Treponema pallidum Strains Among Women and Men Who Have Sex With Women in Amsterdam, the Netherlands and Antwerp, Belgium Between 2014 and 2020 : Sexually Transmitted Diseases

Secondary Logo

Journal Logo


Treponema pallidum Strains Among Women and Men Who Have Sex With Women in Amsterdam, the Netherlands and Antwerp, Belgium Between 2014 and 2020

Zondag, Hélène C.A. Msc∗,†; Zwezerijnen-Jiwa, Florine H. Msc∗,‡; de Vries, Henry J.C. MD, PhD∗,§; De Baetselier, Irith PhD; Bruisten, Sylvia M. PhD∗,∥

Author Information
Sexually Transmitted Diseases 50(6):p e5-e7, June 2023. | DOI: 10.1097/OLQ.0000000000001779

Treponema pallidum subspecies pallidum (TPA), causing syphilis, is found globally. The circulating TPA strains have mainly been investigated in men who have sex with men (MSM), a disproportionally affected population in high-income countries, and showed to be geographically similar regarding the major strains.1 To gain insight in the genetic diversity of TPA strains several molecular typing schemes for TPA have been developed in the last two decades. In 2018, a TPA multilocus sequence typing (MLST) scheme2 was set up, and soon after the TPA pubMLST database was implemented to record all detected TPA allelic profiles worldwide.3 Limited information is available on the prevalence and variation among women, and men who have sex with women (MSW). We aimed to identify TPA strains within MSW and women and compare them to the TPA strain distribution among MSM.

Between 2014 and 2020, 41 TPA PCR positive samples were included; 32 from the Public health laboratory (PHL) of Amsterdam, the Netherlands (16 women and 16 men) and 9 from the Belgian National Reference Centre for STIs (NRC-STI) in Antwerp, Belgium (1 woman and 8 men). Patients had either visited a sexual health center or a general physician for routine surveillance or symptomatic screening.

TPA was characterized using the TPA MLST method based on the partial amplification and sequence analysis of three genetic regions; tp0136, tp0548, and tp0705.2 In addition, the 23S rRNA loci were checked for macrolide resistance-associated mutations (MRAMs). All complete allelic profiles (AP) and available patient data were added to the TPA pubMLST database (ID numbers 745, 780, 788, and 1651–1677).

We compared our data to TPA typed isolates from women and men whose sexual orientation was known from the TPA pubMLST database (extracted on July 5, 2022).3

Possible associations between clinical characteristics and AP were tested using the χ2 test or Fisher exact test with a statistical significance set at P ≤ 0.05 and Simpson's diversity index was calculated for comparison within the sample set of the study using RStudio (v1.2.5033).

The median age of 41 years included patients was 36 years old (interquartile range, 25–48 years). From 23 of 41 (56%) included patients, the human immunodeficiency virus (HIV) status was known, with 4 patients living with HIV and 19 without. Sample material was known for 19 samples with 16 ulcer swabs, 2 biopsies, and 1 serum (Supplementary Table 1,

A total of 30 of 41 (73%) samples, derived from 12 women and 18 MSW, could be typed for all three loci. Nine distinct AP were found (Table 1). Samples taken from tissue (biopsy) or blood serum failed to be typed. There were 3 of 30 (10%) typed samples that originated from Antwerp and all had the same AP, being 1.3.1. The most prevalent AP were 1.3.1 (12/30, 40%) followed by 1.1.1 (17%). Allelic profiles of 3.2.3 and 9.7.3, pertaining to 5 samples (17%), belonged to the Nichols lineage and were found only in men (Table 1).

TABLE 1 - Allelic Profiles Found Among the 30 Fully-Typed Treponema pallidum Subsp. Pallidum Samples From Women, MSW Between 2014 and 2020 in Amsterdam, the Netherlands and Antwerp, Belgium
Allelic Profile Frequency, n (%) Lineage 23S rRNA Mutation, (n) Sex of Patients, (n)
1.3.1 12* (40) SS14 A2058G (11)
Undetermined (1)
Men (8)
Women (4)
1.1.1 5 (17) SS14 A2058G (3)
A2059G (1)
No mutation (1)
Men (1)
Women (4)
1.43.1 3 (10) SS14 A2058G (3) Men (1)
Women (2)
3.2.3 3 (10) Nichols A2058G (1)
Undetermined (2)
Men (3)
1.1.3 2 (7) SS14 A2059G (1)
No mutation (1)
Men (1)
Women (1)
9.7.3 2 (7) Nichols A2058G (2) Men (2)
1.1.10 1 (3) SS14 A2058G (1) Women (1)
19.3.1 1 (3) SS14 Undetermined (1) Men (1)
1.1.8 1 (3) SS14 No mutation (1) Men (1)
*Including all 3 typed samples from the Belgian National Reference Centre for STIs in Antwerp, Belgium.

The presence of a MRAM could be determined in 30 of 41 samples of which 90% contained a MRAM, 26 of these samples were fully typed (Table 1) and 4 were partially typed (not shown). Twenty-three of 26 (88%) fully typed samples contained MRAM (Table 1). The A2058G mutation was present in samples from both MSW and women. However, the A2059G mutation or the absence of a mutation was only found in MSW.

Similar AP diversity was found in samples from men and women; 8 AP were found in 18 of 24 (75%) samples derived from men, resulting in a Simpson diversity index of 0.79, compared with 5 AP from 12 of 17 (70%) samples among women resulting in a diversity index of 0.80.

