Performance of direct detection tests for syphilis

Key Question

What are the performance characteristics of each direct detection test for Treponema pallidum and what are the optimal specimen types for each test (darkfield microscopy, direct fluorescent antibody, PCR and immunohistochemical, or silver staining of tissue)?

Literature Search Terms

(syphilis OR Treponema pallidum) AND (genital ulcer disease OR primary syphilis OR secondary syphilis OR tertiary syphilis OR congenital syphilis OR ocular syphilis) AND (diagnosis OR lesions OR polymerase chain reaction OR PCR OR nucleic acid amplification test OR NAAT OR multiplex test OR silver stain OR silver staining OR immunohistochemistry OR IHC OR rabbit infectivity testing OR RIT OR direct detection OR dark field microscopy OR darkfield microscopy OR dark-field microscopy OR direct fluorescent antibody OR DFA OR direct fluorescent antibody for T. pallidum OR DFA-TP OR direct fluorescent antibody tissue test for T. pallidum OR DFAT-TP). Solely-based international studies were excluded from the literature search.


Four reviewers the evidence as high, medium, and low based on each study’s strengths and weaknesses. Case reports or small case studies were reviewed.

High quality publications: Studies using clinically characterized specimens, stratified by stage, larger sample size, prospective or a well-done cross sectional or retrospective study. Studies with large sample sizes, clinically characterized but not stratified by stage, or characterized but unclear exactly how it was done.

Medium quality publications: Studies with small sample sizes, moderate methodological issues, single lab test as gold standard, or descriptive.

Low quality publications: Studies with major methodological issues or small sample sizes.

I: Case reports or small case studies.

Performance of direct detection tests for syphilis’
Citation Description of study type, design, population, and setting Reported findings, quantitative results related to key question Overall quality; strengths/weaknesses or limitations Relevance to the key question and/or overall importance Rank
Horowitz, H. W., et al. (1994). “Brief report: cerebral syphilitic gumma confirmed by the polymerase chain reaction in a man with human immunodeficiency virus infection.” New England Journal of Medicine 331(22): 1488-1491.



  • Single case report
  • HIV pos man with mental status changes, seizures and difficulty swallowing w/ prior dx and t’ment for Tp.
  • TPPA pos & RPR 1:8
  • Multiple ring enhancing lesions and obstructive hydrocephalus at 4th ventricle
  • Brain gumma biopsy performed at death
  • nPCR for Tp bmp gene, Steiner stain, DFA testing with mAb to Tp 37kDa and and polyclonal Tp Ab
  • Steiner stain and polyclonal DFA positive on deparaffinized, fixed brain tissue
  • mAb to Tp 37 kD surface protein negative
  • bmp nPCR on all 5 paraffin embedded, formalin fixed sections was positive


  • Positive (rabbit testes tissue) and negative (deceased pt due to heart attack) controls used for PCR
  • PCR confirmed by DNA hybridization
  • anti-B. burgdorferi antibody control for DFA
  • Single case study included due to rarity of condition
  • Targeted Tp bmp nPCR may be useful on FFPE brain biopsy material for diagnosis of cerebral gummas
  • Silver stain and DFA pos in gumma
Handsfield, H. H., et al. (1983). “Demonstration of Treponema pallidum in a cutaneous gumma by indirect immunofluorescence.” Archives of Dermatology 119(8): 677-680.


  • Single case report
  • 79 yo male with penile ulcer, VDRL (1:2) and FTA-ABS pos; no exudate from ulcer.
  • Dx with cutaneous penile gumma based on clinical and lab findings
  • DFM performed on ulcer scrapings
  • Edge of ulcer biopsied and stained w/ Warthin-Starry
  • Bx immunofluorescence testing
  • Other causes of ulcer were ruled out
  • Indirect IFA performed on fixed, deparaffinized tissue sections using anti-Nicols strain Tp rabbit antiserum
  • 5 DFM exams from lesion scrapings were negative
  • Lesion biopsies were negative by Warthin-Starry
  • Indirect IFA showed many spirochetes consistent w/ Tp
  • Controls for indirect IFA included (Gt anti-Rb alone, normal Rb serum) and positive control (Nichols strain) used.
  • Additional neg. control from pts w/o syphilis included
  • Anti-serum not pre-absorbed with w/ nonpathogenic treponemes to exclude cross-reactivity
  • Single case reported included due to rarity of diagnosis
  • Warthin-Starry staining of tertiary cutaneous gumma is insensitive
  • Indirect IFA on cutaneous lesion biopsy may be helpful for detection/dx of tertiary syphilitic gumma
Benzick, A. E., et al. (1999). “Pituitary gland gumma in congenital syphilis after failed maternal treatment: a case report.” Pediatrics 104(1): e4.


