Literature review > Issue 1 > Review Semeniuk et al. 

This study evaluated the performance of 4 assays for chlamydia: two were non-amplified (Access Enzyme Immunoassay and Pace 2) and two were amplified (COBAS PCR and AMT-CT). The authors undertook this study to determine the feasibility and performance of two different sequential testing strategies, using a non-amplified test, followed by a nucleic acid amplified test (NAAT) for their particular laboratory. They considered work-flow, staffing, space, and through-put volume capabilities of the different algorithms. Enrolled were 504 women, aged 15 to 75, who were being tested for cervical C. trachomatis infection. The Access EIA had the lowest sensitivity (71.4%), and the PACE 2 assay was less sensitive (85.7%) than either of the NAATs (both 100%). The COBAS assay had the lowest PPV because of the assay's high false positive rate and the highest repeat factor due to the presence of inhibitory substances detected by the internal control.

Several issues are important in this study. First, is the prospective comparison of the tests that were performed. Secondly, is the issue of whether a combination of a non-amplified assay, followed by an amplified assay for gray zone results can improve diagnostic accuracy. Lastly, are the important local issues of costs, staffing, turn around times, space, and technical feasibility, when choosing a testing method or algorithm for chlamydia detection.

As expected from previous studies, the NAATs detected more true positives than did either of the non-amplified tests [1-6]. However, they also detected more false positives. There were 28 true positives as defined by the definition of two positive cervical amplified tests. It is interesting that the authors did not a priori define a true positive as two positive urine amplified tests also. The false positive results (5 for Pace2, 2 for AMP-CT, 7 for COBAS CT for cervical samples and 3 for Cobas CT for urines) underscore the importance for laboratory managers to consider the necessity for confirming positive chlamydia test results, especially for amplified tests. CDC guidelines now recommend confirmation of a positive amplified test, when screening low prevalence populations, where the positive predictive value (PPV) is less than 90% [7]. Satisfactory approaches are to 1) test a second specimen with a different test that uses a different target or format, 2) test the original specimen with a different test that uses a different target or format, 3) repeat the original assay on the original specimen with a blocking antibody or competitive probe or 4) repeat the original test on the original specimen [7]. Decisions as to whether to confirm any or all positive NAATs and how to do it are widely under consideration by scientists presently.

Use of NAATs after patients have received appropriate antibiotics for chlamydia, as noted in the manuscript, deserves a word of caution. NAATs can remain positive for two or more weeks after appropriate therapy even though patients are probably not still infected or infectious. DNA, which is likely not infectious, has been demonstrated to remain amplifiable in sequential samples after adequate therapy, probably reflecting the time required for cellular turn over [8,9]. It has been recommended that repeat NAAT testing not be performed for at least three weeks after treatment [7,9]. NAAT retests performed before this interval may be falsely positive with regard to infectiousness of the patient.

The 5 false positive Pace2 results, occurring early in this study, also indicate the necessity for making sure that personnel are adequately trained and practiced before being given the responsibility for turning out clinical results. Adequate training and fastidious attention to the procedural details of the assay are especially paramount for NAAT testing. Additional methods to reduce false positive NAATs include adherence to rigorous cleaning protocols with 10% bleach and periodic environmental "swipe" assays by using the NAAT for testing wet swabs of multiple sites in the processing and testing areas. Any positive results dictate that meticulous cleaning should be performed.

The issue of a sequential strategy of performing a NAAT after resulting a non-amplified test that has a gray zone result (a test value close to the positive cut-off value) is theoretically appealing and has been used by some laboratories [10]. Semeniuk et al. recommend that when testing low risk (low prevalence) populations, this approach might serve to increase the PPV. There will be a tradeoff for decreased sensitivity of the non-amplified test however, because approximately half of the samples negative by non-amplified test and positive by NAAT will fall below the negative gray zone and would be completely missed using this algorithm. It appears to be more accurate to utilize the increased sensitivity capability of a NAAT and then confirm positive tests according to CDC recommendations for samples that come from low prevalence populations. The feasibility of any algorithm like the former or latter depends on the ability of the laboratory to know the type of population from which a particular sample comes. Often this is problematic. The solution may be to confirm all positive NAATs, if the population prevalence is unknown, as might be the case for a commercial or reference laboratory. This NAAT confirmation dilemma may not be easily solved.

