Monkeypox viral detection in semen specimens of confirmed cases: A systematic review and meta‐analysis

The current literature shows increasing concerns about potential seminal transmission of monkeypox virus (MPXV). Accordingly, we aimed to understand better the potential presence of MPXV in the seminal fluids and others specimens obtained from MPX cases. On June 26, 2022, a systematic search of the literature was conducted to find articles that examine the presence of MPXV in the seminal fluid of confirmed cases. The search was updated once on August 12 and another on October 12, 2022, to include newly published articles. The prevalence of MPXV DNA presence in the seminal fluid and other specimens was pooled in a meta‐analysis (from studies with sample size > 5 to reduce overestimation) and results were presented as effect sizes (ES) and their corresponding 95% confidence intervals (CI). Nine articles were included. Only five studies were eligible for a meta‐analysis, and the pooled prevalence of MPXV DNA in semen specimens was 72.4% (95% CI: 55.7%−84.5%) among 115 patients. The positive rate of MPXV viral polymerase chain reaction (PCR) was higher among skin samples (89%; 95% CI: 78.2%−94.8%; N = 62; studies = 2), followed by anogenital/rectal samples (74.3%; 95% CI: 60.4%−84.5%; N = 54; studies = 2). On the other hand, the positivity rate was lower in nasopharyngeal (62.4%; 95% CI: 20.4%−91.5%; N = 587; studies = 3), urine (21.1%; 95% CI: 4.3%−61.1%; N = 617; studies = 4), and blood/plasma (14.3%; 95% CI: 11.3%−18.1%; N = 609; studies = 3) samples. Besides, MPXV can be detected in semen early from Day 1 and up to 19 days after symptoms onset. Finally, two articles investigated the infectivity of MPXV particles detected in seminal specimens by testing their replication competence. Culturing MPXV was successful in two out of four patients included in these studies. MPXV is highly prevalent in seminal specimens of MPX cases, further corroborating the role of sexual transmission of the disease. However, further evidence is still needed to shed more light on the replication competence of these particles.

Evidence from previous MPXV outbreaks shows that the disease is characterized by the widespread of characteristic rash with multiple lesions affecting different body parts, including the legs, arms, and face, and less commonly, in the soles, palms, and genitalia. [2][3][4] On the other hand, evidence from the current outbreak shows that the rash shows an atypical pattern to the previously reported one, spreading mainly on the genital and perianal regions. 5,6 Such events have been concerning since different reports indicated that most MPXV cases are individuals that identify themselves as men who have sex with men (MSM). [7][8][9] Therefore, the widespread of rashes and MPXV lesions in the genital and perianal regions might suggest viral transmission in this population. The transmission of MPXV has been recorded in different ways, including close contact with MPXV cases (mainly when contacting an MPXV active lesion), contacting animals directly or infected materials, and prolonged contact with infected individuals by droplet transmission. [9][10][11][12] Moreover, there have been concerns about the potential viral transmission among seminal fluids since most cases were reported among MSM. However, no cumulative evidence was found in the literature to indicate this hypothesis, and data is scattered among single reports with insufficient highlights regarding positive MPXV DNA in seminal specimens. 6 2022. Additionally, we adopted a manual search strategy to retrieve any relevant article that was missed during the electronic database search. This strategy was conducted by: (1) screening the references of included studies, (2) searching "similar articles" of finally included studies on PubMed, and (3) conducting a random search on Google engine with the keywords "monkeypox" + "semen." No restrictions regarding year, country, or the language of publication were applied.

| Screening and study selection
After completing the search strategy, a senior member collected all the search results into a single Endnote library, which was used to remove all duplicates among the various databases considered in the search strategy. An excel sheet was then drafted with article reference, title, abstract, DOI, URL, journal, and link to full-text to start the screening process, which was done in two steps: title/ abstract and full-text screening. These steps were done in a blind approach by at least two reviewers who discussed their differences to reach a final decision under the supervision of the senior authors.
Finally, all included studies were grouped and prepared for data extraction.