  • Case report on congenital syphilis case with pituitary gland gumma
  • tpp47 PCR and RIT performed
  • Specimens: endotracheal aspirate, blood, CSF collected after 2 doses of ampicillin, pituitary gumma on autopsy
  • PCR on tpp47 S. blot confirmation (Sanchez JID 1993) positive on endotracheal aspirate and gumma
  • RIT performed on blood and CSF was negative
  • Steiner silver stain demonstrated numerous spirochetes in gumma
  • Case report
  • Included b/c infrequence of congenital syphilis with gumma
  • PCR and RIT performed
  • Variable sensitivity of PCR depending on specimen source.
  • Demonstration of spirochetes in biopsy material may lead to higher sensitivity of PCR
  • Abx treatment may  decrease yield of PCR and RIT
Lin, R. J., et al. (2017). “Syphilis masquerading as mass lesions: The utility of modern-day imaging and molecular sequencing in clinical diagnosis.” Sexually Transmitted Diseases 44(5): 303-305.
  • 2 syphilitic gumma case reports (anorectal and spinal)
  • Both pts HIV+, pos by trep serology and RPR in bld (1:32 – 1:128)
  • Lesion biopsies submitted for Warthin-Starry and bacterial 16S rRNA PCR/sequencing
  • Both gummas were negative by Warthin-Starry
  • Anorectal gumma was 16S rRNA neg; spinal gumma 16S rRNA RT-PCR generated 800bp amplicon which when sequenced matched 100% to Tp
  • Case reports included due to rarity of presentation
  • Broad-range bacterial 16S rRNA sequencing performed; not Tp specific
  • 16S rRNA RT-PCR has limited sensitivity in syphilitic gummas; though positive result in right patient can be used to confirm diagnosis
Wendel, G. D., Jr., et al. (1991). “Identification of Treponema pallidum in amniotic fluid and fetal blood from pregnancies complicated by congenital syphilis.” Obstetrics & Gynecology 78(5 Pt 2): 890-895.
  • 2 case reports of congenital syphilis
  • Amniotic fluid collected from both cases and fetal blood from one case
  • Specimens subjected to DFM, indirect IFA w/ mAb to Tp and RIT
  • DFM positive in amniotic fluid from both cases
  • Amniotic fluid and fetal blood positive by RITs
  • Indirect IFA performed on case 2 only and was positive
  • Case 2: Rabbit seropositivity and dark-field pos orchitis at 5 weeks post inoculation
  • Amniotic fluid from control patients used
  • Fetal blood not previously used for direct detection methods – first report of positivity by RIT/Dark-field/IFA
  • Case reports
  • RIT (amniotic fluid and blood) may be helpful in cases of congenital syphilis, though lack of availability and slow turnaround time
  • Dark-field and indirect IFA , if available, on amniotic fluid may be helpful in cases of congenital disease
Rawstron, S. A., et al. (1997). “Congenital syphilis: detection of Treponema pallidum in stillborns.” Clin Infect Dis 24(1): 24-27.
  • 17 cases of fetal demise due to congenital syphilis in RPR+/FTA-ABS+ mothers and CDC criteria identified
  • Original tissue from autopsies restained with Dieterle and indirect IFA using rabbit α-Tp sera (S. Lukehart)
  • Additional 116 sections from multiple organs from 16 fetuses with CS stained with Dieterle and IFA
  • Of original specimens, Dieterle was pos in 7/17 (41%) and IFA pos in 15/17 (88%)
  • Of 116 tissue sections from 16 fetuses with CS, IFA pos in 63/116 (54%) and Dieterle pos in 43/116 (37%); IFA significantly more sens than Dieterle (p<0.01)
  • Tissue most common to have treponemes was lung and umbilical cord
  • Statistics performed
  • Large number of tissue sections from multiple organs in 16 cases of CS
  • Negative controls used from stillbirth fetuses in RPR neg mothers
  • All stains read blindly
  • Detailed information regarding organ sections from stillbirths was lacking
  • IFA more sensitive than Diterle stain in tissue sections from stillbirth fetuses with CS
  • Most common sites of treponemes in lung and umbilical cord
Hollier, L. M., et al. (2001). “Fetal syphilis: clinical and laboratory characteristics.” Obstetrics & Gynecology 97(6): 947-953.
  • Prospective, single site enrollment of VDRL/MHA-TP positive pregnant women at least at 24 wks gestation
  • Amniotic fluid (AF) collected for RIT, DFM and PCR for tpp47 using Grimprel et al 1991 assay
  • Funipuncture performed for VDRL and IgM by WB
  • Maternal syphilis staged by clinical criteria (CDC 1985 guidelines)
  • 24 women met criteria; 6 primary, 12 secondary, 6 early latent
  • 16/24 (67%) fetuses determined to have CS based on sonography and testing; therefore 67% transmission rate
  • 3/6 in primary cases
  • 8/12 in secondary case
  • 5/6 in early latent cases
  • Amniotic Fluid:
  • 5/21 (24%) DFM pos
  • 12/20 (60%) RIT pos
  • 10/20 (50%)PCR pos
  • 5 pos by all 3 assays
  • 14 pos by ≥ 1 assay
  • Compared to RIT, DFM had a sens of 41% (5/12) and PCR a sens of 82% (9/11)
  • RIT used and DFM and PCR evaluated
  • RIT has highest sensitivity for detection of Tp, followed by PCR on amniotic fluid
Nathan, L., et al. (1997). “In utero infection with Treponema pallidum in early pregnancy.” Prenatal Diagnosis 17(2): 119-123.
  • Single-site; 11 pregnant women b/w 14-19 wks gestation w/ untreated early syphilis enrolled
  • Amniotic fluid (AF) tested by RIT and PCR for tpp47 (658 bp) w/ S. Blot confirmation
  • IgM for Tp and VDRL evaluated in all neonates
  • 4/11 (36%) AF samples positive by RIT
  • 3/11 (27%) AF samples positive by PCR
  • All PCR pos were RIT pos; 1 RIT pos was PCR neg
  • 9/11 (82%) neonates were negative for IgM to Tp
  • All neonates had VDRLs equal to or less than mom’s at delivery
  • Few studies have evaluated utility of amniotic fluid testing during early gestation for diagnosis of syphilis
  • Evaluated direct detection and serologic testing
  • DNA hybridization used for confirmation of PCR
  • RIT has higher sensitivity for CS in AF fluid compared to PCR, though both low.
Michelow, I. C., et al. (2002). “Central nervous system infection in congenital syphilis.” New England Journal of Medicine 346(23): 1792-1798.


  • Single site study; enrolled 148 newborns whose mothers were diagnosed with syphilis during pregnancy
  • CSF tested for Tp by RIT and compared to clinical findings, radiography, and other laboratory testing:
  • Tpp47 PCR on serum, plasma, blood, CSF
  • RIT on blood, serum
  • IgM blotting on serum, CSF
  • 27/148 (18%) blood/serum RIT positive
  •  19/148 (13%) CSFs positive by RIT
  • CSF collected in 76 babies prior to Abx
  • RIT pos in 17/76 (22%)
  • PCR pos in 11/76 (14%)
  • PCR v. RIT sens: 65%
  • Of 17 babies w/ confirmed CNS CS:
  • RIT+ in 13/14 sera/bld
  • PCR+ in 16/17 sera/bld
  • Blood PCR good predictor of CNS infection (sens 16/17 and spec 53/59) using RIT in CSF as ref. method
  • 4 infants with normal CSF findings and VDRL neg were RIT positive in CSF


  • No information on maternal dx during pregnancy
  • Administration of Abx prior to CSF collection associated with lower RIT sensitivity
  • PCR on blood best predictor of CSF infection (vs. serologic testing) when RIT in CSF used as reference method.


Sanchez, P. J., et al. (1993). “Evaluation of molecular methodologies and rabbit infectivity testing for the diagnosis of congenital syphilis and neonatal central nervous system invasion by Treponema pallidum.” J Infect Dis 167(1): 148-157.
  • Single site; enrolled 19 mother-infant pairs. Mothers w/ untreated early syphilis, HIV neg
  • 7 symptomatic and 12 asymptomatic infants at birth
  • tpp47 PCR and S. blot confirmation
  •  RIT performed on all infant CSF and some serum
  • VDRL and IgM blot on all infant CSF/serum
  • 7 symptomatic infants:
  • sera pos by RIT in 3/3 samples; PCR pos in 6/7 sera, including the 3 RIT pos
  • RIT pos in 6/7 CSF; PCR pos in 5/7


  • 12 asympto infants:
  •  RIT pos in 2/2 sera tested; PCR pos in 2/12; 1 sera RIT and PCR pos
  •  RIT pos in 1/12 CSF; PCR neg in all 12 CSF; VDRL NR in 11/12 CSF


  • Overal PCR vs. RIT in CSF (n=19):
  •  71% (5/7) sens
  • 100% (12/12) spec
  • kappa: 0.76
  • Overall PCR vs. RIT in CSF and sera (n=29 samples):
  • 75%  (9/12) sens
  • 100% (17/17) spec
  • kappa: 0.79
  • 5 infants w/ RIT/PCR pos CSF pre t’ment were CSF neg. 2-8m post t’ment
  • Used RIT as gold standard
  • No mother-infant controls enrolled
  • ost t’ment RIT/PCR data not shown
  • Tp PCR on CSF of symptomatic infants is helpful adjunct for detection of congenital syphilis
  • PCR on CSF of asymptomatic infants w/ congenital syphilis has limited sensitivity, similar to RIT
Grimprel, E., et al. (1991). “Use of polymerase chain reaction and rabbit infectivity testing to detect Treponema pallidum in amniotic fluid, fetal and neonatal sera, and cerebrospinal fluid.” Journal of Clinical Microbiology 29(8): 1711-1718.


