Abstract. Introduction. Editor: S. Cutler

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1 ORIGINAL ARTICLE BACTERIOLOGY Multicentre surveillance of prevalence of the 23S rrna A2058G and A2059G point mutations and molecular subtypes of Treponema pallidum in Taiwan, B.-R. Wu 1, C.-J. Yang 2, M.-S. Tsai 2, K.-Y. Lee 3, N.-Y. Lee 4, W.-C. Huang 5,H.Wu 6, C.-H. Lee 5, T.-C. Chen 7,8, W.-C. Ko 4, H.-H. Lin 9, P.-L. Lu 7, Y.-H. Chen 7, W.-C. Liu 10, S.-P. Yang 11, P.-Y. Wu 11, Y.-C. Su 10, C.-C. Hung 10,12,13 and S.-Y. Chang 1,14 1) Department of Clinical Laborary Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, 2) Department of Internal Medicine, Far Eastern Memorial Hospital, New Taipei City, 3) Department of Internal Medicine, National Taiwan University Hospital Hsin-Chu Branch, Hsin-Chu, 4) Department of Internal Medicine, National Cheng Kung University College of Medicine and Hospital, Tainan, 5) Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, 6) Centres for Disease Control, Taipei, 7) Department of Internal Medicine, Kaohsiung Medical University Hospital and College of Medicine, 8) Department of Internal Medicine, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University, Kaohsiung, 9) Department of Internal Medicine, E-Da Hospital/I-Shou University, Taiwan, 10) Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, 11) Centre for Infection Control, National Taiwan University Hospital, Taipei, 12) Department of Medical Research, China Medical University Hospital, 13) China Medical University, Taichung, and 14) Department of Laborary Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan Abstract Resistance mutations A2058G and A2059G, within the 23S rrna gene of Treponema pallidum, have been reported cause treatment failures in patients receiving azithromycin for syphilis. Genotyping of T. pallidum strains sequentially isolated from patients with recurrent syphilis is rarely performed. From September 2009 August 2013, we collected 658 clinical specimens from 375 patients who presented with syphilis for genotyping examine the number of 60-bp repeats in the acidic repeat protein (arp) gene, T. pallidum repeat (tpr) polymorphism, and tp0548 gene, and detect A2058G and A2059G point mutations by restriction fragment length polymorphism. Treponemal DNA was identified in 45.2% (n = 298) of the specimens that were collected from 216 (57.6%) patients; 268 (40.7%) specimens tested positive for the 23S rrna gene, and were examined for macrolide resistance. Two isolates (0.7%) harboured the A2058G mutation, and no A2059G mutation was identified. A tal of 14 strains of T. pallidum were identified, with 14f/f (57.5%) and 14b/c (10.0%) being the two predominant strains. Forty patients who presented with recurrent episodes of syphilis had T. pallidum DNA identified from the initial and subsequent episodes, with five cases showing strain discrepancies. One patient had two strains identified from different clinical specimens collected in the same episode. Our findings show that 14f/f is the most common T. pallidum strain in Taiwan, where the prevalence of T. pallidum strains that show A2058G or A2059G mutation remains low. Different genotypes of T. pallidum can be identified in patients with recurrent episodes of syphilis. Keywords: Antimicrobial resistance, azithromycin, genotyping, macrolides, molecular epidemiology, syphilis Original Submission: 1 Ocber 2013; Revised Submission: 10 January 2014; Accepted: 12 January 2014 Edir: S. Cutler Article published online: 20 January 2014 Clin Microbiol Infect 2014; 20: / Corresponding author: C.-C. Hung, Department of Internal Medicine, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei 100, Taiwan hcc0401@ntu.edu.tw and S.-Y. Chang, Department of Clinical Laborary Sciences and Medical Biotechnology, National Taiwan University College of Medicine, 1 Chang-Te Street, Taipei, Taiwan sychang@ntu.edu.tw Introduction Syphilis remains a significant public health problem in many developed and developing countries in the era of the human immunodeficiency virus (HIV) epidemic [1 4], although effective antibiotics have been available for more than six decades. In the USA, the number of cases of primary and secondary syphilis reported the CDC increased from in 2010 Clinical Microbiology and Infection ª2014 European Society of Clinical Microbiology and Infectious Diseases

