Circulation of Distinct Treponema pallidum Strains in Individuals with Heterosexual Orientation and Men Who Have Sex with Men

Human treponematosis is caused by various pathogenic Treponema pallidum subspecies, including T. pallidum subsp. pallidum, T. pallidum subsp.

ABSTRACT

Human treponematosis is caused by various pathogenic Treponema pallidum subspecies, including T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, T. pallidum subsp. endemicum, and Treponema carateum. The global prevalence of syphilis has been increasing since the 2000s. Men account for more than 90% of the cases, with the majority being men who have sex with men (MSM). In Japan, the increase in the number of syphilis patients began in 2011, a 10-year delay from the global trend. In 2017, a total of 5,829 syphilis cases (3,934 men and 1,895 women) were reported, with an outstanding increase in cases among young adult women; the number reported for women age 15 to 20 years was 1,100. Hence, a molecular epidemiological study was conducted on circulating T. pallidum strains using two strain typing methods, the enhanced CDC method and sequencing-based molecular typing. Clinical specimens from 95 adults suspected of syphilis were collected from September 2013 to August 2017 in Osaka, Japan. T. pallidum DNA was detected in specimens from 25 males and 11 females, including seven MSM. The majority of the heterosexual patients (66.7% and 90.9% of males and females, respectively) were positive for 14d/f-SSR8. In contrast, the genotypes identified in the MSM group were significantly divergent. T. pallidum subsp. endemicum was notably identified in two MSM patients. Macrolide-sensitive or Nichols-like strains were significantly associated with the MSM group. These data suggest that distinct T. pallidum strains were circulating in the heterosexual and MSM groups. Our findings imply that independent factors may contribute to the increased syphilis prevalence in heterosexual and MSM populations.

INTRODUCTION

Human-pathogenic treponemas include Treponema pallidum subsp. pallidum, T. pallidum subsp. pertenue, T. pallidum subsp. endemicum, and Treponema carateum, causing venereal syphilis, yaws, bejel, and pinta, respectively. Yaws, bejel, and pinta are commonly denoted endemic nonvenereal treponemas (1). Syphilis is a sexually transmitted disease (STD) caused by T. pallidum subsp. pallidum. In 2012, the World Health Organization (WHO) estimated a global incidence of 5.6 million new syphilis cases in women and men age 15 to 49 years (2). Oral administration of a penicillin antibiotic, e.g., amoxicillin, is the first-line treatment for syphilis, as per the current treatment guidelines in Japan, and minocyclines are used as second-line drugs, especially for those allergic to penicillin. However, drug resistance to macrolides has emerged (3). Thus, syphilis remains a serious public health concern. In the 2000s, the number of reported cases increased globally, especially in Europe, the Americas, and Australia (4–7). The majority of syphilis patients are males, with the majority of cases being attributable to men who have sex with men (MSM) (6–10).

In Japan, syphilis had been controlled until its reemergence in 2011, which was delayed for approximately 10 years relative to the global trends (11–14). According to a nationwide surveillance in 2016, the annual incidence of syphilis in Japan was 4,564, representing almost a 7-fold increase compared to the figures reported in 2010 (11, 15). Concomitantly, reported cases of congenital syphilis increased 3.5-fold from 0.4 in 2012 to 1.4 in 2016 per 100,000 live births (15). Reported cases were concentrated in metropolitan areas, such as Tokyo, Osaka, Yokohama, and Nagoya (11, 15). Based on epidemiological data, the majority of reported syphilis patients are in their 20s to 40s for males and high teens to early 20s for females, and approximately half of the male syphilis patients identify with a heterosexual orientation (11, 15). Thus, the relative contribution of MSM to the rise in the prevalence of syphilis in Japan seems to be less extensive than that for other countries (11, 15). The driving force behind the increase of syphilis in Japan remains unclear.

