Advanced Techniques in Diagnostic Microbiology

22. Molecular Techniques for STDs Diagnosis 355
male urethral smears ranges 90–95% while that of female endocervical smears
is 50–70% (Janda et al., 2003). Owing to colonization by other Gram-negative
coccobacillary organisms, endocervical smears as well as rectal specimens require
extra care in interpretation. Enzyme immunoassay (EIA) (Gonozyme, Abbott
Laboratories, Abbott Park, IL, USA) is a presumptive diagnostic method for
gonococcal antigen detection. The sensitivity and specificity is comparable to direct
smear examination, although it is less sensitive for endocervical specimens
(Knapp, 1988). Isolation and identification of N. gonorrhoeae from cultures is
still considered the gold standard for the diagnosis of gonococcal infections. Both
nonselective (chocolate agar) and selective media such as modified Thayer–Martin
(MTM), Martin–Lewis (ML), or New York City (NYC) are often used for primary
isolation.
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 plasmid
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 chemiluminescent
substrate with alkaline phosphatase–labeled antibodies. However, there