Introduction

Mpox, formerly named monkeypox, is a disease caused by the infection of a DNA virus from the Orthopoxvirus genus within the Poxviridae family [1], described as a cause of disease in humans in 1970 in the Democratic Republic of Congo [2]. It is considered a zoonosis, whose hosts are more probably rodents and mammals that inhabit tropical forests in Africa. However, it could be periodically transmitted to humans through direct contact via bodily fluids, skin lesions, or mucous membranes [3]. Humans could also acquire the virus from person to person through close contact with infectious skin lesions until the lesions crust over. These outbreaks have generally been self-limited, usually confined to the geographical area of central and west Africa, with limited secondary spread among humans [4]. Nonetheless, starting in May 2022, a very high number of cases were described worldwide, in countries where no documented cases had previously existed [5–7], and the World Health Organization (WHO) declared the mpox outbreak a global health emergency [8]. In this outbreak, the predominant pattern of transmission occurred during intimate contact in sexual intercourse, although any close contact can result in transmission [9,10]. Most of the cases have been described in gay, bisexual, or other men who have sex with men (GBMSM) and have presented with a self-limiting clinical course, characterized by different symptoms and signs such as skin lesions, fever, adenopathy, or proctitis [11–14].

Since the start of the 2022 outbreak, Spain has consistently been the third country in the world with the highest number of cases diagnosed, behind the United States and Brazil [5]. The peak incidence coincided with most festive gatherings and overcrowding in the country. Despite later cases decreased, outbreaks and isolated cases continued, so there may be some low-grade transmission in asymptomatic people, as has happened in the past with other viral infections that have led to epidemics [15]. The presence of asymptomatic cases could highlight the variability in how individuals respond to infection according to their immune status [16,17]. Despite the resurgence of the mpox outbreak in Africa in 2024, the hidden circulation of mpox is a question with no answer. Some studies have attempted to address this question; however, they have generally involved small sample sizes and heterogeneous methodologies [18]. New studies with larger cohorts are needed to evaluate the usefulness of screening measures at specific times and among certain at-risk population groups [19,20].

There is data available showing that co-infections of bacterial sexually transmitted infections and mpox have been common, and screening for these infections is recommended in patients with suspected mpox [21–23]. A substantial proportion of these bacterial infections is transmitted by asymptomatic individuals, especially at extragenital sites such as the pharynx and rectum [24]. Consequently, guidelines recommend systematic screening of asymptomatic sexually transmitted infections (STIs) in certain populations [25].

In this study, we determined the prevalence of mpox infection in asymptomatic individuals who attended a STI clinic and its referral hospital. Furthermore, we described the sociodemographic and clinical characteristics of this population, including the prevalence of bacterial STIs, and evaluated the prevalence of mpox vaccination and prior infection status.

Materials and methods

Results

Characteristics of participants

A total of 344 participants were included in the study. The power to detect a proportion of 6.5% of our sample was 99%. One participant who subsequently reported symptoms compatible with proctitis at the time of sampling was excluded from the study, leaving a total of 343 participants. The basal characteristics of the participants are shown in Table 1, including potential protective factors such as vaccination and previous mpox immunity due to an earlier mpox infection. Median time since the last dose of vaccine was 320 days (IQR 169-382). In those who had had the disease, the median number of days since diagnosis was 403 days (IQR 367-443).

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Table 1. Sociodemographic and clinical characteristics of all participants recruited for this study.

https://doi.org/10.1371/journal.pone.0335729.t001

Discussion

This observational, cross-sectional study described the prevalence of asymptomatic mpox in a population at risk for STIs in Spain, which is the third country in the world with the highest number of mpox cases diagnosed so far [5]. The study setting is an STI clinic and the follow-up unit of people living with HIV in the partner hospital. A sample of nearly 350 participants was recruited, making this one of the largest mpox screening studies published in the literature to date.

We aimed to identify mpox healthy carriers because these individuals can transmit pathogens without showing any symptoms, essentially acting as reservoirs of infection. Even in these subjects, pathogens can evolve or adapt, leading to the emergence of new strains or variants that may be more transmissible, virulent, or resistant to treatment, which may complicate disease management further.

