Diagnostic Value of Molecular Confirmation Assays for Neisseria gonorrhoeae

The COBAS Amplicor system (Roche Diagnostics, Rotkreuz, Switzerland) is a widely used screening test for infections with Neisseria gonorrhoeae due to its excellent sensitivity and ease of use. However, it has been found to cross-react with certain commensal Neisseria species (1, 3, 4). Therefore, an independent confirmation assay is mandatory. In the May 2007 issue of the Journal of Clinical Microbiology, Mangold et al. (2) presented a new confirmation assay which is capable of distinguishing between N. gonorrhoeae and Neisseria spp. in comparison to confirmation assays targeting the porA pseudogene and the cppB gene. They obtained positive results in their in-house assay for 102/181 (56%) COBAS Amplicor-positive samples; the porA confirmation assay was positive for 69/181 (38%) samples, and the cppB assay detected 115/181 (64%) positive samples. Additionally, Mangold et al. reported 15%, 2%, and 4% positive results for COBAS Amplicor-negative samples with the cppB, porA, and in-house assays, respectively. The presented data are discrepant from observations at our institution. Upon optimization of published detection protocols, we have compared the performances of three assays targeting the porA pseudogene (5), the cppB gene (6), and the 16S rRNA gene (in-house) for quality assurance. In samples positive for all three targets, the cppB confirmation assay was the most sensitive (mean crossing point [Cp], 25.9), followed by the porA assay (mean Cp, 26.2) and the 16S rRNA gene assay (mean Cp, 32.8). Over a period of 6 months, 398 samples yielded a positive COBAS Amplicor result (optical density [OD] of >0.2, as recommended by the manufacturer). Of these 398 samples, 258 samples (64.8%) were negative in all three confirmation assays. The remaining 140 samples (35.2%) yielded a positive result in at least two of the confirmation assays, with the majority being positive in all three assays (128/140 [91.4%]). Eight of 140 samples (5.7%) were negative in the 16S rRNA gene assay, probably due to small DNA amounts in the sample, as this assay is less sensitive than the other two confirmation assays, which is corroborated by the fact that 2/8 samples showed an OD value lower than 2.0 when initially tested in the COBAS Amplicor system. Four specimens were positive in the 16S rRNA gene and porA detection assays only and were interpreted as gonococci lacking the cppB-harboring plasmid (2.9%); the COBAS Amplicor value was above 2.0 OD in all four samples. Taken together for 136/140 (97.1%) specimens, the porA and cppB confirmation assays were concordantly positive. In contrast to the findings of Mangold et al., we did not obtain positive results for the confirmation assays when applied to COBAS Amplicor-negative samples. The conflicting results might in part be explained by an epidemiological distinct pool of N. gonorrhoeae isolates examined and an unfortunate study design, as modified interpretation guidelines for the COBAS Amplicor assay were used by Mangold et al. Samples were considered positive if two out of three runs gave a positive signal above an OD of 2.0, as opposed to one sample above an OD of 0.2 being sufficient. This decreases the number of positive samples and inevitably has a negative impact on the sensitivity of the screening assay. Presumably, this protocol accounts for the “false”-positive results of the cppB plasmid assay (33/227). It would be interesting if these samples were initially positive in the COBAS Amplicor system (OD of >0.2) by use of the manufacturer's interpretation criteria. Our analysis further cannot confirm the low sensitivity of the porA detection assay, as the majority of our specimens yielded positive results in all confirmation assays. Regarding the in-house NsppID assay published by Mangold et al., the design of the test itself is not completely conclusive: N. gonorrhoeae displays 100% homology with strains of Neisseria polysaccharea at the binding site of the detection probe (referred to as Npolysaccharea1 in the publication). N. polysaccharea was not tested for cross-reactivity in the NsppID assay. By use of an NCBI BLAST search, strains of Neisseria meningitidis also display 100% homology in this region of the 16S rRNA gene. Additionally, coamplification in PCRs should be avoided if no important information is obtained by the coamplified product, as it is likely to decrease sensitivity. A confirmation assay for N. gonorrhoeae that coamplifies apathogenic Neisseria spp. does not provide any additional information of clinical relevance. The studies indicate that the use of molecular diagnostic assays for the detection of N. gonorrhoeae infection remains challenging and that the characterization of the prevalence of target genes in distinct epidemiological pools is important to ensure accurate diagnosis.