Literature review > Issue_12 > Review 

Gonococcal opa genes are an attractive target choice for nucleic acid amplification tests (NAATs) because all strains of N. gonorrhoeae express the outer membrane opacity (Opa) proteins, and each strain contains multiple (11-13) opa gene copies. However, N. meningitidis strains also contain multiple (3-4) opa genes, and hybridization studies conducted using a cloned gonococcal opa gene probe showed that the genomes of some commensal Neisseria species isolates (80% of N. lactamica strains and 36% of N. cinerea strains) also contain homologous sequences [6] (gonococcal opa genes in this reference are referred to by their former designation as PII genes). Thus, the benefits of the universal distribution of opa genes among gonococcal strains and the sensitivity afforded by the relatively high copy number are inevitably accompanied by issues of species specificity. As a case in point, the Abbott LCx assay for N. gonorrhoeae (discontinued in 2003 due to reagent supply problems) was an opa-based ligase chain reaction (LCR) test with primers that targeted nucleotides within the Opa-coding sequence [11]. The LCx NG primers could also hybridize to meningococcal opa genes, and sufficient analytical specificity was obtained only by omitting from the reaction mix one of the nucleotides that was required to generate the meningococcal LCR product, but not the gonococcal product (H. Lee, personal communication).

The GCopa primers and probe for real time PCR described by Tabrizi et al. hybridize in the promoter region upstream of the opa translational start site. In a paper published shortly after this one, Geraats-Peters and colleagues described a similar assay using primers and probes targeting the same opa promoter region [7]. Both opa-based real time PCR assays produced negative results with an array of non-Neisseria species commonly isolated from the urogenital tract and, most importantly, with multiple non-gonococcal Neisseria isolates including N. meningitidis, N. cinerea, N. lactamica and N. subflava [7, 8]. Thus, the analytical specificity of real-time PCR targeting the gonococcal opa promoter region appears to be satisfactory and superior to that of the AMPLICOR NG PCR assay.

As expected with a multicopy target, the analytical sensitivities were very high for both the Tabrizi et al. [8] and Geraats-Peters et al. [7] opa-based real-time PCR assays; the tests were positive with nucleic acids from < a single gonococcal organism per reaction. All 100 N. gonorrhoeae strains analyzed by Tabrizi et al. yielded positive PCR results with a single reported primer and probe combination [8]. However, in their analysis of 448 N. gonorrhoeae isolates, Geraats-Peter et al. discovered that real time PCR with a single probe including the last 13 bases of the Tabrizi et al. probe failed to detect 24 (5%) gonococcal strains [7]. DNA sequence analysis demonstrated that the amplified opa promoter regions from all of those strains contained a single variant sequence with mismatches at 5 of the 19 nucleotides in the original probe. To increase the sensitivity of their real-time PCR assay, Geraats-Peter et al. included a second opa probe matching this variant sequence. The Tabrizi et al. probe sequence extended further towards the 5' end of the amplicon than the original Geraats-Peter et al. probe. It is not clear whether the opa genes in the Australian N. gonorrhoeae strains analyzed by Tabrizi and colleagues lacked the variant sequence identified by Geraats-Peter and co-workers in strains isolated in The Netherlands, or if hybridization of the additional bases in the Tabrizi et al. probe was sufficient to overcome potential mismatches. With a smaller sample size and the apparently low prevalence of strains with the variant sequence, Tabrizi and co-workers may have missed the problem simply by chance. Whatever the explanation, it seems prudent to routinely include probes containing both known sequences in opa promoter-based detection assays in the future.

As a confirmatory test for clinical specimens analyzed by the AMPLICOR NG assay, the opa-based real-time PCR assay described by Tabrizi et al. correctly identified all AMPLICOR NG false-positives that had been confirmed as negative with other N. gonorrhoeae NAATs. Similar results were obtained by Geraats-Peters et al. [7]. In addition, Tabrizi et al. showed that opa-based PCR correctly identified as positive one sample that was positive by the AMPLICOR NG assay, positive by a N. gonorrhoeae rRNA PCR assay and negative by another confirmatory PCR that targets the cppB gene on the gonococcal cryptic plasmid [1, 5, 9, 10]. Thus, opa-based PCR may be more appropriate than a cppB-based assay for detection and/or confirmation of N. gonorrhoeae, particularly in populations where circulating gonococcal strains lack the cryptic plasmid.

References:

1. Tabrizi, S.N., et al., Evaluation of real time polymerase chain reaction assays for confirmation of Neisseria gonorrhoeae in clinical samples tested positive in the Roche Cobas Amplicor assay. Sex Transm Infect, 2004. 80(1): p. 68-71.

2. Diemert, D.J., M.D. Libman, and P. Lebel, Confirmation by 16S rRNA PCR of the COBAS AMPLICOR CT/NG test for diagnosis of Neisseria gonorrhoeae infection in a low-prevalence population. J Clin Microbiol, 2002. 40(11): p. 4056-9.

3. Van Der Pol, B., et al., Enhancing the specificity of the COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae by retesting specimens with equivocal results. J Clin Microbiol, 2001. 39(9): p. 3092-8.

4. Farrell, D.J. and T.J. Sheedy, Urinary screening for Neisseria gonorrhoeae in asymptomatic individuals from Queensland, Australia: an evaluation using three nucleic acid amplification methods. Pathol, 2001. 33(2): p. 204-5.

5. Farrell, D.J., Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR. J Clin Microbiol, 1999. 37(2): p. 386-90.

6. Aho, E.A., G.L. Murphy, and J.G. Cannon, Distribution of specific DNA sequences among pathogenic and commensal Neisseria species. Infect Immun, 1987. 55(4): p. 1009-1013.

7. Geraats-Peters, C.W., et al., Specific and sensitive detection of Neisseria gonorrhoeae in clinical specimens by real-time PCR. J Clin Microbiol, 2005. 43(11): p. 5653-9.

8. Tabrizi, S.N., et al., Evaluation of opa-based real-time PCR for detection of Neisseria gonorrhoeae. Sex Transm Dis, 2005. 32(3): p. 199-202.

9. Leslie, D.E., et al., An assessment of the Roche Amplicor Chlamydia trachomatis/Neisseria gonorrhoeae multiplex PCR assay in routine diagnostic use on a variety of specimen types. Commun Dis Intell, 2003. 27(3): p. 373-9.

10. Ho, B.S.W., et al., Ploymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples. J Clin Pathol, 1992. 45: p. 439-442.

11. Abbott Laboratories LCx probe system Neisseria gonorrhoeae assay Product Insert, 1996.

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