Syphilis is a chronic systemic sexually transmitted infection caused by Treponema pallidum, a pathogen that cannot be subcultured in the laboratory on any biochemical medium.1 Early diagnosis of T. pallidum infection is helpful for disease management. Serum-specific treponemal antibody arrays (e.g. T. pallidum particle agglutination [TPPA]), T. pallidum Western blot, chemiluminescent magnetic microparticle immunoassays (CMIAs), and nonspecific treponemal antibody arrays (e.g. rapid plasma regain [RPR]), are suitable for the identification and management of suspected syphilis infections.2 However, nontreponemal antibody arrays may give false positives, not only for cases of nonvenereal treponematosis, but also in the event of concurrent pregnancy, autoimmune disease, or other infections. Furthermore, a positive treponemal or nontreponemal test result may indicate serostate, not active syphilis.3
With serum treponemal antibodies, electrochemiluminescent immunoassays and CMIAs have high sensitivity and specificity and can be used to screen for T. pallidum infection.2 Marra's group reported that a CSF-TPPA titer threshold of 1:640 was sufficient for neurosyphilis diagnosis, and that CSF-TPPA might be useful for identifying patients with neurosyphilis when a CSF venereal disease research laboratory (VDRL) test is nonreactive.4 The conventional test for T. pallidum DNA using PCR is suitable for lesion swab specimens from lesions of patients with probable early syphilis.5,6 Indeed, conventional PCR testing has a sensitivity of up to 78.4% for lesion swabs of primary genital or anal chancres, and up to 83.0% for blood from neonates with congenital syphilis; the specificity of both tests is approximately 95%.7 However, the sensitivity of PCR was low for T. pallidum DNA in peripheral blood specimens in adult.8,9 There is an optimal window for PCR-based diagnosis of neurosyphilis, although nested and real-time PCR with T. pallidum polA or tpp47 is preferable.10 Grange's group found no agreement between T. pallidum in blood using nested PCR and a syphilis serological diagnosis; nested PCR had a sensitivity of only 29% for peripheral blood mononuclear cells and 14.7% for serum.3 Conventional PCR is more likely to contaminate DNA leading to unstable results, and real-time quantitative PCR can omit an electrophoresis step, decreasing contamination in a sealed reaction environment. However, its sensitivity for T. pallidum DNA array is low.10,11 The nested PCR, after several initial cycles, can amplify the target gene and increase accuracy.3,10 Thus, we tested the sensitivity and specificity of nested real-time PCR (NR-PCR), which is sensitive to a minimum of 2 T. pallidum strain Nichols cells/mL,12 which is greater than 103T. pallidum strain Nichols cells/mL using real-time quantitative PCR11 and 20 T. pallidum strain Nichols cells/mL of nested PCR.3
Available studies of T. pallidum DNA in biospecimens of syphilis patients are limited, so we investigated the feasibility of NR-PCR for identifying T. pallidum DNA from biological samples from syphilitic patients.
MATERIALS AND METHODS
Participants and Specimens
Inclusion Criteria for Participants
All patients with suspected syphilis visiting the out-patient dermatology or sexually transmitted disease (STD) departments of seven hospitals in Guangzhou, China between July 2012 and June 2014 were asked to participate in this study if they met the following criteria: a history of high-risk sexual behavior or more than 1 partner in the past; a reactive treponemal serological antibody test, with or without manifestations consistent with syphilis; and no prior syphilis treatment. Patients gave informed consent for participation prior to enrollment. All procedures used in this study were approved by the medical ethics committee of the Guangzhou Institute of Dermatology (N20121A031001_1).