2 CMI Wu et al. Macrolide resistance and genotyping of Treponema pallidum in Taiwan in 2011, an increase of 1.4%, with men who have sex with men (MSM) accounting for 83% and 72% in 2010 and 2011, respectively [1]. Recurrences of syphilis are not uncommon in patients with previous syphilis, ranging from 10% 42.7% [5 7]; however, without reliable genes for phylogenetic analysis of Treponema pallidum, outbreak investigation and surveillance of syphilis are difficult. Recently, a molecular typing method that combined the repeat numbers of the 60-bp acidic repeat protein (arp) gene with the restriction patterns of the T. pallidum repeat (tpr) gene and the sequence of tp0548 was introduced determine T. pallidum strains [8,9]. This technique may make epidemiological moniring of T. pallidum infection possible in different geographical regions [8,10 14]. Benzathine penicillin G, at a dose of 2.4 million units administered intramuscularly, has been the recommended treatment for all stages of syphilis other than neurosyphilis; alternative treatments have included azithromycin and doxycycline for patients with penicillin allergy or inlerance [2]. Of the alternatives, azithromycin has the advantages of single oral dosing, a long tissue half-life, and treatment of concurrent chlamydial infection [2,15]. However, the emergence of T. pallidum with macrolide resistance mutations, A2058G and A2059G, on the 23S rrna gene is a major concern [16,17], especially as a high prevalence of macrolide-resistant T. pallidum has been increasingly reported in several developed and developing countries [13,18,19], which makes surveillance of T. pallidum with macrolide resistance mutation an integral part of syphilis control programmes. In this multicentre surveillance study, we aimed investigate the molecular epidemiology of T. pallidum and the prevalence of macrolide resistance mutations (A2058G and A2059G) in Taiwan. We used the newly proposed enhanced molecular typing methods genotype the T. pallidum strains sequentially isolated from the patients with recurrent syphilis. Materials and Methods Study setting and population From September 2009 August 2013, we enrolled patients who presented with untreated syphilis at the infectious diseases clinics of eight major designated hospitals for HIV care around Taiwan, including National Taiwan University Hospital (Taipei), Far Eastern Memorial Hospital (New Taipei City), National Taiwan University Hospital Hsin-Chu Branch (Hsin-Chu), National Cheng Kung University Hospital (Tainan), Kaohsiung Chang Gung Memorial Hospital, Kaohsiung Medical University Hospital, Kaohsiung Municipal Ta-Tung Hospital, and E-Da Hospital (Kaohsiung). Patients who had received antibiotic treatment prior enrolment and rapid plasma reagin (RPR) titres of <1 : 4 were excluded. Information was collected on the demographics, RPR titre, HIV serostatus and stage of syphilis in all patients, and on combination antiretroviral therapy (cart), CD4 lymphocyte count and plasma HIV RNA load (PVL) in HIV-infected patients. This study was approved by the Research Ethics Committees of the participating hospitals (registration number, R), and all subjects gave written informed consent. Although there is no standard definition for recurrent syphilis, we defined recurrent syphilis as a four-fold increase in RPR titre after achievement of a decline of RPR titres after 6 12 months of treatment plus a hisry of