Molecular Detection Methods

The commercial detection kits currently available for N. gonorrhoeae include those using probe hybridization (Pace 2, GenProbe, San Diego, CA, USA), PCR (COBAS AMPLICOR; Roche Molecular Systems, Branchburg, NJ, USA), ligase chain reaction (LCR; Abbott Laboratories, Abbott Park, IL, USA), and strand displacement amplification (SDA; Becton Dickinson, Sparks, MD, USA). Besides, two PCR assays have been published that target the gene encoding outer membrane protein III (ompIII) (Liebling et al., 1994) and the cppB gene (Ho et al., 1992) of N. gonorrhoeae.

Probe Hybridization

No amplification of nucleic acid takes place in the probe hybridization method. This method is based on annealing of complementary nucleic acid strands on a stable double-strand. There are two nucleic acid probe assays: the GenProbe PACE 2 and PACE 2C assays (GenProbe) and the Hybrid Capture II assay (Digene Corp., Gaithersburg, MD, USA) (Modarress et al., 1999). Both assays have been approved by the Food and Drug Administration (FDA) in the United States for detecting N. gonorrhoeae. In the GenProbe assay, target sequence of ribosomal RNA of N. gonorrhoeae is hybridized by an acridinium ester-labeled complementary DNA probe (Kluytmans et al., 1991). After adsorption of DNA-RNA hybrids to magnetic particles and removal of the unbound probe, the acridinium ester-DNA-RNA hybrid is measured in a luminometer. PACE 2C is a single-tube assay that can screen for presence of both Chlamydia trachomatis and/or N. gonorrhoeae (Iwen et al., 1995). If positive result is initially obtained, individual organism can be identified by performing separate tests. The initial positive result can also be verified by probe competition assay with unlabeled probe. In Hybrid Capture II assay, specific RNA hybridization probes are used to detect both genomic DNA and cryptic plas-mid DNA sequences of C. trachomatis and N. gonorrhoeae (Girdner et al., 1999; Schachter et al., 1999). The RNA-DNA hybrids are captured by hybrid-specific antibodies in microtiter plates and detected in luminometer by adding chemilu-minescent substrate with alkaline phosphatase-labeled antibodies. However, there is no supplemental test to verify the initial positive results. Sensitivity and specificity of probe hybridization ranged from 96.3% to 100% and 98.8% to 99.6%, respectively (Girdner et al., 1999; Schachter et al., 1999).

Molecular techniques using direct nucleic acid amplification from clinical samples are so powerful that theoretically a single copy of target DNA or RNA in the sample can be detected. These techniques obviate the requirement for presence of viable organisms in specimens, much like other nonculture approaches, in the diagnosis of infections as well as for molecular epidemiological typing studies. The COBAS AMPLICOR (Roche Molecular Systems) is one of three commercial molecular detection tests that are FDA approved for N. gonorrhoeae. The COBAS AMPLICOR NG test targets a 201-bp sequence in the cytosine methyltransferase gene of N. gonorrhoeae. Sensitivity and specificity of PCR ranged from 94.2% to 98.1% and 98.4% to 100%, respectively (Martin et al., 2000; van Doornum et al., 2001). However, it has been reported that the COBAS AMPLICOR NG test for N. gonorrhoeae cross-reacts with certain strains of nonpathogenic Neisseria species, such as N. subflava and N. cinerea (Farrell, 1994). Approximately 26% of COBAS AMPLICOR NG-positive results were false positives in one test population and corresponded to approximately 3% of the total population (Farrell, 1994). However, the same laboratory observed less than 1% false-positive results among urogenital specimens from a second study population (Farrell, 1994).

Two PCR assays targeting the gene encoding outer membrane protein III (ompIII) (Liebling et al., 1994) and the cppB gene (Ho et al., 1992) of N. gonorrhoeae have been published. Specificity of the ompIII assay was 96.4% and sensitivity 78.6% (Liebling et al., 1994). No false-positive or false-negative results have been described in ompIII PCR assay (Palmer et al., 2003), whereas cppB PCR can give false-positive results from several different Neisseria spp. (Palmer et al., 2003). Less frequent genetic exchange events possibly occur at ompIII, making it a potentially good target for use in molecular diagnostic tests (Palmer et al., 2003).

Ligase Chain Reaction

The N. gonorrhoeae ligase chain reaction (LCR) assay was developed by Abbott Laboratories as LCx Neisseria gonorrhoeae assay (Abbott Laboratories). This uses a ligase chain reaction amplification in the LCx probe system for detection of a specific 48-bp nucleotide sequence in the opa-encoding gene (opa-1) of N. gonorrhoeae (Stern et al., 1996). The assay uses four probes complementary to opa genes that may be present in up to 11 copies per bacterial cell. Sensitivity and specificity of the LCx assay ranged from 81% to 97.3% and 99.4% to 99.8%, respectively (Ching et al., 1995; Kehl et al., 1998). A study demonstrated that significant reproducibility problems can occur in the LCx probe system that would not be detected by the manufacturer's quality control procedures (Gronowski et al., 2000). Procedures for detecting and preventing contamination and possible solutions to reproducibility problems have been suggested (Gronowski et al., 2000). In early 2003, the Abbott LCx for N. gonorrhoeae (and also for Chlamydia trachomatis) was globally withdrawn due to continuing batch to batch problems with the performance of the test.

Strand Displacement Amplification

The BDProbeTec amplified DNA assay (Becton Dickinson) uses strand displacement amplification (SDA) and fluorescent resonance energy transfer probes that target DNA sequences homologous to genomic DNA of N. gonorrhoeae (Koenig et al., 2004). The system uses sealed microwells to minimize the release of am-plicons to the environment. Sensitivity and specificity of the BDProbeTec assay ranged from 84.9% to 98.5% and 92.5% to 98.6%, respectively (Little et al., 1999; Van Der Pol et al., 2001). The closed system design and assay flow are advantages when compared with other systems. Assay is performed on samples collected without transport medium, and transport at room temperature provides a significant advantage over other nucleic acid amplification tests.

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