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Evaluation of opa-Based Real-Time PCR for Detection of Neisseria gonorrhoeae

Tabrizi, Sepehr N. PhD*; Chen, Shujun MS*; Tapsall, John MD; Garland, Suzanne M. MD*

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Sexually Transmitted Diseases: March 2005 - Volume 32 - Issue 3 - p 199-202
doi: 10.1097/01.olq.0000154495.24519.bf
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Neisseria gonorrhoeae is the cause of one of the most prevalent bacterial sexually transmitted infections (STIs) in men and women worldwide.1 Accurate diagnosis of gonorrhea depends on laboratory tests that are reliable, reproducible, and robust because of nonspecific or absent clinical manifestations in both men and women.2 Nucleic acid amplification technology (NAAT) has a number of recognized advantages, including those of increased sensitivity of detection and ease of sample collection and transport, when compared with culture-based methods.3–7

A number of commercial NAATs are currently available, and their use has seen many of the anticipated benefits of NAAT in the diagnosis of gonococcal disease realized in practice. However, increasing experience with commercial and “in-house” NAATs over time and in different settings has shown a need for both a considered approach to the application of NAATs under these different conditions and for an awareness of their limitations. These considerations include those of sensitivity and specificity of primary and, where used, supplemental “confirmatory” tests and the prevalence of infection in various populations. Some assays, for example, have been shown to cross-react with other Neisseria species.8,9 The CDC has proposed a number of testing algorithms for confirmation of STI by NAAT, which require use of additional or supplemental assays.10 Supplemental assays based on the detection of the cppB gene of the multicopy 4.2-kb cryptic plasmid have also been described, and their use has been explored.9,11–13 However, in some gonococcal populations, plasmidless strains of N gonorrhoeae can result in false-negative results in the cppB-based NAAT.14

This study describes identification of a 90-bp conserved region of N gonorrhoeae specific multicopy opa gene by a real-time 5′-nuclease PCR assay that can be utilized for rapid detection and confirmation of N gonorrhoeae.

Methods

Bacterial Strains and Clinical Samples

A total of 173 microorganisms, including 100 isolates of N gonorrhoeae, 10 isolates of N meningitis (groups A, B, C, W, and Y), 15 isolates of N subflava, 5 isolates of N lactamica, 6 isolates of other Neisseria species (including N cinerea, N dentrificans, N flavescens, N mucosa, and N sicca) and 37 isolates of non-Neisseria species commonly isolated from the urogenital area were evaluated. Strains of N subflava and N cinera evaluated previously to cross-react with some commercial assays9 were included in the above strains evaluated. Strains used included ATCC strains and clinical isolates from the departmental culture collection. Included in the 100 N gonorrhoeae isolates were 20 clinical strains, which produced a negative PCR result for the cppB gene. In addition a total of 50 clinical samples positive for N gonorrhoeae by consensus NAA methods (including Roche COBAS and 16S, Abbott LCx, and cryptic plasmid PCR), 50 negative samples by all NAA methods, and 35 positive by Roche COBAS were selected from a specimen pool of more than 3000 women tested previously and for whom ethics committee approval had been obtained for STI testing, including the detection of N gonorrhoeae. A 10-fold serial dilution of quantitated N gonorrhoeae DNA (ABI, Columbia, MD) was also utilized and tested to assess sensitivity of the assays.

Sample Preparation

DNA was extracted from cultured bacteria and clinical samples using the automated MagNA Pure LC (Roche Diagnostics) with the associated DNA Isolation Kit I protocol. DNA was eluted in a final volume of 100 μL of MagNA Pure Elution Buffer (Roche Molecular Biochemical, Mannheim, Germany). Approximately 109 bacteria per isolation were utilized. Clinical samples were tested by real-time amplification of β-globin gene sequences to assess presence of adequate amplifiable DNA as described previously.12

Opa Real-Time N gonorrhoeae PCR

Target was chosen from a conserved region of the N gonorrhoeae multicopy cell-surface opacity protein gene. Amplification reaction consisted of 2-μL aliquot of extracted DNA, 1 × Lightcycler Fast Start Reaction Mix (Roche) containing 2 mmol/L MgCl2 (final concentration), and 1 μmol/L of each primer GCopaF-LNA 5′-TTGAAACACCGCCCGGAA 3′ and GcopaR-LNA 5′-TTTCGGCTCCTTATTCGGTTTAA 3′ directed at amplifying a 90-bp fragment of opa gene in a total of 10 μL. The reactions also included 0.2 μmol/L of 27mer FAM and TAMARA labeled locked nucleic acid (LNA) Taq human hybridization probes Gcopa-LNA 5′ CCGATATAATC+CGTC+CTTCAA+CATCAG 3′ (+ indicates the position of LNA bases). LNA fluorescent probes were synthesized by Proligo (Lismore, Australia) and were utilized to increase thermal stability and hybridization specificity. The samples were heated at 95 °C for 10 minutes and cycled 45 times using parameters of 95 °C for 0 seconds, 50 °C for 10 seconds, and 60 °C for 60 seconds. Data for each sample were plotted as derivative of fluorescence versus temperature. All samples which yielded linear increases in their fluorescence readings relative to the negative control sample that had no N gonorrhoeae DNA were considered positive.