exposure patients with syphilis or clinical manifestations of syphilis. Treatment of syphilis was administered by following the Sexually Transmitted Diseases Treatment Guidelines [2,15]. Collection of clinical specimens, DNA extraction, and PCR method All specimens were collected before treatment was administered. Serum and plasma were obtained from anticoagulant-free and EDTA-coated containers (BD Vacutainer tubes; Becn Dickinson, Plymouth, UK), respectively. The cerebrospinal fluid and vitreous fluid specimens were transported in sterile containers. Sampling of multiple specimens was performed when patients presented with multiple ulcers or concurrent lesions in cases of primary and secondary syphilis. Chancres and exudates were collected by swabbing. All specimens were transported the research laborary at the National Taiwan University Hospital for molecular typing and detection of resistance mutations. Treponemal DNA was extracted with the QIAamp DNA mini Kit (Qiagen, Hildens, Germany), according the manufacturer s procol. The diagnostic PCR assay was used amplify a 377-bp fragment of the T. pallidum pol Ι gene, as described previously [20], with confirmation by agarose-gel electrophoresis. Detection of macrolide resistance mutations A2058G and A2059G mutations were detected with restriction fragment length polymorphism (RFLP). Briefly, the 23S rrna gene of T. pallidum was amplified and subjected MboII and BsaI digestion (New England Biolabs, Beverly, MA, USA). The mutations were confirmed by comparison with previous studies after gel electrophoresis [16,17]. Molecular typing of T. pallidum Molecular typing was performed by using a combination of the results for the 60-bp repeat numbers of the acidic repeat protein (arp) gene, the RFLP patterns of T. pallidum repeat (tpr)

3 804 Clinical Microbiology and Infection, Volume 20 Number 8, August 2014 CMI genes with MseI (New England Biolabs), and sequencing of the short region ( bp) within the tp0548 gene [8,9]. All thermocycling was performed on a T3000 thermocycler (Biomatra, Leipzig, Germany). The numbers of the repeat were estimated by comparing the size of the PCR product with that amplified from the Nichols strain, which contains 14 repeats. The RFLP patterns of the tpr gene were determined by comparing the electrophoresis patterns with previous data [9]. The sequences of the tp0548 PCR products were determined with an aumatic sequencer (ABI 3730; Applied Biosystems Instruments, Foster City, CA, USA), and the derived sequences in the region of bp were aligned with the Clustal W programme in the MEGA version 6.0 [21]. Statistical analysis All statistical analyses were performed with SPSS software, version 16.0 (SPSS, Chicago, IL, USA). Associations with categorical variables were determined by use of the Pearson chi-square test or Fisher s exact test, and Student s t-test or the Mann-Whitney U-test was used for non-categorical variables. All p-values were two-tailed, and results with a p-value of <0.05 were considered be statistically significant. Results Characteristics of the study subjects During the 4-year study period, a tal of 658 specimens were collected from 375 patients; 40 (10.7%) returned for retreatment because of recurrent syphilis, and 195 (Fig. S1) provided more than one specimen from the same episode of syphilis (median number, 2; range, 1 6). The clinical characteristics of the patients are shown in Table 1. Of the 375 patients, 306 (81.6%) were HIV-infected, and all except one were male. HIV-infected patients were older (32.7 vs years) and more likely be MSM (98.4% vs. 92.3%) than HIV-uninfected patients. Although the RPR titres (1 : 64) were similar for HIV-infected and HIV-uninfected patients, HIV-infected patients were more likely present with early latent syphilis (36.9% vs. 10.3%) and less likely present with secondary syphilis (35.9% vs. 56.5%) than HIV-uninfected patients. Of