Validation of a laboratory-developed real-time PCR protocol for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine.

ABSTRACT

OBJECTIVE: To evaluate a sensitive and specific, real-time PCR assay with internal control for Chlamydia trachomatis and Neisseria gonorrhoeae DNA detection in urine specimens. METHODS: The diagnostic performance of a laboratory-developed quadruplex assay (LDQA) targeting the cryptic plasmid and MOMP genes of C trachomatis, the porA pseudogene of N gonorrhoeae and a synthetic internal control was assessed using 1028 urine specimens. The LDQA was compared with the Roche COBAS Taqman CT test and the COBAS Amplicor NG assay with supplemental confirmation tests. The subsequent performance of the LDQA in detecting N gonorrhoeae was monitored in comparison with bacterial culture from swabs. RESULTS: 88 (8.6%) urines were determined as C trachomatis positive in the diagnostic evaluation. LDQA sensitivity and specificity were calculated to be 100% and 99.9%, respectively, for C trachomatis. The LDQA showed high specificity with isolates of other Neisseria species and gave complete concordance with resolved data for N gonorrhoeae detection. However, the incidence of N gonorrhoeae infection was low, with 17 (1.7%) positive patients. A post-implementation audit of 14 316 patients gave the LDQA N gonorrhoeae urine PCR protocol (porA, OPA, 16s rDNA) a sensitivity of 96.9% and specificity of 99.8% in comparison with bacterial culture from swabs. CONCLUSIONS: The LDQA was found to be an effective method for the detection of C trachomatis and N gonorrhoeae DNA in urine samples, and the PCR protocol has replaced bacterial culture for the screening of N gonorrhoeae in asymptomatic men and women in the laboratory.