Epidemiology of herpes simplex virus type 2 in the Middle East and North Africa: Systematic review, meta‐analyses, and meta‐regressions

Herpes simplex virus type 2 (HSV‐2) infection is a prevalent, sexually transmitted infection with poorly characterized prevalence in the Middle East and North Africa (MENA) region. This study characterized HSV‐2 epidemiology in MENA. HSV‐2 reports were systematically reviewed as guided by the Cochrane Collaboration Handbook and findings were reported following PRISMA guidelines. Random‐effects meta‐analyses and meta‐regressions were performed to estimate pooled mean outcome measures and to assess predictors of HSV‐2 antibody prevalence (seroprevalence), trends in seroprevalence, and between‐study heterogeneity. In total, sixty‐one overall (133 stratified) HSV‐2 seroprevalence measures and two overall (four stratified) proportion measures of HSV‐2 detection in laboratory‐confirmed genital herpes were extracted from 37 relevant publications. Pooled mean seroprevalence was 5.1% (95% confidence interval [CI]: 3.6%–6.8%) among general populations, 13.3% (95% CI: 8.6%–18.7%) among intermediate‐risk populations, 20.6% (95% CI: 5.3%–42.3%) among female sex workers, and 18.3% (95% CI: 3.9%–39.4%) among male sex workers. Compared to Fertile Crescent countries, seroprevalence was 3.39‐fold (95% CI: 1.86–6.20) and 3.90‐fold (95% CI: 1.78–8.57) higher in Maghreb and Horn of Africa countries, respectively. Compared to studies published before 2010, seroprevalence was 1.73‐fold (95% CI: 1.00–2.99) higher in studies published after 2015. Pooled mean proportion of HSV‐2 detection in genital herpes was 73.8% (95% CI: 42.2%–95.9%). In conclusion, MENA has a lower HSV‐2 seroprevalence than other world regions. Yet, 1 in 20 adults is chronically infected, despite conservative prevailing sexual norms. Seroprevalence may also be increasing, unlike other world regions. Findings support the need for expansion of surveillance and monitoring of HSV‐2 infection in MENA.


| INTRODUCTION
Herpes simplex virus type 2 (HSV-2) infection is a chronic, incurable, and globally prevalent sexually transmitted infection (STI), 1-3 known for persistent reactivation and frequent shedding. 4,5 When individuals are symptomatic, HSV-2 infection presents in the form of recurrent painful genital lesions that are associated with sexual and psychological comorbidities. 1,6-8 HSV-2 can also be passed vertically from mother to child, leading to neonatal herpes, a serious condition for neonates that causes high morbidity and mortality. 9 HSV-2 infection has overlapping epidemiology with HIV infection, 10 with evidence suggesting an epidemiologic synergy between these two infections. [11][12][13] HSV-2 infection is associated with increased HIV acquisition and transmission, and people living with HIV may suffer from increased severity of HSV-2 infection. [13][14][15] Despite burdensome sequelae, inadequate understanding of the epidemiology of STIs in the global context and their impact on sexual, reproductive, and psychosocial health lowered their priority on health policy agendas. [16][17][18] To address this concern, the World Health Organization (WHO) and global partners are leading efforts to control or eliminate STIs as a public health concern by 2030 by integrating preventive, therapeutic, and control frameworks, 16,17 one of which is developing an HSV-2 vaccine, a long overdue public health priority. 19,20 To inform these efforts, and as part of a global project to understand the epidemiology of HSV-1 and HSV-2 infections, 21-30 a systematic review was conducted to characterize HSV-2 epidemiology in the Middle East and North Africa (MENA) region. HSV-2 antibody prevalence (seroprevalence) and proportions of HSV-2 detection in clinically diagnosed genital ulcer disease (GUD) and in laboratory-confirmed genital herpes were identified and synthesized, pooled means were estimated, and predictors of high seroprevalence and temporal trends were investigated.