the HIV-infected patients, 220 (71.9%) were receiving cart when syphilis was diagnosed, with a mean CD4 count of 514 cells/ mm 3 and a PVL of 2.63 log 10 copies/ml. The details of clinical characteristics of the 40 patients with recurrent syphilis are shown in Table S1, and are summarized in Table 2. All of the patients with recurrent syphilis were male and MSM, and >90% were also HIV-infected; one patient seroconverted for HIV when he presented with the second TABLE 1. Characteristics of the study subjects who presented with syphilis and underwent sampling for PCR detect Treponema pallidum Variable HIVinfecteuninfected HIV- All patients (n = 375) (n = 306) (n = 69) p-value a Age (years), mean 31.8 (7.7) 32.7 (7.5) 28.1 (7.5) <0.002 (SD) Male gender, n (%) 374 (99.7) 305 (99.7) 69 (100) >0.999 MSM, n (%) 349 (97.5) 301 (98.4) 49 (92.3) RPR titre (log 2 ), 6(2 11) 6 (2 11) 6 (2 10) median (range) Syphilis stage, n (%) Primary 64 (17.1) 52 (17.0) 12 (17.4) Secondary 149 (39.7) 110 (35.9) 39 (56.5) Primary and 27 (7.2) 20 (6.5) 7 (10.1) secondary Early latent 122 (32.5) 113 (36.9) 9 (10.3) Late latent 5 (1.3) 4 (1.3) 1 (1.4) Neurosyphilis 8 (2.1) 7 (2.3) 1 (1.4) CD4 count in HIVinfected 514 (403) NA patients (cells/mm 3 ), mean (SD) (n = 301) Plasma HIV RNA 2.63 (1.51) NA load (log 10 copies/ml), mean (SD) (n = 301) On cart, n (%) 220 (71.9) NA cart, combination antiretroviral therapy; HIV, human immunodeficiency virus; MSM, men who have sex with men; NA, not applicable; RPR, rapid plasma reagin; SD, standard deviation. a p-value for the HIV-infected and HIV-uninfected patients. episode of syphilis. The median interval between the two episodes of syphilis was 17.6 months (range, months) (Table 2). The percentage of patients with early syphilis was 97.5% for both first and second episodes of infection. No statistically significant differences were found between the two episodes, except for the mean PVL, which significantly decreased from log 10 copies/ml (p 0.013); the mean CD4 count increased from cells/mm 3 (p 0.244). Composition of molecular strains in Taiwan T. pallidum DNA was detected in 298 specimens collected from 216 patients. Of those 298 specimens, 95 collected from 80 patients were fully typed (Fig. S1). Of all typeable specimens, 73.7% were isolated from swab specimens of chancres, whereas 26.3% were from plasma. A tal of 14 strains were identified from 80 patients, including 14f/f (n = 46; 57.5%), 14b/c (8; 10.0%), 14k/f (5; 6.2%), 14a/f (4; 5.0%), 10b/a (3; 3.8%), 14f/c (3;3.8%), 14b/f, 14e/f and 14j/f (two each; 2.5%), and 13b/a, 13b/c, 13f/f, 14j/c and 9f/f (one each; 1.2%) (Fig. 1). In our study, 14f/f was consistently the predominant strain from ; 14b/c first appeared in 2011, and remained as the second most common strain until 2013 (Fig. 1). Forty patients developed recurrent syphilis during the 4-year study period (Table 2; Table S1); 14 (35.0%) and six (15.0%) patients had T. pallidum strains identified that were fully typeable in the first and second episodes, respectively

4 CMI Wu et al. Macrolide resistance and genotyping of Treponema pallidum in Taiwan 805 TABLE 2. Characteristics of the 40 patients who presented with recurrent episodes of syphilis Recurrent syphilis First episode Second episode p-value a Age (years), median (range) 29 (22 28) 31 (23 48) Male gender, n (%) 40 (100) MSM, n (%) 40 (100) Interval b (months), median (range) 17.6 ( ) RPR titre (log 2 ), median (range) 6 (3 9) 7 (2 10) Syphilis stage, n (%) Primary 12 (30.0) 11 (27.5) Secondary 16 (40.0) 8 (20.0) Primary and secondary 3 (7.5) 2 (5.0) Early latent 8 (20.0) 18 (45.0) Late latent 0 (0) 1 (2.5) Neurosyphilis 1 (2.5) 0 (0) HIV infection, n (%) 38 (95.0) 39 (97.5) CD4 count in patients with HIV infection 471 (285) 548 (281) (cells/mm 3 ), mean (SD) Plasma HIV RNA load (log 10 copies/ml), 2.58 (1.38) 1.84 (1.09) mean (SD) On cart, n (% of HIV patients) 