Introduction The causative agents of venereal syphilis ( Treponema subsp. pallidum), yaws (subsp. pertenue), and bejel (subsp. endemicum) are indistinguishable on the basis of morphology, syphilis serology and existing diagnostic PCR. Our objective was to develop a real-time (rt) multiplex PCR assay that can simultaneously detect all three organisms in a single tube. In addition, we evaluated the potential use of FTA Elute cards for collection of skin lesion specimens for molecular testing.

Methods Serial dilutions of purified genomic DNA from T. pallidum were spotted onto FTA cards and air dried. Cards were stored at ambient temperature (∼23^oC) for up to 1 month. DNA was eluted from a 6-mm disc and tested by a polAPCR, a rt PCR for macrolide point mutations in the 23S rRNA gene, and the CDC typing method (PCR/RFLP analysis of tpr and arp). The rt triplex PCR targets the tp858 and tp0620 genes and the assay was evaluated using DNA from 25 strains of T. pallidum, 14 strains of T. pertenue, and 2 strains of T. endemicum.

Results The rt triplex PCR distinguished all three subspecies and had an analytical sensitivity of 1–10 genomic copies using purified DNA from T. pallidum strain Nichols. The detection limit of polA rt PCR was approximately 10–100 genomic copies/6-mm disc. The molecular subtype and azithromycin susceptibility genotype was easily determined using the spiked FTA cards.

Conclusions The rt triplex PCR is a specific and sensitive assay for differentiation of the T. pallidum subspecies and should be useful in areas where both syphilis and yaws or bejel are endemic and, in determining the extent of yaws worldwide. The FTA Elute card provides a simple way to collect, store and transport specimens at ambient temperature in the absence of a cold chain and involves minimal sample processing prior to molecular testing.