A Multiplexed Microfluidic PCR Assay for Sensitive and Specific Point-of-Care Detection of Chlamydia trachomatis

Abstract

Background Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted diseases (STD) worldwide. While commercial nucleic acid amplification tests (NAAT) are available for Ct, none are rapid or inexpensive enough to be used at the point-of-care (POC). Towards the first Ct POC NAAT, we developed a microfluidic assay that simultaneously interrogates nine Ct loci in 20 minutes.Methodology and Principal FindingsEndocervical samples were selected from 263 women at high risk for Ct STDs (∼35% prevalence). A head-to-head comparison was performed with the Roche-Amplicor NAAT. 129 (49.0%) and 88 (33.5%) samples were positive by multiplex and Amplicor assays, respectively. Sequencing resolved 71 discrepant samples, confirming 53 of 53 positive multiplex samples and 12 of 18 positive Amplicor samples. The sensitivity and specificity were 91.5% and 100%, and 62.4% and 95.9%, respectively, for multiplex and Amplicor assays. Positive and negative predictive values were 100% and 91%, and 94.1% and 68.6%, respectively.ConclusionsThis is the first rapid multiplex approach to Ct detection, and the assay was also found to be superior to a commercial NAAT. In effect, nine simultaneous reactions significantly increased sensitivity and specificity. Our assay can potentially increase Ct detection in globally diverse clinical settings at the POC.

Diagnosis and Management of UncomplicatedChlamydia trachomatisInfections in Adolescents and Adults: Summary of Evidence Reviewed for the 2015 Centers for Disease Control and Prevention Sexually Transmitted Diseases Treatment Guidelines

“…The mean incremental cost-effectiveness ratio indicated that the POC strategy would save $28 in total and avert 14 PID cases. Dean et al evaluated the performance of a novel microfluidic multiplex PCR POC test on cervical swabs compared with the Roche Amplicor NAAT (PCR) and found that the sensitivity and specificity for the multiplex assay was 91.5% and 100% vs 62.4% and 95.9% for NAAT; discordant results between the multiplex assay and NAAT were evaluated using microfluidic Sanger sequencing [66]. Pearce et al evaluated the performance of a novel CT POC test using an electrochemical detection method on 306 samples previously pretyped as either CT positive (n = 107) or CT negative (n = 199) by TMA or PCR and found that the sensitivity and specificity for the novel POC test were 98.1% and 98.0%, respectively [67].…”

“…Recent studies reported good performance of novel investigational CT POC tests that appear promising [65][66][67].…”

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

“…Cost, simplified operating procedures and selection of classifier cut-off values that are compatible with programmatic goals are probably more important criteria on which to base the selection of a test for use in trachoma control programs. Some technical improvement can be achieved for these DNA-based tests by targeting multiple target sequences, which can enhance sensitivity at low analyte concentrations [8] and this needs to be explored in the ddPCR system. Indeed, the Abbott RealTime CT/NG assay targets two CT sequences, which is likely to increase its sensitivity compared with single target assays; however, the potential enhancement should only be measured by an evaluation that compares equivalent aliquot sizes.…”

Frequency of Chlamydia trachomatis in Endocervical Samples of Women Referred to a Gynecology Hospital in Qazvin, Iran

“…These techniques however, fail to detect a substantial proportion of infections. Newer tests are accordingly required to be developed that could amplify and detect C. trachomatis-specific DNA or RNA sequences, with greater sensitivity than first generation non-culture-based tests (10)(11)(12)(13)(14). In many studies, the target gene for nucleic acid amplification tests (NAATs) has been the cryptic plasmid.…”

“…Thus, infections caused by plasmidfree variants will be undetected if the plasmid is used as the target gene (12). Targets for amplification have included both cryptic plasmid (7, 8, 10, 18, 19, 22 -24, 27) and chromosomal genes, including those for the major outer membrane protein (MOMP) (2,11,23,25,33), gyrA and phospholipase D endonuclease superfamily gene (PRPHA).…”