  • Single site
  • Amniotic fluid (AF) from 11 pregnant w/untreated syphilis
  • Sera & CSF from 20 neonates w/ probable or suspected congenital syph
  • Pregnant mothers Dx using conventional criteria; all neonates evaluated at birth
  • Control patients from preg. women and neonates w/o syph
  • Goal: a) evaluate different methods to minimize PCR inhibition and b) compare PCR to RIT on diff sources
  • DFM on AF
  • RIT performed on AF, CSF and sera
  • 4 different DNA prep. methods for PCR evaluated
  • PCR for Tpp47 gene with S. blot confirmation


  • 9/11 (82%) AF samples from untreated preg women w/ syphilis pos by PCR using low-spin extraction method; 6 AFs DFM pos. 9 had RIT on AF and 7 were RIT Pos
  • RIT and PCR performed on CSF of 18 neonates and pos in 5 and 3, respectively
  • RIT performed in sera from 5 neonates and pos in all 5; PCR performed in all 20 sera and pos in 9.  All but 1 RIT pos sera was PCR pos
  • Compared to RIT, PCR sensitivity in AF, neonate CSF and neonate sera was 100% (7/7), 60% (3/5) and 67% (4/6), respectively; overall sensitivity of 78 (14/18)%.  Specificity was 100%
  • PCR in sera 2-7m post t’ment neg by PCR in nenoates previously RIT or PCR pos in sera
  • RIT performed and used as gold standard
  • Negative patient controls incorporated
  • Sensitivity difference may be due to different volumes used for RIT (0.5-2 mL) vs PCR (10-100ul)
  • Specimen storage conditions were not optimal following collection and DNA may have degraded (not immediately placed in -70
  • PCR may be useful adjunct to Dx and management of congenital syph pre-treatment


Buffet, M., et al. (2007). “Diagnosing Treponema pallidum in secondary syphilis by PCR and immunohistochemistry.” Journal of Investigative Dermatology 127(10): 2345-2350.
  • Prospective
  • Skin bx from 12 pts w/ secondary syphilis dx based on serology and clinical presentation and 24 control pts w/ dermatologic lesions
  • DFM performed on lesions
  • PCR tpp47 (260bp) and human alb internal control on immediately frozen fresh tissue
  • PCR amplicon detected by Southern Blot for 25 bp region and sequenced
  • IHC on FFPE using avidin-biotin peroxidase complex technique w/ polyclonal Abs (BioCare)
  • Semi-quantitative eval in epidermal, superficial and deep dermal layers
  • IHC pos in 11/12 (91.7%) biopsies from syph pts; spirochetes predominantly in epidermis and superficial to medium dermis
  • PCR pos in 9/12 (75%) syph pts
  • PCR LoD determined 1 ng DNA
  • DFM pos in 7/12 (58%) pts
  • 1 pt DFA pos/IHC and PCR neg
  • All negative controls were negative by all assays
  • alb detected in all PCR assays
  • LoD studies not described well
  • Well characterized secondary syphilis samples (clinical and serology)
  • Low number of samples
  • DFM not well described
  • IHC higher sensitivity than tpp47 PCR
  • Higher PCR sensitivity than other studies – due to fresh snap frozen (-80C) tissue?
  • Recomend both IHC and PCR for secondary syph dx
  • T. pallidum localized to epidermis/upper dermis
Romanowski, B., et al. (1987). “Detection of Treponema pallidum by a fluorescent monoclonal antibody test.” Sex Transm Dis 14(3): 156-159.


  • Single site, STD clinic, 1984-1985 enrollment Edmonton, Canada
  • Anogenital lesion exudate collected from 128 patients and evaluated by DFM and DFA testing using monoclonal H9-1 Ab to 47-48kDa Tp protein
  • Diagnosis of syphilis determined by:
  • DFM  pos, or
  • New serology pos, or
  • 4X increase in RPR
  • 66 patients with syphilis based on indicated criteria
  • 52/66 (78.8%) DFM pos
  • 48/66 (72.7%) DFA positive with H9-1 mAb
  • 48/52 (92%) sensitivity of H9-1 mAb compared to DFM
  • 62 patients without syphilis; dark-field microscopy and H9-1 mAb were negative in all 62 exudate samples
  • Large number of dark-field positive samples to compare to
  • Well defined methods for assessment of DFA and dark-field slides
  • Only one sample from each patient evaluated – does multi-sampling improve sensitivity?


  • High correlation of mAb H9-1 DFA positivity with DFM positive from anogenital lesion exudate
Lee, W. S., et al. (1991). “Detection of Treponema pallidum in tissue: a comparative study of the avidin-biotin-peroxidase complex, indirect immunoperoxidase, FTA-ABS complement techniques and the darkfield method.” Yonsei Medical Journal 32(4): 335-341.


  • Retrospective identification of 30 patients with confirmed syphilis (clinical and serologic)
  • 37 FFPE archived tissue biopsies (5 primary, 31 secondary, 1 gastric mucosa)
  • Biopsies tested by:
    • 1) Avidin-biotin-peroxidase complex (ABC)
    • 2) Indirect Immunoperoxidase (IIP)
    • 3) FTA-ABS Complement (FAC)
  • DFM performed in 3 primary and 14 secondary lesions
  • Results of staining methods compared to clinical diagnosis
  • Compared to clinical Dx, ABC method most sensitive (35/37; 95%), followed by IIP (33/37; 89%), FAC (26/37; 70%) and Dark-field (12/17; 71%)
  • Statically significant difference between ABC and FAC, DFM for secondary syphilis lesions
  • Included method-specific negative controls (omitting primary Ab or replacement with normal serum)
  • Detailed methods provided
  • Not all patients had dark-field performed (N=17)
  • ABC more sensitive due to multiple biotin molecules associated w/ secondary Ab and strong biotin-avidin affinity (vs. other conjugated Ab techniques)
  • Immunoperoxidase methods in general more sensitive than DFM or FAC
Hoang, M. P., et al. (2004). “Secondary syphilis: a histologic and immunohistochemical evaluation.” Journal of Cutaneous Pathology 31(9): 595-599.


