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.

Diagnosing sexually transmitted infections in resource‐constrained settings: challenges and ways forward

“…POCTs that are inexpensive, rapid and fulfil the ASSURED criteria are under development. These molecular assays include: the microwave‐accelerated metal‐enhanced fluorescence test, which needs to be simplified and standardized for basic laboratories ; a low‐cost NAAT called MobiNAAT, which uses a portable device where results are analysed in an automated smartphone diagnostic ; a POCT paper‐fluidic platform to diagnose gonorrhoea that is a highly sensitive molecular assay with visual lateral flow detection and an 80‐minute run time ; and a rapid multiplex microfluidic CT PCR‐based POCT comparable to laboratory‐based NAATs . A 15‐minute run‐time recombinase polymerase amplification‐based prototype POCT (TwistDx) for CT/NG has been reported to be comparable to laboratory‐based NAAT .…”

“…The potential use of molecular diagnostic assays in resource‐constrained settings is driving the development of lower‐cost solutions. Several new industry players have entered, or are entering the development space; however, these tests, previously mentioned , are mostly in the early stages of development – and it remains to be seen how these assays perform, and what the global access pricing strategies will be.…”

2019 European guideline on the management of lymphogranuloma venereum

“…There are multiplex NAATs for genital ulcerative disease that detect LGV but these have not yet been appropriately evaluated in the context of rectal LGV. Different in‐house or laboratory‐developed NAATs have been designed and used . The sensitivities of these NAATs are generally lower than the commercially available C. trachomatis screening NAAT …”

Differential Markers of Bacterial and Viral Infections in Children for Point-of-Care Testing

“…Multiplex microfluidic LAMP has been used in POCT to differentiate between several types of bacteria and viruses by amplifying and detecting their specific DNA sequences [ 93. , 94. , 95.…”