The genetic characteristics of T. pallidum subsp. pallidum have been of increasing interest over the past years for use as a tool to better understand the epidemiology of syphilis, as well as macrolide resistance (13, 16, 17). The Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA) developed a method of subtypes based on the analysis of the number of 60-bp repeats in the acidic repeat protein (arp) gene and on restriction fragment length polymorphism of the Treponema pallidum repeat (tpr) gene family, including tprE, tprG, and tprJ (18). Recently, sequencing of a portion of the tp0548 gene was added to the CDC method for molecular typing of T. pallidum strains and is referred to as the enhanced CDC typing (ECDCT) method, which is based on analyses of three genes (18, 19). An alternative method for genotyping T. pallidum strains is sequence-based molecular typing (SBMT), which is based on sequence analyses of selected portions of the tp0136, tp0548, and 23S rRNA genes (17, 20–24). One of the benefits of SBMT is that it enables the creation of a phylogenetic tree that can be used to analyze potential routes of T. pallidum transmission. In addition, macrolide resistance can be analyzed by sequencing the 23S rRNA gene (21, 23).

Since 1992, we have established a network between regional public health institutes and primary sexually transmitted disease (STD) clinics, and for the past 25 years, we have conducted epidemiological surveillance of human immunodeficiency virus (HIV) infections and STDs in Osaka prefecture (25–27). The network, in part, enabled us to collect clinical specimens from syphilis patients in order to genetically characterize the circulating T. pallidum subsp. pallidum strains. The findings may allow us to identify and better understand the underlying factors that had been driving the reemergence of syphilis in Japan.

MATERIALS AND METHODS

Specimens.

Clinical specimens, including genital lesion swabs, urine, blood, and serum, were collected from 95 patients suspected of syphilis at the STD-related clinics in Osaka, Japan, from September 2013 to August 2017. Demographics and T. pallidum DNA status of the patients are summarized in Tables 1 and 2. All the T. pallidum DNA-positive patients were serologically positive for T. pallidum according to results from a treponema antibody test (TPLA; Mediace, Sekisui Medical Co. Ltd., Tokyo, Japan) or nontreponemal lipid antibody test (rapid plasma reagin [RPR]; Mediace, Sekisui Medical Co. Ltd.).

TABLE 1.

Clinical characteristics of patients with suspected syphilis who tested positive or negative by the screening PCR assay

Classification (n)	Age (mean, range) (y)	Sex (n, %)		Stage of syphilis (n, %)		Notea
		Male	Female	Primary	Secondary
All (95)	39.3, 20–81	68, 71.6	27, 28.4
T. pallidum DNA negative (59)	42.3, 21–81	43, 72.9	16, 27.1
T. pallidum DNA positive (36)	34.1, 20–53	25, 69.4	11, 30.6	34, 94.4	2, 5.6	7 MSM, 1 male SW, 11 female SW

^aMSM, men who have sex with men; SW, sex worker(s).

TABLE 2.