The prevalence of mpox found in this study was 0.3%, lower than initially expected (6.5% in the study by Ferré et al. [26]) or identified in previous studies with fewer participants recruited (1% in a Swiss cohort of 201 participants [27]).

Additionally, the participant with positive pharyngeal and rectal samples developed mucocutaneous lesions 48 hours after inclusion in the study, indicating that he did not have an asymptomatic infection, which was the primary focus of this investigation. This information was consistent with previously published literature in which a search for mpox was performed exclusively in asymptomatic subjects, revealing an absence of mpox subclinical circulation [28]. Other studies that searched for subclinical mpox found higher prevalences (6.19% [29]; 1.78% [30]). However, these studies included people without symptoms associated with mpox, but they might have had other symptoms at the time of sampling. One Japanese study by Mizushima et al. with a large cohort (1,346 participants) reported an asymptomatic mpox prevalence of 0.29%, which is very similar to the findings of our study [31].

In this study, mpox PCR tests were positive at extragenital sites before the disease’s defining symptoms appeared, similar to one case in the Mizushima et al. study, where symptoms developed three days after testing. This suggests there is an asymptomatic period before symptoms, during which the individual could transmit the infection. It may be useful for reinforcing preventive behaviours in asymptomatic contacts of infected persons on days of the incubation period [32]. Nevertheless, our findings indicate that this does not seem to be the primary mechanism of transmission.

The absence of asymptomatic mpox in the studied population is not due to a selection bias towards a low-risk population; rather, the proportion of STIs is higher than that reported in other studies about STI screening in asymptomatic subjects (19.9% [33], 14.6% [34], and between 16.2% and 22.1% [35]).

Another possible explanation for the absence of cases in our series could be the low incidence of the disease in our region during the study period. In Spain, after the 2022 epidemic, mpox cases have appeared intermittently. However, during the study recruitment period, a total of 66 cases were reported, with 14 out of 66 (21.2%) diagnosed in our clinic during that time. This indicates that active transmission was occurring [36].

We have not identified any asymptomatic mpox carriers in our extensive series of individuals studied, despite having a high percentage of STIs as well as having other mpox cases diagnosed in our centre. This finding suggests that the healthy mpox carrier state is rare and does not explain the sporadic cases observed in the months following the 2022 outbreak. The emergence of new cases in populations with inadequate immune responses to this pathogen, driven by significant human mobility in a globalized world, could be the cause for the cases noted. This is consistent with previous data on genomic variability between cases diagnosed in the same country [37]. Therefore, it is advisable to strengthen preventive measures and vaccination efforts among potentially vulnerable people.

This study presented several limitations. First, the participants were individuals who sought screening tests through the National Health System (NHS), which may introduce selection bias concerning those who do not usually utilize the NHS and could be more susceptible to STIs. Second, the study was conducted in a centre specializing in infectious diseases, which may also introduce a selection bias regarding persons evaluated at non-specialized centres. Consequently, the population included in the study consisted only of a specific group of subjects and might not represent the entire population at risk. Another limitation to consider is that only a single sample was collected from each participant, so infections that may have developed later could not be excluded, given the incubation period of up to 21 days. Moreover, this study was conducted under specific epidemiological circumstances, with a low incidence of cases, so the results may not be extrapolated to other epidemiological settings with a higher incidence rate. Finally, having only one case prevented us from analyzing associated factors, representing a study limitation.

The strengths of our study include a large number of patients, making it one of the most extensive series of mpox screening in asymptomatic subjects reported to date, along with comprehensive clinical information collected.

Conclusions

It is suggested that mpox Clade IIb may manifest with no symptoms or only mild symptoms [38,39]; however, the prevalence of asymptomatic mpox in the population studied, considered at risk for STIs, was almost null. Testing for various asymptomatic STIs is recommended in many situations, while testing for asymptomatic mpox may differ depending on location and population. There is insufficient evidence to confirm that ongoing cases of mpox in Spain are attributable to asymptomatic carriers of the infection.

Acknowledgments

We greatly appreciate all individuals who participated in this study.