Because there is no way to culture Treponema pallidum (T. pallidum) in vitro, molecular methods for strain typing are critical.1 The first molecular typing system introduced by the US Centers for Disease Control and Prevention is based on the interstrain variability of acidic repeat protein gene (arp [tp0433]) and T. pallidum repeat subfamily II genes (tprE [tp0313], tprG [tp0317], and tprJ [tp0621], hereinafter referred to as tpr genes).2 An enhanced typing method has recently been developed that uses tp0548 gene in addition to the arp and tpr genes, yielding a greater discriminatory power.3 Molecular typing is a powerful tool for determining diversity and epidemiology of syphilis infection, and has the potential to enhance clinical care and prevention and control efforts by contributing to a better understanding of T. pallidum acquisition and transmission.4 Globally, molecular typing of T. pallidum has been conducted in United States, South Africa, Portugal, Scotland, Canada, Madagascar, Ireland, and Colombia.2,3,5–14
National sexually transmitted disease (STD) surveillance program has demonstrated a resurgence of syphilis in China,15,16 but molecular epidemiology of this outbreak has been limited to only a few published studies confined to Shanghai, Guangdong, and South Hunan with small sample sizes.17–20 This study aimed to investigate strain type distribution of T. pallidum causing early syphilis across diverse areas in China, using the enhanced method.
MATERIALS AND METHODS
Study Population and Data Collection
The study was conducted in the STD clinics from 8 cities during the years 2008 to 2010: namely, Nanjing (East China), Guangzhou, Jiangmen, Fuzhou, Nanning, and Chengdu (South China), and Tianjin and Harbin (North China). Outpatients, who had typical genital ulcer of primary stage or skin lesion of secondary stage, and rapid plasma reagin (RPR) and T. pallidum particle agglutination (TPPA) positive result, were recruited. This study was approved by the Medical Ethics Committee of Chinese Academy of Medical Sciences and Peking Union Medical College Institute of Dermatology. Informed written consent was obtained from subjects before participation. All participants were interviewed using a short structured questionnaire on sociodemographic background, commercial sex behavior, and STD history. Serum RPR titer was recorded. All participants were provided with a routine physical examination, health education, necessary counseling, and treatment, according to the national guidelines.
Clinical samples were collected from moist genital ulcers, condyloma lata, papulae, and mucosal patches. Necrotic materials or crusts on the lesion were gently removed with a sterile gauze. The intact papular lesions were gently scarified by a blunt scalpel. Then clear exudates were obtained using a sterile swab by gently rubbing the base of the lesion. The swab was swirled vigorously in a tube containing 1 mL of sample transport buffer (10 mmol/L Tris-HCl PH = 8.0, 0.1 mol/L EDTA PH = 8.0, and 0.5% SDS) for 15 seconds and then was pressed against the side of the tube to express the excess liquid. The swab was withdrawn and discarded. The tube was capped, labeled with a serial number, and temporarily stored at −20°C in the local laboratory before being transported to the National STD Reference Laboratory in Nanjing for molecular typing.
DNA Extraction and Detection of T. pallidum DNA by Polymerase Chain Reaction
DNA was extracted using QIAamp DNA mini Kit (Qiagen, GmbH, Germany) according to the manufacturer's instructions. All the extracted samples were screened for T. pallidum DNA by a diagnostic polymerase chain reaction (PCR) assay of polA gene, which is unique to T. pallidum, as described by Liu et al.21
Analysis of the arp Gene
Briefly, 5 μL of extracted DNA was added in a 50 μL reaction mixture, containing 10 μL of 5 × buffer, 1.0 mmol/L MgCl2, 1 μL deoxynucleoside triphosphates (dNTPs, 200 umol/L each of dTTP, dCTP, dATP, and dGTP), 500 nmol/L of each primer (5′-CAAGTCAGGACGGACTGTCC-3′ and 5′-GGTATCACCTGGGGATGC-3′), 2.5 U of Taq polymerase (Promega, Madison, WI).2,3 The PCR was performed using the GeneAmp system 9700 under the following conditions: 4 minutes of denaturation at 94°C, then 40 cycles of amplification at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute, followed by 7 minutes of final extension at 72°C. The amplicon was electrophoresed on a 1.5% agarose gel together with 100 base pair (bp) DNA ladder (NEB, Beverly, USA) at 80V for 200 minutes and visualized by ethidium bromide (EB) staining. Then, the sizes of the products were analyzed using Quantity One software (Version 4.6.2, Bio-Rad).
