Getting the measure of syphilis: qPCR to better understand early infection

Abstract

ObjectivesUntil 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 kDa membrane lipoprotein gene DNA (tpp47).MethodsAssay 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.ResultsThe 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.ConclusionsQuantifying 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.

Syphilis: using modern approaches to understand an old disease

“…T. pallidum can be identified by PCR in the bloodstream of patients with all stages of syphilis, and the quantity of treponemes in blood is highest during early syphilis (2,3). Individuals with lesions of early syphilis are most likely to transmit T. pallidum.…”

Macrolide Resistance in the Syphilis Spirochete, Treponema pallidum ssp. pallidum: Can We Also Expect Macrolide-Resistant Yaws Strains?

“…28,29 In human TPA infections, a reported number of bacteria/mL of whole blood reached 1 × 10 3 and 3 × 10 4 . 30,31 The total blood volume (~5 L) therefore likely contains in one point of TPA infection at least 10 8 treponemes. Assuming a relatively low number of infecting spirochetes, the number of DNA replications approaches a number of microorganisms in human body.…”

Multiplex nested polymerase chain reaction for Treponema pallidum using blood is more sensitive than using serum

“…(8,18) The proportion of positives of the PCR tests vary among a number of Table 2. Treponema pallidum multiplex nested PCR test in blood and serological tests of the patients (n=61) studies on patients with secondary syphilis, which comprise the studies conducted by Martin et al, (18) Tipple et al, (8) and Grange et al (10) with a proportion of positives of 44%, 58% and 38%, respectively. In these studies the PCR test with the target gene PolA resulted in the detection of Treponema pallidum using blood in patients with reinfection of syphilis, primary syphilis, secondary syphilis, and latent syphilis.…”

“…(8,9) The Treponema pallidum PCR test may use various types of clinical specimen depending on the course of the disease. The sensitivity and specificity of the molecular PCR test results are affected by the type of specimen, the PCR method, and the target gene used.…”