Background The goal of the study was to evaluate two sequence-based subtyping methods for their ability to further differentiate Treponema pallidum strains characterised by the CDC typing system.

Methods 12 clinical specimens with the 14d strain type obtained from patients with GUD in Cape Town in 2000; 14 specimens with the 14d strain type from patients with primary or secondary syphilis in San Francisco collected between 2004 and 2007; and 12 clinical specimens with the 14d strain type collected between 2000 and 2002 from a syphilis outbreak in Vancouver were included in the study. Specimens were previously characterised using the CDC typing method which involves analysis of the sequence variability with tprE, G, and J and, the variable number of 60-bp tandem repeats within the arp gene. Subtyping was performed by PCR amplification of a homonucleotide G" tandem repeat within tp0279 and a variable region within tp0548. PCR amplicons were purified and directly sequenced using the BigDye® Terminator v3.1 cycle sequencing kit and an ABI 3130 sequencer.

Results Of the 12 samples from South Africa, sequence analysis of tp0279 revealed three subtypes (9G, 10G, 11G) and combining this data with the CDC typing method produced strain subtypes 14d9, 14d10, and 14d11. Sequence analysis of the variable region within tp0548 resulted in five subtypes designated a, c, f, k, and l among these samples and, combination with strain type using the CDC method produced subtypes 14d/a, 14d/c, 14d/f, 14d/k, 14d/l. All 14 samples from San Francisco had 9G tandem repeats within tp0279 resulting in subtype 14d9. Sequence analysis of the variable region within tp0548 resulted in 3 subtypes designated f, g and j among these 14 samples and incorporation of the CDC typing method produced subtypes 14d/f, 14d/g, and 14d/j. Of the 12 samples from Vancouver, sequence analysis of tp0279 produced two subtypes (8G, 9G) resulting in strain subtypes 14d8 (1/12) and14d9 (11/12). Sequencing of tp0548 also produced two subtypes (e, f) resulting in strain subtypes 14d/e (1/12) and14d/f (11/12).

Conclusions The tp0279 subtyping method further differentiated 14d strains into four subtypes while the tp0548 method differentiated 14d strains into eight subtypes. Subtyping of strains from the initial syphilis outbreak in Vancouver suggests clonal spread of T pallidum. Both subtyping methods enhanced the original CDC typing method and appear to be promising tools for molecular epidemiological studies on syphilis.