  • Retrospective identification of 17 patients with secondary syphilis Dx confirmed by serology and histologic examination; 19 bx from 17 patients
  • Bx stained with avidin-biotin peroxidase complex (ABC) technique (BioCare Medical Tp Ab) and Steiner stain
  • 14 control biopsies presenting similar to secondary syphilis also evaluated
  • 12/17 (71%) positive by ABC and 7/17 (41%) positive by Steiner stain
  • All controls negative by ABC; unclear if also negative by Steiner stain
  • Statistically significant higher sensitivity of ABC method shown vs. Steiner (p=0.084)
  • High level of melanin granule staining by Steiner stain
  • Limited number of positive samples
  • Secondary syphilis cases only
  • Controls included!
  • Statistical analysis performed!
  • Detailed description of histologic appearance of lesions and location of treponemes
  • ABC method more sensitive than Steiner Silver stain
Daniels, K. C. and H. S. Ferneyhough (1977). “Specific direct fluorescent antibody detection of Treponema pallidum.” Health Laboratory Science 14(3): 164-171.


  • Single site, prospective
  • Study A: Compared DFM vs. DFA in 350 lesion exudates
  • Study B: Compared DFA from lesion exudate vs. FTA-ABS in serum from 95 patients
  • Syphilis diagnosed using the following criteria:
  • Case history
  • Clinical manifestations
  • Serologic test results
  • DFM results


  • 89.1% (312/350) agreement b/w DFM and DFA in Study A
  • DFA sensitivity and specificity from both Study A and B was 85.9% (115/134) and 100% (311/311), respectively
  • DFM sensitivity and specificity from Study A was 73.8% (87/118) and 97.4% (226/232), respectively
  • 7 samples from clinically diagnosed primary syphilis patients were DFM and DFA negative; 24 samples were DFM negative/DFA positive
  • Large number of samples evaluated
  • Definition/criteria for diagnosis of syphilis (serology results, etc.) not well defined
  • No statistics
  • DFA on acetone fixed lesion exudate is a sensitive method for direct detection of syphilis
  • Lower sensitivity of DFM for detection of spirochetes in lesion exudate compared to DFA
Hook, E. W., 3rd, et al. (1985). “Detection of Treponema pallidum in lesion exudate with a pathogen-specific monoclonal antibody.” Journal of Clinical Microbiology 22(2): 241-244.
  • Hybridomas created to make mAb specific to Tp.
  • H9-1 mAb specific to T. pallidum and T. peritenue, but not other human commensal treponemes
  • C2-1 mAb also used, reacts with all spirochetes (cont.)
  • 30 patients with syphilis based on DFM and serologic criteria and 31 patients without syphilis enrolled
  • Lesion exudate collected from all for DFM and DFA w/ H9-1 mAb.
  • Serum collected for FTA-ABS and VDRL
  • H9-1 mAb DFA was pos in 30/30 pts w/ syphilis and neg in 31/31 patients w/o syphilis
  • DFM was positive in 29/30 pts w/ syphilis and 2/31 patients without syphilis.  Commensal spirochetes confirmed in the 2 patients using the C2-1 mAb


  • Detailed description of methods and criteria used for syphilis diagnosis
  • Use of C2-1 mAb for spriochetes to confirm commensals
  • H9-1 mAb has high sensitivity and specificity for detection of T. pallidum from acetone fixed lesion exudate.
Muller, M., et al. (2007). “Detection of Treponema pallidum in the vitreous by PCR.” British Journal of Ophthalmology 91(5): 592-595.
  • 2 case reports of syphilis ocular chorioretinitis detected by tpp47 PCR (melt temp analysis) on vitreous fluid
  • Patients with serology confirmed syphilis


  • 2/2 patients with ocular disease and positive serology for syphilis were PCR pos for tpp47.  All other viral PCR testing negative. Improved on t’ment.
  • tpp47 PCR LoD determined (100 organsims/mL = 1IFU/rxn)
  • Case report
  • Included due to rarity of diagnosis
  • No negative controls


  • Tpp47 PCR on vitreous fluid may be useful in patients with chorioretinitis who are positive by syphilis serology
  • Consider performing if other common causes of uveitis/retinitis excluded and patient is positive by syphilis serology
Cornut, P. L., et al. (2011). “Detection of Treponema pallidum in aqueous humor by real-time polymerase chain reaction.” Ocular Immunology & Inflammation 19(2): 127-128.


  • Prospective (2009-2010) ,  France enrolling patients with presumed secondary ocular syphilis based on syphilis serology results in serum and CSF
  • 5 patients included and aqueous humor collected
  • PCR for Tp pol I (polA)
  • PCR inhibition control included
  • 3/5 patients were Tp pol I positive; all three were serology positive in serum and CSF; 1 patient was PCR positive in CSF
  • All 3 positive patients had bilateral panuveitis
  • 2 PCR negative patients were also serology positive in serum and CSF with similar titer; presented with optic neuritis and placoid choroiditis
  • Authors suggest lack of direct intraocular infection  for 2 PCR neg patients and that aqueous humor may have been wrong specimen to test
  • Limited number of patients enrolled and positive samples
  • No indication of what the internal control was
  • No positive/negative controls or LoD
  • Tp pol1 PCR on aqueous humor may be useful in cases of panuveitis  due to syphilis
  • Sensitivity may be affected by specific site of infection/presentation
Lukehart S. A., et. al. (1988). “Invasion of the Central Nervous System by Treponema pallidum: Implications for Diagnosis and Treatment.” Annals of Internal Medicine 109: 855-862

(Added, not part of original literature search)

  • Prospective, single site
  • Enrolled pts w/ serologic evidence of syphilis w/o prior treatment for syphilis
  • Sera and CSF tested by VDRL and FTA-ABS
  • 7 P syph
  • 33 S syph
  • 3 EL syph
  • 15 LL syph
  • CSF tested by RIT and compared to clinical stage
  • RIT pos in 12/58 CSF
  • Pos in 2/7 primary and 10/33 secondary syph; neg in all latent cases
  • Tp isolation by RIT higher in pts w/ ≥2 CSF abnormalities (protein, leukocytes, VDRL+)
  • Neurologic signs present in only 6/33 pts w/ SS
  • 7/10 SS pts pos by RIT, were re-tested by RIT post-t’ment
  • 3/3 pts w/ 2-3 penG doses were RIT neg
  • 3/4 pts w/ 1 penG dose were RIT pos
  • Detailed evaluation of RIT performance in comparison to serologic findings, clinical presentation and CSF findings
  • Seminal study on detection of Tp in CSF using RIT
  • Indicates dissemination of Tp into CSF even in asymptomatic individuals w/ primary or secondary infection
  • Impact of treatment on RIT sensitivity
Hay, P. E., et al. (1990). “Detection of treponemal DNA in the CSF of patients with syphilis and HIV infection using the polymerase chain reaction.” Genitourinary Medicine 66(6): 428-432.




