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or endocervical brush is inserted 1 to 2 cm into the endocervical canal, rotated against the wall for 10 to 30 seconds, withdrawn without touching any vaginal surfaces, and then placed in the appropriate transport medium or applied to a slide pre pared for direct fluorescent antibody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 with drawn. 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 pre requisites 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 1). 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.

Table1. Use of Different Laboratory Tests to Diagnose  C. trachomatis Infections

Direct Detection Methods

 Cytologic Examination. Cytologic examination of cell scrapings from the conjunctiva of newborns or persons with ocular trachoma can be used to detect C. trachomatis inclusions, usually after Giemsa staining. Cytology has also been used to evaluate endocervical and urethral scrapings, including those obtained for Pap smears. However, this method is insensitive compared with culture or other methods discussed in the following sections.

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 1). 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.

Fig1. 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 anti bodies 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 anti body 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:

• Culture

• Performing a second nonculture test that identifies a C. trachomatis antigen or nucleic acid sequence that is different from that used in the screening test

 • Using a blocking antibody or competitive probe that verifies a positive test result by preventing attachment of a labeled antibody or probe used in the standard assay

Nucleic Acid Amplification Tests. FDA-approved nucleic acid amplification tests (NAATs) for the laboratory diagnosis of C. trachomatis infection use three different formats: polymerase chain reaction (PCR), strand displacement amplification (SDA), and transcription mediated amplification (TMA). The first two assay formats amplify DNA sequences present in the cryptic plasmid that is present in 7 to 10 copies in the chlamydial EB, whereas the last format amplifies 23S ribosomal RNA sequences. Studies clearly indicate that NAATs are more sensitive than culture and other non-nucleic acid amplification assays. Because of the increased sensitivity of detection, first-voided urine specimens from symptomatic and asymptomatic men and women are acceptable specimens to detect C. trachomatis, thereby affording a noninvasive means of chlamydia testing. NAATs are the preferred methodology for detecting C. trachomatis in most clinical situations because of increased sensitivity, ease of specimen collection, and the availability of automated high volume methods. Table 1 summarizes the possible uses of the different methodologies available for the detection of C. trachomatis; however, NAATs are used almost exclusively for the laboratory detection of C. trachomatis.

Serodiagnosis. Serologic testing has limited value for diagnosis of urogenital infections in adults. Most adults with chlamydial infection have had a previous exposure to C. trachomatis and are therefore seropositive. Serology can be used to diagnose LGV. Antibodies to a genus-specific antigen can be detected by complement fixation (CF), and a single-point titer greater than 1 : 64 is indicative of LGV. This test is not useful in diagnosing trachoma, inclusion conjunctivitis, or neonatal infections. The microimmunofluorescence assay (micro-IF), a tedious and difficult test, is used for type-specific antibodies of C. trachomatis and can also be used to diagnose LGV. A high titer of IgM (1 : 32) suggests a recent infection; however, not all patients produce IgM. In contrast to CF, micro-IF may be used to diagnose trachoma and inclusion conjunctivitis using acute and convalescent phase sera. Detection of C. trachomatis–specific IgM is useful in the diagnosis of neonatal infections. Negative serology can reliably exclude chlamydial infection.

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مواضيع ذات صلة

Chlamydia psittaci

Spectrum of Disease of Chlamydia trachomatis

Epidemiology and Pathogenesis of Chlamydia trachomatis

Antigenic Structure, Pathogenesis, and Pathology of Bordetella pertussis

Haemophilus influenzae

Campylobacter jejuni

Conventional Phenotypic Tests for Mycobacterial Infections

Genetic Sequencing and Nucleic Acid Amplification for Mycobacterial Infections

Acid-Fast Stains for Mycobacterial Infections

Vibrio Parahaemolyticus and Vibrio vulnificus

Vibrio Cholerae

Burkholderia cepacia Complex