Molecular subtyping of Treponema pallidum subspecies pallidum

Background and Objectives: Epidemiologic studies on syphilis have been hampered by the fact that strains of Treponema pallidum subspecies pallidum (T, pallidum), the causative agent of this disease, cannot be differentiated by either protein-based or deoxyribonucleic acid-based methods. Syphilis is endemic in many developing countries and is common in some industrialized nations, In addition, the disease has been shown to increase the risk of infection with the human immunodeficiency virus. Goal: To develop a molecular subtyping method for T, pallidum. Study Design: Two genes exhibiting intrastrain variability were identified as potential targets for strain differentiation: the acidic repeat protein (arp) gene, which contains a variable number of 60 base pair repeats, and a member of the treponema pallidum repeat (tpr) gene family. Polymerase chain reaction amplification and restriction endonuclease digestion of polymerase chain reaction products from laboratory strains and clinical specimens were used to develop a molecular subtyping scheme for T, pallidum. Results: Determining the number of repeats in the alp gene by polymerase chain reaction resulted in 12 different subtypes among the 63 isolates that were studied. Among those, most (54.2%) had alp genes with 14 repeats. The other II subtypes had mp genes with 7 to 21 repeats, each accounting for 2% to 14% of the isolates. Polymerase chain reaction amplification of a member of the tpr gene family from a subset of 46 isolates followed by digestion of the polymerase chain reaction product with MseI resulted in seven restriction fragment length polymorphism patterns designated a to g, Strains with 14 repeats could be grouped into five restriction fragment length polymorphism subtypes, By combining the two systems we observed 16 subtypes among 46 isolates examined. This typing system is stable, reproducible, and easy to perform. In addition, the use of the ABI Genetic Analyzer for the determination of fragment size and banding patterns makes the results unbiased, Conclusion: This is the first molecular subtyping system that distinguishes among clinical isolates of T, pallidum.