The export from the TPA BIGSdb pubMLST database of fully typed samples from women, and men whose sexual orientation was recorded contained 501 typed isolates derived from 55 women (11%) and 446 men (89%). Of these, 76% (379/501) isolates were derived from MSM and 24% (122/501) from women and MSW. The most prevalent AP in the pubMLST database among women and MSW was 1.1.8 (51/122, 42% records), followed by 1.3.1 (30/122, 25% records). However, in this study, AP 1.1.8 was only found in one MSW (Table 1).

No associations between sex and AP were found (P = 0.208). In addition, no associations were found between HIV status, age, ethnicity and AP (respectively P = 0.096, P = 0.885 and P = 0.420).

This study shows an overall distribution of TPA strains among women and MSW in Amsterdam and Antwerp similar to the distribution found among MSM in Amsterdam.4 The lineage distribution in this study was 83% SS14 and 17% Nichols. Samples with types belonging to the Nichols lineage were found only in samples from MSW. Similar distributions were found in previous studies conducted in The Netherlands, mostly among MSM4,5 and worldwide.1,6

The most prevalent allelic profile in this study was 1.3.1 occurring in 40% of the samples from MSW, as well as in women in the two cities from our study. This was consistent with the data from the TPA pubMLST database. However, the most prevalent strain among women, being 1.1.8, according to the TPA pubMLST database, was only found once in the present study in a sample derived from a MSW. Also, the MSW in our study showed more similarities with the MSM group from the pubMLST database with regard to heterogeneity of strains and a higher percentage of Nichols lineage. This may suggest that MSW, although denying sex with men, contracted their syphilis strain from other men. This is a common finding associated with the stigma associated with homosexuality.7

Interestingly, a Japanese study discovered a higher prevalence of macrolide resistance mutations in MSW (97%) than in MSM (38%).8 The 90% MRAM rate among this population is higher than in samples from 2006–2018 among mostly MSM in Amsterdam, where 81% of the samples contained a MRAM.5 However, a temporal increase of MRAM has been reported before in TPA isolates.5,9 Macrolide resistance-associated mutation was present in all 7 successfully determined samples from Antwerp, in concordance with a previous study.10

The small sample size is an important limitation of this study. As a result, the power to detect significant differences between populations is limited. Another limitation is that it was not possible to obtain complete or even partially typed allelic profiles from blood or from tissue samples. This finding has been observed by others as well, supporting that ulcer swabs, which are known to have the highest bacterial load, are the best source for molecular typing.11,12

Although this study population is too small to make clear statements about circulating TPA strains in relation to different sexual preferences, further research is warranted to gain a better understanding of the transmission patterns of TPA within specific populations and help public health surveillance. A better understanding of the transmission patterns of TPA within specific populations could be used to inform policy makers on who should best be targeted for preventive measures.


1. Beale MA, Marks M, Cole MJ, et al. Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis. Nat Microbiol 2021; 6:1549–1560.
2. Grillova L, Bawa T, Mikalova L, et al. Molecular characterization of Treponema pallidum subsp. pallidum in Switzerland and France with a new multilocus sequence typing scheme. PLoS One 2018; 13:e0200773.
3. Grillova L, Jolley K, Smajs D, et al. A public database for the new MLST scheme for Treponema pallidum subsp. pallidum: surveillance and epidemiology of the causative agent of syphilis. PeerJ 2019; 6:e6182.
4. Zondag HCA, Nieuwenburg SA, Himschoot M, et al. Treponema pallidum subspecies pallidum intrapatient homogeneity at various body locations in men with infectious syphilis. Microbiol Spectr 2022; 10:e0248221.
5. Zondag HCA, Bruisten SM, Vrbova E, et al. No bejel among Surinamese, Antillean and Dutch syphilis diagnosed patients in Amsterdam between 2006-2018 evidenced by multi-locus sequence typing of Treponema pallidum isolates. PLoS One 2020; 15:e0230288.
6. Taouk ML, Taiaroa G, Pasricha S, et al. Characterisation of Treponema pallidum lineages within the contemporary syphilis outbreak in Australia: A genomic epidemiological analysis. Lancet Microbe 2022; 3:e417–e426.
7. de Vries HJ. Sexually transmitted infections in men who have sex with men. Clin Dermatol 2014; 32:181–188.
8. Kanai M, Arima Y, Nishiki S, et al. Molecular typing and macrolide resistance analyses of Treponema pallidum in heterosexuals and men who have sex with men in Japan, 2017. J Clin Microbiol 2019; 57:e01167–e01118.
9. Beale MA, Marks M, Cole MJ, et al. Contemporary syphilis is characterised by rapid global spread of pandemic <em>Treponema pallidum</em> lineages. medRxiv 2021; 2021.03.25.21250180.
10. Mikalova L, Grillova L, Osbak K, et al. Molecular typing of syphilis-causing strains among human immunodeficiency virus-positive patients in Antwerp, Belgium. Sex Transm Dis 2017; 44:376–379.
11. Gayet-Ageron A, Lautenschlager S, Ninet B, et al. Sensitivity, specificity and likelihood ratios of PCR in the diagnosis of syphilis: A systematic review and meta-analysis. Sex Transm Infect 2013; 89:251–256.
12. Peng RR, Wang AL, Li J, et al. Molecular typing of Treponema pallidum: A systematic review and meta-analysis. PLoS Negl Trop Dis 2011; 5:e1273.

Supplemental Digital Content

Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Sexually Transmitted Diseases Association.