Literature review > Issue 8 > Review on Tabrizi et al. 

This manuscript reports the use of two real time PCR assays to confirm results of Cobas Amplicor PCR assay N. gonorrhoeae using positive and negative clinical samples and ATCC isolates, as well as using the LCR assay by Abbott and the 16S rRNA primers by Roche for confirmation. (Neither of the two latter methods is available commercially now, however). It is an important article, which confirms earlier articles reporting that false positive results for N. gonorrhoeae occur with the Amplicor assay and underscores the importance of the use of a confirmatory assay for N. gonorrhoeae Amplicor positive results. For this purpose the authors offer two very sensitive and specific real time research PCR assays using two different methods, molecular beacons (MB) and fluorescence resonance energy transfer (FRET) probes.

The performances of these two real-time LightCycler PCR assays, which were based on amplification of a nested region of the cppB gene of the multicopy 4.2 kb cryptic plasmid of N. gonorrhoeae were excellent with the FRET and MB probe assays having a sensitivity of 99% and 100% respectively, and a specificity of 98% and 100%, respectively, compared to the consensus results as the gold standard. Among the 122 Cobas specimens positive for N. gonorrhoeae, 73, 68, 71, and 72 were positive by the 16S, LCx, FRET, and MB assays, respectively. Only 59.8% of the 122 Roche positive samples for gonorrhea were confirmed as true positives using all optical densities (O.D.) >0.2-2.0, while 51% of those with O.D.s >2.0-3.5 confirmed. Thus, the confirmation rate was proportional to the optical density of the Cobas PCR, with the highest confirmation of 93.5% obtained with samples having optical density readings >3.5.

The authors clearly stated their objective and provided a large group of both clinical isolates and ATCC isolates on which they performed a large battery of assays. The methods were well described with excellent detail so that other laboratories wishing to use these confirmatory assays could perform them accurately.

In conclusion, more than 40% of the Cobas Amplicor positive samples were not confirmed as positive by the consensus criteria and are presumed false positive results. This high false positive rate for the Cobas Amplicor N. gonorrhoeae PCR assay highlights the importance of confirmation of all positive results by a supplemental assay. Users of the Amplicor assay for the diagnosis of N. gonorrhoeae should examine their procedures for use of O.D. cutoffs (>3.5 O.D. seems safe), and/or procedures for confirmatory tests, especially those results 2.0-3.5. This study provides valuable information for laboratorians who use the Amplicor PCR assay.

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