| METHODS
Methods used in this study are described in Box 1 and briefly below.
The methods for investigating epidemiology of HSV-2 infection were adapted based on a series of published systematic reviews and metaanalytics assessing HSV-2 epidemiology in other WHO regions. 26

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 prevalence (seroprevalence) as detected by a type-specific diagnostic assay. 3. Proportion of HSV-2 in GUD as detected by standard viral detection and subtyping methods. 4. Proportion of HSV-2 in laboratory-confirmed genital herpes (as opposed to HSV-1), as detected by standard viral detection and subtyping methods. -Exclusion criteria were: o Case reports, case series, reviews, editorials, commentaries, and qualitative studies. o Measures reporting seroprevalence in infants <6 months-old 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.

Detailed description
Data extraction and data synthesis -Extracted variables included: author(s), publication title, year(s) of data collection, publication year, 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, HSV-2 outcome measures, and diagnostic assay. -Overall outcome measures and their stratified measures were extracted, provided the 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 in descending order of preference were: 1. Population type as defined in Supporting Information: Box S1. 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 subregion, age group, year of data collection category, and year of publication category. -Pooled proportions of HSV-2 detection in GUD and in genital herpes cases were estimated.
-Heterogeneity assessment was based on three complementary metrics: o Cochran's Q statistic to assess existence of heterogeneity in effect size (p-value < 0.1 indicated heterogeneity). o I 2 heterogeneity measure to assess the percentage of between-study variation in effect size that is due to actual differences in effect size rather than chance. o 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. -Factors in the univariable model with a p-value < 0.1 were included in the multivariable analyses.
-Factors in the multivariable model with a p-value ≤ 0.05 were deemed to be significant predictors.

| Quality assessments
Due to known limitations in diagnostic performance of HSV-2 serological assays, [35][36][37] validity and reliability of each assay in each study was assessed by Professor Rhoda Ashley-Morrow-a leading expert in HSV-2 serological assays who has investigated and evaluated different HSV assays for three decades. Details of each assay used in each study were shared with Professor Ashley-Morrow, who then used her expert judgment to identify whether each assay was valid and reliable to be included. Only assays deemed as valid and reliable were included. Precision and risk of bias (ROB) assessments, as informed by the Cochrane approach 38 and described in Box 1, were then conducted on each included study.

| Meta-analyses
Extracted measures were described using summary statistics, including medians, ranges, and pooled mean estimates and their 95% confidence intervals (CI). The DerSimonian-Laird random-effects model 39 was used to conduct meta-analyses accounting for both sampling variation and heterogeneity in effect size between studies (Box 1). 39,40 The variance of each outcome measure was stabilized using the Freeman-Tukey arcsine square-root transformation, 41 after ensuring its applicability. 42 Heterogeneity was assessed based on Cochran's Q statistic, I 2 heterogeneity measure, and prediction interval. All meta-analyses were conducted with R version 4.41.3 43 using the "meta" package, 44 by adapting existing codes to conduct these analyses.

| Meta-regressions
Univariable and multivariable meta-regressions of log-transformed seroprevalence measures were conducted in Stata/SE version 16 45 using the "metareg" package, 46 as described in Box 1, by adapting existing codes to conduct these analyses. All stratified seroprevalence measures in all population types, as defined in Supporting Information: Box S1, were utilized in these meta-regressions.
Variables in the univariable analyses with a p-value < 0.1 were included in the multivariable meta-regression models.

| Search results and scope of evidence
The study selection process followed PRISMA guidelines

| HSV-2 seroprevalence overview
Overall extracted HSV-2 seroprevalence measures (n = 61) are listed in Supporting Information: Definitions of these populations can be found in Box S1.
F I G U R E 1 Flow chart of article selection for this systematic review of herpes simplex virus type 2 (HSV-2) epidemiology in the Middle East and North Africa, per PRISMA guidelines. 33,34 MENA, Middle East and North Africa; PRISMA, preferred reporting items for systematic reviews and meta-analyses.

