Presymptomatic viral shedding in high‐risk mpox contacts: A prospective cohort study

The risk of infection after exposure to clade IIb mpox virus (MPXV) is unknown, and potential presymptomatic shedding of MPXV remains to be demonstrated. High‐risk contacts of mpox patients were followed‐up in a prospective longitudinal cohort study. Individuals reporting sexual contact, >15 min skin‐to‐skin contact, or living in the same household with an mpox patient were recruited in a sexual health clinic in Antwerp, Belgium. Participants kept a symptom diary, performed daily self‐sampling (anorectal, genital, and saliva), and presented for weekly clinic visits for physical examination and sampling (blood and oropharyngeal). Samples were tested for MPXV by PCR. Between June 24 and July 31, 2022, 25 contacts were included, of which 12/18 (66.0%) sexual and 1/7 (14.0%) nonsexual contacts showed evidence of infection by MPXV‐PCR. Six cases had typical mpox symptoms. Viral DNA was detected as early as 4 days before symptom onset in 5 of them. In 3 of these cases, replication‐competent virus was demonstrated in the presymptomatic phase. These findings confirm the existence of presymptomatic shedding of replication‐competent MPXV and emphasize the high risk of transmission during sexual contact. Sexual contacts of mpox cases should abstain from sex during the incubation period, irrespective of symptoms.


| INTRODUCTION
Since May 2022, an outbreak of mpox (formerly monkeypox) has caused more than 80 000 laboratory-confirmed cases across the world, primarily among men who have sex with men (MSM). 1 This epidemic is caused predominantly by variant B.1 of the subclade IIb of monkeypox virus (MPXV), and, in contrast to previous outbreaks, is driven uniquely by human-to-human transmission, especially through sexual contact. 2 Highest MPXV DNA loads are demonstrated in skin lesion and anorectal samples. 3,4 In addition, the clinical presentation in this global outbreak differs from what was commonly reported before 2022. Lesions often predominate or first appear at the presumed site of inoculation and frequently involve mucosal membranes, resulting in proctitis, urogenital symptoms, or tonsillitis. 2,5 Based on data from previous outbreaks, most guidelines, including those issued by WHO, ECDC, and US CDC, considered mpox patients infectious from the start of symptoms until the complete healing of skin lesions. 6,7 For that reason, public health messaging has mainly focused on awareness of symptoms, early diagnosis, and isolation of symptomatic cases.
However, it was recently demonstrated that asymptomatic MPXV infections could play an important role in transmission. [8][9][10][11] Furthermore, recent epidemiological data suggest that presymptomatic transmission also occurs and could be responsible for about half of all infections. 12 Presymptomatic transmission can take place when viral shedding precedes clinical symptoms and often has a major impact on epidemic dynamics, as observed during the COVID-19 outbreak. 13 Nevertheless, presymptomatic shedding of MPXV, the body sites from which it may occur and its timing in relation to the onset of symptoms remain elusive. 14 To study the risk of infection after exposure to MPXV and the natural history of the early phase of MPXV infection, we performed a detailed follow-up of high-risk contacts of clade IIb MPXV infected patients. Here, we describe their clinical and virological characteristics from exposure until potential diagnosis.

| Study design and participants
In this study, we prospectively followed-up individuals who had highrisk contact with a confirmed mpox patient. Participants were recruited in two ways: either by referral through their index cases, or when they presented for postexposure vaccination (PEV). Highrisk contact was defined as either sexual contact (exposure of mucosal membranes through receptive or insertive penetrative or oral sex, irrespective of exposure time), prolonged (>15 min) skin-toskin contact with an mpox patient with skin lesions, or living in the same household as an mpox patient. Adult individuals were included in this study if their contact occurred in the 21 days before recruitment and if they provided written informed consent for study participation.
Study participants attended a predefined schedule of clinic visits, including one baseline visit and weekly follow-up visits ( Figure 1A). At baseline, we recorded medical history including smallpox vaccination status, and date and type of contact with the index case. At every visit, symptoms were recorded through a standardized questionnaire, clinical signs of mpox were assessed by a thorough physical examination, and the following samples were collected: blood, saliva, oropharyngeal swabs, genital swabs (skin swab from the coronal sulcus for men or vaginal swab for women), anorectal swabs, and swabs from skin lesions if applicable.
Between study visits, participants were asked to keep a symptom diary and to perform daily self-sampling of anorectal swabs, genital swabs, and saliva at home. [15][16][17] Study follow-up was ceased maximum 21 ± 2 days after inclusion or as soon as any sample was MPXV-PCR positive with C t -value <34 in a participant with typical mpox symptoms which were defined as characteristic mpox skin lesions, proctitis, urethritis, or tonsillitis. Confirmed mpox cases were further managed and followed up in routine clinical care.