Analysis of the tpr Genes
In brief, 5 μL of extracted DNA was added in a 50 μL reaction mixture, containing 10 μL of 5 × buffer, 2.5 mmol/L MgCl2, 1 μL dNTPs (200 umol/L each of dTTP, dCTP, dATP, and dGTP), 600 nmol/L of each primer (5′-CAGGTTTTGCCGTTAAGC-3′ and 5′-AATCAAGGGAGAATACCGTC-3′), 2.5 U of Taq polymerase.3 The PCR conditions were the same as that of the arp gene. In all, 10 μL of amplicon was digested for 16 hours in a 20 μL reaction mixture, containing 2 μL of 10 × buffer, 0.2 μL of 100 × BSA, and 10 U MseI (NEB, Beverly, MA).3 Digestion products were separated by 2% agarose gel electrophoresis at 100 V for 150 minutes and visualized by EB staining. The banding patterns were compared with that identified by Pillay et al.2
Analysis of the tp0548 Gene
Briefly, 5 μL of extracted DNA was added in a 50 μL reaction mixture, containing 10 μL of 5 × buffer, 1.5 mmol/L MgCl2, 1 μL dNTPs (200 umol/L each of dTTP, dCTP, dATP, and dGTP), 600 nmol/L of each primer (5′-GGTCCCTATGATATCGTGTTCG-3′ and 5′-GTCATGGATCTGCGAGTGG-3′), 2.5 U of Taq polymerase.3 Cycling conditions were 2 minutes of denaturation at 95°C, then 40 cycles of amplification at 95°C for 1 minute, 62°C for 1 minute 15 seconds, and 72°C for 1 minute, followed by 10 minutes of final extension at 72°C. The amplicons were sequenced, and the portion of the gene ∼130 bp downstream of the start codon of tp0548 gene was examined to determine the sequence pattern.3
In each PCR assay, DNA from T. pallidum Nichols strain and double distilled water instead of DNA were used as positive and negative control, respectively. The strain type was determined by combining results of the above 3 genes as described by Marra et al.3
Statistical package for the Social Sciences for Windows (SPSS, version 18.0, Chicago, IL) was used for statistical analysis. Descriptive statistics were used to calculate median, interquartile range (IQR), and 95% confidence interval (CI). χ2 tests were used to compare the distribution of strain types across the 3 geographic areas, and to analyze the relationship between strain type and characteristic of patients with fully typed samples.
A total of 324 samples were collected from 324 outpatients with clinically suspected early syphilis, including 211 with primary syphilis and 113 with secondary syphilis. Specifically, 112 were from East China, 164 from South China, and 48 from North China. The median age of patients was 38 with IQR of 30 to 46. Among the 324 patients, 79% were male, and 73% were married.
T. pallidum DNA existed in 218 samples by polA PCR assay, including 127 primary syphilis samples and 91 secondary syphilis samples. The numbers of successful amplification of the arp, tpr, and tp0548 genes were 199 (91%), 197 (90%), and 207 (95%), respectively. Overall, 197 (90%) had sufficient DNA for full molecular typing. The characteristics of patients providing fully typed samples are shown in Table 1.
Overall, 27 strain types were identified among 197 fully typed samples. A range of 3 to 20 repeats (except 4, 11, and 19 repeats) and 25 repeats were found for the 60 bp tandem repeats presenting in the arp gene. This was the first time the 9 and 25 repeats were detected. For the restricted fragment length polymorphism (RFLP) analysis of the tpr genes, patterns a, d, h, j, and l were identified. This was the first time the h, j, and l patterns were observed in China. For the sequence analysis of the tp0548 gene, sequences c, e, and f were identified. Overall, strain type 14d/f was predominant (39% 76/197, 95% CI = 32%–46%), and its distribution trend was consistent among areas, ranging from 34% in the South to 43% in the East. Strain types 13d/f, 15d/f, 16d/f were the next most common types (each 13% 25/197, 95% CI = 9%–18%). We did not find any significant change in strain type distribution over time period. The detailed distribution of strain types is shown in Figure 1.