29 (76.3) 36 (92.3) Fully typed, n (% of all patients) 14 (35.0) 6 (15.0) Strain distribution 14f/f (n = 9), 14k/f (3), 14a/f (2), 14f/c (1), /f (5), k/a, 14 /f, j/f, f/f, c/f, a/c, /c, f/ (1 for each) Strain discrepancy, n (%) c 5 (12.5) 14f/f (n = 4), 13f/f (1), 10b/a (1), /f (5), /a (2), f/f, b/c, a/f, /c (1 for each) cart, combination antiretroviral therapy; HIV, human immunodeficiency virus; MSM, men who have sex with men; RPR, rapid plasma reagin; SD, standard deviation. a p-value for the first episode and the second episode of infection. b The period between the first episode and the second episode of infection. c Strain discrepancy indicates that any of the three loci was different. Molecular typing was performed by using a combination of the results for the 60-bp repeat numbers of the acidic repeat protein (arp) gene, the RFLP patterns of T. pallidum repeat (tpr) genes, and sequencing of the short region ( bp) within the tp0548 gene. Once either one of the three genes was unable amplify, blank is used f/f 14b/c 14a/f 14f/c 14k/f 10b/a Others* Number of cases FIG. 1. Distribution of genotypes of the Treponema pallidum strains identified between September 2009 and August *Detected once in each given year. 0 September 2009 August 2010 September 2010 August 2011 September 2011 August 2012 September 2012 August 2013 (Table 2). It is of note that five patients (12.5%) had T. pallidum genotype changes identified in the first and second episodes of syphilis; the genotypes of cases 5, 14, 17, 25, and 39 changed from 14k/f /a, 14f/f 13f/f, 14f/c /f, /c /f and c/f /c, respectively (Table S1). In one patient, two strains were identified in the same episode of secondary syphilis. The strain of T. pallidum from an oral swab specimen had a full typing result (14b/c), but that from a plasma specimen was only partly typed (/f) (Fig. S2). Detection of macrolide resistance mutations During the study period, the 23S rrna gene of T. pallidum was detected in 268 specimens (40.7%). The numbers of specimens in which the 23S rrna gene was detected during the study periods of September 2009 August 2010, September 2010 August 2011, September 2011 August 2012 and September 2012 August 2013 were 19, 72, 41, and 136, respectively. Only two strains of T. pallidum that were identified in 2013 were shown harbour the A2058G mutation, giving an overall prevalence of the macrolide

5 806 Clinical Microbiology and Infection, Volume 20 Number 8, August 2014 CMI resistance mutation (A2058G) of 0.7% (2/268), and no A2059G mutation was identified. These two isolates of macrolide-resistant T. pallidum were identified from two patients presenting with primary syphilis, who reported that they might have acquired syphilis in Hong Kong and Shanghai, China, respectively. Furthermore, in one of these two patients, a macrolide-resistant strain was identified in the second episode of syphilis in 2013, whereas the isolate in the first episode in 2010 had no such mutation. Discussion In this multicentre surveillance study, we found that the predominant strain type of T. pallidum was 14f/f, followed by 14b/c, which was circulating stably over the 4-year study period. The prevalence of T. pallidum harbouring macrolide resistance mutations (A2058G or A2059G) remained very low, which suggests that azithromycin could be an alternative for the treatment of early syphilis in Taiwan. The predominant strain circulating in the USA, France and China [8,10,12 14] was 14d/f. The 14f strain, for which the typing scheme used was in accordance with US CDC methods, was identified in China, Portugal, and the USA [22 24]; however, we previously found the differentiation of 14f in 14f/f by the use of enhanced typing methods [11]. Grimes et al. [13] have noted that 14d/g replaced 14d/f become the most common strain in the USA after This replacement was not observed in our study in Taiwan over the study