Consensus positivity among the clinical samples tested by the above methods was defined as positive by 4 other methods, including COBAS AMPLICOR PCR, Roche 16S assay, Abbott LCx, cppB gene PCR.12

All amplification reactions contained a positive and negative control. Specimen contamination and carryover was prevented by using positive-displacement pipettes, prior aliquotting of all reagents, and by performing pre- and post-PCR steps in different rooms specifically allocated for PCR.

Results

DNA extracts from all bacterial strains tested by the opa real-time PCR demonstrated positive results only in 100 N gonorrhoeae strains examined, including the 20 strains which lacked the cppB cryptic plasmid gene. All other Neisseria species and bacterial species tested showed a negative result by this assay.

The assays produced real-time amplification curves allowing rapid identification of positive samples with an analytical sensitivity (measured by a serial 10-fold dilution of the N gonorrhoeae bacterial DNA) of 1 copy per reaction (Fig. 1).

F1-12
Fig. 1:
Amplification curves generated from dilutions of N gonorrhoeae on LightCycler using detection of fluorescence at 530 nM. Figure is marked with N gonorrhoeae copy numbers included in each amplification reaction.

Clinical sensitivity and specificity of the assay were assessed in 50 samples previously determined to be positive for N gonorrhoeae by consensus NAA methods (including Roche COBAS and 16S, Abbott LCx, and cryptic plasmid PCR) and 50 negative samples from a specimen pool of more than 3000 women tested previously.12 Extracted DNA from all samples was assessed with detection of human β-globin as an indicator of adequacy of sample and detection of inhibitors. All clinical samples tested positive for the human β-globin, indicating the presence of adequate amplifiable DNA in the specimen. All samples yielded similar quantities of extracted DNA. All 50 samples previously assessed to be positive for N gonorrhoeae were also positive by the opa real-time assay (Table 1). None of the 50 negative samples showed an amplification curve. In addition, 35 clinical samples which showed a positive result by the COBAS assay and had determined to be negative by confirmation cppB PCR were tested. Analysis of these samples demonstrated only 1 sample being positive by the opa PCR. Analysis of these COBAS-positive samples by an alternative 16S PCR (data not shown) demonstrated a positive confirmation of the result obtained by opa PCR assay.

T1-12
TABLE 1:
Comparative Results opa Real-Time Assays on Clinical Samples for Detection of N gonorrhoeae to Previously Samples Tested by Other NAA Assays

Discussion

Sensitive and specific detection of infection with N gonorrhoeae is important in clinical management of patients with STIs. Currently, there are a number of commercial NAATs available for the laboratory diagnosis of gonorrhea. However, some assays have been shown to cross-react with some isolates of certain nonpathogenic Neisseria species (N subflava and N cinerea),8,9,14 while other studies have reported cross-reaction with lactobacilli, especially in specimens with heavy bacterial loads.8 Consequently, laboratories performing N gonorrhoeae NAATs need either to select or develop a supplementary assay for their own confirmation assays or else report all positives without confirmation.10

In this study, a 90-bp fragment of the conserved region of the chromosomally located gene encoding for the cell surface opacity proteins (opa) gene was targeted for amplification and detection by a real-time 5′-nuclease assay. In some strains, this gene has been mapped to up to 11 loci throughout 60% of the gonococcal genome.15,16 This target was chosen for its increased copy numbers, which can enhance sensitivity, as well as provide a much-reduced probability of false-negative reactions due to deletion or strain variability in all the copies of the opa gene.16 The evaluation of sensitivity of this assay demonstrated amplification of a single copy of N gonorrhoeae. This assay did not show amplification with any of the 173 nongonococcal or non-Neisseria species evaluated. However, all 100 strains of gonococcus examined produced a positive result in this NAAT.

Included in the examination were 20 gonococci which previously demonstrated a negative result by the cppB PCR. Several studies have described the 4.2-kb cryptic plasmid, which has been shown to be present both as integrated and episomal states at multiple sites as a sensitive target for confirmatory assay.9,11,12 A recent study evaluating a cppB-based PCR assay found positive reactions with all clinical isolates examined and with gonococci from various geographical locations.12 However, some strains of N gonorrhoeae have been reported to lack the cryptic plasmid,14,17,18 and these subtypes may at times expand to constitute a significant proportion of a gonococcal population.19,20 The results here indicate that confirmation of the N gonorrhoeae by the opa-based assay may be more appropriate in populations where the plasmidless strains are circulating. In addition, lack of detection of closely related Neisseria species such as N meningitidis is important since the latter is also present in clinical samples that are generally obtained for detection of N gonorrhoeae.21

To further evaluate the sensitivities and specificities, 135 clinical samples previously assayed by 4 different NAAT methods were selected (i.e., 50 positive by a consensus method, 50 negative by all methods, and 35 positive only by COBAS Roche PCR).12 Overall, the 100 selected samples which had consensus-positive and -negative results gave identical results with the opa PCR assay. Among 35 samples initially assessed to be positive by Roche COBAS assay and which had produced a negative cppB PCR result, only 1 sample generated a positive amplification with the opa assay. Confirmation of this sample by an alternative PCR targeting the 16S gene showed only a positive 16S amplicon (data not shown) in the opa PCR positive, indicating that this sample contained a plasmidless strain of N gonorrhoeae.

This study has described a very sensitive and specific gonococcal real-time PCR which can be used not only for screening but also for offering a supplemental assay for further affirmation of positive samples tested by commercial amplification assays which are prone to false-positive results.

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