  • Single site, prospective
  • Tp tmpA (45kDa) and 4D (19kDa) PCR on CSF with confirmation by S. blot
  • Tp distinguished from T. pertenue at 1 bp in 4D gene, but this bp not in amplified product
  • CSF positive for Tp only if both PCR rxns positive
  • 3 grps of patients tested:
  • A (N=30: No hx of syphilis/HIV
  • B (N=19): Syph pos pts (TPHA and FTA pos on serum, w/ or w/o pos VDRL), concern for neurosyphilis
  • C (N=28): HIV+ pts undergoing LP for ? of CNS disease
  • Neurosyph Dx by symptoms and pos VDRL in CSF
  • Possible neurosyph Dx if abnormal CSF, VDRL neg, but FTA/TPHA CSF pos
  • 1/30 (3.3%) pts in Grp A w/o Hx of Tp were PCR pos
  • Grp B: 10/19 (53%) PCR pos in CSF, including both pts w/ VDRL pos CSF, 1/2 pts w/ possible neurosyph, and 7/14 pts w/ latent syph (late and tertiary syph)
  • Grp C: 14/28 pts w/ CNS disease of these 14, 7 were Tp PCR pos and all 14 pts w/o CNS disease were PCR neg
  •  Overall:
    • 59% sens, 97% spec, 91% PPV, 83% NPV in pts w/o prior syph tment
    • 38% sens, 87% spec, 87% PPV, 62% NPV in HIV+ pts w/ Hx of syph
    • CSF stored in liquid N2 until testing
    • LoD: 65 org/0.5 mL
  • Retrospectively determined that primer pairs used for testing were not 100% specific. 2/3 false positives were negative using nested PCR for 4D gene
  • No other comparator assays (e.g., RIT)
  • PCR on CSF may help confirm diagnosis of neurosyphilis in suspicious cases, though a negative result does not r/o infection
Castro, R., et al. (2016). “Detection of Treponema pallidum Sp. Pallidum DNA in Cerebrospinal Fluid (CSF) by Two PCR Techniques.” Journal of Clinical Laboratory Analysis 30(5): 628-632.
  • Single site, enrollment of 124 pts w/ syph reactive blood tests
  • Neurosyph (NS) Dx based on European guidelines (French et al. 2009)
  •  + CSF TPHA and/or FTA and increased CSF WBC (>5/mm3) or RPR/VDRL+
  • Syph staging based on 2014 European guidelines (Janier et. al)
  • 100 CSF from pts syph seroneg included as controls
  • tpp47 (Orle) and polA (Liu) PCRs performed on all CSFs; products visualized by EtBr on gel
  • All 100 CSF controls were neg by both PCRs
  • 37/124 (29.8%) CSFs pos by tpp47 PCR
  • Of 33 w/ NS criteria, 25  (75.6%) were tpp47+
  • Of 91 w/o NS criteria, 12 (13%) were tpp47+
  • 30/124 (24.2%) CSFs pos by polA PCR
  • Of 33 w/ NS criteria, 23 (69.7%) were polA+
  • Of 91 w/o NS criteria,  7 (7.7%) were polA+


  • Majority of PCR discrepancies in pts w/ latent syph
  • Specific ‘syph reactive bld test’ results not defined for enrollment criteria
  • Ref. stand. used included clinical criteria and serology
  • No further analysis of discrepant samples
  • tpp47 PCR higher sens vs. polA PCR higher spec in CSF
  • Overall sens/spec for NS PCRs:
  • tpp47: 75.6%/87%
  • polA: 69.7%/92.3%
  • PCR on CSF sens/spec in pts w/ NS symptoms meeting European criteria for NS
  • Utility/sensitivity of PCR in asymptomatic NS cases is questionable
Orle, K. A., et al. (1996). “Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers.” J Clin Microbiol 34(1): 49-54.
  • Single site, USA STD clinic, collection of genital lesion exudate (N=298)
  • Multiplex PCR for tpp47, HSV and H. ducreyi
  • Colorimetric detection w/ biotinylated-olgiont probes
  • DFM and RPR/VDRL used as comparators
  • Individual Tp PCR to basic memb protein gene used for discrepant analysis


  • 65/298 samples DFM pos, of which 58 also pos by M-PCR for Tp.
  • 17 samples M-PCR +/DFM -, of which 15 also pos by individual Tp PCR.  2 sampes were M-PCR false+
  • 91% (66/73) sens of M-PCR to DF/individual Tp PCR
  • 88% sens of DFM compared to M-PCR/individual Tp PCR
  • 99% and 100% spec for M-PCR  and DF, respectively
  • Comparison to RPR/VDRL not included since no treponemal serology used to confirm testing
  • Combination of reference standards used to evaluate M-PCR
  • Extensive analytical specificity panel performed
  • 7 M-PCR false negatives may have been due to sampling error due to multiple swabs of same lesion
  • LoD studies performed (10 org)
  • M-PCR and individual PCR for Tp from genital lesions more sensitive than dark field microscopy


Zoechling, N., et al. (1997). “Molecular detection of Treponema pallidum in secondary and tertiary syphilis.” British Journal of Dermatology 136(5): 683-686.
  • Retrospective selection of 13 FFPE from secondary (N=6) and tertiary (N=7) cases of syphilis confirmed clinically, by histo path findings and serology
  • 5 neg control biopsies, 6 non-Tp spirochete and 1 Tp culture slide controls included
  • tpp47 nPCR from 10um FFPE cuts used; beta-actin internal amp control used
  • All 13 were Warthin-Starry negative
  • 4/6 secondary syph bx were PCR+
  • 1/7 tertiary syph bx were PCR+; pos samples was from gumma
  • Blades and gloves changed b/w each cut and reaction
  • No comparison to DFM, IHC or other
  • Superior sensitivity of tpp47 nPCR vs. traditional means (silver stains) in biopsy material
  • Higher sensitivity in secondary vs. tertiary syph
Mertz, K. J., et al. (1998). “Etiology of genital ulcers and prevalence of human immunodeficiency virus coinfection in 10 US cities. The Genital Ulcer Disease Surveillance Group.” Journal of Infectious Diseases 178(6): 1795-1798.
  • Multi-site, 10 STD clinics in US cites w/ highest rates of primary syphilis (50-55 pts/site)
  • Enrolled pts w/ GUs w/ open sores; base of largest ulcer swabbed
  • RPR, DFM, Clinical Dx, T’ment recorded
  • M-PCR for tpp47 (Orle 1996) used
  • 516 GU swabs tested, 51 (10%) tpp47+
  • 25/51 (45%) DFM+
  • 40/51 (79%) RPR+
  • 75 pts RPR+, of whom27 (36%) were tpp47 negative
  • Limited clinical data (duration of symptoms, Abx use, etc.)
  • Data for DFM/serology not shown
  • Biased towards highest syphilis prevalence cities
  • PCR used as gold std. for comparison of DFM/serology
  • No discrepant resolution
  • DFM has lower sens vs. tpp47 PCR in primary syphilis
  • PCR on lesion swabs offers higher sensitivity for detection of primary syphilis
Risbud, A., et al. (1999). “The etiology of genital ulcer disease by multiplex polymerase chain reaction and relationship to HIV infection among patients attending sexually transmitted disease clinics in Pune, India.” Sex Transm Dis 26(1): 55-62.
  • Cross-sectional, prospective, 2 STD clinics, India
  • Enrolled pts evaluated for GUD; tested for HIV and syph (RPR/VDRL and FTA-ABS)
  • Base of GU swabbed with Dacron (Amplicor) swabs; in transpo media/stored -70C
  • 2nd swab and direct slide application to lesion evaluated by DFM
  • All GU swabs assessed by M-PCR (tpp47, HSV, H. ducreyi) (Orle, 1996)
  • M-PCR used as reference std. to assess sensitivity of clinical Dx or DFM
  • Pt staging based on clinical dx:
  • PS: painless ulcer, DFM+
  • SS: multiple painless ulcers, RPR+, DFM+
  • 302 pts enrolled (only 25 F)
  • 67/302 (22%) HIV+
  • 29/302 (10%) tpp47+, 79 (26%) HSV+, 69 (23%) chancroid+
  • 104 M-PCR neg samples, 23 were DFM+ and 12/23 were RPR+
  • Compared to tpp47 PCR:
  • DFM had S/Sp/PPV of: 39%/82.1%/25.8%
  • PS Dx criteria had S/Sp/PPV: 7.3%/98%/37.5%
  • Painless lesion had S/Sp/PPV: 22%/82%/28.8%
  • RPR+ S/Sp/PPV: 68%/76%/33%
  • RPR+/FTA-ABS+ had S/Sp/PPV: 65.9%/90%/52%
  • Duration of symptoms and prior Abx use not impactful on M-PCR pos rates


