Evaluation of Macrolide Resistance and Enhanced Molecular Typing ...

5 downloads 0 Views 340KB Size Report
Mar 9, 2012 - of Treponema pallidum in Patients with Syphilis in Taiwan: a ... syphilis at six major designated hospitals for HIV care in Taiwan. The T.
Evaluation of Macrolide Resistance and Enhanced Molecular Typing of Treponema pallidum in Patients with Syphilis in Taiwan: a Prospective Multicenter Study Hsiu Wu,a Sui-Yuan Chang,b,c Nan-Yao Lee,d Wen-Chi Huang,e Bing-Ru Wu,b,c Chia-Jui Yang,f Shiou-Haur Liang,g Chen-Hsiang Lee,e Wen-Chien Ko,d Hsi-Hsun Lin,g Yen-Hsu Chen,h Wen-Chun Liu, i Yi-Ching Su,b,c Chia-Yin Hsieh,i Pei-Ying Wu,i and Chien-Ching Hungi Centers for Disease Control, Taipei, Taiwana; Department of Laboratory Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwanb; Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwanc; Department of Internal Medicine, National Cheng-Kung University Hospital, Tainan, Taiwand; Department of Internal Medicine, Kaohsiung Chung-Gang Memorial Hospital, Kaohsiung, Taiwane; Department of Internal Medicine, Far Eastern Memorial Hospital, New Taipei City, Taiwanf; Department of Internal Medicine, E-Da Hospital/I-Shou University, Taiwan, Kaohsiung, Taiwang; Department of Internal Medicine, Kaohsiung Medical University Hospital and Kaohsiung Medical University, Kaohsiung, Taiwanh; and Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwani

Studies of macrolide resistance mutations and molecular typing using the newly proposed enhanced typing system for Treponema pallidum isolates obtained from HIV-infected patients in the Asia-Pacific region are scarce. Between September 2009 and December 2011, we conducted a survey to detect T. pallidum using a PCR assay using clinical specimens from patients with syphilis at six major designated hospitals for HIV care in Taiwan. The T. pallidum strains were genotyped by following the enhanced molecular typing methodology, which analyzed the number of 60-bp repeats in the acidic repeat protein (arp) gene, T. pallidum repeat (tpr) polymorphism, and the sequence of base pairs 131 to 215 in the tp0548 open reading frame of T. pallidum. Detection of A2058G and A2059G point mutations in the T. pallidum 23S rRNA was performed with the use of restriction fragment length polymorphism (RFLP). During the 2-year study period, 211 clinical specimens were obtained from 136 patients with syphilis. T. pallidum DNA was isolated from 105 (49.8%) of the specimens, with swab specimens obtained from chancres having the highest yield rate (63.2%), followed by plasma (49.4%), serum (35.7%), and cerebrospinal fluid or vitreous fluid (18.2%) specimens. Among the 40 fully typed specimens, 11 subtypes of T. pallidum were identified. Subtype 14f/f (18 isolates) was the most common isolates, followed by 14f/c (3), 14b/c (3), and 14k/f (3). Among the isolates examined for macrolide resistance, none had the A2058G or A2059G mutation. In conclusion, we found that type 14 f/f was the most common T. pallidum strain in this multicenter study on syphilis in Taiwan and that none of the isolates exhibited 23S rRNA mutations causing resistance to macrolides.

S

yphilis is a multistage, sexually transmitted disease, caused by the spirochete Treponema pallidum, which may lead to severe complications such as neurosyphilis, cardiovascular syphilis, and adverse pregnancy outcomes if left untreated. Furthermore, syphilis has also been documented to facilitate both infectivity of and susceptibility to HIV infection (10, 28, 33). The incidence of syphilis, especially primary and secondary syphilis, is rising again in several developed countries since 2000, after a steady decline over the past 60 years following the introduction of penicillin (11, 33). In the United States, the rate of primary and secondary syphilis reported increased annually after 2001 and reached 4.6 cases per 100,000 population in 2009 (4); overall increases in rates have been observed primarily among men (increasing from 3.0 cases per 100,000 population in 2001 to 7.9 cases per 100,000 population in 2010) (4). In addition, outbreaks of syphilis have been noted among men who have sex with men (MSM) in many cities in the United States, Europe, and Australia (5, 12, 29). In Taiwan, 6,482 cases of syphilis (diagnosed by elevated titers of rapid plasma reagin [RPR] or the Venereal Disease Research Laboratory [VDRL] test and positivity for Treponema pallidum hemagglutination [TPHA]) were reported to the Taiwan CDC in 2010, which resulted in an annual incidence of 28.0 per 100,000 population (3). Under such circumstances, early diagnosis of syphilis, effective treatment of patients with the early stage of syphilis, and detection of outbreaks are essential components for disease control.

