Blind evaluation of the microwave-accelerated metal-enhanced fluorescence ultrarapid and sensitive Chlamydia trachomatis test by use of clinical samples

Abstract

Accurate point-of-care (POC) diagnostic tests for Chlamydia trachomatis infection are urgently needed for the rapid treatment of patients. In a blind comparative study, we evaluated microwave-accelerated metal-enhanced fluorescence (MAMEF) assays for ultrafast and sensitive detection of C. trachomatis DNA from vaginal swabs. The results of two distinct MAMEF assays were compared to those of nucleic acid amplification tests (NAATs). The first assay targeted the C. trachomatis 16S rRNA gene, and the second assay targeted the C. trachomatis cryptic plasmid. Using pure C. trachomatis, the MAMEF assays detected as few as 10 inclusion-forming units/ml of C. trachomatis in less than 9 min, including DNA extraction and detection. A total of 257 dry vaginal swabs from 245 female adolescents aged 14 to 22 years were analyzed. Swabs were eluted with water, the solutions were lysed to release and to fragment genomic DNA, and MAMEF-based DNA detection was performed. The prevalence of C. trachomatis by NAATs was 17.5%. Of the 45 samples that were C. trachomatis positive and the 212 samples that were C. trachomatis negative by NAATs, 33/45 and 197/212 were correctly identified by the MAMEF assays if both assays were required to be positive (sensitivity, 73.3%; specificity, 92.9%). Using the plasmid-based assay alone, 37/45 C. trachomatis-positive and 197/212 C. trachomatis-negative samples were detected (sensitivity, 82.2%; specificity, 92.9%). Using the 16S rRNA assay alone, 34/45 C. trachomatis-positive and 197/212 C. trachomatis-negative samples were detected (sensitivity, 75.5%; specificity, 92.9%). The overall rates of agreement with NAAT results for the individual 16S rRNA and cryptic plasmid assays were 89.5% and 91.0%, respectively. Given the sensitivity, specificity, and rapid detection of the plasmid-based assay, the plasmid-based MAMEF assay appears to be suited for clinical POC testing.

Fig 1

(Top left) Cartoon depicting microwave-accelerated heating above MEF substrates. (Top right) MAMEF assay construct for detection of chlamydial DNA. (Bottom) Anchor and fluorescent probe sequences. SH, sulfhydryl group for attachment of DNA to the silver surface; TMR, TAMRA NHS ester dye; Mw, microwave heating; MEF, metal-enhanced fluorescence; F, fluorophore.

Fig 2

Flow chart showing the sequence of events and timeline for a MAMEF-based C. trachomatis test.

Fig 3

Classification of MAMEF assay results according to fluorescence intensity. CT, C. trachomatis; AU, arbitrary units; 16S, 16S rRNA MAMEF assay; plasmid, cryptic plasmid MAMEF assay; A and B, duplicate tests; 2W, second wash. Sample 1 showed low fluorescence intensity below the threshold of positivity (dashed line), characteristic of a C. trachomatis-negative sample. For sample 2, elevated signal intensity was detected initially but intensity decreased below the threshold of positivity after a second washing step. Sample 3 showed high signal intensity before and after the second washing step, indicative of strong binding of the target C. trachomatis DNA to the DNA capture probes.

Fig 4

Inner configuration of the optical reader for the C. trachomatis assays. The bitruncated fiber both excites and collects metal-enhanced fluorescence emission from the silver-coated wells.

Fig 5

Serial dilution plot for the cryptic plasmid-based MAMEF assay. The log(IFU/ml) values for C. trachomatis were plotted against fluorescence intensity (R² = 0.9801).

Fig 6

Outer configuration of the optical reader for the C. trachomatis MAMEF assays. The inner workings of the reader are shown in Fig. 4.