| Extraction and quality assessment
Data extraction was conducted in a similar approach to that of screening. A senior author drafted a pilot sheet on Excel that included three main tabs, a baseline characteristics tab, another one for intended outcomes, and the third one for quality assessment. Extracted baseline characteristics included reference, country, study design, sample size, age, gender, symptoms, travel history, previous smallpox vaccination, sexual transmission (through MSM or not), and diagnostic method. The outcome tab included events of sexually transmitted infections, lesion site, and diagnostic sample (including semen, urine, blood/plasma, skin, genital/anal, fecal, and oral/nasopharyngeal). Events of positive MPXV DNA, time of onset of symptoms, and viral loads were extracted for each of these samples.
The quality of included case series was assessed using the National Institute of Health (NIH) quality assessment tool. The tool assesses the quality of each study at the level of seven domains/ questions. Each domain, as well as the overall quality, is given a rating of good, fair, or poor. Two reviewers assessed the quality of included studies, and any discrepancies among them were solved by consulting one of the senior authors.

| Data synthesis
The quantitative synthesis was conducted using STATA Software (Version 17). The overall prevalence of positivity rate of MPXV viral PCR in different specimens was estimated using the metaprop command. The random-effects and fixed-effects models were used according to the presence or absence of heterogeneity, respectively. Heterogeneity was measured using the I 2 statistic, where a value of >50% or a p-value of <0.05 indicates significant heterogeneity. The exact cimethod was used to pool the ES along with its 95% confidence interval (CI). The assessment of publication bias was not feasible due to the lack of a sufficient number of studies (10 studies).

| Search results
The results of the initial and updated database searches are illustrated in Figure 1. The initial database search yielded 573 articles, out of which 70 duplicates were identified and excluded through EndNote Software. The title and abstracts of 503 articles were screened, resulting in 13 articles eligible for full-text screening.
Three studies were included with the initial database search, 8,14,17 five were added with the updated search on August 12, 2022, 6,13,15,18,19 one was added on October 12, 2022, 20 and none was added through manual search. We furtherly found that Angelo et al. 21 and Hornuss et al. 22 reported positive seminal samples in their cohort. However, the exact rate of these samples was not provided.
So, we excluded them from our study. Finally, nine studies were included in the qualitative synthesis and five studies were included in the quantitative synthesis.

| Baseline characteristics
The baseline characteristics of included studies are presented in Table 1. Six studies were case series 6,8,14,15,18,20 and three were case reports. 13,17,19 The number of included patients in each study ranged from one 13,17,19 to as high as 528 MPX-confirmed cases. 6

| Quality assessment
The quality of included studies across different domains is presented in Table 3. Overall, six case series were assessed, 6,8,14,15,18,20 all of which had good quality.

| The positivity rate of MPXV DNA in seminal specimens
Out of nine studies, five were eligible for a meta-analysis (examined at least 5 semen specimens of MPXV cases). 6,13,15,18 The remaining studies were not analyzed because of their design (case reports and series < 3 cases), as they all reported a 100% positivity rate. 8,13,14,17 Among included studies, the individual positivity rate of MPXV DNA in semen specimens ranged from 54.17% 20 to as high as 90.63%. 6 The meta-analysis included a total number of 115 patients (82 showed positivity), revealing an overall positivity rate of MPXV in seminal specimens of 72.4% (95% CI: 55.7−84.5%; I 2 = 60.62%) (Figure 2). Of note, the analyzed population is not reflective of the overall population included in these studies (Table 1) since only a minority of these populations were examined and analyzed.

| Replication competence of MPXV detected in seminal specimens
Only two included studies, which included four MPX patients reported this outcome. Lapa et al. 13    T A B L E 3 The quality of included studies using the NIH tool for case series Note: Q1: Was the study question or objective clearly stated? Q2: Was the study population clearly and fully described, including a case definition? Q3: Were the cases consecutive? Q4: Were the subjects comparable? Q5: Was the intervention clearly described? Q6: Were the outcome measures clearly defined, valid, reliable, and implemented consistently across all study participants? Q7: Was the length of follow-up adequate? Q8: Were the statistical methods well-described? Q9: Were the results well-described? Moreover, the prevalence rates of MPXV viral PCR in saliva 100%, and feces were 100% and 66.66%, respectively. However, we could not conduct a meta-analysis because reported data were limited to an individual report per specimen site (Table 5).

| DISCUSSION
The main aim of the current study is to provide more insight into