July 2012 Volume 50 Number 7

Because the causative agent, T. pallidum, cannot be cultivated on standard bacteriological media, the diagnosis of syphilis is usually made by either detection of spirochetes by dark-field microscopy or immunostaining with fluorescent-antibody-conjugated reagents of clinical specimens or by serology. PCR methods to detect T. pallidum have been developed since 1990 for the purpose of improving the sensitivity and specificity of laboratory diagnosis. A recently developed assay in which primers are rationally designed based on two unique characteristics of the DNA polymerase I gene of T. pallidum can detect pathogenic T. pallidum subspecies in clinical specimens with a sensitivity of 95.8% and a specificity of 95.7% (14). Moreover, with the use of PCR assays, researchers are able to perform molecular typing and detect macrolide resistance of T. pallidum. A molecular subtyping scheme for T. pallidum was first developed by Pillay et al. in 1998 using the number of 60-bp tandem

Received 9 February 2012 Returned for modification 9 March 2012 Accepted 10 April 2012 Published ahead of print 18 April 2012 Address correspondence to Chien-Ching Hung, [email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.00341-12

Journal of Clinical Microbiology

p. 2299 –2304

jcm.asm.org

2299

Wu et al.

repeats in the acidic repeat protein (arp) gene and restriction fragment length polymorphism (RFLP) analysis of the T. pallidum repeat (tpr) gene (25). With the use of this method, several studies have described the most common subtypes in different countries: 14d in the United Kingdom, Canada, South Africa, China, and Madagascar; 14f in the United States; and 14a in Portugal (7, 9, 13, 17–19, 26, 27, 30). However, its inadequate power to discriminate the most common subtypes limited the use of this typing method in outbreak investigations. Given this limitation, Marra and colleagues developed an enhanced molecular typing method to improve discrimination by analyzing a short region of the tp0548 gene in addition to analysis of the arp and tpr genes (17). Studies on macrolide resistance mutations and molecular types of isolates of T. pallidum obtained from MSM in the Asia-Pacific region using the newly proposed method are limited (18, 24, 34). In this study, we aimed to investigate the molecular epidemiology and prevalence of azithromycin-resistant T. pallidum among patients with early syphilis in Taiwan. (Preliminary analyses of these data were presented at the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2011 [11a].) MATERIALS AND METHODS Setting and study population. This was a multicenter, prospective survey that was conducted at the infectious diseases clinics of six major hospitals designated for HIV care between September 2009 and December 2011; two of the hospitals are located in northern Taiwan, while the other four hospitals are in southern Taiwan. Adult patients who presented with primary, secondary, or early latent syphilis, neurosyphilis, and uveitis with a rapid plasma reagin (RPR) titer of ⭌1:4 and positive Treponema pallidum particle hemagglutination (TPHA) assay results were enrolled. Patients who had received antibiotics for syphilis before enrollment were excluded. Data collected included the patient’s demographics, risk for HIV infection, HIV serostatus, CD4 count and plasma HIV RNA load (for HIV-infected patients), coinfection, stage of syphilis, treatment of syphilis, and serological response. The stages of syphilis were defined by following the treatment guidelines for sexually transmitted diseases of the U.S. Centers for Disease Control and Prevention (32). The study was approved by the Research Ethics Committee from each participating hospital, and written informed consent was obtained from each patient. Collection of clinical specimens. All appropriate clinical specimens were collected before antibiotics were administered. Specimens of ulcer lesions were collected by swabs that were pressed and rolled over the ulcers. The exudate expressed on the lesion was absorbed onto Dacron swabs (CultureSwab EZ; BD, Franklin Lakes, NJ). Blood specimens were obtained from patients with primary, secondary, and early latent syphilis. The whole-blood specimens from the two hospitals in northern Taiwan were collected in EDTA-coated containers (BD Vacutainer or BD Hemogard) and centrifuged within 1 day. The cerebrospinal fluid (CSF) and vitreous fluid specimens were collected in sterile containers. Multiple specimens were obtained from patients with multiple ulcers or concurrent primary and secondary lesions. All specimens from northern Taiwan were transported to the central laboratory at the National Taiwan University Hospital within 1 day. The blood specimens from the other four hospitals in southern Taiwan were collected in serum containers (BD Vacutainer SST tubes) and transported at 4°C to the laboratory within 2 days. Laboratory investigations. Treponemal DNA was extracted from clinical specimens using the Qiagen DNA minikit (Qiagen, GmbH, Germany) according to the manufacturer’s instructions. PCR assay was performed to detect the presence of T. pallidum by amplifying a 378-bp fragment of the T. pallidum polymerase A gene (polA) as described by Liu et al. (14). RFLP of the 23S rRNA was performed to detect macrolide resistance as described by Lukehart et al. (15). Because azithromycin treat-