Lastly, making the decision of how to test for chlamydia is the responsibility of each laboratory manager relative to the local environment. This includes the type of laboratory, its funding source, volume of the work load, staffing considerations, turn around time requirements, space issues, feasibility of knowing the population types being tested, and re-imbursement concerns. Semeniuk et al. have thoughtfully addressed these issues in this manuscript, including the possibility of pooling multiple samples, as a cost saving plan for NAATs. Importantly, choice of the most appropriate testing modality may require a local study and careful deliberation about these complex factors on an individual laboratory basis.

References:

1. Crotchfelt KA, Pare B, Gaydos C, Quinn TC. Detection of Chlamydia trachomatis by the Gen-Probe AMPLIFIED Chlamydia trachomatis assay (AMP-CT) in urine specimens from men and women and endocervical specimens from women. J Clin Microbiol 1998; 36:391-94.

2. Crotchfelt, K. A., Pare, B., Gaydos, C., and Quinn, T. C. Detection of Chlamydia trachomatis by the Gen-Probe Transcription Mediated Amplification (TMA) Assay in male urine specimens and female endocervical and urine specimens. Proceedings Euope.Soc.Chlamydia Research , 317. 1996.
Ref Type: Abstract

3. Ferrero DV, Meyers HN, Schultz DE, Willis SA. Performance of the Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay in Detecting Chlamydia trachomatis in Endocervical and Urine Specimens from Women and Urethral and Urine Specimens from Men Attending Sexually Transmitted Disease and Family Planning Clinics. J Clin Microbiol 1999; 35:3230-3233.

4. Goessens WHF, Mouton JW, Van Der Meijden WI et al. Comparison of three commercially available amplication assays, AMP CT, LCx, and COBAS AMPLICOR, for detection of Chlamydia trachomatis in first-void urine. J Clin Microbiol 1997; 35:2628-33.

5. Van Der Pol B, Quinn T.C., Gaydos CA et al. Evaluation of the AMPLICOR and Automated COBAS AMPLICOR CT/NG Tests for the Detection of Chlamydia trachomatis. J Clin Microbiol 2000; 38 :1105-12.

6. Gaydos CA, Quinn TC, Willis D et al. Performance of the APTIMA Combo 2 assay for the multiplex detection of Chlamydia trachomatis and Neisseria gonorrheae in female urine and endocervical swab specimens. J Clin Microbiol 2003; 41:304-9.

7. Centers for Disease Control and Prevention. Screening Tests To Detect Chlamydia trachomatis and Neisseria gonorrhoeae Infections--2002. MMWR 2002; 51 (No. RR-15):1-38.

8. Workowski KA, Lampe MF, Wong KG, Watts MB, Stamm WE. Long-term eradication of Chlamydia trachomatis genital infection after antimicrobial therapy: Evidence against persistent infection. JAMA 1993; 270:2071-75.

9. Gaydos, C. A., Crotchfelt, K. A., Howell, M. R., Kralian, S., Hauptman, P., and Quinn, T. C. Molecular amplification assays to detect Chlamydia trachomatis infections in urine specimens from high school female students and to monitor the persistence of chlamydial DNA after therapy. Journal of Infectious Diseases 177, 417-424. 1998.

10. Dean, D., Ferrero, D., and McCarthy, M. Comparison of performance and cost-effectiveness of direct fluoresent-antibody, ligase chain reaction, and PCR assays for verification of chlamydial enzyme immunoassay results for populations with a low to moderate prevalence of Chlamydia trachomatis. J Clin Microbiol 36, 94-99. 1998.

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