By categorizing strain types into 13d/f, 14d/f, 15d/f, 16d/f, and others, the distribution was statistically different between East, South, and North China (χ2 = 20.6, P = 0.006). There were no relationship between strain type and migrant residence (χ2 = 6.6, P = 0.89), between strain type and commercial sex behavior (χ2 = 5.1, P = 0.27), and between strain type and having STD history (χ2 = 1.7, P = 0.79). The relationship between strain type and men who have sex with men (MSM) behavior was not analyzed because the number of patients having MSM behavior was small (2 participants acknowledged MSM behavior).
China is a country with elimination of syphilis in 1964 and resurgence of this infectious disease in 1980s.15 Epidemic of syphilis has been experienced all over the country, although most of cases have been reported in areas along coastal regions.15 The spread of syphilis in China has been driven by many factors, including rural-to-urban migration; high-risk behaviors and sexual networks of heterosexual, homosexual, and bisexual contacts; limited routine screening and treatment; incomplete partner notification; and stigma associated with health-seeking behaviors among high-risk populations.22 Our study found an abundant diversity of strain types (27 types out of 197 fully typed samples), which is similar to that identified in South Africa where syphilis has been endemic (35 types out of 161 fully typed samples).8 The high level of diversity and broad distribution of strain types may reflect a relatively long-term prevalence of infectious syphilis, allowing widespread transmission from untreated individuals to susceptible contacts.
Overall, 14d/f accounted for 39% of fully typed samples, indicating that it may be the most prevalent circulating strain type in China. As all the study subjects came from patients attending STD clinics in different areas, the discordant distribution of strain types in geographic locations related to East, South, and North China may imply that the sexual network was relatively independent from each other in the study areas. Moreover, predominance of few strain types (14d/f, 13d/f, 15d/f and 16d/f) from our study may indicate a linked transmission network. The predominance of strain type 14d/f in our study coincided with the finding in Seattle (51% of fully typed samples).3 Among previous studies using the arp and tpr genes for molecular typing of T. pallidum causing early syphilis in other countries, 14d was also a most common subtype in Scotland (76% of fully typed samples),12 Canada (83% of fully typed samples),13 South Africa (27% of fully typed samples),8 and Colombia (2 of 6 fully typed samples).14 However, 14a was most frequent in Portugal.10,11 Moreover, both studies in Arizona and North and South Carolina in the United States identified 14f as most common.5,6
In China, some small-scale molecular studies, which were based only on the arp and tpr genes, have described the subtype distribution of T. pallidum in Shanghai, Guangdong, and South Hunan.17–20 Compared with these studies, our report described the first identification of h, j, and l patterns for the tpr genes in China. Moreover, our use of the enhanced typing method described by Marra et al3 has improved the typing discrimination among T. pallidum strains circulating in China. Previously, 10 subtypes were identified among 63 fully typed samples from 2002 to 2004 in South Hunan,19,20 12 subtypes in 79 fully typed samples from 2002 to 2004 in Guangdong,18,19 and 4 subtypes from 36 subjects in Shanghai from 2007 to 2008.17 Subtype 14d was predominant in South Hunan (44%) and Guangzhou (57%), which is consistent with our study.18–20 Of interest, the study at an STD clinic in Shanghai reported a different subtype distribution, where 14f (83%) was largely predominant, and 14d accounted for 11% of 36 fully typed samples.17 Our study did not recruit subjects in Shanghai and did not find the 14f subtype. Shanghai is China's largest city and may have its own circulating pattern that needs to be explored using a larger sample size.
Some limitations of this study should be acknowledged. First, the number of clinical samples collected in the 3 geographic areas was not well proportional and may result in lack of recognition of some strain types in the underrepresented regions. Second, Tibet, Inner Mongolia, and Xinjiang, where the minorities are gathering, were not included in the study sites. Thus, the strain type distribution in minorities was not addressed in this report. Third, we were unable to find pairs of mutual sexual contacts, and it was not possible to identify clusters associated with specific transmission networks. However, to our knowledge, this report is the largest molecular typing study to date, and is the first cross-sectional study to investigate strain type distribution of T. pallidum causing early syphilis in multiple geographic areas of China. This study can be used as a baseline survey for setting up a periodic molecular surveillance program in China to monitor the trends of strain type distribution of T. pallidum in different populations, and as a supplement for better understanding of epidemiology and transmission of syphilis in China.