2300

jcm.asm.org

ment failure was reported in a case of syphilis in which T. pallidum harbored an A2059G point mutation (20), we also investigated the presence of this point mutation in archived specimens. PCR amplification of a 60-bp tandem repeat region within the arp gene was carried out in a final volume of 50 ␮l containing 20 mM TrisHCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates (dNTPs), 0.3 ␮M each primer (Arp1A/Arp2A), 2.5 U platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), and 10 ␮l DNA template. The primer pair used was Arp1A (5=-CAA GTC AGG ACG GAC TGT CCC TTG C-3=) and Arp2A (5=-GGT ATC ACC TGG GGA TGC GCA CG-3=). The amplification conditions were 94°C for 5 min, 45 cycles of 94°C for 1 min, 61°C for 1 min, and 72°C for 2.5 min, and a final extension at 72°C for 10 min. The number of arp gene repeats was determined by comparing the sizes of the PCR amplicons with those of the Nichols strain of T. pallidum, which has 14 repeats. A nested PCR assay was used to amplify the tpr gene. The first primer pair used was B1 (5=-ACT GGC TCT GCC ACA CTT GA-3=) and A2 (5=-CTA CCA GGA GAG GGT GAC GC-3=). The amplification conditions were 94°C for 5 min, 40 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2.5 min, and a final extension at 72°C for 10 min. A 1-␮l aliquot of the first-round PCR product was used for the second-round PCR, for which the conditions were the same as in the first round except that annealing was done at 59°C. The second primer pair used was Tpr-F (5=CAG GTT TTG CCG TTA AGC-3=) and Tpr-R (5=-AAT CAA GGG AGA ATA CCG TC-3=). The tpr amplicons were digested with MseI, and the digestion patterns were compared with published data (25). One sense primer, Tp0548-F (5=-GGT CCC TAT GAT ATC GTG TTC G-3=), and two antisense primers, Tp0548-R1 (5=-CGT TTC GGT GTG TGA GTC AT-3=) and Tp0548-R2 (5=-GTC ATG GAT CTG CGA GTG G-3=), were used to amplify DNA from blood specimen (Tp0548-F and Tp0548-R1) and lesion exudates (Tp0548-F and Tp0548-R2). The amplification conditions were 94°C for 2 min, 40 cycles of 95°C for 1 min, 62°C for 70 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The amplicons were sequenced for genotyping (17). Statistical analysis. Statistical Package for the Social Sciences (SPSS) statistics 17.0 software (IBM Corporation, Somers, NY) was used for statistical analysis. Associations between categorical variables were determined by the ␹2 test or the Fisher exact test. The Student t test was used to determine differences between continuous variables. Results with P values of ⬍0.05 were considered to be statistically significant.