  • Enrollement criteria not well defined
  • DFM methods not detailed (time to exam, assessment, swab vs. slide ‘touch prep’ on lesion, etc.)
  • PCR used as ref std to asses sens/spec of clinical assessment, DFM and serology
  • Discordant PCR vs. DFM or serology not addressed/discussed



  • Compared to tpp47 PCR, DFM has low sensitivity (39%) and specificity of 82% in swabs of GU exudate
Marfin, A. A., et al. (2001). “Amplification of the DNA polymerase I gene of Treponema pallidum from whole blood of persons with syphilis.” Diagn Microbiol Infect Dis 40(4): 163-166.
  • Single site, outbreak investigation
  • WB collected from 32 pts w/ either signs of syphilis or who were a sexual partner of someone w/ syphilis
  • Samples originally collected for molecular typing via tpr and apr genes
  • WB tested for Tp polA and amplicons visualized by EtBr stain of gel
  • 7 pts w/ incubating, 7 pts w/ primary, 1 w/ secondary, 13 w/ latent and 4 w/ non-syph lesions
  • 13/28 (46%) WB samples from pts w/ syph were polA+; all 4 WB samples from non-syph pts were neg by PCR
  • polA+ in
  • 8/13 latent cases
  • 3/7 incubating cases
  • 1/7 and 1/1 PS and SS
  • No correlation b/w RPR titer and MHA-TP reactivity and PCR positivity
  • No confirmation of PCR amplicons using S. blot
  • Small number of pts
  • No RIT
  • Tp polA PCR on WB may be used to detect syphilis in pts w/ latent infection
  • PCR may be useful regardless of serologic level of reactivity
  • Spirochetemia occurs throughout course of infection and may be detected by PCR
Liu, H., et al. (2001). “New Tests for Syphilis: Rational Design of a PCR Method for Detection of Treponema pallidum in Clinical Specimens Using Unique Regions of the DNA Polymerase I Gene.” Journal of Clinical Microbiology 39(5): 1941-1946.
  • Development of a Tp polA PCR and amplicon detection by the ABI 310 Genetic Analyzer
  • Analytic specificity evaluated using culture
  • polA PCR compared to the M-PCR from Orle et. al. 1996 using 112 genitourinary lesion specimens
  • Amplicons only observed with pathogenic T. pallidum subspecies
  • LoD with gel analysis was 10 organisms; LoD with ABI instrument was 1 organism
  • Using the M-PCR as the reference standard, the polA PCR had a positive agreement of 96% (22/23), negative agreement of 96% (85/89), overall agreement of 96% (107/112)
  • Retrospective comparison to Orle M-PCR study, which had dark field results but those not included in this study
  • No details on clinical specimens (other than genitourinary lesion) or collection included
  • No RIT
  • PCR for Tp polA has similar performance characteristics in genitourinary lesions for Tp as the M-PCR for Tp, HSV and H. ducreyi (Orle 1996)
Palmer, H. M., et al. (2003). “Use of PCR in the diagnosis of early syphilis in the United Kingdom.” Sex Transm Infect 79(6): 479-483.
  • Prospective enrollment of pts w/ ulcers at STD clinics
  • Swabs (dry or in viral transpo media) tested by tpp47 PCR (Orle 1996) and viewed by EtBr
  • 98 pts evaluated, 100 specimens tested
  • Results compared to clinical diagnosis/staging and serologic results
  • DFM performed on 34/100 (34%) samples
  • 18/19 (95%) of pts w/ PS were PCR+; 2/10 (20%) were DFM+
  • 8/10 (80%) of pts w/ SS were PCR+; 0/3 were DFM+
  • 23/23 pts w/ HSV or past, treated syph were PCR-; 9/9 were DFM-
  • 48 pts w/ Dx of ‘Not syphilis’, 1/48 PCR+ and 1/12 DFM+
  • Compared to clinical/serologic Dx, PCR had sens/spec/PPV/NPV for PS and SS of 95%/99%/95%/99% and 80%/99%/89%/97%, respectively
  • Comparison of PCR to clinical diagnosis (presentation and serologic findings), guidelines not cited
  • Limited number of ulcer samples tested by DFM; none tested by DFA for Tp
  • Amplicon visualization using EtBr only
  • No RIT
  • tpp47 PCR helpful in distinguishing HSV vs Tp ulcers in primary disease



