Development and Evaluation of a Next-Generation Digital PCR Diagnostic Assay for Ocular Chlamydia trachomatis Infections

Abstract

Droplet digital PCR (ddPCR) is an emulsion PCR process that performs absolute quantitation of nucleic acids. We developed a ddPCR assay for Chlamydia trachomatis infections and found it to be accurate and precise. Using PCR mixtures containing plasmids engineered to include the PCR target sequences, we were able to quantify with a dynamic range between 0.07 and 3,160 targets/μl (r2 = 0.9927) with >95% confidence. Using 1,509 clinical conjunctival swab samples from a population in which trachoma is endemic in Guinea Bissau, we evaluated the specificity and sensitivity of the quantitative ddPCR assay in diagnosing ocular C. trachomatis infections by comparing the performances of ddPCR and the Roche Amplicor CT/NG test. We defined ddPCR tests as positive when we had ≥95% confidence in a nonzero estimate of target load. The sensitivity of ddPCR against Amplicor was 73.3% (95% confidence interval [CI], 67.9 to 78.7%), and specificity was 99.1% (95% CI, 98.6 to 99.6%). Negative and positive predictive values were 94.6% (95% CI, 93.4 to 95.8%) and 94.5% (95% CI, 91.3 to 97.7%), respectively. Based on Amplicor CT/NG testing, the estimated population prevalence of C. trachomatis ocular infection was ∼17.5%. Receiver-operator curve analysis was used to select critical cutoff values for use in clinical settings in which a balance between higher sensitivity and specificity is required. We concluded that ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections.

Sensitive detection of Porcine circovirus-2 by droplet digital polymerase chain reaction

“…Because absolute quantification and enhanced detection sensitivity can be achieved, ddPCR has been used in various research applications, such as in microorganism detection 12 and biomedical research. 3 In our study, a specific and sensitive diagnostic method based on ddPCR for the detection of PCV-2 was established.…”

Clinical Utility of Droplet Digital PCR for Human Cytomegalovirus

“…Several studies have previously investigated the potential of droplet digital PCR (ddPCR) in molecular diagnostics for pathogens, including assays for chlamydia (8), HIV (9, 10), CMV (11), and chromosomally integrated human herpesvirus 6 (HHV-6) (12). In the previous CMV study, investigators concluded that quantitative PCR showed greater sensitivity and less variability than ddPCR in clinical samples (11).…”

Will droplet digital PCR become the test of choice for detecting and quantifying ocularChlamydia trachomatisinfection? Maybe

“…However, we suggest for the reasons outlined below that the sensitivity estimates presented in our evaluation are likely misleading. The issues behind calculating sensitivity in ddPCR and the reasons why this appeared 'low' given the technical superiority of digital PCR have been discussed [1,3]. The low sensitivity that we reported is most likely explained simply by sampling error, which is a problem that is unavoidable at low analyte concentrations in any test system [4], as reported by Schachter et al in 2006 [5].…”

“…Julius Schachter offers a provocative title in his expert review of the diagnostic Chlamydia trachomatis droplet digital PCR (CT ddPCR) assay developed by Roberts et al [1,2]. He put forward four main objections to the utility of this test in the face of other next-generation commercial nucleic acid amplification test platforms.…”