Comparison of COBAS AMPLICOR Neisseria gonorrhoeae PCR, Including Confirmation with N. gonorrhoeae- Specific 16S rRNA PCR, with Traditional Culture

Abstract

A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased the positive predictive value from 54.8 to 96.6%.For the detection of Neisseria gonorrhoeae, culture is still used by many laboratories. However, recent studies showed that nucleic acid amplification-based N. gonorrhoeae tests (NAATs) are much more sensitive (1,2,4,6,7,10). The Roche COBAS AMPLICOR (CA) N. gonorrhoeae test is probably the most widely used amplification test for N. gonorrhoeae. It was shown that certain strains of N. subflava, N. cinerea, N. flavescens, N. lactamica, N. sicca, and Lactobacillus species may produce false-positive results with the CA N. gonorrhoeae test (3, 4, 5, 9, and Manual for COBAS AMPLICOR Neisseria gonorrhoeae PCR test, version 1.0, Roche Diagnostics) and some other N. gonorrhoeae NAATs (8). Therefore, it is clear that positive results obtained with the CA N. gonorrhoeae test should be confirmed by another test method (2,3,4,8,9). In the present study we evaluated the reliability of N. gonorrhoeae testing in a low-prevalence population, using the CA N. gonorrhoeae test as a screening assay and an N. gonorrhoeae-specific 16S rRNA PCR for confirmation.N. gonorrhoeae PCR and culture were performed on 3,023 specimens from nonselected patients (2,415 female and 608 male) obtained by visiting general practitioners (48%), general hospitals (32%), or venereal disease (VD) clinics (20%). The vast majority of the patients were symptomatic, and 75% of them were between 15 and 35 years of age. Over 95% of the specimens were urogenital swabs, and the remaining 5% were rectal, throat, or eye swabs. Swabs for PCR and culture were sent to the laboratory in 2-sucrose-phosphate medium and in charcoal transport medium, respectively. The CA N. gonorrhoeae PCR (Roche) was tested according to the manufacturer's instructions (Roche Diagnostics manual, version 1). A specimen was considered negative for N. gonorrhoeae if the optical density (OD) was Ͻ0.200 and the OD of the inhibition control (IC) was Ն0.200. The specimen was considered positive for N. gonorrhoeae if the OD was Ն0.200, regardless of the IC result. All specimens with an initial positive result were retested in the CA N. gonorrhoeae PCR, and the result of this repeat CA test was conclusive for the final interpretation of the CA N. gonorrhoeae test. For the 16S rRNA N. gonorrhoeae PCR test, new DNA extracts from the original clinical specimens were prepared. The 16S rRNA N. gonorrhoeae PCR test (Roche) was also performed according to the manufacturer's instructions (manual for Neisseria gonorrhoeae 16S rRNA PCR test, version 2, Roche Diagnostics) and included hybridization with an N. gonorrhoeae-specific probe added to microwell plates. Specimens repeatedly positive by CA N. gonorrhoeae PCR and positive by 16S rRNA N. gonorrhoeae PCR were con...

Aptima Combo 2 Testing Detected Additional Cases of Neisseria gonorrhoeae Infection in Men and Women in Community Settings

“…There were no culture-positive, NAAT-negative findings. These findings are similar to those of a previous study (10) comparing Cobas Amplicor (AMP) PCR to culture of 3,023 clinical specimens from woman and men, which demonstrated an increase from 58 to 85 positives, an increase of 46% due to PCR testing. Of 142 extra positive samples, 65 were available for confirmatory testing by AGC and 64 (98.5%) were confirmed to be positive.…”

“…Of 142 extra positive samples, 65 were available for confirmatory testing by AGC and 64 (98.5%) were confirmed to be positive. Confirmatory testing of GC Amplicor (AMP)-positive results in a previous study (10) showed a 56.1% rate of confirmation (87/155) using a 16S rRNA N. gonorrhoeae PCR (Roche), and only 57 were culture positive. This lower rate of confirmation may have been due to the AMP PCR crossreacting with nongonorrhoea Neisseria (NGN), a phenomenon which has been widely reported (6,7,17).…”

Neisseria Species Identification Assay for the Confirmation of Neisseria gonorrhoeae -Positive Results of the COBAS Amplicor PCR

“…Still, several cross-reacting nonpathogenic organisms, primarily other Neisseria spp., with homologous sequences are known to contribute to false-positive results (6,7,13,24,25). As recommended by the Centers for Disease Control and Prevention (5), confirmation of N. gonorrhoeae infection using independent testing for a different N. gonorrhoeaespecific target greatly improves the positive predictive value (14).…”

Loop-Mediated Isothermal Amplification Test for Detection of Neisseria gonorrhoeae in Urine Samples and Tolerance of the Assay to the Presence of Urea

“…NAAT offers unparalleled specificities and sensitivities, with PCR assays typically boasting sensitivities and specificities of Ͼ95% and 100%, respectively, compared with sensitivities of ϳ68% obtained via culture (3).…”