| Sampling and sample handling
Blood (BD Vacutainer ® ; BD Benelux NV), saliva (15 mL Safe-Lock Tubes; Eppendorf Belgium NV-SA) and all study swabs (Eswab; Copan Diagnostics) were collected during clinic visits by a trained physician or nurse and were processed immediately. Home-based samples were collected with the same type of swabs and tubes prelabelled for this purpose. Home-based samples were packaged in appropriate packaging material for storage in the participant's refrigerator and were brought to the clinic at the next study visit.

| Laboratory procedures
The MPXV-PCR used in this study was an in-house PCR targeting the MPXV-TNF receptor gene carried out on the Applied Biosystems QuantStudio PCR system, as described previously. 8,18 Viral culture was performed as described previously, on a subset of MPXV-PCR positive samples with cycle threshold (C t ) value <30, from participants with presymptomatic viral shedding. 8 Orthopoxvirus serology was performed at the end of follow-up for all participants and at baseline for participants with positive end-offollow-up serology (IgG titer ≥1:20). An in-house assay detecting antiorthopoxvirus IgG at the Bundeswehr Institute of Microbiology was used. 19

| Cut-offs and definitions
Considering the reports of false positive MPXV-PCR results in patients with a low clinical suspicion of MPX, 20 we defined two C t -value cutoffs: one highly specific (C t 34) and another highly sensitive (C t 37). The latter  Figure 1). A cutoff C t -value of 37 was chosen to provide an additional margin of 2 C t -values to preserve adequate specificity.
Samples with C t -values between 34 and 37 underwent confirmation testing by repeating the MPXV-PCR and a PCR melting curve analysis.
Unconfirmed results were reported as negative.
Based on the two MPXV-PCR C t -value cutoffs, the infection status of individual participants was defined as one of three outcomes: definitely infected, possibly infected, or uninfected (Box 1).

| RESULTS
Recruitment started June 24, 2022 and ended July 31, 2022, due to the waning mpox epidemic. During this period, 25 high-risk contacts of 23 confirmed mpox cases were included. All participants except 1 (96.0%) self-identified as MSM; the median age was 43 years (IQR: 36-51, Table 1). Eighteen (72.0%) participants reported having had sexual contact with an index case. Seven (28.0%) were nonsexual high-risk contacts: 5 were household contacts, and 2 had prolonged skin-to-skin contact with an mpox confirmed case. The median time between the last exposure and inclusion was 4 (IQR: 3-7) days. Five participants received PEV, and 6 reported being vaccinated against smallpox during childhood. Overall, participants were followed-up for a median of 16

| DISCUSSION
Our extensive follow-up of high-risk contacts provides unique and detailed insights into the early stages of mpox disease. First, we detected presymptomatic MPXV DNA and even replicationcompetent virus in 5 out of 6 participants with typical mpox symptoms, as early as 4 days before symptom onset. In reality, presymptomatic shedding might start even earlier, as 7 cases in our study were already PCR-positive at inclusion. The existence of presymptomatic transmission was suggested by an epidemiological study of surveillance and contact tracing data in the United Kingdom, which found that the median serial interval in 79 case-contact pairs was shorter than the median incubation period of 54 cases in the data set and that exposure of the contact took place during the presymptomatic phase of the index case in 10 out of 13 casecontact pairs. 12 We now provide biological evidence of pre-

ACKNOWLEDGMENTS
We thank our participants for their willing contribution, the study staff and all clinical and laboratory personnel that had part in sample T A B L E 2 Clinical presentation and viral DNA detection by outcome. L.). The funder had no role in the collection, analysis, and interpretation of data, nor in the writing of the report or the decision to submit the manuscript for publication. All authors vouch for the accuracy and completeness of the data.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Deidentified participant data collected for the study will be made available from the corresponding authors on reasonable request.

ETHICS STATEMENT
The study was conducted in accordance with the International