Summary of 36 cases and the classification of the Treponema pallidum strains

Case no.	Age (yr)	Sexa	Specimen	TPLA (T.U.)b	RPR (R.U.)c	Syphilis stage	ECDCTd	SBMTe	Strain similarity	Sexual orientationf	HIV statusg	Notesh
1	20	M	Swab	7.2	<1.0	Primary	XX/c	XXR8	T. pallidum subsp. endemicum	MSM	Negative
2	20	M	Swab	20.3	4.2	Primary	14d/f	SSS	SS14-like	MSM	Negative
3	20	M	Swab	106.2	34.5	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative
4	21	M	Swab	584.8	351.5	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative
5	23	M	Swab	46.6	1.7	Primary	14X/c	U3U14S	Nichols-like	MSM	NT
6	25	M	Swab	92.8	22.4	Primary	14d/f	SSS	SS14-like	MSM	Negative
7	26	M	Swab	8,180.0	187.5	Primary	14d/X	XXS	Unknown	MSM	positive
8	27	M	Swab	139.4	<1.0	Primary	14d/f	SSR8	SS14-like	Heterosexual	NT	SW
9	28	M	Swab	400.3	<1.0	Primary	14X/f	SSR8	SS14-like	Heterosexual	NT
10	29	M	Swab	63.4	24.5	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative
11	30	M	Swab	20,768.0	39.1	Secondary	XX/c	XXR8	T. pallidum subsp. endemicum	MSM	Negative
12	36	M	Swab	211.9	1.3	Primary	14a/e	SU11S	SS14-like	Heterosexual	Negative
13	42	M	Swab	74.6	64.6	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative
14	43	M	Swab	30.1	21.0	Primary	13d/f	SSR8	SS14-like	Heterosexual	Negative
15	44	M	Swab	40.9	14.4	Primary	13X/X	SXR8	Unknown	Heterosexual	NT
16	45	M	Swab	1,564.7	25.9	Primary	XX/f	XXR8	Unknown	Heterosexual	Negative
17	47	M	Swab	1.9	<1.0	Primary	11d/f	SSR8	SS14-like	Heterosexual	Negative
18	48	M	Swab	73.4	8.7	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative
19	48	M	Urine	76.6	16.2	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative
20	49	M	Swab	953.1	32.6	Primary	14d/f	SSR8	SS14-like	Heterosexual	NT
21	49	M	Swab	1,141.4	199.0	Primary	14d/f	SSR8	SS14-like	Heterosexual	NT
22	52	M	Swab	95.7	<1.0	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative
23	52	M	Swab	114.0	<1.0	Primary	14d/f	SSR8	SS14-like	Heterosexual	NT
24	52	M	Swab	3,352.9	59.7	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative
25	53	M	Swab	191.5	<1.0	Primary	13d/f	SSR8	SS14-like	MSM	Negative
26	21	F	Swab	19.9	<1.0	Primary	14d/f	SXR8	SS14-like	Heterosexual	Negative	SW
27	24	F	Swab	20.6	4.3	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative	SW
28	24	F	Swab	224.5	<1.0	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative	SW
29	24	F	Swab	8,230.0	337.0	Secondary	14d/f	SSR8	SS14-like	Heterosexual	Negative	SW
30	25	F	Swab	772.8	75.2	Primary	14d/f	SSR8	SS14-like	Heterosexual	NT	SW
31	26	F	Swab	2,587.0	13.1	Primary	14d/f	SSR8	SS14-like	Heterosexual	NT	SW
32	27	F	Swab	6,905.6	76.6	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative	SW
33	28	F	Swab	7,685.6	73.9	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative	SW
34	31	F	Swab	78.9	<1.0	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative	SW
35	34	F	Swab	27.4	15.3	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative	SW
36	47	F	Swab	94.4	29.0	Primary	14d/f	SSR8	SS14-like	Heterosexual	Negative	SW

^aM, male; F, female.

^bTPLA, T. pallidum latex agglutination; T.U., titer units for antitreponema antibody. One T.U. equals 2 mIU with WHO reference material. A positive result is >10 T.U.

^cRPR, rapid plasma reagin; R.U., RPR units, based on the WHO standard. One R.U. is equal to 0.4 IU and is equivalent to the value using the RPR card method. A positive result is >1 R.U.

^dECDCT, enhanced CDC typing.

^eSBMT, sequencing-based molecular typing.

^fMSM, men who have sex with men.

^gNT, not tested.

^hSW, sex worker.

Nucleic acid-based diagnosis of T. pallidum infection.

Total DNA was extracted from the clinical specimens using a QIAamp DNA minikit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. For detection of treponemal DNA, PCR tests that specifically targeted the polA and TpN47 genes were performed as described previously (28, 29). The sequences of all the primers used in this study are shown in Table 3. T. pallidum infection was considered positive when more than one gene was successfully amplified.

TABLE 3.

