Development, validation and testing costs of an in-house real-time PCR assay for the detection of Chlamydia trachomatis

Purpose. To improve the screening of Chlamydia trachomatis (C. trachomatis) in Brazil, an accurate and affordable method is needed. The objective of this study was to develop and assess the performance and costs of a new in-house real-time PCR (qPCR) assay for the diagnosis of C. trachomatis infection. Methodology. Asymptomatic women aged 14-25 years who attended primary health services in Manaus, Brazil, were screened for C. trachomatis using the Digene Hybrid Capture II CT-ID (HCII CT-ID) DNA test. A subset of cervical specimens were tested using an in-house qPCR and a commercial qPCR, Artus C. trachomatis Plus RG PCR 96 CE (Artus qPCR) kit, as a reference test. A primer/probe based on the sequence of cryptic plasmid (CP) was designed. An economic evaluation was conducted from the provider's perspective. Results. The primers were considered specific for C. trachomatis because they did not amplify any product from non-sexually transmitted bacterial species tested. Overall, 292 specimens were tested by both the commercial kit (Artus qPCR) and the in-house qPCR. Of those, one resulted in no amplification and was excluded from the analysis. The sensitivity, specificity, and positive and negative predictive values of the in-house qPCR were 99.5% [95% confidence interval (CI): 97.1-100], 95.1% (95% CI: 89-98.4), 97.4% (95% CI: 94-99.1) and 99.0% (95% CI: 94.5-100), respectively. The cost per case of C. trachomatis was 0.44 pound ($0.55) for HCII CT-ID, 1.16 pound ($1.45) for Artus qPCR and 1.06 pound ($1.33) for in-house qPCR. Conclusion. We have standardized an in-house qPCR to detect cervical C. trachomatis targeting CP. The in-house qPCR showed excellent accuracy and was more affordable than the commercial qPCR kit.