Epidemiological, clinical and virological characteristics of four cases of monkeypox support transmission through sexual contact, Italy, May 2022

<jats:p>Since May 2022, an outbreak of monkeypox has been ongoing in non-endemic countries. We report four cases in Italy in young adult men reporting condomless sexual intercourse. The patients are in good clinical condition with no need for specific antiviral drugs. Biological samples from seminal fluid were positive for monkeypox viral DNA. For many other viruses found in semen there is no evidence of sexual transmission. The possibility of sexual transmission of monkeypox virus needs to be investigated.</jats:p>

section_content: Since May 2022, an outbreak of monkeypox has been ongoing in non-endemic countries. We report four cases in Italy in young adult men reporting condomless sexual intercourse. The patients are in good clinical condition with no need for specific antiviral drugs. Biological samples from seminal fluid were positive for monkeypox viral DNA. For many other viruses found in semen there is no evidence of sexual transmission. The possibility of sexual transmission of monkeypox virus needs to be investigated. 

Human monkeypox virus (MPXV) is a doublestranded DNA virus of the Orthopoxvirus genus of the Poxviridae family. Two genetic MPXV clades have been characterised: West African and Central African. Outside of Africa, the first cases of monkeypox were reported in 2003 in the United States (US) [1] when an outbreak occurred following the importation of rodents from Africa. Over the past five decades, monkeypox outbreaks have been reported in 10 African countries and four countries outside of Africa [2]. To date, in the ongoing outbreak in 2022, 118 cases of monkeypox have been reported in non-endemic countries [3]. We report four monkeypox cases observed in Italy between 17 and 22 May 2022.

section_content: All four monkeypox patients were young adult men who have sex with men (MSM). Two of them (Patients 1 and 3) had an HIV infection and received effective antiretroviral therapy (ART); the other two (Patients 2 and 4) were on antiretroviral pre-exposure prophylaxis (PreP). 

All patients travelled in the first 2 weeks of May 2022: three patients participated in a mass gathering event in Gran Canary island (GCI) and one travelled for sex work. During the travel, they had condomless sexual intercourse with different male partners. All four patients had a history of sexually transmitted infections (STIs) (Table 1 ). 

All four monkeypox patients were in good clinical conditions and were admitted to two different hospitals in central Italy, in combined droplet and contact isolation measures plus filter face piece-2 (FFP2) for clinical care management. 

The first case was a male in his 30s who referred to our centre after accessing an emergency department where an MPXV infection was suspected. During travel made in mid-May, he had been treated with oral ciprofloxacin and acyclovir and one single dose of benzylpenicillin for the appearance of skin lesions. At admission, multiple asynchronous deep-seated and well-circumscribed lesions with central umbilication were present on the genital area, with inguinal lymphadenopathy; a single lesion was present on the anterior and posterior thorax and on the left calf. 

Patient 2 was a male in his 30s, taking daily-PreP. He was admitted for fever and asthenia starting mid-May. Three days later, perianal lesions appeared as raised, itchy papules secreting serous, with concomitant painful inguinal lymphadenopathy. Multiple anal lesions appeared over the next 3 days, followed by few single lesions with different timing on the skin of the back, legs and the sole of one foot. Patient 3 was a male in his 30s. He was admitted for a 2-day history of fever, followed by the appearance of clustered itchy papular lesions in the anal region and single lesions on head, thorax, legs, arms, hand and penis. He reported smallpox vaccination during childhood, 30 years earlier. 

Patient 4 was a male in his 30s, taking event-driven PreP. After a 2-day history of myalgia, vesicular-papular genital lesions appeared, followed by further skin lesions that appeared 6 days later in the suprapubic area and chest. 

In all patients, skin lesions progressed with an asynchronous evolution (Figure 1 ). 

Samples obtained from skin, genital and anal lesions, serum, plasma, seminal fluid, faeces, and nasopharynx were all positive for MPXV DNA in real-time PCR (Table 2 ). The patients did not experience any systemic symptoms during clinical observation, and they recovered spontaneously, without any specific antiviral therapy. In Patient 2 only, anti-inflammatory and antihistaminic drugs were used for perianal pain and general itch.
section_name: Case description

section_content: Viral DNA was extracted by Qiamp Viral RNA mini kit (Qiagen), and two real-time PCRs were used to assess the presence of MPXV DNA. Real-Star Orthopoxvirus PCR Kit (Altona Diagnostics GmbH) was used as screening PCR. This method recognises a region common to all Orthopoxviruses without distinction of species. The second PCR (G2R_G assay) published by Li et al. targets the tumour necrosis factor (TNF) receptor gene and was used as confirmatory PCR [4]. The Rotor Gene Q (Qiagen) platform was used to run both assays. We also measured viral quantification cycle (Cq) in positive samples.
section_name: Laboratory diagnosis and virus identification

section_content: We performed Sanger sequencing of the amplicon obtained from a third end point PCR (in-house method), targeting the complete gene for the viral haemagglutinin (HA) to identify which of the two known MPXV clades, the virus in the patient samples belonged to. We obtained the HA gene sequence only for the first three patients. The sequence of the fourth patient is in progress. 