Molecular typing of T. pallidum provides the opportunity to evaluate the distribution of macrolide resistant strains, to identify neuroinvasive strain types, and to trace sexual networks in order to guide public health interventions. Further studies with more representative samples and careful collection of clinical and epidemiologic data are needed to investigate these research and public health questions.
1.Lafond RE, Lukehart SA. Biological basis for syphilis. Clin Microbiol Rev 2006; 19:29–49.
2.Pillay A, Liu H, Chen CY, et al. Molecular subtyping of Treponema pallidum
. Sex Transm Dis 1998; 25:408–414.
3.Marra CM, Sahi SK, Tantalo LC, et al. Enhanced molecular typing of Treponema pallidum
: Geographical distribution of strain types and association with neurosyphilis. J Infect Dis 2010; 202:1380–1388.
4.Morshed MG, Lee MK, Jorgensen D, et al. Molecular methods used in clinical laboratory: Prospects and pitfalls. FEMS Immunol Med Microbiol 2007; 49:184–191.
5.Sutton MY, Liu H, Steiner B, et al. Molecular subtyping of Treponema pallidum
in an Arizona County with increasing syphilis morbidity: Use of specimens from ulcers and blood. J Infect Dis 2001; 183:1601–1606.
6.Pope V, Fox K, Liu H, et al. Molecular subtyping of Treponema pallidum
from North and South Carolina. J Clin Microbiol 2005; 43:3743–3746.
7.Katz KA, Pillay A, Ahrens K, et al. Molecular epidemiology of syphilis—San Francisco, 2004–2007. Sex Transm Dis 2010; 37:660–663.
8.Pillay A, Liu H, Ebrahim S, et al. Molecular typing of Treponema pallidum
in South Africa: Cross-sectional studies. J Clin Microbiol 2002; 40:256–258.
9.Molepo J, Pillay A, Weber B, et al. Molecular typing of Treponema pallidum
strains from patients with neurosyphilis in Pretoria, South Africa. Sex Transm Infect 2007; 83:189–192.
10.Florindo C, Reigado V, Gomes JP, et al. Molecular typing of Treponema pallidum
clinical strains from Lisbon, Portugal. J Clin Microbiol 2008; 46:3802–3803.
11.Castro R, Prieto E, Aguas MJ, et al. Molecular subtyping of Treponema pallidum
in Lisbon, Portugal. J Clin Microbiol 2009; 47:2510–2512.
12.Cole MJ, Chisholm SA, Palmer HM, et al. Molecular epidemiology of syphilis in Scotland. Sex Transm Infect 2009; 85:447–451.
13.Martin IE, Tsang RS, Sutherland K, et al. Molecular typing of Treponema pallidum
strains in western Canada: Predominance of 14d subtypes. Sex Transm Dis 2010; 37:544–548.
14.Cruz AR, Pillay A, Zuluaga AV, et al. Secondary syphilis in Cali, Colombia: New concepts in disease pathogenesis. PLoS Negl Trop Dis 2010; 4:e690.
15.Chen ZQ, Zhang GC, Gong XD, et al. Syphilis in China: Results of a national surveillance programme. Lancet 2007; 369:132–138.
16.Tucker JD, Chen XS, Peeling RW. Syphilis and social upheaval in China. N Engl J Med 2010; 362:1658–1661.
17.Martin IE, Gu W, Yang Y, et al. Macrolide resistance and molecular types of Treponema pallidum
causing primary syphilis in Shanghai, China. Clin Infect Dis 2009; 49:515–521.
18.Zheng HP, Ou ZY, Hu YS, et al. Detection and genotyping of Treponema pallidum
by a nested PCR. Chin J Dermatol 2005; 38:546–548.
19.Zeng TB, Wu YM, Huang SJ, et al. Preliminary study on molecular subtyping of Treponema pallidum
in Hengyang and Jiangmen regions. Chin J Dermatol 2004; 37:692–694.
20.Zhan LS, Zeng TB, Yan JL, et al. Preliminary study on molecular subtyping of Treponema pallidum
in South Hunan area. Pract Prevent Med 2005; 12:486–488.
21.Liu H, Rodes B, Chen CY, et al. 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. J Clin Microbiol 2001; 39:1941–1946.
22.Tucker JD, Cohen MS. China's syphilis epidemic: Epidemiology, proximate determinants of spread, and control responses. Curr Opin Infect Dis 2011; 24:50–55.