Reduced-cost Chlamydia trachomatis-specific multiplex real-time PCR diagnostic assay evaluated for ocular swabs and use by trachoma research programmes

Abstract

Introduction: Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), is the leading infectious cause of preventable blindness. Many commercial platforms are available that provide highly sensitive and specific detection of Ct DNA. However, the majority of these commercial platforms are inaccessible for population-level surveys in resource-limited settings typical to trachoma control programmes. We developed two low-cost quantitative PCR (qPCR) tests for Ct using readily available reagents on standard real-time thermocyclers.

Methods: Each multiplex qPCR test targets one genomic and one plasmid Ct target in addition to an endogenous positive control for Homo sapiens DNA. The quantitative performance of the qPCR assays in clinical samples was determined by comparison to a previously evaluated droplet digital PCR (ddPCR) test. The diagnostic performance of the qPCR assays were evaluated against a commercial assay (artus C. trachomatis Plus RG PCR, Qiagen) using molecular diagnostics quality control standards and clinical samples. We examined the yield of Ct DNA prepared from five different DNA extraction kits and a cold chain-free dry-sample preservation method using swabs spiked with fixed concentrations of human and Ct DNA.

Results: The qPCR assay was highly reproducible (Ct plasmid and genomic targets mean total coefficients of variance 41.5% and 48.3%, respectively). The assay detected 8/8 core specimens upon testing of a quality control panel and performed well in comparison to commercially marketed comparator test (sensitivity and specificity>90%). Optimal extraction and sample preservation methods for research applications were identified.

Conclusion: We describe a pipeline from collection to diagnosis providing the most efficient sample preservation and extraction with significant per test cost savings over a commercial qPCR diagnostic assay. The assay and its evaluation should allow control programs wishing to conduct independent research within the context of trachoma control, access to an affordable test with defined performance characteristics.

Keywords: Chlamydia trachomatis; Diagnosis; Quantitative PCR; Trachoma.

Graphical abstract

Fig. 1

Comparison of plasmid load estimate from qPCR compared to artus cycle threshold. (A). ABI 7900HT. (B). Rotor-Gene 3000. The main plots show concordant results (ABI n = 24, Rotor-Gene n = 20), side panels show discordant results. ND: Not detected.

Fig. 2

Agreement between load estimates from ddPCR and qPCR. (A) ABI 7900HT. (B) Rotor-Gene 3000. Main panels show concordant results, side bars show discrepant results. ND: Not detected.

Fig. 3

Recovery of (A) omcB and (B) pORF2 from Ct-spiked swabs by five different extraction kits. Boxes represent median, inter-quartile range and range of all swabs for each treatment. Circles represent swabs spiked with high-load elementary bodies, triangles represent medium-load spiking, and crosses represent low-load spiking. There is variation in the number of targets recovered by different extraction kits. Biochain and Qiagen cador kits appear to yield more Ct DNA than comparators.

Fig. 4

Change in recovered load of (A) Ct plasmid and (B) genomic targets following long-term storage frozen and at room temperature. Points represent mean of four swabs per time point per condition. Dashed lines represent linear regression model between load and time.