RESULTS

During the 2-year study period, a total of 211 specimens were collected from 136 patients. The demographic data and clinical presentations of the patients enrolled are shown in Table 1. Of the 132 patients with available data, all were male, with an age ranging from 19.6 to 57.9 years (mean, 30.1 years), and 93 (70.5%) were HIV infected. Of the 116 patients who reported sexual orientation, 109 (94.0%) were MSM. Of the 132 patients with reported stages of syphilis, 38 (28.8%) were diagnosed with primary syphilis, 81 (61.4%) with secondary syphilis, 17 (12.9%) with concurrent primary and secondary syphilis, 26 (19.7%) with early latent syphilis, 4 (3.0%) with neurosyphilis and syphilitic uveitis, and 1 (0.8%) with concurrent primary and secondary syphilis, neurosyphilis, and syphilitic uveitis. Only one patient reported use of a macrolide antibiotic within 1 year prior to the diagnosis of syphilis. Compared with HIV-uninfected patients, HIV-infected patients had a significantly higher proportion of concurrent primary and secondary syphilis (62.0 versus 15.0%; P ⬍ 0.001). Each patient provided one to six clinical specimens (mean, 1; median, 1.5). Of the 211 specimens, 125 (59.2%) were blood samples, 75 (35.5%) swab samples, 9 (4.3%) CSF, and 2 (0.9%) vitre-

Journal of Clinical Microbiology

Treponema pallidum Infection in Taiwan

TABLE 1 Clinical characteristics of patients with syphilis who tested positive or negative by the screening PCR assay Value for:

Variable

All patients (n ⫽ 132)

Patients testing positive (n ⫽ 73)

Patients testing negative (n ⫽ 59)

Mean age (SD), yr Male sex, n (%) MSM, n (%)a RPR titer, mean (SD) log2 HIV infection, n (%)

31.0 (7.5) 132 (100) 109 (94.0) 6.3 (1.7) 93 (70.5)

30.4 (7.0) 73 (100) 61 (95.3) 6.3 (1.7) 51 (69.9)

31.8 (8.2) 59 (100) 48 (92.3) 6.3 (1.8) 42 (71.2)

0.70 ⬎0.99 ⬎0.99

Stage of syphilis, n (%) Primary Secondary Primary and secondary Early latent Neurosyphilis and uveitis

38 (28.8) 81 (61.4) 17 (12.9) 26 (19.7) 5 (3.8)

29 (39.7) 51 (69.9) 14 (19.2) 7 (9.6) 1 (1.4)

9 (15.3) 30 (50.8) 3 (5.0) 19 (32.2) 4 (6.8)

0.002 0.03 0.02 0.002 0.17

P value 0.29

a Sixty-four and 52 patients testing positive and negative in the PCR assay, respectively, identified their sexual orientation.

ous fluid samples. Of the 125 blood samples, 45 (33.6%) were serum samples and 80 (64.0%) plasma samples. At screening, 105 (49.8%) and 102 (48.5%) were positive for polA and 23S rRNA PCR, respectively. Of specimens that were amplifiable for polA, 47 (44.8%), 64 (61.0%), and 83 (79.0%) were positive for the arp, tpr, and tp0548 genes, respectively. The proportion of specimens that were positive for polA varied among the specimens analyzed: swab, 63%; plasma, 49%; serum, 36%; and CSF and vitreous fluid, 18%. Figure 1 shows the yield rates of PCR assays for the polA, arp, tpr, and tp0548 genes in different types of clinical specimens. Blood samples from the patients with secondary syphilis had a significantly higher yield rate in screening PCR of blood samples than those from patients with primary or latent syphilis (58.6% versus 7.7%; P ⫽ 0.001). In patients with secondary syphilis and primary syphilis, there were no significant differences in the

FIG 2 Distribution of molecular types of 36 nonduplicated fully typeable strains of Treponema pallidum by arp-tpr (horizontal axis) and tp0548 (different colors).

screening yield rates of blood samples (62.2% versus 64.3%; P ⬎ 0.99) and swab samples (65.2% versus 66.7%; P ⬎ 0.99) between HIV-infected and HIV-uninfected patients. All typeable molecular types were consistent in each patient who provided more than one specimen. Of the 105 amplifiable specimens, 40 (38.1%) tested positive for all tpr, arp, and tp0548 genes to produce 11 full subtypes. The distribution of the typeable specimens is shown in Fig. 2. Of the 36 nonduplicated, fully typeable strains of T. pallidum, 14f/f was the most common subtype (18/36; 50%), followed by 14f/c, 14b/c, and 14k/f (all 3/36; 8.3%). Comparisons of clinical characteristics between patients infected with 14f/f and those infected with non-14f/f subtypes are shown in Table 2. Patients infected with subtype 14f/f were more likely to present with primary syphilis. In patients with secondary syphilis, more patients infected with the 14f/f subtype developed a JarischHerxheimer reaction following benzathine penicillin treatment (5/6 [83.3%] versus 2/9 [22.2%]; P ⫽ 0.04) (data not shown). Of all specimens with amplifiable 23S rRNA by PCR, none were detected to have an A2058G or A2059G mutation, which confers resistance to macrolides.

