A multiplex real-time PCR subtyping assay combining chromosomal (pmpH) and plasmid (pgp3) markers for differentiation of LGV and non-LGV Chlamydia trachomatis.

Lymphogranuloma venereum (LGV), caused by invasive Chlamydia trachomatis genovars L1-L4, requires timely differentiation from non-LGV strains to guide appropriate treatment and to strengthen case-based surveillance. We aimed to develop and validate a multiplex real-time PCR assay for simultaneous detection and differentiation of LGV and non-LGV C. trachomatis, targeting an LGV-associated chromosomal pmpH deletion, an LGV-associated single-nucleotide polymorphism in the plasmid pgp3 gene, and the human RNaseP gene as an internal control. Primers and probes were adapted from published assays and optimized for multiplex performance. Analytical validation was performed using synthetic gBlocks, cultured LGV and non-LGV strains, and Quality Control for Molecular Diagnostics (QCMD) proficiency panels. Clinical evaluation was conducted using 239 C. trachomatis NAAT-positive specimens submitted to the National Microbiology Laboratory for LGV detection and previously characterized using a pmpH-based assay. All targets demonstrated efficient amplification (PCR efficiency ~100%) with no cross-reactivity. The limit of detection was 5-50 copies/reaction, and analytical specificity for LGV/non-LGV differentiation was 100%; results were fully concordant with QCMD panels. Clinical testing showed complete agreement with the original pmpH assay for LGV-positive, non-LGV, and dual-positive specimens, while improving classification of indeterminate and previously pmpH-negative samples. This assay enables improved, single-reaction LGV detection to support routine public health laboratory workflows and surveillance.IMPORTANCEAccurate differentiation of lymphogranuloma venereum (LGV) from non-LGV Chlamydia trachomatis is essential for appropriate treatment and effective surveillance. Current typing methods have reduced sensitivity in low-bacterial-load specimens, resulting in missed cases and reduced surveillance accuracy. We developed a multiplex real-time PCR assay that simultaneously detects and differentiates LGV and non-LGV C. trachomatis using chromosomal and plasmid targets. This assay provides a streamlined and reliable approach for LGV detection. Implementation of the assay will improve turnaround time for actionable results, support appropriate treatment decisions, and reduce non-typeable results.