Methodology	Description
Data source and search strategy	-Search was first conducted on January 12, 2021, and then updated on February 20, 2022, in PubMed and Embase. -Search strategies included exploded MeSH/Emtree terms and broad terms with no language or time restrictions. - The definition of Europe included 53 countries classified by European subregion/country: ○ Eastern Europe: Belarus, Bulgaria, Czech Republic, Hungary, Poland, Republic of Moldova, Romania, Russian Federation, Slovakia, and Ukraine. ○ Northern Europe: Denmark, Estonia, Finland, Iceland, Ireland, Latvia, Lithuania, Norway, Sweden, and United Kingdom of Great Britain and Northern Ireland. ○ Southern Europe: Albania, Andorra, Bosnia and Herzegovina, Croatia, Greece, Italy, Malta, Montenegro, Portugal, the former Yugoslav Republic of Macedonia, San Marino, Serbia, Slovenia, and Spain. ○ Western Europe: Austria, Belgium, France, Germany, Luxembourg, Monaco, Netherlands, and Switzerland. ○ Intersection of Europe and Asia: Armenia, Azerbaijan, Cyprus, Georgia, Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, and Uzbekistan. ○ Israel. ○ Turkey.
Study selection and inclusion and exclusion criteria	-Search results were imported into the reference manager Endnote (Thomson Reuters, USA). - Screening was performed in four stages: 1. Duplicate publications were identified and excluded. 2. Titles and abstracts were screened for relevant and potentially relevant publications. 3. Full texts of relevant and potentially relevant publications were retrieved and screened for relevance. 4. Bibliographies of relevant publications and reviews were checked for additional potentially relevant publications. - Inclusion criteria were any publication, including a study with a minimum sample size of 10, reporting primary data on any of the following outcome measures: 1. HSV-2 antibody incidence as detected by a type-specific diagnostic assay. 2. HSV-2 antibody seroprevalence as detected by a type-specific diagnostic assay. 3. Proportion of HSV-2 detection in clinically diagnosed GUD as detected by standard viral detection and subtyping methods. 4. Proportion of HSV-2 detection in laboratory-confirmed genital herpes (as opposed to HSV-1) as detected by standard viral detection and subtyping methods. - Exclusion criteria were: ○ Case reports, case series, reviews, editorials, commentaries, and qualitative studies. ○ Measures reporting seroprevalence in infants aged <6 months as their antibodies can be maternal in origin. -In this study, the term “publication” refers to a document reporting one or several outcome measures. “Study” or “measure” refers to a specific outcome measure and its details.
Data extraction and data synthesis	-Extracted variables included: author(s), publication title, year of publication, year(s) of data collection, subregion, country of origin, country of survey, city, study site, study design, study sampling procedure, study population and its characteristics (e.g., sex and age), sample size, response rate, HSV-2 outcome measures, and diagnostic assay (Box S2). -Overall outcome measures and their stratified measures were extracted, provided sample size in each stratum is ≥10. -For studies including overall sample size, but no individual strata sample sizes, the sample size of each stratum was assumed equal to overall sample size divided by the number of strata in the study. - Stratification hierarchy for incidence and seroprevalence measures in descending order of preference were: 1. Population type as defined in Box S1. 2. Sex. 3. Age group classified as (groups optimized to best fit reported data): ○ <20 years old. ○ 20–29 years old. ○ 30–39 years old. ○ 40–49 years old. ○ 50–59 years old ○ ≥60 years old. ○ Mixed ages. - Stratification hierarchy for GUD and genital herpes included genital herpes episode status and study site: 1. Genital herpes episode status classified as: ○ First episode genital herpes. ○ Recurrent genital herpes. 2. Study site stratification classified as: ○ Hospital. ○ STI clinic. -Measures reporting any HSV-2 outcome among children <15 years old were only reported but not included in the analyses.
Quality assessments	The Cochrane-informed approach for risk of bias assessment included: -Study's precision classification into low versus high was based on the sample size (<200 versus ≥200). - Study's appraisal into low versus high risk of bias was determined using two quality domains: ○ Sampling method: probability-based versus non-probability-based. ○ Response rate: ≥80% versus <80% or unclear.
Meta-analyses	-Meta-analyses were conducted using DerSimonian-Laird random-effects models with inverse variance weighting. The variance of each outcome measure was stabilized using the Freeman-Tukey arcsine square-root transformation. -Pooled mean HSV-2 seroprevalence was estimated for each population type by sex, and for general populations by European country, European subregion, age group, year of data collection range, and year of publication range. -Pooled proportions of HSV-2 detection in GUD and in genital herpes were estimated. - Heterogeneity assessment was based on three complementary metrics: ○ Cochran's Q statistic to assess existence of heterogeneity in effect size (p-value < 0.1 indicated heterogeneity). ○ I2 heterogeneity measure to assess the percentage of between-study variation in effect size that is due to actual differences in effect size rather than sampling variation. ○ Prediction interval to describe the distribution of true outcome measures around the pooled mean.
Meta-regressions	-Univariable and multivariable random-effects meta-regression analyses using log-transformed proportions were carried out to identify predictors of HSV-2 seroprevalence and proportion of HSV-2 detection in genital herpes. -Factors in the univariable analyses with a p-value <0.1 were included in the multivariable analysis. -Factors in the multivariable analyses with a p-value ≤0.05 were deemed to be significant predictors. - Variables included in the univariable meta-regression models for HSV-2 seroprevalence were: ○ Population type. ○ Age group. ○ Sex. ○ European subregion/country. ○ Country's income. ○ Assay type (western blot, ELISA, and monoclonal antibody). ○ Sample size. ○ Sampling method. ○ Response rate. ○ Year of data collection. ○ Year of publication. ○ Year of data collection category (<1995, 1995–2005, >2005). ○ Year of publication category (<2000, 2000–2010, >2010). - Variables included in the univariable meta-regression models for proportion of HSV-2 detection in genital herpes were: ○ Age group. ○ Sex. ○ Genital herpes episode status. ○ European subregion/country. ○ Sample size. ○ Year of data collection category (<1995, 1995–2005, >2005). ○ Year of publication category (<2000, 2000–2010, >2010). -The year of data collection had missing variables that were imputed by adjusting the year of publication using the median difference with the year of data collection in studies with reported year of data collection.

Abbreviations: ELISA = Enzyme-linked immunosorbent type-specific assay, GUD = Genital ulcer disease, HSV-1 = Herpes simplex virus type 1, HSV-2 = Herpes simplex virus type 2, STI = Sexually transmitted infection.