Castro, R., et al. (2007). “Detection of Treponema pallidum sp pallidum DNA in latent syphilis.” International Journal of STD & AIDS 18(12): 842-845.
  • Single site in Lisbon
  • 69 pts w/ indeter. latent syphilis (no symptoms, RPR/MHA-TP/FTA-Abs+)
  • 18 pts treated for syph, tested 6m post-t’ment
  • Collected:
    • WB, Plasma, S, Ear lobe scrapings
    • Orle 1996 tpp47 PCR and Lui 2001 polA PCR used; EtBr gel detection of amplicons
    • tpp47 and polA PCRs also used as multiplex
  • PCR results for each target for the 69 pts meeting CDC criteria for latent syphilis are as follows:
  • tpp47 PCR+
  • WB+: 27/69 (39.1%)
  • Plasma+: 31/69 (44.9%)
  • Serum+: 18/69 (26.1%)
  • Ear lobe+: 16/28 (57.1%)
  • polA PCR+
  • WB+: 19/69 (27.5%)
  • Plasma+: 25/69 (36.2%)
  • Serum+: 14/69 (20.3%)
  • Ear lobe+: 15/28 (53.8%)
  • Tpp4 and/or polA M-PCR+
  • WB+: 26/69 (37.7%)
  • Plasma+: 29/69 (42%)
  • Serum+: 19/69 (27.5%)
  • Ear lobe+: 16/28 (57.1%)
  • tpp47 was the only PCR pos in 3 patients
  • No sample polA or M-PCR pos only; both PCRs were pos in 2 pts neg by tpp47
  • -tpp47 PCR+ in 39.1% (92/235 samples) of pts w/ latent syph; M-PCR pos in 38.3% (90/235 samples)
  • Tp Nicols strain pos cont and syph seroneg pt DNA for neg controls used
  • No RIT
  • Tp not detected by PCR post complete treatment of syphilis
  • Highest sensitivity for latent syphilis PCR achieved in earlobe scrapings>plasma>WB>serum
  • tpp47 PCR had higher sens vs. polA PCR
Martin, I. E., et al. (2009). “Molecular characterization of syphilis in patients in Canada: azithromycin resistance and detection of Treponema pallidum DNA in whole-blood samples versus ulcerative swabs.” J Clin Microbiol 47(6): 1668-1673.
  • Two-site, STD clinic prospective enrollment of pts w/ suspected syph; 87 specimens from 68 pts
  • Pts staged based on clinical and serologic findings according to Canadian STI guidelines
  • WB, serum CSF and swab from ulcers/lesions
  • nPCR for bmp and tpp47; std PCR for tpp47
  • Beta-glob amp control included
  • EtBr visualization of amplicons
  • 41/68 pts dx w/ syph (53 specimens from 41 pts)
  • 19 primary; 9 secondary; 10 latent; 3 congenital
  • 14/41 (34%) pts and 19/53 (36%) of samples PCR+
  • 9/19 primary syph PCR+
  • 9/12 (60%) swabs with Beta-glob amplification were PCR+
  • 0/9 WB+
  • 4/9 secondary syph PCR+
  • 4/9 (44%) WB+
  • 1/1 swab+
  • 1/3 WB from congenital case was PCR+
  • 0/10 latent cases was PCR+ in WB, swab or CSF
  • All 27 nonsyph cases were PCR-


  • No differentiation b/w performance of each PCR rxn
  • Small number of cases
  • No RIT, DFM
  • PCR on swabs from primary syph lesions highest sensitivity; WB not sens source for primary syph cases
  • WB better PCR source for secondary syph, though still <50% sens
  • PCR in WB/swab/CSF not sense for latent syph
Gayet-Ageron, A., et al. (2009). “Assessment of a real-time PCR test to diagnose syphilis from diverse biological samples.” Sex Transm Infect 85(4): 264-269.
  • Multi-site, prospective enrollment of pts w/ suspected syphilis and matched case-controls (!!)
  • Comparison of tpp47 RT-PCR (Taqman) performance in WB, urine, swabs and/or CSF for HIV+ and HIV- pts
  • Syph cases staged based on clinical and serologic data/CDC guidelines
  • 74 pts enrolled/126 specimens collected
  • 38 pts/47 specimens tpp47 RT-PCR+ and discussed
  • Overall sensitivity in:
    • Primary 17/26 (65%)
    • Secondary 21/40 (53%)
    • Latent 0/8
  • No diff in sensitivity in HIV+ vs. HIV- pts, though HIV+ pts w/ secondary syph had lower Ct values (high spirochetemia)
  • Primary syph sens by source:
    • Lesion swab 8/10 (80%)
    • WB: 5/18 (28%)
    • Serum: 6/11 (55%)
    • Urine 2/7 (29%)
  • Secondary syph sens by source:
    • Lesion swab 1/5 (20%)
    • WB: 11/31 (36%)
    • Serum: 7/15 (47%)
    • Plasma: 2/2 (100%)
    • Urine 4/9 (44%)
    • CSF: 3/6 (50%)
  • High specificity of assay – all controls negative.
  • No RIT, DFM, etc.
  • First to evaluate urine
  • No control of Abx use prior to collection
  • Not all specimens received from all pts
  • Urine sens 29-44%
  • Highest sensitivity from lesion swabs for primary syphilis (80%)
  • Highest sensitivity from serum, WB, plasma for secondary syph (36-100%)
  • PCR off any source not useful in latent syph
  • No difference in PCR performance in HIV- vs. HIV+
Behrhof, W., et al. (2008). “PCR testing for Treponema pallidum in paraffin-embedded skin biopsy specimens: test design and impact on the diagnosis of syphilis.” J Clin Pathol 61(3): 390-395.


  • Retrospective study using 39 archived FFPE collected between 1954-2006 in Germany
  • 36 skin bx from pts with secondary syphilis (33 confirmed by serology and 3 not confirmed)
  • Nested (Tp1; 228 bp) and semi-nested (Tp2; 125 bp) PCR for DNA polymerase I performed on all samples.
  • Gel and direct seq. Internal bcl-1 control tested.
  • Samples tested by Dieterle silver stain and IHC using rabbit polyclonal antibody (Biocare)
  • Tp1 and Tp2 nested PCRs positive in 7 and 14 of the 36 FFPE, respectively
  • 23/36 FFPE had positive internal bcl-1 control by PCR
  • 11/23 pos by Tp2 and   6/23 pos by Tp1 PCRs
  • 17/35 FFPE positive by IHC (pos defined as presence of >3 spirochetes)
  • 9/35 FFPE positive by Dieterle
  • Internal controls for PCR and positive/negative controls for staining used
  • Nested PCR, amplicons run on gel and direct sequencing for interpretation = risk for sample contamination
  • 13 samples failed to amplify internal control on PCR and were unusable
  • No statistics performed b/w sources/methods
  • PCR more sensitive than Dieterle staining
  • In cases where DNA quality is good, PCR has similar sensitivity to IHC


Cruz, A. R., et al. (2010). “Secondary syphilis in cali, Colombia: new concepts in disease pathogenesis.” PLoS Neglected Tropical Diseases [electronic resource] 4(5): e690.
  • Multi-site enrollment at STI clinics in Colombia of pts w/ suspected secondary syphilis (57 enrolled)
  • Cases defined using serologic (RPR>=1:4 and FTA-ABS+) and clinical criteria (charc. lesions)
  • WB and bx collected and tested by RT-PCR for Tp polA (TaqMan); inhib control (human RNase P gene) tested for samples neg for Tp
  • Bx also stained w/ Warthin-Starry
  • Skin bx from 11 pts with secondary syph; Warthin-Starry pos in 1 (9%)
  • Tp polA PCR LoD was 15-150 spirochetes/ml; LoD 1 log higher in samples stored at 4C for 26hrs vs. RT for 1hr
  • WB collected in 26 pts; 12/26 (46%) Tp polA+
  • Copies/mL ranged from 195-1954; not correlated w/ RPR titer
  • No inhibition control in PCR neg samples
  • Tp polA+ in 8/12 (66%) biopsies tested
  • No RIT
  • Not all enrolled patients tested by PCR in WB or biopsied
  • Study focused on epi of SS in Colombia
  • Small numbers; no statistics performed comparing sources
  • Tp polA RT-PCR higher sens in skin bx from pts w/ SS (66%) vs WB (46%)
Grange, P. A., et al. (2012). “Evaluation of a PCR test for detection of treponema pallidum in swabs and blood.” Journal of Clinical Microbiology 50(3): 546-552.
  • Multi-site (2 STD clinics in Paris) enrolling pts w/ suspected syphilis and 35 healthy volunteers
  • Diagnosis of syph and staging based on CDC criteria
  • DFM on lesions from primary and secondary
  • nPCR for tpp47 on lesion exudate collected by sterile swab and all blood fractions (WB, PBMCs, serum, plasma)
  • LoD: 20 org/mL
  • 65 pts w/ primary syph
  • 49 (75%) DFM+ vs. 52 (80%) nPCR+; p=0.011
  • 44 pts w/ secondary syph
  • 31 (70%) DFM+ vs. 38 (86%) nPCR+; p=0.339
  • Sn/Sp/YI/PV/NV/LR+/LR for DFM in 1st and 2nd syph:
    • 72/88/0.61/94/57/6.3/0.31
  • Sn/Sp/YI/PV/NV/LR+/LR for nPCR in 1st and 2nd syph:
    • 82/95/0.78/98/68/18/0.18
  • nPCR concordance w/ dx was 82.6%
  •  DFM/nPCR concordance rate 77%; kappa 0.53


