Evaluation of conventional and real-time PCR assays using two targets for confirmation of results of the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae test for detection of Neisseria gonorrhoeae in clinical samples.

Two conventional PCR-enzyme immunoassays (PCR-EIAs) and two real-time PCR assays (LightCycler system; Roche Diagnostics) were evaluated as confirmation assays with cppB and 16S rRNA genes as targets. Of 765 male and female genitourinary and nasopharyngeal specimens positive for Neisseria gonorrhoeae in the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR test (Roche Diagnostics), 229 (30%) were confirmed positive; 13 of these (5.7%) were lacking the cppB gene. Of the 534 samples (70%) that could not be confirmed, 81 (15%) showed a positive crossing point. However, melting curve analysis revealed an aberrant melting temperature in the LightCycler 16S rRNA assay; therefore, these samples were considered non-N. gonorrhoeae Neisseria species. Both of the 16S rRNA assays performed well, with positive predictive values of 99.1% and 100% for the PCR-EIAs and the real-time assays, respectively, and a negative predictive value of 99.8% for both. The cppB assays were compromised by the absence of the cppB gene in 5.7% of the N. gonorrhoeae-positive samples, resulting in negative predictive values of 96.8% and 97.6% for the PCR-EIAs and the real-time assays, respectively. Therefore, the 16S rRNA gene is preferable to the cppB gene as a target for confirmation assays. The melting curve analysis of the real-time assays provides useful additional information.

Evaluation of an in-house polymerase chain reaction for detection of Neisseria gonorrhoeae in urogenital samples.

AIM: To develop and evaluate a one day in-house polymerase chain reaction (PCR) assay for the detection of Neisseria gonorrhoeae DNA in urogenital samples. METHODS: 429 urogenital specimens were tested for the presence of N gonorrhoeae by in-house PCR and by Gen-Probe. The PCR assay amplifies target sequences within the N gonorrhoeae cppB gene on the 4.2 kb cryptic plasmid, after which amplicons are detected by a streptavidinbiotin based enzyme immunoassay using an internal probe. Discordant specimens were further evaluated by repeating the PCR and the Gen-Probe assay, and by an additional PCR using another set of 16S primers followed by radioactive detection of amplicons on a Southern blot. RESULTS: Of the 429 samples tested, 15 were found positive by in-house PCR, eight of which were confirmed by Gen-Probe. Of the seven discrepant samples, five were confirmed by 16S PCR and are also considered true positive. The remaining two samples were positive in the in-house PCR only, and are considered false positive. After resolution of discrepant samples, the sensitivities of the N gonorrhoeae assays were 100% and 61.5% for the in-house PCR and Gen-Probe, respectively, while specificities were comparable at 99.5% and 100%. CONCLUSIONS: The in-house PCR for the detection of N gonorrhoeae DNA is at least comparable to Gen-Probe in performance. An extended evaluation period should elucidate if the additional five GO-PCR positive specimens, confirmed by 16S PCR, are caused by persistence of DNA or whether the method is indeed more sensitive.

One-day detection of PCR amplified Chlamydia trachomatis DNA in clinical samples: ELISA versus Southern blot hybridisation.

AIMS: To compare ELISA and Southern blot hybridisation for the detection of PCR amplified Chlamydia trachomatis DNA extracted from clinical samples; to assess the value of the ELISA method in a clinical setting. METHODS: DNA was extracted from urogenital samples of 508 patients, purified and amplified using C trachomatis specific primers, one of which was endlabelled with biotin. Amplification products were detected by Streptavidin biotin based ELISA and non-radioactive Southern blotting. RESULTS: Of the 508 samples, 29 were positive and 479 negative by both methods. No discrepant results were observed. CONCLUSION: Streptavidin biotin based ELISA and Southern blotting were equally sensitive for detecting PCR amplified C trachomatis DNA. Using ELISA, test results could be generated within a single day.