TABLE 2 Comparisons of demographic and clinical characteristics between patients infected with 14f/f and non-14f/f subtypes Value for patients with subtype: Variable

14f/f (n ⫽ 18)a Non-14f/f (n ⫽ 18) P value

Mean age (SD), yr MSM, n (%)b RPR titer, mean (SD) log2 HIV infection, n (%)c

31.2 (5.8) 12 (92.3) 6.1 (1.9) 13 (81.3)

30.4 (6.4) 15 (100) 6.1 (1.7) 15 (83.3)

0.70 0.46 0.98 ⬎0.99

FIG 1 Yield rates of RCR assay to detect the polymerase A (screening), tpr, arp,

Stage of syphilis, n (%) Primary Secondary Primary and secondary Early latent Neurosyphilis and uveitis

14 (87.5) 10 (62.5) 8 (50.0) 0 (0) 1 (6.3)

4 (22.2) 12 (66.7) 0 (0) 2 (11.1) 0 (0)

⬍0.001 ⬎0.99 0.001 0.49 0.47

and tp0548 genes in different clinical specimens. “Other” specimens included 9 cerebrospinal fluid (CSF) and 2 vitreous fluid specimens. Of two specimens (one CSF and one vitreous fluid) that were positive for screening PCR, tp0548 was amplifiable from one by PCR but arp and tpr were not amplifiable from either.

For reported stage of syphilis, n ⫽ 16. Thirteen and 15 patients infected with 14f/f and non-14f/f subtypes, respectively, identified their sexual orientation. c Sixteen and 15 patients infected with 14f/f and non-14f/f subtypes, respectively, had available information on HIV serostatus.

July 2012 Volume 50 Number 7

a b

jcm.asm.org 2301

2302

jcm.asm.org

U.S., Madagascar, South Africa

U.S.

U.S.

U.S.

Canada

Colombia

China

South Africa

South Africa Portugal

Portugal

UK

Australia

Pillay et al. (25)

Sutton et al. (30)

Pope et al. (27)

Katz et al. (13)

Martin et al. (19)

Cruz et al. (8)

Martin et al. (18)

Pillay et al. (26)

Molepo et al. (22) Florindo et al. (9)

Castro et al. (2)

Cole et al. (7)

Azzato et al. (1)

U.S., China, Ireland, Madagascar China Taiwan

Study site(s)

2004-2009

2006-2007

2003-2005

1999-2000 2004-2007

1996-2000

2007-2008

2003-2009

2007-2009

2004-2007

1999-2003

1998-1999

NA

NA

74.7

NA

NA NA

NA

NA

NA

NA

84.3

NA

0

NA

23.9

13.3

NA

NA NA

NA

2.6

0

NA

43.5

NA

NA

NA

0 70.5

86.9a

1999-2008 1.0 94.0

83.3a

MSM (%)

Study yr

2008-2010 2009-2011

HIV infection (%)

Among patients in Seattle with available demographic data. CNS, central nervous system. c NA, not available. d —, typing not done.

b

a

Conventional

Marra et al. (17)

Enhanced

Peng et al. (24) Wu et al. (present report)

Authors (reference)

Molecular typing method

NA

Primary, secondary

Primary, secondary, latent

CNS Primary, secondary

Primary

Primary

Secondary

Primary, secondary, congenital

Primary, secondary

Primary, secondary, latent Primary, secondary

Primary, secondary, early latent, CNSb Primary, secondary Primary, secondary, early latent, CNS, uveitis Primary

Stage(s) of syphilis

CSF Primary ulcer, secondary lesion, whole blood Primary ulcer, secondary lesion, whole blood, serum, ear lobe scraping Genital ulcer, anal ulcer, oral ulcer Oral, genital, and anorectal ulcer, lesion swabs, CSF, tissue biopsy specimen

Primary ulcer

Primary ulcer, whole blood Primary ulcer, secondary lesion Primary ulcer, secondary lesion Primary ulcer, secondary lesion, whole blood, plasma, serum, vitreous fluid Secondary lesion, whole blood Primary ulcer

