the eyelashes follow (trichiasis), resulting in rubbing and scratching of the cornea. The combined effects of the mechanical damage to the cornea and inflammation result in ulceration, scarring, and loss of vision.



Lymphogranuloma Venereum.

Lymphogranuloma venereum (LGV) is a sexually transmitted disease rarely identified in North America but relatively frequent in Africa, Asia, and South America. It is reemerging in Europe, especially in homosexual males. C. trachomatis serovars L1, L2, L2b, and L3 are invasive causing LGV, in contrast to C. trachomatis serovars A-K, leaving the mucosa to spread to the regional lymph nodes. The disease is characterized by a brief appearance of a primary genital lesion at the initial infection site. This lesion is often small and may be unrecognized, especially by female patients. The second stage, acute lymphadenitis, often involves the inguinal lymph nodes, causing them to enlarge and become matted together, forming a large area of groin swelling, or bubo. During this stage, infection may become systemic and cause fever or may spread locally, causing granulomatous proctitis. In a few patients (more women than men), the disease progresses to a chronic third stage, causing the development of genital hyperplasia, rectal fistulas, rectal stricture, draining sinuses, and other manifestations.



Oculogenital Infections.

C. trachomatis can cause acute inclusion conjunctivitis in adults and newborns. The organism is acquired when contaminated genital secretions get into the eyes via fingers or during passage of the neonate through the birth canal. Autoinfection rarely occurs. The organism can also be acquired from swimming pools, poorly chlorinated hot tubs, or by sharing eye makeup. Inclusion conjunctivitis is associated with swollen eyes and a purulent discharge. In contrast to trachoma, inclusion conjunctivitis does not lead to blindness in adults (or newborns).


Genital tract infections caused by C. trachomatis have surpassed gonococcal (Neisseria gonorrhoeae) infections as a cause of sexually transmitted disease in the United States. Similar to gonococci, C. trachomatis causes urethritis, cervicitis, bartholinitis (Bartholin glands or greater vestibular glands), proctitis, salpingitis (infection of the fallopian tubes), epididymitis, and acute urethral syndrome in women. In the United States, 60% of cases of nongonococcal urethritis are caused by chlamydiae. Both chlamydiae and gonococci are major causes of PID, contributing significantly to the rising rate of infertility and ectopic pregnancies in young women. Following a single episode of PID, as many as 10% of women may become infertile because of tubal occlusion. The risk increases dramatically with each additional episode.


Many genital chlamydial infections in both sexes are asymptomatic or not easily recognized by clinical criteria; asymptomatic carriage in both men and women may persist, often for months. As many as 50% of men and 70% to 80% of women identified as having chlamydial genital tract infections have no symptoms. Of significance, these asymptomatic infected individuals serve as a large reservoir to sustain transmission of the organism within a community.


When symptomatic, patients with a genital chlamydial infection will have an unusual discharge and pain or a burning sensation, symptoms similar to those for gonorrhea.




Laboratory Diagnosis


C. trachomatis can be diagnosed by cytology, culture, direct detection of antigen or nucleic acid, and serologic testing.



Specimen Collection and Transport.

The organism can be recovered from or detected in infected cells of the urethra, cervix, conjunctiva, nasopharynx, rectum, and material aspirated from the fallopian tubes and epididymis. The endocervix is the preferred anatomic site to collect screening specimens from women. The specimen for C. trachomatis culture should be obtained following collection of all other specimens (e.g., those for Gram-stained smear, Neisseria gonorrhoeae culture, or Papanicolaou [Pap] smear). A large swab should first be used to remove all secretions from the cervix. The appropriate swab (for nonculture tests, use the swab supplied or specified by the manufacturer) or endocervical brush is inserted 1 to 2 cm into the endocervical canal, rotated against the wall for 10 to 30 seconds, withdrawn without touching any vaginal surfaces, and then placed in the appropriate transport medium or applied to a slide prepared for direct fluorescent antibody (DFA) testing.


Urethral specimens should not be collected until 2 hours after the patient has voided. A urogenital swab (or one provided or specified by the manufacturer) is gently inserted into the urethra (females, 1 to 2 cm; males, 2 to 4 cm), rotated at least once for 5 seconds, and then withdrawn. Again, swabs should be placed into the appropriate transport medium or onto a slide prepared for DFA testing. Screening of rectal or pharyngeal specimens for C. trachomatis by nucleic acid tests has proven useful in homosexual male patients. Urine specimens in appropriate transport media provided by manufacturers of nucleic acid testing methodologies are also available for both men and women. Because chlamydiae are relatively labile, viability can be maintained by keeping specimens cold and minimizing transport time to the laboratory. For successful culture, specimens should be submitted in a chlamydial transport medium such as 2SP (0.2 M sucrose-phosphate transport medium with antibiotics); a number of commercial transport media are available. Specimens should be refrigerated upon receipt, and if they cannot be processed for culture within 24 hours, they should be frozen at –70° C.