| Pooled mean estimates for HSV-2 seroprevalence
Pooled mean HSV-2 seroprevalence stratified by sex among the different populations is provided in  A measure that assesses the magnitude of between-study variation that is due to actual differences in seroprevalence across studies, rather than sampling variation. c Prediction interval: A measure that estimates the distribution (95% interval) of true seroprevalence around the estimated mean. pooled mean across all populations. Forest plots of these metaanalyses confirmed substantial heterogeneity in seroprevalence measures (Supporting Information: Figure S1).
Pooled mean HSV-2 seroprevalence among subgroups of the general populations is provided in Virtually all meta-analyses showed evidence of heterogeneity (p-value < 0.01), as confirmed by wide prediction intervals (Tables 1   and 2). Heterogeneity was due to true differences in seroprevalence across studies rather than sampling variation (I² > 50%).

| Predictors of HSV-2 seroprevalence and between-study heterogeneity
Results of univariable and multivariable meta-regression analyses for HSV-2 seroprevalence are described in

| HSV-2 detection in genital herpes
Overall extracted proportion measures of HSV-2 detection in laboratory-confirmed genital herpes (n = 2) are listed in Supporting Information: Table S4. The proportion of HSV-2 detection in genital herpes among the four stratified measures had a median of 75.1% and a pooled mean of 73.8% (95% CI: 42.2%-95.9%; Table 4).
The meta-analysis showed evidence of high heterogeneity (p-value < 0.01, I² > 50%, and wide prediction interval). A forest plot of this meta-analysis confirmed substantial heterogeneity (Supporting Information: Figure S2).

| Quality assessments
Quality assessments of diagnostic assays used excluded 38 publications due to potential validity and/or reliability issues with the employed diagnostic method in relation to sensitivity, specificity, and importantly, potential cross-reactivity with HSV-1 antibodies 35,37,47-49 ( Figure 1). For validity, it is essential for any assay to be able to detect antibodies to the type-2 specific glycoprotein, G-2 (gG-2). 50  A measure that assesses the magnitude of between-study variation that is due to actual differences in seroprevalence across studies, rather than sampling variation. c Prediction interval: A measure that estimates the distribution (95% interval) of true seroprevalence around the estimated mean.  and may reflect differences in sexual networking across the region. 10,[60][61][62] This study demonstrated classic attributes in HSV-2 epidemiology that are also observed in other world regions. [26][27][28]30,[56][57][58][59] These include strong hierarchy in seroprevalence by level of sexual risk behavior (Tables 1 and 3), increasing seroprevalence with age, reflecting cumulative exposure to the infection (Table 2), and larger seroprevalence among women than men (Table 1). [26][27][28]30,[56][57][58][59] The latter supports a higher bio-anatomical susceptibility to HSV-2 acquisition among women. 63,64 Though this study clarified aspects of HSV-2 epidemiology in MENA, it also identified major gaps in evidence. Seroprevalence among FSWs and MSWs was surprisingly low, being only 20%, lower than in other world regions. 26 sampling method, and response rate, yet these factors did not appear to affect observed seroprevalence in the meta-regression analyses.
Unexpectedly, there was no evidence for a statistically significant association between seroprevalence and age, sex, and sampling method, but this finding likely reflected the insufficient number of seroprevalence measures to precisely quantify these associations.
Included studies exhibited heterogeneity, but nearly half of this heterogeneity was subsequently explained through the metaregression analyses in terms of effects of subregion and population type on seroprevalence.

| CONCLUSIONS
MENA has a lower HSV-2 seroprevalence than other world regions. Yet, about one in twenty adults is chronically infected with HSV-2 despite conservative prevailing sexual norms. Seroprevalence may also be T A B L E 4 Pooled mean proportion of HSV-2 detection in laboratory-confirmed genital herpes cases in the Middle East and North Africa. A measure that assesses the magnitude of between-study variation that is due to actual differences in proportion of HSV-2 virus detection across studies, rather than sampling variation. c Prediction interval: A measure that estimates the distribution (95% interval) of true proportion of HSV-2 virus detection around the estimated mean.