Treponema pallidum DNA oligonucleotide primers

Target gene	Polarity	Nucleotide sequence (5′–3′)	Reference
polA	Sense	TGC GCG TGT GCG AAT GGT GTG GTC	Liu et al. (28)
	Antisense	CAC AGT GCT CAA AAA CGC CTG CAC G
TpN47	Sense	GAA GTT TGT CCC AGT TGC GGT T	Orle et al. (29)
	Antisense	CAG AGC CAT CAG CCC TTT TCA
arp	Sense	CAA GTC AGG ACG GAC TGT CCC TTG CAT CT	Pillay et al. (18) (modified)
	Antisense	GGT ATC ACC TGG GGA TGC GCA CGT CCT TTC
tprE, tprG, and tprJ	Sense	CAG GTT TTG CCG TTA AGC	Pillay et al. (18)
	Antisense	AAT CAA GGG AGA ATA CCG TC
tp0548	Sense	GGT CCC TAT GAT ATC GTG TTC G	Marra et al. (19)
	Antisense	GTC ATG GAT CTG CGA GTG G
tp0136	Sense	GGT GTG TCT ACT CAG CAC GA	This study
	Antisense	TTA CTC GCG GTT CCA GGA GC
23S rRNA	Sense	GTA CCG CAA ACC GAC ACA G	Lukehart et al. (30)
	Antisense	AGT CAA ACC GCC CAC CTA C
tp0548 1st	Sense	TGG GGC ACT AAA CCG GAA GA	Matějková et al. (17)
	Antisense	TAC GGG CAT TTG CGG ATA GG
tp0548 2nd	Sense	GCG GTC CCT ATG ATA TCG TGT	Woznicová et al. (31)
	Antisense	GAG CCA CTT CAG CCC TAC TG
tp0136 1st	Sense	AAC CCG TTA GCG CCC AAC AT	Matějková et al. (17)
	Antisense	TCC CAG CTC AGC CGA ATC TC
tp0136 2nd	Sense	AGT GTC TTC CTC GTC CGT TC	Woznicová et al. (31)
	Antisense	CAC GTG GTG GTG TCA AAC TT
tp0856 1st	Sense	GCG GCG AAG ACT CGC TC	Cameron (35)
	Antisense	CTT GCG TCC GAG CAG G
tp0856 2nd	Sense	ATG ACG CAC AGT GAG CTA	This study
	Antisense	ACG GGA GAA GTT CAC AAT

Molecular typing of treponemal DNA.

T. pallidum genotyping using the ECDCT and SBMT protocols was performed on all PCR-positive specimens. The ECDCT method is based on the analyses of three genomic regions (arp, tprEGJ, and tp0548 genes) (18, 19). Samples were considered typeable when at least one of three target genes was successfully analyzed. The SBMT method is based on the sequences of the tp0136, tp0548, and 23S rRNA genes (17, 21, 23, 24, 30, 31). Samples were considered typeable when at least one target locus (either tp0136 or tp0548) was successfully analyzed. T. pallidum subsp. pallidum strain Nichols was used as the positive control for T. pallidum DNA amplification. PCR products for the tp0136, tp0548, and 23S rRNA genes were sequenced using an ABI3130 automated sequencer (Thermo Fisher Scientific, Waltham, MA, USA). Sequence analysis was performed using GENETYX-MAC version 17 software (Genetyx, Tokyo, Japan). For macrolide susceptibility analysis, nucleotide positions 2058 and 2059 of the 23S rRNA gene were evaluated for the A-to-G mutation that is associated with drug resistance (17, 32), as demonstrated experimentally (33). T. pallidum subsp. pallidum and T. pallidum subsp. endemicum were distinguished by phylogenetic analysis (see below) based on tp0136, tp0548, and tp0856 in comparison to representative T. pallidum subsp. pallidum, T. pallidum subsp. endemicum, and T. pallidum subsp. pertenue strains.

Phylogenetic analysis.

A phylogenetic tree was generated using the MEGA6 software, with the bootstrapping maximum likelihood algorithm and the Tamura-Nei model (34).

Statistical analysis.

Fisher’s exact test was used to test whether any of the ECDCT subtypes coincided with a specific SBMT genotyping and whether any of the genotypes were more frequent in any particular risk group compared to that in other groups. Results were considered statistically significant when the P value was less than 0.01.