Cultivation.

Cultivation of C. trachomatis is discussed before methods for direct detection and serodiagnosis because all nonculture methods for the diagnosis of C. trachomatis are compared with culture. Culture is being performed less often, however, with nucleic acid amplification tests (NAAT) being used almost exclusively for genital tract infections. For example, in a survey taken in 2007 of public health laboratories, 89.7% of tests for Chlamydia were NAAT.


Several different cell lines have been used to isolate C. trachomatis in cell culture, including McCoy, HeLa, and monkey kidney cells; cycloheximide-treated McCoy cells are commonly used. After shaking the clinical specimens with 5-mm glass beads, centrifugation of the specimen onto the cell monolayer (usually growing on a coverslip in the bottom of a vial, commonly called a “shell vial”) presumably facilitates adherence of elementary bodies. After 48 to 72 hours of incubation, monolayers are stained with a fluorescein-labeled monoclonal antibody that is either species specific, targeting the MOMP of C. trachomatis, or genus specific, targeting the LPS. The monolayers are examined microscopically for inclusion. Use of iodine to detect inclusions is less specific and not recommended.


Although its specificity approaches 100%, the sensitivity of culture has been estimated at between 70% and 90% in experienced laboratories. Limitations of Chlamydia culture contributing to the lack of sensitivity include prerequisites to maintain viability of patient specimens by either rapid or frozen transport and to ensure the quality of the specimen submitted for testing (i.e., endocervical specimens devoid of mucus and containing endocervical epithelial or metaplastic cells or urethral epithelial cells). In addition, successful culture requires a sensitive cell culture system and a minimum of at least 2 days turnaround time between specimen receipt and the availability of results. Despite these limitations, culture is still recommended as the test of choice in some situations (Table 44-3). As of this writing, only chlamydia cultures should be used in situations with legal implications (e.g., sexual abuse) when the possibility of a false-positive test is unacceptable. Local and state requirements may vary.




Direct Detection Methods



Antigen Detection and Nucleic Acid Hybridization.

To circumvent the shortcomings of cell culture, antigen detection methods are commercially available.


Direct fluorescent antibody (DFA) staining methods use fluorescein-isothiocyanate conjugated monoclonal antibodies to either MOMP or LPS of C. trachomatis to detect elementary bodies in smears of clinical material (Figure 44-2). The sensitivity and specificity of DFA are similar to those of culture. Chlamydial antigen can also be detected by enzyme immunoassays (EIA). Numerous U.S. Food and Drug Administration (FDA)-approved kits are commercially available. These assays use polyclonal or monoclonal antibodies that detect chlamydial LPS. These tests are not species-specific for C. trachomatis and may cross-react with LPS of other bacterial species present in the vagina or urinary tract and thereby produce a false-positive result.


image
Figure 44-2 Appearance of fluorescein-conjugated, monoclonal antibody–stained elementary bodies in direct smear of urethral cell scraping from a patient with chlamydial urethritis. (Courtesy Syva Co, San Jose, California)

Nucleic acid hybridization tests for Chlamydia were first available for the clinical microbiology laboratory in the late 1980s. Two hybridization tests are currently available, Gen-Probe PACE 2C (Hologic-Gen-Probe, San Diego, California) and Digene Hybrid Capture II assay (Digene, Silver Spring, Maryland). The Gen-Probe PACE 2C assay uses a chemiluminescent-labeled DNA probe complementary to a sequence of ribosomal RNA (rRNA) in the chlamydial genome. If chlamydial rRNA is present in the sample, the labeled DNA probe will bind. In a unique hybridization protection assay, only bound label is detected by measuring chemiluminescence in a luminometer. The Digene Hybrid Capture II assay uses an RNA probe to detect chlamydial DNA in a sample. The DNA/RNA hybrids are captured using monoclonal antibodies imbedded on the side of the well that recognize the unique structure produced by the DNA/RNA hybrid. A second enzyme labeled anti-DNA/RNA hybrid antibody binds to captured hybrids, and enzyme activity is measured by chemiluminescence. Both assays are species specific for C. trachomatis.


Based on numerous studies, these nonculture tests are more reliable for the detection of infection in patients who are symptomatic and shedding large numbers of organisms than in those who are asymptomatic and most likely shedding fewer organisms. For the most part, these assays have sensitivities of greater than 70% and specificities of 97% to 99% in populations with a prevalence of C. trachomatis infection of 5% or more. In a low-prevalence population—that is, less than 5%—a significant proportion of positive tests will be falsely positive. Therefore, a positive result in a low-prevalence population should be handled with care, and a positive result should be verified. Positive results can be validated by the following methods:


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Aug 25, 2016 | Posted by in MICROBIOLOGY | Comments Off on Obligate Intracellular and Nonculturable Bacterial Agents

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