As C. trachomatis is an obligate intracellular parasite, isolation of the organism must be performed in tissue culture, with culture confirmation by staining with species-specific fluorescent monoclonal antibodies and identification of the characteristic intracytoplasmic inclusions with microscopy. Until recently, the gold standard of diagnosis of C. trachomatis infection in women was isolation by culture from the cervix (2). Culture requires careful specimen collection and stringent transport conditions with maintenance of the cold chain and requires 48-72 hours to perform. In addition, culture methods for C. trachomatis are not standardized; therefore, there can be significant variation in performance from laboratory to laboratory. Serology, using either the microimmunofluorescence test or enzyme immunoassays (EIAs), is not suitable for the diagnosis of genital C. trachomatis infection (19).
Several types of nonculture tests for detection of C. trachomatis in clinical specimens have been available in the past. These include EIAs, specifically Chlamydiazyme (Abbott Diagnostics, Chicago, IL) and MicroTrak EIA (Genetic Systems, Seattle, WA) and direct fluorescent antibody (DFA) tests, including Syva MicroTrak (Genetic Systems, Seattle, WA) and Pathfinder (Sanofi-Pasteur, Chaska, MN) and a deoxyribonucleic acid (DNA) hybridization assay, GenProbe Pace II (GenProbe, San Diego, CA), were introduced in the 1980s (20).
The DFA test was less sensitive than culture (70-90% depending on the site and clinical status) but highly specific (>95%). The DFA was best suited for high-prevalence populations when culture was not available; however, the very nature of the test made it difficult to use for screening large numbers of specimens.
Unlike the DFA, EIAs were semiautomated and suitable for processing large numbers of specimens. EIAs use either a polyclonal or a monoclonal antibody directed against the Chlamydia genus-specific lipopolysaccharide antigen. These tests will detect other chlamydia species, specifically C. pneumoniae, if used for respiratory specimens.
The direct nucleic acid probe was until recently the most commonly used nonculture test for detection of C. trachomatis in many hospital and public health laboratories in the United States. Using chemiluminescence, the probe hybridizes to a species-specific sequence of chlamydial 16S ribosomal RNA. The performance of the probe assay has been similar to available EIAs, with sensitivities of 75-80% and specificities greater than 99%. This assay is not a nucleic acid amplification test. Neither EIAs nor DNA probe are sensitive enough to detect C. trachomatis in urine from women.
A major advance in the diagnosis of C. trachomatis infection during the past decade has been the introduction of nucleic acid amplification tests (NAATs [20,21]). These tests have high sensitivity, perhaps even detecting 10-20% greater than culture, while retaining high specificity (>98-99%). There are currently three commercially available NAATs approved by the Food and Drug Administration (FDA): polymerase chain reaction (PCR; Amplicor, Roche Molecular Diagnostics, Nutley, NJ), transcription-mediated amplification (TMA; GenProbe), and strand displacement amplification (SDA; ProbeTec, Becton Dickson, Sparks, MD). A fourth assay, ligase chain reaction (LCR)-LCx Chlamydia trachomatis Assay (Abbott Diagnostics), was withdrawn from the market by Abbott in June 2002 because of performance problems. PCR and SDA are DNA amplification tests; all use primers that target gene sequences on the cryptic C. trachomatis plasmid, which has approx 10 copies per cell. TMA is an RNA amplification assay that uses reverse transcriptase and a T7 polymerase to produce anywhere from 1 million to 1 billion copies of an RNA target. Unlike the DNA amplification assays, TMA is isothermal; that is, it does not require the use of a thermocycler, which is necessary for PCR or LCR. As shown in Table 2, EIAs can detect a minimum of 104-105 organisms; culture can detect 10-100 organisms, and NAATs can detect 1-10 organisms.
Amplification tests are more sensitive than currently available EIAs and nonamplification DNA probe assays. One such study found PCR to be 37% more sensitive than the DNA probe. However, these tests are not all equivalent. False-negatives caused by inhibitors of DNA polymerase are more of a problem than false-positives caused by amplicon carryover. Inhibitor appears to be more frequent in cervical specimens and urine. Inhibitory substances in urine from pregnant women include P-human
Relative Limits of Detection of Different Technologies Used to Diagnose C. trachomatis
Number of organisms/samplea a
1-101 10*-102 101-103 103-105 103-104
'Log number of chlamydial elementary bodies.
chorionic gonadatropin, crystals, nitrites, and hemoglobin. Of note, there are no inhibition controls included with any of the currently available kits. Also, there are rare strains of C. trachomatis that lack the cryptic plasmid and thus would not be detected by PCR.
Currently available commercial NAATs have FDA approval for cervical swabs from women, urethral swabs from men, and urine from men and women. However, the sensitivity for detection of C. trachomatis in urine from women is less than for the cervix (78-96%). The use of noninvasive specimens such as urine is especially useful in high-prevalence populations such as sexually active adolescents (9). A second-generation TMA was just recently approved for vaginal specimens from women.
Urine specimens for NAATs should be collected as directed by the test manufacturer. The patient should not have urinated within the previous hour, and women should be instructed not to clean the perineum prior to voiding. The first catch of 10-20 mL urine should be collected in a clean collection cup and refrigerated immediately at 2-8°C. However, a midstream catch urine has been reported to be effective for testing with LCR. Longer times (>3 hours) since the last void appeared to reduce the sensitivity of antigen tests for urine from women. This appears to be less of an issue with the amplification tests. The time the urine specimen is left at room temperature should be minimized because the low pH and high urea content can rapidly denature DNA present in the specimen, especially at temperatures greater than 25°C. Although PCR is currently not approved for use with frozen urine specimens, freezing and thawing may actually improve the sensitivity of PCR by eliminating transient inhibitory factors that are present in some specimens.
NAATs should probably not be used as a test of cure within 3 weeks of treatment. More than 40% of follow-up urine specimens from female patients who were initially positive by PCR and 73.3% of those initially positive by LCR were still positive 1-3 days after treatment with single-dose azithromycin. Only at 15 days posttherapy did all specimens test negative (10).
Isolation by culture remains the preferred method for detection of C. trachomatis from the conjunctiva, nasopharynx, vagina, or rectum of infants. Several nonculture methods have FDA approval for diagnosis of chlamydial conjunctivitis. They include EIAs, specifically Chlamydiazyme and MicroTrak, and DFAs, including Syva MicroTrak and Pathfinder. These tests appear to perform well with conjunctival specimens with sensitivities greater than or equal to 90% and specificities greater than or equal to 95% compared with culture (22,23). The performance with nasopharyngeal specimens has not been as good, with sensitivities ranging from 33 to 90%. The DNA probe does not have approval for any site in children. Data on use of NAATs in children are limited. Preliminary data suggest that PCR (Amplicor) is equivalent to culture for detection of C. trachomatis in the conjunctiva and nasopharynx of infants with conjunctivitis (18). However, no NAATs are currently approved by the FDA for this indication.
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