Ethics statement.

This study was approved by the ethics review board of the Osaka Institute of Public Health (approval no. 0810-05-4). Specimens were collected primarily for the purpose of diagnosing T. pallidum infection. The molecular epidemiological study of T. pallidum was performed using the residual specimens. All the patients gave informed verbal consent prior to specimen collection by the clinician, which was documented in the medical record of the clinic.

Data availability.

Unique sequences identified in this study were deposited in GenBank under accession numbers LC383799 to LC383801.

RESULTS

Detection of T. pallidum DNA in clinical specimens from suspected syphilis patients.

A total of 95 clinical specimens were collected from 95 patients suspected of syphilis. Treponemal DNA was detected in 36 patients (37.9%) by PCR (Tables 1 and 2). All the clinical specimens positive by PCR, except one urine specimen, were swabs of urogenital lesions. Of the PCR-positive patients, 69.4% (25/36) were males age 20 to 53 years old. The 11 PCR-positive female patients (11/36) were 21 to 47 years old. Among the males, 28.0% (7/25) were MSM. The rest were considered heterosexual. One of the heterosexual males and all of the females were sex workers (SW). Of note, one MSM was HIV positive. The majority of the PCR-positive patients had primary syphilis (94.4% [34/36]), and only two cases involved patients with secondary syphilis (5.6% [2/36]). Molecular typing of the T. pallidum strains by the ECDCT and SBMT methods was performed on the 36 PCR-positive specimens. T. pallidum subsp. endemicum was identified in two of the specimens (2/36 [5.6%]). Molecular typing of the T. pallidum subsp. pallidum strains by ECDCT and SBMT methods was performed on the 34 PCR-positive specimens.

T. pallidum subsp. pallidum subtyping using the ECDCT method.

Of the 34 PCR-positive specimens, 29 were fully subtyped and 5 were partially subtyped using the ECDCT method (Table 4). At least 5 different subtypes were detected, 14d/f, 13d/f, 11d/f, 14a/e, which were fully subtyped, and 14X/c, which was partially subtyped. Subtype 14d/f was the most prevalent (25/34 [73.5%]), followed by 13d/f (2/34 [5.9%]) and the seven other subtypes (1/34 each).

TABLE 4.

Summary of Treponema pallidum subsp. pallidum molecular typing by ECDCT^a

Subtype	Typing	arp	tprE, tprG, and tprJ	tp0548	No. (%) of isolates
14d/f	Complete	14	d	f	25 (73.5)
13d/f	Complete	13	d	f	2 (5.9)
11d/f	Complete	11	d	f	1
14a/e	Complete	14	a	e	1
13X/X	Partial	13	X	X	1
14d/X	Partial	14	d	X	1
14X/c	Partial	14	X	c	1
14X/f	Partial	14	X	f	1
XX/f	Partial	X	X	f	1

^aX, undetermined.

T. pallidum subsp. pallidum genotyping using the SBMT method.

Of the 34 PCR-positive specimens, 30 were fully genotyped, 2 were partially genotyped, and 2 (XXR8-XX/f and XXS-14d/X) were not genotyped using the SBMT method (Table 5) (1, 35–37). A maximum of 4 different genotypes were detected, SSR8, SSS, SU11S, and U3U14S. Genotype SSR8 was the most prevalent (26/34 [76.5%]), followed by SSS (2/34 [5.9%]). Many of the SSR8 strains were 14d/f (22/26 [84.6%]).

TABLE 5.

Summary of Treponema pallidum subsp. pallidum molecular typing by SBMT^a

Genotype	Typing	tp0136	tp0548	23S rRNA	Strain similarity	No. (%) of isolates
SSR8	Complete	Identical to SS14	Identical to SS14	A2058G	SS14-like	26 (76.5)
SSS	Complete	Identical to SS14	Identical to SS14		SS14-like	2 (5.9)
SU11S	Complete	Identical to SS14	Unique 11		SS14-like	1
U3U14S	Complete	Unique 3	Unique 14		Nichols-like	1
SXR8	Partial	Identical to SS14	X	A2058G	SS14-like	2 (5.9)
XXR8 (−XX/f)	X	X	X	A2058G	Unknown	1
XXS	X	X	X		Unknown	1

^aX, undetermined.