All participants were tested using TPPA and RPR. Syphilis was diagnosed based on previously published methods.13 Briefly, based on a history of high-risk sexual behavior in the past 12 months, patients were diagnosed with confirmed primary syphilis if they had one or more chancres and if they were positive for T. pallidum based on dark field microscope regardless of result of RPR and/or TPPA test (RPR could be negative if the T. pallidum infection is less than 2 to 3 weeks, and TPPA could be negative at an early stage). Patients were diagnosed with confirmed secondary syphilis if they manifested syphilitic polymorphic skin eruptions and/or mucosa condylomata lata, and if both TPPA and RPR were positive, regardless of DFM results. We diagnosed patients with confirmed tertiary syphilis if they had either primary or secondary syphilis for more than 2 years; or if syphilitic skin nodules or gummas were observed; and if the patient presented with bone, eye, or other visceral syphilis, or with cardiovascular syphilis, such as simple aoristic, aortic regurgitation, or aortic aneurysm. We diagnosed patients with neurosyphilis based on serum TPPA and RPR positivity, and a routine CSF test recovered protein greater than 500 mg/L and white blood cell counts greater than 5 × 106/L without another explanation. Also, diagnoses were made if CSF fluorescent treponemal antibody absorption and/or VDRL were positive, and neurosyphilis was classified according to symptoms. For example, patients were considered to have a diagnosis of asymptomatic neurosyphilis if they had been without symptoms but had a positive serum TPPA and RPR test, a positive CSF routine test, and a positive CSF treponemal antibody test. Patients with meningeal neurosyphilis presented with fever, headache, stiff neck, and papilledema. Patients with cerebrovascular syphilis presented with cerebral vascular occlusive syndrome (e.g., hemiplegia aphasia) and epilepsy. Patients were diagnosed with brain parenchyma syphilis with additional mental symptoms, such as apathy and disorientation, and/or with nervous system symptoms, such as ataxia, thrill, and myasthenia. Confirmed early latent syphilis was diagnosed if the patients were positive for TPPA and RPR, and had no obvious CSF abnormality, and if patient had a sexual partner with a history of confirmed early syphilis within the past 2 years, or if patient had a history of high-risk sexual behavior or a blood transfusion within the past 2 years. Also, if the patient had untreated manifestations consistent with either primary or secondary syphilis within the past 2 years, a diagnosis of confirmed early latent syphilis was made. Late latent syphilis was considered if asymptomatic patient were TPPA- and RPR-positive, and had a syphilis history of more than 2 years. All syphilis patients were treated according to STD guidelines.13 The “first visit” was defined as the visit when syphilis was identified for the first time.
All biospecimens were collected according to prescribed procedures.12
For patients with ulcerate eruptions, the lesion exudate was collected with a lesion swab. For secondary syphilis with skin lesions, such as macules or papules without mucosal lesions, lesions were scraped gently with a lesion swab to obtain serous exudate. For DFM analysis, lesion exudate was collected with a bacteriology loop and transferred immediately to a glass slide for examination. For DNA extraction, lesion exudate was collected with a lesion swab, then immediately placed in 1-mL sterile PBS.
Whole Blood, Serum, and CSF Collection
We collected 3-mL whole blood in dry tubes from patients with primary, secondary, tertiary, and latent syphilis. We divided 1 mL of the collected blood into five 0.2-mL aliquots, and centrifuged the remaining 2 mL for 10 minutes at 2500g at room temperature. We then collected the supernatant (sera) and subpackaged it in 0.2-mL aliquots. We collected 50- to 100-μL earlobe peripheral blood from latent syphilis and neurosyphilis patients, and to the blood, we added 200-μL 0.9% NaCl. We collected CSF via lumbar puncture from suspected neurosyphilis patients without contraindications. Patients who received lumber punctures were given 500 mL of 10% glucose plus 2 g vitamins C (intravenously guttae), and were asked to lie flat for 6 hours of observation. Irrespective of syphilis type or stage, we attempted to amplify T. pallidum DNA in all collected samples which were stored at −80°C until NR-PCR was performed. We also collected 38 samples from nonsyphilitic patients as negative controls for DNA amplification. Table 1 depicts the characteristics of these collections.
Preparation for Reference T. pallidum DNA
Rabbit testis cultures containing T. pallidum strain Nichols were donated by Dr. Yin Yueping (National Venereology Reference Laboratory, Nanjing STD Research Center, Nanjing, Jiangsu province, China) to be prepared for reference DNA. We isolated T. pallidum DNA with a TIAN Amp Micro DNA kit (TianGen Biotech Co., Ltd., Beijing, China), following the manufacturer's instructions. We used Nano Drop ND-1000 Ultraviolet spectrophotometers (Thermo Scientific Co., Guangzhou, China) to quantify extracted DNA. We amplified T. pallidum DNA using Taq Man Master Mix (ABI Co., Ltd, Guangzhou, China) for PCR.
Patients DNA Extraction
We extracted DNA from 200 μL each of lesion swabs, whole blood, serum, earlobe peripheral blood, and CSF using a TIAN Amp Micro DNA Kit mentioned above, following the manufacturer's instructions. A UV spectrophotometer was used for quantitating DNA from patient samples, which were stored at −80°C until amplification.
