A multiplexed real-time PCR assay for rapid detection of Chlamydia trachomatis and identification of serovar L-2, the major cause of Lymphogranuloma venereum in New York

Lymphogranuloma venereum (LGV) is caused by a rare form of Chlamydia trachomatis that is difficult to diagnose, since culture is not readily available, and since other methods are not reliable or lack sensitivity. We report here a rapid, sensitive, and specific real-time multiplex polymerase chain reaction (PCR) assay capable of detecting C. trachomatis and identifying serovar L-2 in the same reaction, directly from rectal swabs. The analytical sensitivity of the assay was 25 genome copies for C. trachomatis, and 50 genome copies for L-2. The analytical specificity was 100%, as demonstrated with a diverse range of C. trachomatis serovars and other site-specific bacterial pathogens. With the use of a rapid DNA extraction method, a blinded validation of spiked rectal swabs correctly identified 30 samples containing C. trachomatis cells, L-2 DNA, or negative samples. The multiplexed PCR assay also identified serovar L-2 in 13 of 70 rectal swab samples taken from symptomatic patients. Twelve additional samples were positive for C. trachomatis only, and omp1 sequencing determined these samples as either serovar D, E, G, J, or K. This assay represents the first real-time PCR method capable of detecting C. trachomatis DNA, and of simultaneously identifying C. trachomatis infection as serovar L-2.