According to the analysis of the tp0136 and/or tp0548 genes, 86.1% (31/34) were SS14-like strains, and 2.8% (1/34) were Nichols-like strains.

The 23S rRNA gene was PCR amplified, and the nucleotides at positions 2058 and 2059 were evaluated. Of the 36 specimens analyzed, the A-to-G mutation at these locations, which is known to lead to macrolide resistance, was found at nucleotide 2058 in 86.1% (31/36) of the specimens. No mutation was detected at position 2059. The mutation for macrolide resistance was identified in SS14-like strains (28/31 [90.3%]), Nichols-like strains (0/1 [0%]), and T. pallidum subsp. endemicum (2/2 [100%]).

Risk group analysis of the T. pallidum strains.

The distribution of T. pallidum strains was assessed relative to patient sex and sexual orientation. To increase the precision of the assessment, genotypes from both the ECDCT and SBMT approaches were analyzed concomitantly, and T. pallidum subsp. endemicum was considered an independent group (Fig. 1). Regarding the genotype 14d/f-SSR8 (n = 22) versus non-14d/f-SSR8 (n = 11), the 14d/f-SSR8 genotype was frequently detected in females (10/10) and less frequently in males (12/23), and this difference was statistically significant (P = 0.01290). However, the association of 14d/f-SSR8 with the heterosexual population (22/26) was notable compared to that with the MSM group (0/7), with the variation between the two groups being highly statistically different (P < 0.01). The numbers of independent genotypes identified were 7 in 25 male patients and 1 in 11 female patients. However, the variety of genotypes detected in the MSM group of patients (4 genotypes/7 patients) was much greater than that in the heterosexual population (4 genotypes/29 patients). Nichols-like strains were found only in the MSM group (1/7) and not in the heterosexual group (0/29), and this difference was not statistically significant (P = 0.19). The percentage of macrolide-sensitive strains was 57.1% (4/7) in the MSM group, which was dramatically higher than that in the heterosexual group (1/29 [3.4%]), with the difference being statistically significant (P < 0.01).

FIG 1.

Treponema pallidum strains in relation to the sex and sexual orientations of the subjects. Blue and red represent male and female, respectively. Green highlights macrolide susceptibility. Wearing a pair of glasses indicates a Nichols-like strains, otherwise, they are SS14-like strains and T. pallidum subsp. endemicum (TEN). A hat indicates the incomplete T. pallidum genotyping using the sequence-based molecular typing (SBMT) method (XXS and XXR8).

DISCUSSION

Increased prevalence of syphilis among MSM has been reported globally (6–10). This is assumed to be due to increased high-risk sexual behaviors, including unprotected sex, because successful reduction in HIV transmission has been reported in association with preexposure prophylaxis (PrEP) (10, 38). A prescription of antiretroviral drugs for PrEP is not officially supported in Japan. However, antiretroviral drugs prescribed to HIV-positive individuals can be used for PrEP. The 10-year delay in the rise of syphilis prevalence in Japan may be partly attributed to the awareness of this information in Japan among MSM.

The genotypes of T. pallidum strains among the MSM group were diverse, consistent with the multiple partnerships and the high-risk behaviors among MSM, which can be facilitated by social networking platforms on mobile devices (39–41). The diversity is also attributable to the geographic broadness of the MSM community. Similar findings have been reported in several countries (7, 20, 21). The strain diversity of T. pallidum subsp. pallidum in some countries, such as China, Scotland, or the United States, has not been reported to be as diverse as that in our findings, probably due to distinct social situations (42). It was unclear whether there were any epidemiological links between Japan and other countries where the rise in the prevalence of syphilis in the general population was preceded by a prevalence increase in the MSM population. To address this, cooperative efforts should be made regarding the global molecular epidemiology of syphilis. It seems that performing both SBMT and ECDCT analyses may be beneficial for improving the genetic resolution. It was surprising that T. pallidum subsp. endemicum was identified only among MSM, which also indicated the relatively closed circulation of T. pallidum strains in the MSM network. T. pallidum subsp. endemicum causes bejel, a nonvenereal disease endemic to the Sahel region of Africa and the Arabian Peninsula (43). T. pallidum subsp. endemicum can be transmitted via sexual contact (37). Further studies are needed to clarify whether T. pallidum subsp. endemicum has been locally transmitted among MSM in Japan.