PCR Primer Design
Primers and TaqMan Minor Groove Binder Probe
All PCR was performed at the School of Public Health, Sun Yat-sen University, Guangzhou, China. For the NR-PCR assay, we designed two pairs of primers specific to the target T. pallidum gene, polA (partial coding sequence (CDS); GenBank accession no. TPU57757.1; Table 2). We renewed additional sets of primers for PCR and real-time PCR using published primer sequences.14,15 Primers and the TaqMan Minor Groove Binder probe (Table 2) were synthesized by Applied Biosystems (Guangzhou, China).
We amplified T. pallidum DNA using Taq Man Master Mix (ABI Co., Ltd, Guangzhou, China) for PCR. Each 25 μL initial PCR volume contained 3 μL extracted DNA as the template, 1 μL of each outer primer (F1/R1, 0.4 μM), and 20 μL Platinum PCR Super Mix (Invitrogen, Guangzhou, China). Initial PCR was performed in a Thermal Cycle PTC-200 (Bio-Rad, Shanghai, China), with the following cycling program: degeneration at 94°C for 5 minutes; 30 cycles of 94°C for 30 seconds, 68°C for 30 seconds, and 72°C for 2 minutes; and a final extension at 72°C for 7 minutes. The product of initial amplification was diluted in 1:2, We then added 4 μL of dilution as template to each well of a 384-well plate, the remaining dilution was stored in −20°C. Plates were dried in an incubator at 65°C for 30 minutes. We next added 2.5 μL TaqMan Master Mix (ABI, Guangzhou, China) and 1 μL of each inner primer (F2/R2, 0.3 μM) to each well. PCR cycling conditions were as follows: incubation 50°C for 2 minutes, followed by 40 cycles of 95°C for 15 minutes, 95°C for 15 seconds, and 60°C for 1 minute. For each specimen, we also amplified negative and positive controls (negative sample, sterile water and T. pallidum strain Nichols, respectively). For all specimens, Ct values were derived using Sequence Detection System (SDS, ABI) software. We considered Ct values less than 40 positive for T. pallidum DNA.
We used χ2 tests and SPSS 17.0 (SPSS, Chicago, IL) to assess the relationship between positive T. pallidum PCR results and syphilis type (P < 0.05 was considered statistically significant).
There were 266 participants in our study, between the ages of 21 and 65 (median: 36) (Table 1). Of these participants, 228 patients with suspected syphilis, with an average disease duration of 6.8 months (range, 3 days to 52 months), and 38 nonsyphilitic individuals used as controls of these patients. Of the 228 patients, 200 were eventually diagnosed with confirmed infections (Table 1). All 19 of the patients diagnosed with primary syphilis presented with chancres. Of the 23 patients diagnosed with secondary syphilis, 12 presented with a rose rash and 11 presented with condyloma lata. The remaining 28 patients, without confirmed infections, were considered probably syphilitic: 11 were diagnosed with probable primary syphilis, 12 were TPPA-positive with no other symptoms, indicating nonreactive syphilis, and 5 were suspected to have latent syphilis or be in a syphilitic serostate (RPR positive with a history of syphilis treatment within the last 2 years).
Positive T. pallidum Tests in Different Specimen Types From Patients at Various Stages of Syphilis Progression
Our NR-PCR identified 71.7% of the syphilitic patient samples (264/368) as positive for T. pallidum DNA (Table 3); all 38 control samples were negative for T. pallidum DNA. In patients diagnosed with neurosyphilis, there were no significant differences between the results for the serum and whole-blood samples (P = 0.2), or between the earlobe blood and CSF samples (p = 0.74). When serum and whole-blood samples were combined (to generate venous whole blood samples [VWB]), earlobe blood samples tested positive significantly more often than did the VWB samples (Fisher exact test, P = 0.02), but no significant difference was observed between the CSF and VWB samples (P = 0.14). In the patients diagnosed with latent syphilis, there were no significant differences between the number of serum and whole blood samples positive for T. pallidum DNA (P = 0.18); all earlobe blood and CSF samples tested positive (Table 3). Of the 84 biospecimens considered negative for the NR PCR test, 1 serum sample from a patient with serological RPR positive was positive for T. pallidum DNA.
