Rapid detection and strain typing of Chlamydia trachomatis using a highly multiplexed microfluidic PCR assay

Abstract

Nucleic acid amplification tests (NAATs) are recommended by the CDC for detection of Chlamydia trachomatis (Ct) urogenital infections. Current commercial NAATs require technical expertise and sophisticated laboratory infrastructure, are time-consuming and expensive, and do not differentiate the lymphogranuloma venereum (LGV) strains that require a longer duration of treatment than non-LGV strains. The multiplexed microfluidic PCR-based assay presented in this work simultaneously interrogates 13 loci to detect Ct and identify LGV and non-LGV strain-types. Based on amplified fragment length polymorphisms, the assay differentiates LGV, ocular, urogenital, and proctocolitis clades, and also serovars L1, L2, and L3 within the LGV group. The assay was evaluated in a blinded fashion using 95 clinical swabs, with 76 previously reported as urogenital Ct-positive samples and typed by ompA genotyping and/or Multi-Locus Sequence Typing. Results of the 13-plex assay showed that 51 samples fell within urogenital clade 2 or 4, 24 samples showed both clade 2 and 4 signatures, indicating possible mixed infection, gene rearrangement, or inter-clade recombination, and one sample was a noninvasive trachoma biovar (either a clade 3 or 4). The remaining 19 blinded samples were correctly identified as LGV clade 1 (3), ocular clade 3 (4), or as negatives (12). To date, no NAAT assay can provide a point-of-care applicable turnaround time for Ct detection while identifying clinically significant Ct strain types to inform appropriate treatment. Coupled with rapid DNA processing of clinical swabs (approximately 60 minutes from swab-in to result-out), the assay has significant potential as a rapid POC diagnostic for Ct infections.

Fig 1. Generated 13-plex PCR electropherograms from six representative Ct strains: LGV strains L1/440, L2/434 and L3/404, urogenital strain E/Bour, proctocolitis strain H/UW-43/Cx and ocular strain A/Har-13.

The FAM (blue), TMR (yellow but presented as black in the output profiles) and ROX (red) labeled-FLTs are aligned to illustrate amplicon sizes from the set of six strains. Targets and amplicon sizes in bases (b) are indicated to show variations observed among strains in the TMR and ROX channels (Peak 1 = IGS-101; Peak 2 = IGS-102; Peak 3 = IGS-103; Peak 4 = mdhC; Peak 5 = IGS-104; Peak 6 = IGS-105; Peak 7 = ompA; Peak 8 = IGS-106; and Peak 9 = IGS-107). Amplicon sizes in FAM are conserved and not numbered. The internal control amplicon is JOE-labeled (green). X-axis: fragment size in bases; Y-axis: relative fluorescence units (RFU).

Fig 2. Specificity of the 13-plex Ct amplification assay using L1/440 Ct strain in the presence (A) or absence of Neisseria gonorrhoeae and human genomic DNAs as background DNAs (B).

Strain L1/440 was tested at 100 genomic equivalents, and Neisseria gonorrhoeae and human genomic DNAs at 100,000 and 10,000 genomic equivalents, respectively. Background DNA had no significant effect on the characteristic Ct signature based on amplicon and internal control peak heights, and no nonspecific peaks were observed. X-axis: fragment size in bases; Y-axis: RFU.