  • nPCR in blood fractions:
    • 12-38% sens range
    • 24% overall sens; 97% overall spec, 39.9% concordance btw nPCR and dx
    • PBMCs highest sens in primary syph 32% (23/72)
    • WB highest sens in secondary syph 38% (26/69)
  • Oral lesions not excluded from dark field microscopy
  • Amplicons visualized by EtBr on gel
  • No RIT
  • nPCR may be used as replacement/in addition to DFM for assessment of lesions suspicious for syph
  • nPCR on blood has low sensitivity; serum is worst source overall
Pinto, M., et al. (2017). “A retrospective cross-sectional quantitative molecular approach in biological samples from patients with syphilis.” Microbial Pathogenesis 104: 296-302.
  • Retrospective, cross-sectional; aim to quantify Tp load at various sites to correlate to stage
  • Previously Tp nested-PCR (bmp, polAI) or Tp Real-TM (Sacace Biotech, Italy) pos samples retrieved.
  • All 309 pts symptom and originally dx w/ syph based on PCR and serology
  • Diverse sample set (swabs, blood fractions, vitreous, CSF, placenta) stored at -80C
  • tprB (single copy) quant RT-PCR and beta-actin quant RT-PCR
  • – Relative load of Tp determined as ratio b/w tprB and beta-actin copies
  • Primary syph exudates (swabs) provide significantly higher bacterial load and widest distribution vs. secondary syph swabs
  • Secondary syph blood samples provide significantly higher bacterial load and widest distribution vs. primary
  • Lowest Tp/beta-actin ratio in WB and PBMCs and sign. Lower than ratio from genital exudates
  • Ext. low Tp loads in vitreous fluid/placenta vs. other sample types
  • Samples assoc. w/ primary syph had highest Tp loads
  • Lowest Tp loads from early latent syph
  • No difference in Tp loads in pts w/ or w/o HIV or other co-infecting STIs
  • Small amplicon which overcomes possible issues of long term storage degradation
  • No info regarding duration of symptoms at time of collection
  • Wide array of Tp loads throughout lesion types = heterogeneous duration of primary syph
  • Higher Tp loads in bld from pts w/ secondary syph
Yang, C. J., et al. (2015). “Unexpectedly high prevalence of Treponema pallidum infection in the oral cavity of human immunodeficiency virus-infected patients with early syphilis who had engaged in unprotected sex practices.” Clinical Microbiology & Infection 21(8): 787.e781-787.
  • Single site, prospective enrolling 240 pts (267 episodes) w/ any stage of syphilis defined by CDC STD T’ment guidelines, RPR >/=1:4 and TPPA+
  • Oral swabs on all pts, w/ or w/o oral ulcers; swabs of genital lesions, plasma from pts w/ secondary or early latent syph, CSF from pts w/ neurosyph
  • Samples collected b4 Abx
  • polA PCR (Liu 2001 assay) on all samples
  • Typing using arp, tpr, tp0548
  • Macrolide resistance by 23S PCR
  • All pts were male, 242/267 (91%) of tests in HIV+
  • 22/267 (8.2%) episodes had oral syphilitic ulcers
  • 38 cases of primary syphilis, 17 had oral ulcers, polA+ in 13/38 (34%) of samples and 13/17 oral ulcer cases
  • polA+ in 113/267 (42%) oral swabs
  • 64.5% of oral swabs from pts w/ secondary syph were polA+, regardless of lesion presence/absence
  • polA+ in 28% and 40% of oral swabs from pts w/ early or late latent syph, resp.
  • Detection of polA+ in oral swabs assoc w/ younger age, secondary syph, oral lesion, RPR >1:32
  • Higher yield rates for polA, arp, tpr, tp0548 in pts w/ oral ulcers vs. those w/o
  • 45/113 oral swabs amplified all 3 serotyping genes
  • Tp serotype matched in 31/33 pts where DNA recovered from multiple sites
  • Staging based on clinical criteria; lesions may be overlooked/subtle
  • Biased towards HIV+ males


  • Rate of polA detection from oral swabs is high, even among pts w/o lesions, particularly in secondary syph
Heymans, R., et al. (2010). “Clinical value of Treponema pallidum real-time PCR for diagnosis of syphilis.” Journal of Clinical Microbiology 48(2): 497-502.
  • Single site, prospective STI Clinic, Amsterdam
  • Enrolled pts w/ suspected primary or secondary syph
  • DFM on all anogenital lesions
  • RT-PCR (TaqMan) for polA on all lesion swabs or skin scraping (secondary syph)
  • 3 Reference Stds for PCR utility in primary syph:
    • 1.  PCR vs. DFM
    • 2.  PCR vs. clinic-based dx which incorporates DFM, prior syph episodes, serology results
    • 3. PCR vs. clinic-based dx w/o DFM
  • Criteria for secondary syph
  • ‘Skin manifestation possibly related to secondary syph (macular/popular rash, condyloma latum, roseolas, mucous patches, alopecia)’ and RPR >/=1:8
  • 93/716 (13%) and 34/133 (26%) suspected primary and secondary polA+
  • polA vs. DFM sens/spec:
  • 87% (47/54) and 94% (616/662); kappa 0.61
  • polA vs. primary ref. std. #2 sens/spec:
  • 73% (83/114) and 98% (592/602), kappa 0.769, PPV 89%, NPV 95%
  • polA vs. primary ref. std. #3 sens/spec:
  • 74% (76/102) and 97% (597/614); kappa 0.745
  • polA vs. secondary syph sens/spec:
  • 43% (33/77) and 98% (55/56); kappa 0.372


  • Unclear if DFM from swab (probably swab) vs/ directly from lesion
  • Limitations of clinical dx, no mention of serologic results; no use of std diagnostic guidelines
  • Limited discussion/explanation of PCR discordant samples
  • Fair agreement b/w polA and DFM
  • polA PCR better correlation w/ disease when compared to clinical/serologic assessment for primary syphilis
  • Low PCR sensitivity for secondary syphilis