In contrast to the MSM population, T. pallidum genotype diversity was limited in the heterosexual population in both males and females. The dominant genotype associated with female patients in our study was 14d/f, a genotype frequently identified around the globe, including in China (19, 44, 45), Peru (46), Russia (47), and the United States (19). It was assumed that certain T. pallidum subsp. pallidum strains should have been passed over among heterosexual groups globally. Given that T. pallidum strains identified in two different groups do not overlap, the “spillover” of syphilis from MSM to young adult females did not fully explain the epidemiological feature of syphilis in Japan. It seems more likely that independent factors are responsible for contributing to the reemergence of syphilis in the two populations. The increasing prevalence of syphilis among young adult females is likely attributable to the advancement of mobile networking devices, which increasingly allows for anonymous casual dating (48). In past years, commercial sex work was truly occupational. However, current advancement of social networking tools allows for sex work by young women to be a “casual part-time job” (49). They may have limited knowledge about sexually transmitted infections (STIs), resulting in them being placed at increased risk (50). This may have coincided with changes in the sexual behaviors of the MSM cohort.

Recently, the prevalence of syphilis in females in the United States has been increasing. Rates for reported congenital syphilis cases also increased during 2015 and 2016 (www.cdc.gov/std/stats16/Syphilis.htm). It is a concern that syphilis may be spreading to females from MSM who identify with a bisexual orientation. This could accelerate the prevalence of syphilis among women in the near future, which is a particular concern because this would likely result in an increased prevalence of congenital syphilis.

As the prevalence of syphilis increases, social awareness regarding syphilis also improves, in large part through mass media. This may motivate individuals to examine their STI status. At the same time, STI specialists become increasingly aware of syphilis being a differential during diagnosis. This feedback boosts the number of reported cases of syphilis.

In contrast to syphilis, nationwide surveillance indicates that the prevalence of other STDs in Japan has not been trending upward in parallel to syphilis, including genital chlamydial infections, genital herpesvirus infections, condyloma acuminata, or gonococcal infections. The reason for this divide remains to be clarified. One concern is that not all the patients with the above-mentioned STDs are reported to the national surveillance system because they are under fixed-point surveillance. This should be addressed in future studies.

We detected macrolide resistance-associated mutations in 86.1% of the specimens analyzed, which is the second highest prevalence previously reported, following only China (51, 52). Interestingly, the resistance rate was significantly higher in the heterosexual group than in the MSM group (96.6% versus 42.9%, respectively; P < 0.01). This is in sharp contrast to the finding from many countries, including England, Australia, and the United States (53–55), suggesting that T. pallidum strains do not readily spread globally but are more likely to circulate regionally. In our data, macrolide resistance was predicted by the A2058G mutation, similar to previous reports. However, in some countries, the A2059G mutation is attributable to 40% of the macrolide-resistant T. pallidum subsp. pallidum strains (23). If macrolide resistance is induced independently from place to place, the antibiotic usage may affect the mutation site preference. This possibility should be addressed in future studies.

In conclusion, we demonstrated that different T. pallidum strains circulate among individuals with different sexual orientations in Japan, suggesting that independent factors drove, or at least greatly influenced, the spread of syphilis in each population. Serology is unable to distinguish between syphilis and other endemic treponematoses (37). Molecular identification of T. pallidum strains is emphasized to reveal the current spread of this pathogen in the human population.