For the first case, next generation sequencing was performed on an Ion Torrent GSS5 platform (ThermoFisher), using a metagenomic approach starting from 100 ng extracted DNA. The library was prepared using the Ion Xpress Plus Fragment Library Kit (Thermofisher). After quality filter, the reads were de-novo assembled using SPAdes [5]. Major contigs were selected and aligned with BWA v. 0. 7. 12 [6], to recently published MPXV sequences, and merged in a unique contig. The final length resulted in 190,280 nt, with a mean coverage of 159 reads. At the end, the assembled genome of MPXV-INMI-Pt1 was annotated, transferring the annotation from the sequence MPXV-UK_P1 (MT903343. We performed phylogenetic analyses using INMI sequences (HA and full genome) (Figure 2 ) and sequences related to the HA gene of different Orthopoxviruses retrieved from GenBank (Belgium, Germany, Portugal and the United States), including MPXV sequences belonging to the two clades West Africa and Central Africa. The phylogenetic trees were built using the maximum likelihood method and evolutionary distances were computed using the Tamura 3-parameter model for single gene tree and Kimura three parameters for the full genome analysis. Bootstraps were generated using 1,000 replicates.
section_name: Sequencing, bioinformatic and phylogenetic analysis

section_content: The clinical description of, to our best knowledge, the first four monkeypox cases detected in Italy warrants closer investigation for several reasons. Firstly, it has been suggested that the MPXV in the currently ongoing outbreak is transmitted from human to human [7], even though this route of transmission has been reported as poorly efficient in previous outbreaks caused by the West African clade [8]. Secondly, the clinical picture seems to be different from the available information in the literature because the skin lesions in our patients were asynchronous, ranging from single or clustered spot to umbilicated papule with progressive central ulceration and, finally, to scabs. Lesions were mostly located in genital and perianal sites. Finally, the individuals affected by monkeypox are mostly men [9]. In endemic areas, most of the cases were observed in men [10], probably related to hunting practices, whereas in the current outbreak, most individuals are MSM, people with multiple sexual partners or people who practice condomless sex. 

Sexual behaviour of the cases in the present series and the initial appearance of lesions mostly in the anal and genital areas all suggest that close contact during sexual intercourse was important for virus transmission. The seminal fluid obtained from three monkeypox patients at the time closest (5-7 days) to symptoms onset, was found to be positive for MPXV DNA in all four patients, with a Cq range from 27 to 30. The correlation between Cq value and infectious viral load in MPXV infections is not yet known, but the range of Cq values we measured in semen makes viral isolation unlikely. However, detection of viral DNA in the three seminal fluid samples we analysed, excludes the hypothesis of biological sample contamination. Although these findings cannot be considered definitive evidence of
section_name: Discussion

section_content: Pictures from 2 patients with the horizontal disease progression from the same patient for each site, monkeypox cases, Italy, May 2022

Lesions appeared 1-3 days after systemic symptoms, clustered (anal area 1A) or isolated (skin or penis 1B-1C). They typically began as raised, itchy papules secreting serous, with a central umbilication; over days, the central umbilication widened until the lesion opened and the scab formed about 2 weeks after symptom onset. 

infectivity, they demonstrate viral shedding whose efficiency in terms of transmission cannot be ruled out. It has been observed that it is likely that MPXV can be transmitted through substances of human origin [11, 12] and it needs to be emphasised that the Cq values in semen of our patients were in the range of those measured in their nasopharyngeal swabs. 

We need to consider that many other viruses causing viraemia can be found in semen [13, 14] with no direct evidence of sexual transmission. Indeed, viral seeding in the male reproductive tract can frequently occur in the context of viraemia, as blood-testis barriers are imperfect for viruses, especially in the presence of systemic or local inflammation [15]. Since testes are an immunologically privileged sanctuary site, the virus may persist even though unable to replicate within the reproductive tract.
section_name: Figure 1

section_content: The characteristics of these four patients reflect those described in other European countries in the current outbreak. The phylogenetic characteristics of the virus could support the hypothesis of a recent introduction of the West African clade of the MPXV into the community in non-endemic countries. Moreover, the characteristics of the population involved, as well as reported exposure to multiple, condomless sexual contacts, suggest that human-to-human transmission through close physical contact in sexual networks plays a key role in the current outbreak. Further studies are needed to assess presence, persistence and contagiousness of MPXV in different body fluids.
section_name: Conclusion

section_name: Acknowledgements

section_content: The authors gratefully acknowledge the patients for providing consent to have this study published and the nurse staff for their contribution in collecting the samples.
section_name: Acknowledgements

section_name: Funding statement

section_content: This work was supported by Line 1 and 2 of the Ricerca Corrente, funded by the Italian Ministry of Health.
section_name: Funding statement

section_name: Ethical statement

section_content: Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.
section_name: Ethical statement

section_name: Conflict of interest

section_name: Authors' contributions