Clinical Validation of Loop-Mediated Isothermal Amplification Assays for the Rapid Detection of Neisseria gonorrhoeae and Chlamydia trachomatis

Abstract

Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) are the predominant causes of bacterial sexually transmitted diseases. While nucleic acid amplification testing (NAATs), primarily polymerase chain reaction (PCR), is regarded as the gold standard for identifying these two pathogens, it usually takes a prolonged turnaround time and requires sophisticated equipment with considerable expense. In this study, we developed novel loop-mediated isothermal amplification (LAMP) assays for rapid detection (< 30 min) of GC and CT in clinical urine and swab specimens. We analysed 208 clinical samples with three different pre-treatment techniques including heating inactivation, centrifugation, and DNA extraction. LAMP results were compared with clinical results from the FDA-approved BD ProbeTec ET assay. After heating inactivation, LAMP detected merely 41% and 65% of BD-identified GC- and CT-positive samples, respectively. Introducing centrifugation as an affordable and rapid pre-treatment step increased detection rates to 81% and 91% for GC and CT, respectively. DNA extraction further enhanced the detection rates to 96% and 95% for GC- and CT-LAMP, respectively. All these LAMP assays exhibited clinical specificity of ≥ 98%, underscoring the specificity of the chosen target genes (the porA pesudogene for GC and the ftsK gene for CT). Discrepant samples were verified by real-time PCR; results were consistent with our LAMP findings. The overall LAMP performance met the WHO criteria for sensitivity and specificity for GC/CT point-of-care testing.

Keywords: Chlamydia trachomatis; Neisseria gonorrhoeae; LAMP assay; Sexually Transmitted Infectious; diagnosis; point‐of‐care testing (POCT).

FIGURE 1

Three different sample pre‐treatments were employed before LAMP detections.

FIGURE 2

The amplification efficiency of primer sets targeting porA pseudogene in GC (a) qLAMP assay using the porA gene fragment with two different primer sets, porA_1 and porA_2. For each template concentration, the LAMP reaction was performed in triplicates, and the results were presented as mean ± SEM. (b) The analytical sensitivity of porA_1 primer set. The porA_1 primer set can detect 36 copies of gene fragments per reaction within 20 min, but only one out of three replicates was detected.

FIGURE 3

The amplification efficiency of primer sets targeting ftsK gene in CT (a) qLAMP assay using the ftsK gene fragment with two different primer sets, ftsK_1 and ftsK_2. For each template concentration, the LAMP reaction was performed in triplicates, and the results were presented as mean ± SEM. (b) The analytical sensitivity of ftsK_1 primer set. The ftsK_1 primer set can detect 22 copies of gene fragments per reaction within 20 min, but only two out of three replicates were detected.

FIGURE 4

The distribution of different specimen types in the total clinical samples, in female and male specimens.