Development of a Multiplex Real-Time Polymerase Chain Reaction Test for Detection of Chlamydia Trachomatis and Neisseria Gonorrhoeae, and Its Adaption to a Point of Care Nucleic Acid Amplification and Detection System

Background:  Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are two of the most prevalent sexually transmitted infections (STIs). Nucleic acid amplification testing (NAAT) is recommended for diagnosis due to high sensitivity and specificity. Maxim has developed a multiplex real time PCR test (MqPCR) for detection of CT/NG in a single reaction. Utility of NAAT at point of care (POC) has been limited by complexity of technique and equipment.  We additionally developed this assay with a portable device: “POCKIT” that utilizes insulated isothermal PCR (iiPCR). POCKIT is rapid (<1 hour), limited in complexity (no sample preparation required) and no data analysis, results are automatically reported as plus/minus.

Methods: Analytical sensitivity studies were performed for MqPCR on AB7500 and on POCKIT using purified DNA and titered CT and NG strains diluted in negative matrix and heated at 95°C.  A specificity panel of 26 closely related microorganisms/normal flora were also tested.  Clinical performance evaluation was done using 65 stored urogenital specimens previously characterized and de-identified.

Results:  The sensitivity of MqPCR and POCKIT, were equivalent detecting 25 copies/reaction or < 1 IFU or CFU/ml for CT and NG. Specificity testing utilizing >1X10⁵ copies/reaction of each organism demonstrated 100% specificity. Clinical sensitivity of the MqPCR test was 95% and 92.8% for CT and NG, respectively, while clinical specificity was 95.3% and 100% for CT and NG, respectively. For POCKIT, clinical sensitivity and specificity was 93.3% and 86.6%, respectively for CT, while for NG clinical sensitivity and specificity were equivalent to the MqPCR assay.

Conclusions: Preliminary studies indicated equivalent sensitivity and specificity for CT and NG utilizing either MqPCR or POCKIT. Further study examining a larger number of clinical samples is required.  POCKIT can serve as a rapid, affordable tool for detection of CT/NG and other STIs in point of care and low resource settings.