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For the detection of Neisseria gonorrhoeae, culture is still the reference diagnostic test. Nucleic acid amplification tests (NAATs) are becoming more popular for research and screening programs with centralized batch processing of clinical specimens. Because of their more recent introduction, the accuracy of NAATs for N. gonorrhoeae is less well documented than that of NAATs for Chlamydia trachomatis, although high sensitivities of N. gonorrhoeae NAATs with female genital specimens have been reported (1-3, 8, 10). It is proven that clinical isolates of Neisseria subflava and Neisseria cinerea, belonging to the commensal flora of the human respiratory or genital tract, may exhibit cross-reactivity in the AMPLICOR PCR (5, 11). Accurate confirmation assays include other commercially available NAATs as well as noncommercial PCRs targeting the ccpB gene on the 2.6-MDa cryptic plasmid or DNA encoding the 16S rRNA (a prototype of the 16S rRNA PCR was developed by Roche Diagnostics Systems, Branchburg, N.J.) (3, 5-7).

We used the AMPLICOR C. trachomatis-N. gonorrhoeae manual coamplification assay to diagnose chlamydial and gonococcal infection in endocervical specimens obtained from cohorts of female commercial sex workers who were participating in a multicenter study conducted in Hat Yai (HA), Thailand; Cotonou (CO), Benin; and Durban (DU), South Africa. For management of infected women, each participating center performed C. trachomatis enzyme immunoassay (MicroTrak; Syva, San Jose, Calif.) and N. gonorrhoeae culture on modified Thayer-Martin medium. An additional endocervical swab was kept dry in an empty tube, stored frozen at 20°C within 5 h, and shipped on dry ice to the Institute of Tropical Medicine, Antwerp, Belgium. After arrival, swabs were stored at 20°C until testing. We used dry swabs to avoid problems of incompatibility of commercial collection kits and transport media for multiple testing of specimens with different NAATs (12). It has been shown that samples transported on dry swabs may give higher positive rates than swabs swirled in transport media (9).

The preparation of the swabs was modified during the study. The first 3,712 swabs were suspended directly in 500 µl of AMPLICOR lysis buffer, and further treatment and AMPLICOR PCR were performed by strictly following the instructions of the manufacturer (method A). The next 1,166 swabs were suspended in 1.0 ml of 2-sucrose phosphate (2-SP) medium. After vortexing at maximum speed for 20 s, swabs were discarded, 250 µl of each sample suspension was pipetted into a small conical tube and centrifuged at 12,000 × g for 10 min, and the pellet was resuspended in 250 µl of AMPLICOR lysis buffer; further treatment and PCR were similar to those for method A (method B). The last 2,195 swabs were suspended in 1.0 ml of 1:10-diluted phosphate-buffered saline (PBS) (containing 9 parts saline and 1 part PBS). Samples were vortexed at maximum speed for 20 s. After removal of the swab, 250 µl was treated in the same way as for the 2-SP suspensions (method C). The remaining samples treated with lysis buffer were stored at 20°C for later testing. The frozen DNA extracts of all AMPLICOR-positive samples were thawed and kept at room temperature for 2 h, and 50 µl was then transferred to a small conical tube for the performance of the 16S rRNA PCR according to a standard procedure provided by Roche Diagnostics Systems. Samples with weakly positive AMPLICOR or 16S rRNA results as well as samples showing inhibition were retested, and results were interpreted according to instructions of the manufacturer.

Statistical comparison of variables was done by chi-square analysis. Yates-corrected P values of <0.05 were considered statistically significant. Ninety-five percent confidence intervals were calculated based on the binomial distribution of the observed test results.

The data for the specimens treated by method A are shown in Table 1. The sensitivities of the AMPLICOR PCR were quite similar for the three centers. The sensitivity of culture was extremely low in HA and DU and was significantly higher in CO (P < 0.0001). The overall sensitivity of the 16S rRNA PCR for AMPLICOR PCR-positive samples was significantly lower for samples from HA (P < 0.00001) but was also lower than expected for samples from the other sites. Because of the large differences between AMPLICOR PCR and culture results, suggesting poor culture performance, major efforts were immediately made in all three centers to try to improve N. gonorrhoeae culture performance. The low sensitivity of the 16S rRNA PCR was striking and could not be explained by high numbers of false-positive AMPLICOR PCR results (12). It was decided to modify the specimen processing by using 2-SP medium (also recommended by Roche).

The test results for specimens treated by method B are shown in Table 2. The overall sensitivity of the AMPLICOR PCR was similar to that for specimens treated by method A. The sensitivity of the 16S rRNA PCR was lower than that for method A for samples from DU (P < 0.00001) but higher for samples from HA (P = 0.05) and CO (P = 0.57) The overall sensitivity of the 16S rRNA PCR was still remarkably low. The performance of culture improved greatly in all centers: HA, P = 0.003; CO, P = 0.02; and DU, P = 0.0003. 

The data for the specimens treated by method C are shown in Table 3. The AMPLICOR PCR performance did not differ from that for specimens treated by method A or B. The sensitivity of the 16S rRNA PCR, however, was significantly higher than that for methods A and B (P < 0.00001). The performance of the 16S rRNA PCR was not biased by time lapses and storage periods between AMPLICOR PCR and 16S rRNA PCR testing (data not shown). The sensitivity of culture, however, decreased from 77.8 to 58.8% (P = 0.004), and a decrease was observed in all centers: from 81.3 to 68.4% in HA (P = 0.46), from 80.6 to 62.2% in CO (P = 0.07), and from 73.7 to 47.7% in DU (p 0.03).

The AMPLICOR C. trachomatis-N. gonorrhoeae PCR has been validated for use with 2-SP medium, but extended evaluation studies have not yet been published (3, 4, 6, 11). Our motivation to switch from 2-SP treatment to diluted PBS was based on our laboratory's extensive experience with PCR testing for other pathogens. Applying this processing to samples collected during the last study period resulted in significantly improved performance of the 16S rRNA PCR. Samples suspended in 1:10-diluted PBS showed a high correlation between AMPLICOR PCR and 16S rRNA PCR: 134 out of 142 (94.4%) AMPLICOR-positive samples were positive on 16S rRNA PCR. On the basis of these data, the 16S rRNA PCR was proven to be a very accurate confirmatory assay.