Evaluation of real time polymerase chain reaction assays for confirmation of Neisseria gonorrhoeae in clinical samples tested positive in the Roche Cobas Amplicor assay

Abstract

Objective: Development of a rapid, sensitive, and accurate assay for confirmation of Neisseria gonorrhoeae in clinical samples. Method: Two real time polymerase chain reaction (PCR) assays, developed on the LightCycler for amplification of the N gonorrhoeae cppB gene, were utilised for confirmation of this bacterial pathogen in samples positive by the Roche Cobas Amplicor assay. Performance characteristics of the two assays were compared with other commercial nucleic acid amplification assays, including the Abbott LCx and Roche 16S rRNA tests. Results: All related Neisseria as well as other bacterial species tested negative by both cppB gene based assays, whereas 120 N gonorrhoeae clinical isolates from various geographical regions gave in positive results. Both assays had a sensitivity of one copy per reaction. 122 clinical samples positive and another 50 samples negative for N gonorrhoeae by Roche Cobas Amplicor were selected from a specimen pool of more than 3000 women tested previously. Overall, 73 of 122 (59.8%) samples were confirmed as positive. The two real time assays had sensitivities of 99% and 100% and specificities of 98% and 100%, respectively. The 16S and LCx assays produced similar results to the real time assays, indicating a similar sensitivity to and specificity of both real time assays. Conclusion: The data from this study highlight the need to confirm N gonorrhoeae positive Cobas Amplicor PCR results as an important part of the testing algorithm of all diagnostic laboratories utilising this assay.

Prevalencia de Chlamydia trachomatis y Neisseria gonorrhoeae en adolescentes de colegios de la zona Sabana Centro de Cundinamarca, Colombia

“…En cuanto a la infección por N. gonorrhoeae, solo se encontró un caso en una mujer asintomática, hallazgo que coincide con otros reportes que demuestran una alta proporción de casos asintomáticos de esta infección en las mujeres (12). La ausencia de infección concomitante difiere de lo hallado en otros estudios en los Estados Unidos, en los que se ha encontrado que ésta se presenta hasta en 50 % de las mujeres de 15 a 19 años que consultan en los servicios de salud (14); sin embargo, en otras poblaciones del mismo país se ha informado una proporción más baja de infección concomitante, como es el caso de un grupo de adolescentes asistentes a colegios de Nueva York, en el que la proporción fue de 3,5 % (35).…”

Nucleic Acid Amplification Testing for Neisseria gonorrhoeae

“…Recently, two independent studies investigated the use of N. gonorrhoeae cppB PCR assays on populations in Australia and the Netherlands. 50, 51 Tabrizi et al 51 found no false-negative results and considered the cppB gene assay to be suitable for use in a population in Victoria, Australia, whereas Bruisten et al 50 found a high incidence (5.8%) of false-negative results and concluded that the cppB gene target was not suitable for use in the Dutch population. Interestingly, other Australian studies have revealed a high incidence of cppB-related false-negative PCR results in a patient population in the Northern Territory in Australia, due to the recent appearance and expansion of a non-PAU Ϫ gonococcal subtype.…”

“…57,72 Supplementary tests for the Roche Amplicor assay have included a 16S PCR assay provided by Roche as well as various in-house PCR methods targeting the cppB gene. 51,64,90 Roche later withdrew the 16S assay from the marketplace, so cppB gene-based PCR assays were widely adopted for confirmation. However, cppB gene-based NAAT assays are now no longer considered adequate for some patient populations because of the limitations outlined previously.…”

Validation of Roche COBAS Amplicor Assay for Detection of Chlamydia trachomatis in Rectal and Pharyngeal Specimens by an omp1 PCR Assay

“…An additional PCR for amplification of ␤-globin sequence was conducted for detection of inhibition and sample adequacy (22).…”