Plasma Plasma, serum, primary ulcer, CSF, vitreous fluid Primary ulcer

Blood, lesion exudate, CSF

Type(s) of specimen

28/50 (56.0) 86/NA (NA) 90/212 (42.5)

polA

75/87 (86.2) 303/3652 (8.2)

polA polA

201/1954 (10.3)

38/57 (66.7)

bmp, tpp47, polA tpp47 or polA tpp47 bpm, polA

20/38 (52.6)

49/449 (10.9)

bpm, tpp47, polA polA

71/74 (95.9)

27/61 (44.3)

56/112 (50.0)

NA

67.3 49.8

NA

polA

polA

polA

tpp47

polA polA

NAc

Screening PCR assay

Positive rate (%) of screening assay

57.1, NA, —

81.3, 85.3, —

NA

46.4, 53.6, — NA

80.9, 87.1, —

94.7, 100.0, —

30.0, 40.0, —

100.0, 83.7, —

97.2, 98.6, —

NA

NA

NA

91.3, 90.4, 95.0 44.8, 61.0, 79.0

NA

Yield rate (%) for arp, tpr, tp0548, respectivelyd

90/10

58/6

20/5

13/4 42/3

161/35

36/4

6/4

36/4

69/8

23/7

38/7 (US), 8 (Madagascar), 3 (South Africa) 45/10

197/27 40/11

173/25

No. of specimens typed/no. of types identified

TABLE 3 Summary of the published studies that used enhanced molecular typing and conventional molecular typing of clinical strains of Treponema pallidum

14e (31.1)

14d (75.8)

14a (65.0)

14a (53.8) 14a (50.0)

14d (27.3)

14d (33.3) 16d (33.3) 14f (83.3)

14d (83.3)

14d (75.4)

14f (52.2)

NA

NA

NA

NA NA

NA

NA (100)

NA

7/43 (16.3)

42/62 (67.7)

NA

NA

NA

14d (39.5)

14f (53.3)

NA 0 (0)

NA

A2058G mutation (%)

14d/f (38.6) 14f/f (50.0)

14d/f (50.3)

Most common subtype (%)

Wu et al.

Journal of Clinical Microbiology

Treponema pallidum Infection in Taiwan

DISCUSSION

In this study, using the enhanced molecular typing method proposed by Marra and colleagues (17), we found that subtype 14f/f accounted for 50% of the fully typeable specimens in patients with syphilis in Taiwan, and the A2058G and A2059 point mutations, which are associated with azithromycin resistance, were not detected in this study. Molecular typing can provide additional information in epidemiological investigations. Previous studies have described the geographic distribution of molecular subtypes of T. pallidum (Table 3). Subtype 14f was the most common subtype observed using the conventional typing method in the United States and China in studies reported in 2005 and 2009, respectively (18, 27, 30). In our present study, we found a predominance of subtype 14f/f. This result is different from the findings of the latest two studies that also used the enhanced molecular typing method, both of which showed 14d/f as the most common subtype from 1999 to 2010 in the United States and China (Table 3) (17, 24). In this study, we found two new subtypes, 9f/f and 10b/a, that had not been reported before. These findings suggest that currently circulating T. pallidum strains in Taiwan may be unrelated to those in the United States and China as determined by the enhanced molecular typing methodology. Manifestations of syphilis may be associated with specific T. pallidum subtypes. In the study by Marra et al., subtype 14d/f was found to be associated with neurosyphilis (17). In this study, we found that subtype 14f/f was statistically significantly associated with primary syphilis. The cause of this association remains unclear. One possible explanation for this finding is that subtype 14f/f tends to produce chancres that are more manifest or last longer for patients or physicians to notice. It is also speculated that 14f/f subtypes are more environmentally resistant in swab specimens or during transportation. While the clinical implications remain unclear, more studies are warranted to confirm our preliminary findings. Prior studies reported different prevalences of genotypic resistance of T. pallidum to macrolides, which were 2% in British Columbia during 2000 to 2003 (23), 88% in Dublin in 2002 (15), and 16 to 28.6% in Canada (18, 19), 37 to 68% in San Francisco (13, 21), and 100% in China (18). In this 2-year survey in Taiwan, no A2058G point mutation was found in the 211 clinical specimens, which was similar to the finding of the study by Van Damme and colleagues that demonstrated no evidence of azithromycin resistance of T. pallidum in Madagascar (31). The absence of a A2058G or A2059G point mutation in T. pallidum strains in this study may be partly explained by the findings of studies by Marra et al. and Mitchell et al. that macrolide resistance in multiple strains resulted from antibiotic pressure (16, 21). In our study, only one patient had a history of exposure to macrolide antibiotics prior to the diagnosis of syphilis. Patient-delivered partner treatment with macrolide antibiotics has not been implemented as a control strategy in preventing syphilis or Chlamydia trachomatis infection during and before our study period in Taiwan. Our finding suggests that azithromycin remains an appropriate alternative treatment for early syphilis in nonpregnant patients in Taiwan who are intolerant of penicillins. Nevertheless, caution should be taken and surveillance of drug-resistant T. pallidum be continued because of increased international travel and cross-strait travel between Tai-