Testing for T. pallidum DNA in Serum From Patients With Different RPR Titrations
We divided all syphilis patients with serum samples into 2 groups based on serum RPR titer (≥1:8 or ≤ 1:4). We tested these 159 serum samples independently for T. pallidum DNA (1 serum sample per patient). All 33 patients (23 diagnosed with secondary, and 10 with tertiary syphilis) had RPR titers >1:4 (Table 4). In all 23 cases of secondary syphilis, RPR titer was 1:8 or greater; 62.5% of these samples tested positive for T. pallidum DNA. Of the 10 tertiary syphilis patients with RPR of 1:8 or greater; 50% of these samples tested positive for T. pallidum DNA. There were no significant differences in number of positive NR-PCR results between the RPR groups for primary and neurosyphilis. However, there were significantly more positive T. pallidum DNA tests in the latent syphilis patients with RPR titers of 1:8 or greater, as compared with the patients with RPR titers of 1:4 or less (Table 4).
Nested and real-time PCR to assess T. pallidum DNA in various biospecimens from syphilis patients correctly returned a positive result for 71.7% of samples: lesion swab samples were 74.3% positive, serum samples were 66.9% positive, whole blood samples were 64.2% positive, earlobe blood samples were 92% positive, and CSF samples were 90.2% positive across all patients with confirmed syphilis infections. No significant differences were observed in positive results from lesion swab and whole blood samples from early syphilis cases, perhaps due to small sample size.
Nested and real-time PCR accurately returned more correct positive results than previous PCR approaches for the collection methods used here3,11,14,16; nevertheless, NR-PCR did not return more positive results than previous PCR approaches for lesion swabs.3,7,14 This may be due to insufficient T. pallidum DNA in lesion swab samples. The NR-PCR results also indicated specificity for T. pallidum DNA of 99%—only 1 of 84 control samples was positive for T. pallidum DNA (Table 3), and the positive DNA result was from a patient with positive serological RPR. Increased accuracy of NR-PCR may be explained by three differences between NR-PCR and previous PCR approaches. First, we increased the concentration of the T. pallidum DNA template after amplification with the outer primer pairs, increasing sensitivity. Second, we used a sensitive and specific TaqMan Minor Groove Binder probe. Third, we used a more specific target gene polA, which encodes DNA polymerase I which has a high cysteine content and four unique insertions. The T. pallidum polA gene has low homology with polA from other microorganisms.6
PCR can be used to diagnose early syphilis and asymptomatic neurosyphilis when serum antibodies and CSF VDRL are negative.7,17,18 Currently used PCR methods identify between 1 and 400 T. pallidum cells/mL. For example, Marra's group designed an improved RT-PCR method to identify up to 10 T. pallidum cells/mL in CSF.19 Zeng and Zheng's group reported that regular and nested PCR found 400 and 10 T. pallidum cells/mL, respectively,20,21 whereas Grange's group reported that nested PCR identified 20 T. pallidum strain Nichols cells/mL.3 Except for a congenital syphilis a neonate sample with more greater T. pallidum DNA positivity than an adult sample,22 PCR data for syphilitic samples were the least sensitive for whole blood samples and most sensitive for lesion swab samples.3,9,14,23 Insufficient T. pallidum cells in lesion swabs sample may have resulted in less DNA positivity.18 Meanwhile, NR-PCR assays were positive for 38.5% to 87.2% of all syphilis cases tested, an improvement over previous nested PCR assays.3,24,25 Nested and real-time PCR was the most successful at identifying T. pallidum in earlobe blood of neurosyphilis and latent syphilis patients. In addition, NR-PCR assays of CSF samples from latent syphilis and neurosyphilis patients were more accurate than previous tests using real-time PCR.7T. pallidum DNA positivity was associated with serum RPR titers in latent syphilis patients: NR-PCR was significantly more likely to return a positive result for latent syphilis patients with serum RPR titers of 1:8 or greater, compared to RPR titers of 1:4 or less. The RPR titers 1:8 or greater are typically considered to indicate active syphilis.26,27
Thus, NR-PCR can be used to quantify T. pallidum DNA, but limitations include the following: we used no controls for real-time PCR, and we did not analyze syphilis diagnoses for positive DNA tests using NR-PCR and positive serum treponemal antibody with TPPA arrays. Finally, different sample sizes of syphilis types might have introduced bias, and larger sample sizes are necessary to validate our results.
- (1) NR-PCR is feasible and valuable for identifying T. pallidum DNA from biospecimens of patients at various stages of syphilis disease progression.
- (2) Peripheral earlobe venous blood from latent syphilis and neurosyphilis patients had the greatest T. pallidum DNA positivity.
- (3) Serum T. pallidum DNA positivity for latent syphilis patients with RPR titers ≥ 1:8 was greater than that for RPR titers ≤ 1:4.
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