period, when 14f/f remained the predominant strain from The public health and clinical implications need be further investigated. Clinical information and changes in RPR titres remain the current criteria for determining recurrent syphilis [5,6]. More discriminary molecular typing could assist in the diagnosis of re-infection by different strains of T. pallidum [10], although this requires further validation. In the study described by Grange et al. [10], two different strains were sequentially detected (14d/ f and 14d/g) when the patient initially presented and subsequently returned for recurrent syphilis after 19 months. In our study, we were able identify discrepant subtypes of T. pallidum in five of the 40 patients who presented with recurrent syphilis (Table S1). However, the sensitivity of PCR for the detection of each typing gene varies, which may preclude fully typing each strain of T. pallidum in all recurrent episodes [11]. In our study, 195 subjects provided more than one specimen collected in the same episode of syphilis, and we found two different strains from the oral swab and plasma specimens in one patient. There are two possible explanations for this mismatch: the patient might have had sexual contact with different partners at the same time or sequentially at different time-points before seeking care; or he might have had sex with an individual who also carried different strains of T. pallidum. The prevalence of macrolide resistance mutations varies with geographical regions, and has been shown be associated with a hisry of macrolide use [13,18,19,25]. Our previous study of a smaller number of cases in Taiwan between 2009 and 2011 showed no cases of syphilis caused by macrolide-resistant T. pallidum [11]. However, in this study, two patients who were infected with T. pallidum harbouring the A2058G mutation were found in One of the two patients reported having a hisry of travel Shanghai, where the prevalence of macrolide resistance is 100% [19]. Furthermore, in one patient with recurrent syphilis, the isolate of T. pallidum causing the second episode of syphilis carried a macrolide mutation (A2058G), whereas the first isolate did not. Those findings suggest that continued surveillance and close follow-up of the patients who may receive azithromycin for the treatment of early syphilis are warranted. There are several limitations of our study. First, most of our participants were HIV-infected MSM who sought care at the designated hospitals, and our results may therefore not be generalizable HIV-uninfected patients, heterosexual males or females in Taiwan. Second, the study duration was short, and a longer duration of surveillance and close moniring of treatment response is warranted, given the introduction of two strains of macrolide-resistant T. pallidum and increased international travel. Third, because of different sensitivities of PCR for DNA pol I and macrolide resistance mutation analysis, not all specimens yielded both amplifiable genes. In our study, one-third of the clinical specimens were collected from patients with latent syphilis (Table 1), for which the detection rate of T. pallidum would be lower than for patients with chancres [11,18,20,26]. Similarly, the stage of syphilis and clinical specimens examined further limit the sensitivity of PCR for genotyping [11,26]; consequently, the number of fully typed specimens in our study remains small. As a result, identifiction of strain discrepancies in all patients with multiple clinical specimens or recurrent syphilis is difficult. In conclusion, we found that 14f/f was the predominant stain of T. pallidum and that the prevalence of T. pallidum with macrolide resistance remains low in Taiwan. Different genotypes of T. pallidum can be identified in patients with recurrent episodes of syphilis. Transparency Declaration The authors declare that there are no conflicts of interest.