July 2012 Volume 50 Number 7

wan and mainland China, where 100% of the T. pallidum strains were reportedly resistant to macrolides (18). There are several limitations of our study, and interpretation of the results should be cautious. First, all specimens were obtained from patients in medical centers designated for HIV care, and therefore, the majority of the subjects were HIV-infected MSM. The results of molecular typing may not be representative of the T. pallidum strains that circulate in heterosexual or HIV-uninfected populations. Second, the number of clinical specimens that were fully typed using enhanced molecular typing methods remains small in our study compared to the two previously reported studies (Table 3). Continued surveillance among the participating hospitals is needed. Third, we used the VDRL and TPHA tests but not dark-field microscopy to confirm the diagnosis of syphilis. We might have missed some cases of primary syphilis. Last, this study was designed as a cross-sectional survey, which precluded us from following the patients longitudinally to investigate the changes of subtypes of T. pallidum in those patients with treatment failure. In conclusion, we found that subtype 14f/f was the most common subtype of T. pallidum in Taiwan and that none of the strains harbored an A2058G or A2059G point mutation associated with macrolide resistance in this 2-year survey. ACKNOWLEDGMENT We have no conflicts of interest to declare.

REFERENCES 1. Azzato F, Ryan N, Fyfe J, Leslie ED. 2012. Molecular subtyping of Treponema pallidum during a local syphilis epidemic in men who have sex with men in Melbourne, Australia. J. Clin. Microbiol. 50:1895–1899. 2. Castro R, et al. 2009. Molecular subtyping of Treponema pallidum subsp. pallidum in Lisbon, Portugal. J. Clin. Microbiol. 47:2510 –2512. 3. Centers for Disease Control. 2010. Statistics of communicable diseases and surveillance report, Taiwan CDC. Centers for Disease Control, Department of Health, Taipei. Taiwan. http://www.cdc.gov.tw/uploads/files /47d8b8ba-560e– 486f-8b3f-73e66a29f90b.pdf. 4. Centers for Disease Control and Prevention. 2010. Sexually transmitted disease surveillance 2010. Centers for Disease Control and Prevention, Atlanta, GA. 5. Centers for Disease Control and Prevention. 2004. Trends in primary and secondary syphilis and HIV infections in men who have sex with men—San Francisco and Los Angeles, California, 1998-2002. MMWR Morb. Mortal. Wkly. Rep. 53:575–578. 6. Reference deleted. 7. Cole MJ, Chisholm S, Palmer HM, Wallace LA, Ison CA. 2009. Molecular epidemiology of syphilis in Scotland. Sex. Transm. Infect. 85:447– 451. 8. Cruz AR, et al. 2010. Secondary syphilis in Cali, Colombia: new concepts in disease pathogenesis. PLoS Negl. Trop. Dis. 4:e690. doi:10.1371/ journal.pntd.0000690. 9. Florindo C, et al. 2008. Molecular typing of Treponema pallidum clinical strains from Lisbon, Portugal. J. Clin. Microbiol. 46:3802–3803. 10. Gadkari DA, et al. 1998. HIV-1 DNA shedding in genital ulcers and its associated risk factors in Pune, India. J. Acquir. Immune Defic. Syndr Hum. Retrovirol. 18:277–281. 11. Gerbase A, Rowley J, Heymann D, Berkley S, Piot P. 1998. Global prevalence and incidence estimates of selected curable STDs. Sex. Transm. Infect. 74(Suppl 1):S12–S16. 11a.Huang W-C, et al. 2011. Abstr. 51st Intersci. Conf. Antimicrob. Agents Chemother., abstr. L2-891. 12. Jin F, et al. 2005. Epidemic syphilis among homosexually active men in Sydney. Med. J. Aust. 183:179 –183. 13. Katz KA, et al. 2010. Molecular epidemiology of syphilis—San Francisco, 2004-2007. Sex. Transm. Dis. 37:660 – 663. 14. Liu H, Rodes B, Chen CY, Steiner B. 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. J. Clin. Microbiol. 39:1941–1946.

