title: Getting the measure of syphilis: qPCR to better understand early infection.
abstract: OBJECTIVE
  Until recently, PCR had been used to detect but not quantify Treponema pallidum. To understand infection kinetics of this uncultivable organism, a real-time PCR assay was developed to quantify 47&emsp14;kDa membrane lipoprotein gene DNA (tpp47).
  
  
  METHODS
  Assay specificity was determined against DNA from humans, skin organisms and sexually transmitted pathogens. tpp47 DNA (Nichols strain) was used to construct a standard curve for T pallidum quantification. Blood and ulcer samples were obtained from 99 patients being investigated or screened for syphilis and tpp47 was quantified.
  
  
  RESULTS
  The assay was specific, not cross-reactive with other organisms tested and sensitive, with a detection limit of a single copy of tpp47 DNA. For ulcer samples, the assay was 100% sensitive and 97.14% specific. Sensitivity fell to 34.1% for blood samples but specificity remained high (100%). tpp47 DNA was more commonly detected, and at a higher copy number, in blood of patients with secondary infection (sensitivity 57.89%) compared with primary infection. Quantity of tpp47 DNA was higher in primary infection ulcers, especially in HIV-1-positive patients, than in ulcers persisting into secondary disease.
  
  
  CONCLUSIONS
  Quantifying T pallidum provides insight into syphilis infection kinetics: Ulcers of primary disease in HIV-1-positive patients are perhaps more infectious and the presence and load of T pallidum bacteraemia is variable, with a peak in the secondary stage. Quantitative PCR has the potential to map T pallidum infection and to highlight the impact of HIV on syphilis.
authors.affiliation: Imperial College London, Jefferiss Trust Laboratories, Wright-Fleming Institute, London W2 1PG, UK. c.tipple@imperial.ac.uk
authors.forename: Craig
authors.initials: C
authors.lastname: Tipple
authors.forename: Mariam O F
authors.initials: MO
authors.lastname: Hanna
authors.forename: Samantha
authors.initials: S
authors.lastname: Hill
authors.forename: Jessica
authors.initials: J
authors.lastname: Daniel
authors.forename: David
authors.initials: D
authors.lastname: Goldmeier
authors.forename: Myra O
authors.initials: MO
authors.lastname: McClure
authors.forename: Graham P
authors.initials: GP
authors.lastname: Taylor
mesh_terms: D000328:Adult; D002352:Carrier Proteins / Q000032:analysis*; D003430:Cross-Sectional Studies; D004269:DNA, Bacterial / Q000032:analysis*; D042241:Early Diagnosis; D005260:Female; D015658:HIV Infections / Q000150:complications; D015497:HIV-1; D006801:Humans; D008074:Lipoproteins / Q000032:analysis*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012680:Sensitivity and Specificity; D013587:Syphilis / Q000150:complications / Q000175:diagnosis*; D014210:Treponema pallidum / Q000235:genetics / Q000302:isolation & purification
chemical_list: C090573:47-kDa immunogen, Treponema pallidum; D002352:Carrier Proteins; D004269:DNA, Bacterial; D008074:Lipoproteins