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.

5-Color Multiplexed Microwave-Accelerated Metal-Enhanced Fluorescence: Detection and Analysis of Multiple DNA Sequences from within one Sample Well within a Few Seconds

“…Our MAMEF Chlamydia assays have also been compared to two PCR assays recently in a blinded clinical study using vaginal swabs taken from 270 women. Our CT assay was found to be≈90 % concordant with regard to assay sensitivity and specificity as compared to both PCR tests [12]. In another recent study we have shown the<10 cfu detection of Toxins A and B from the human pathogen Clostridium difficile in 40 seconds using MicrowaveAccelerated Metal-Enhanced Fluorescence from stool samples [13].…”

“…In addition, MAMEF does offer a much quicker speed to assay completion / detection, typically less than 30 s due to microwave-acceleration, and can potentially be performed at a much lower cost. Recently our PLOS 1 paper on the 20 s 1 cfu/ml detection of salmonella and our paper on the ultra-fast ultra-sensitive <10 cfu/ml detection of Chlamydia have been published [10][11][12], which underscores the significant potential of our approach for detecting low levels of disease burden at PCR equivalent levels, the assumption being that we know the target we are looking for. Our MAMEF Chlamydia assays have also been compared to two PCR assays recently in a blinded clinical study using vaginal swabs taken from 270 women.…”

Point of care diagnostics for sexually transmitted infections: perspectives and advances

“…Another new POC CT technology in development is the microwave accelerated metal-enhanced fluorescence (MAMEF) assay [41–43]. The MAMEF assay can detect approximately 10 inclusion-forming units/ml of CT in <9 min, including DNA extraction and detection.…”

“…The MAMEF assay can detect approximately 10 inclusion-forming units/ml of CT in <9 min, including DNA extraction and detection. Using a plasmid-based assay on archived clinical samples, sensitivity and specificity were 82.2% (37/45) and 92.9% (197/212), respectively [43]. In the assay, target DNA sequences bind to a fluorophore-labeled probe and additionally an anchor probe covalently bound to the assay surface.…”

“…If target CT DNA sequence is present, metal enhanced fluorescence occurs through close proximity of the fluorophore to silver metallic nanoparticles. Sample lysis utilizes gold bowtie geometries, which highly focus microwaves onto clinical samples [43]. …”