jcm.asm.org 2303

Wu et al.

15. Lukehart SA, et al. 2004. Macrolide resistance in Treponema pallidum in the United States and Ireland. N. Engl. J. Med. 351:154 –158. 16. Marra CM, et al. 2006. Antibiotic selection may contribute to increases in macrolide-resistant Treponema pallidum. J. Infect. Dis. 194:1771–1773. 17. Marra CM, et al. 2010. Enhanced molecular typing of Treponema pallidum: geographical distribution of strain types and association with neurosyphilis. J. Infect. Dis. 202:1380 –1388. 18. Martin IE, Gu W, Yang Y, Tsang RS. 2009. Macrolide resistance and molecular types ofTreponema pallidum causing primary syphilis in Shanghai, China. Clin. Infect. Dis. 49:515–521. 19. Martin IE, et al. 2010. Molecular typing of Treponema pallidum strains in western Canada: predominance of 14d subtypes. Sex. Transm. Dis. 37: 544 –548. 20. Matejková P, et al. 2009. 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. 58:832– 836. 21. Mitchell SJ, et al. 2006. Azithromycin-resistant syphilis infection: San Francisco, California, 2000 –2004. Clin. Infect. Dis. 42:337–345. 22. Molepo J, Pillay A, Weber B, Morse SA, Hoosen AA. 2007. Molecular typing of Treponema pallidum strains from patients with neurosyphilis in Pretoria, South Africa. Sex. Transm. Infect. 83:189 –192. 23. Morshed MG, Jones HD. 2006. Treponema pallidum macrolide resistance in BC. CMAJ 174:349. 24. Peng RR, et al. 2012. Molecular typing of Treponema pallidum causing early syphilis in China: a cross-sectional study. Sex. Transm Dis. 39:42– 45.

2304

jcm.asm.org

25. Pillay A, et al. 1998. Molecular subtyping of Treponema pallidum subspecies pallidum. Sex. Transm. Dis. 25:408 – 414. 26. Pillay A, et al. 2002. Molecular typing of Treponema pallidum in South Africa: cross-sectional studies. J. Clin. Microbiol. 40:256 –258. 27. Pope V, et al. 2005. Molecular subtyping of Treponema pallidum from North and South Carolina. J. Clin. Microbiol. 43:3743–3746. 28. Sellati TJ, et al. 2000. Virulent Treponema pallidum, lipoprotein, and synthetic lipopeptides induce CCR5 on human monocytes and enhance their susceptibility to infection by human immunodeficiency virus type 1. J. Infect. Dis. 181:283–293. 29. Simms I, et al. 2005. The re-emergence of syphilis in the United Kingdom: the new epidemic phases. Sex. Transm. Dis. 32:220 –226. 30. Sutton MY, et al. 2001. Molecular subtyping of Treponema pallidum in an Arizona County with increasing syphilis morbidity: use of specimens from ulcers and blood. J. Infect. Dis. 183:1601–1606. 31. Van Damme K, et al. 2009. Evaluation of azithromycin resistance in Treponema pallidum specimens from Madagascar. Sex. Transm. Dis. 36: 775–776. 32. Workowski KA, Berman SM. 2006. Sexually transmitted diseases treatment guidelines. MMWR Recomm. Rep. 55(RR-11):1–94. 33. Zetola NM, Klausner JD. 2007. Syphilis and HIV infection: an update. Clin. Infect. Dis. 44:1222–1228. 34. Zhou P, et al. 2010. Azithromycin treatment failure among primary and secondary syphilis patients in Shanghai. Sex. Transm. Dis. 37:726 –729.

Journal of Clinical Microbiology