6 CMI Wu et al. Macrolide resistance and genotyping of Treponema pallidum in Taiwan 807 Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Study flow of surveillance of macrolide resistance mutations and genotyping of Treponema pallidum. Of the 159 patients who tested negative in PCR assays, 11 had recurrent syphilis. Figure S2. The tp0548 sequences ( bp) of two different strains isolated from one patient who presented with secondary syphilis. Table S1. Detailed chacteristics of the 40 patients with recurrent syphilis. References 1. Centres for Disease Control and Prevention. Sexually transmitted disease surveillance Atlanta, GA: CDC, Workowski KA, Berman S, CDC (USA). Sexually transmitted diseases treatment guidelines, MMWR Recomm Rep 2010; 59: Liu J, Huang Y, Wang J et al. The increasing prevalence of serologic markers for syphilis among Chinese blood donors in 2008 through 2010 during a syphilis epidemic. Transfusion (Paris) 2012; 52: Savage EJ, Marsh K, Duffell S, Ison CA, Zaman A, Hughes G. Rapid increase in gonorrhoea and syphilis diagnoses in England in Euro Surveill 2012; 17: pii: Phipps W, Kent CK, Kohn R, Klausner JD. Risk facrs for repeat syphilis in men who have sex with men, San Francisco. Sex Transm Dis 2009; 36: Ogilvie GS, Taylor DL, Moniruzzaman A et al. A population-based study of infectious syphilis rediagnosis in British Columbia, Clin Infect Dis 2009; 48: Long CM, Klausner JD, Leon S et al. Syphilis treatment and HIV infection in a population-based study of persons at high risk for sexually transmitted disease/hiv infection in Lima, Peru. Sex Transm Dis 2006; 33: Marra C, Sahi S, Tantalo L et al. Enhanced molecular typing of Treponema pallidum: geographical distribution of strain types and association with neurosyphilis. J Infect Dis 2010; 202: Pillay A, Liu H, Chen CY et al. Molecular subtyping of Treponema pallidum subspecies pallidum. Sex Transm Dis 1998; 25: Grange PA, Allix-Beguec C, Chanal J et al. Molecular subtyping of Treponema pallidum in Paris, France. Sex Transm Dis 2013; 40: Wu H, Chang SY, Lee NY et al. Evaluation of macrolide resistance and enhanced molecular typing of Treponema pallidum in patients with syphilis in Taiwan: a prospective multicenter study. J Clin Microbiol 2012; 50: Peng RR, Yin YP, Wei WH et al. Molecular typing of Treponema pallidum causing early syphilis in China: a cross-sectional study. Sex Transm Dis 2012; 39: Grimes M, Sahi SK, Godornes BC et al. Two mutations associated with macrolide resistance in Treponema pallidum: increasing prevalence and correlation with molecular strain type in Seattle, Washingn. Sex Transm Dis 2012; 39: Dai T, Li K, Lu H, Gu X, Wang Q, Zhou P. Molecular typing of Treponema pallidum: a 5-year surveillance in Shanghai, China. J Clin Microbiol 2012; 50: Workowski KA, Berman SM, CDC (USA). Sexually transmitted diseases treatment guidelines, MMWR Recomm Rep 2006; 55: Matejkova P, Flasarova M, Zakoucka H et al. Macrolide treatment failure in a case of secondary syphilis: a novel A2059G mutation in the 23S rrna gene of Treponema pallidum subsp. pallidum. J Med Microbiol 2009; 58: Lukehart SA, Godornes C, Molini BJ et al. Macrolide resistance in Treponema pallidum in the United States and Ireland. N Engl J Med 2004; 351: Muldoon EG, Walsh A, Crowley B, Mulcahy F. Treponema pallidum azithromycin resistance in Dublin, Ireland. Sex Transm Dis 2012; 39: Chen XS, Yin YP, Wei WH et al. High prevalence of azithromycin resistance Treponema pallidum in geographically different areas in China. Clin Microbiol Infect 2013; 19: Liu H, Rodes B, Chen CY, Steiner B. 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: Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011; 28: Castro R, Prie E, Aguas MJ, Manata MJ, Botas J, Pereira FM. Molecular subtyping of Treponema pallidum subsp. pallidum in Lisbon, Portugal. J Clin Microbiol 2009; 47: Martin IE, Gu W, Yang Y, Tsang RS. Macrolide resistance and molecular types of Treponema pallidum causing primary syphilis in Shanghai, China. Clin Infect Dis 2009; 49: Pope V, Fox K, Liu H et al. Molecular subtyping of Treponema pallidum from North and South Carolina. J Clin Microbiol 2005; 43: Muller EE, Paz-Bailey G, Lewis DA. Macrolide resistance testing and molecular subtyping of Treponema pallidum strains from southern Africa. Sex Transm Infect 2012; 88: Wu B-R, Tsai M-S, Yang C-J et al. Spirochetemia caused bydue Treponema pallidum using polymerase-chain-reaction assays in patients with early syphilis: prevalence, associated facrs, and treatment response. Clin Microbiol Infect 2013; doi: / [Epub ahead of print]