Monkeypox virus isolation from a semen sample collected in the early phase of infection in a patient with prolonged seminal viral shedding

The unexpected increase in human monkeypox cases in non-endemic countries that began in May, 2022, is raising concerns of a novel global infectious threat. Since the first human case in 1970 in the Democratic Republic of the Congo, the virus has become endemic in several countries in central and western Africa.1Bunge EM Hoet B Chen L et al.The changing epidemiology of human monkeypox-A potential threat? A systematic review.PLoS Negl Trop Dis. 2022; 16e0010141Crossref PubMed Scopus (116) Google Scholar Imported cases have been sporadically reported outside Africa (in England, the USA, Singapore, and Israel), with the majority of cases associated with travellers returning from endemic countries, or due to nosocomial contact or contact with infected imported rodents.2Vaughan A Aarons E Astbury J et al.Two cases of monkeypox imported to the United Kingdom, September 2018.Euro Surveill. 2018; 231800509Crossref PubMed Scopus (80) Google Scholar, 3Reed KD Melski JW Graham MB et al.The detection of monkeypox in humans in the western hemisphere.N Engl J Med. 2004; 350: 342-350Crossref PubMed Scopus (414) Google Scholar As of July 22, 2022, 16 016 laboratory-confirmed monkeypox cases have been reported from 75 countries worldwide, and the WHO Director-General has declared the escalating global monkeypox outbreak to be a public health emergency of international concern.4WHO2022 monkeypox outbreak: global trends.https://worldhealthorg.shinyapps.io/mpx_global/Date: July 28, 2022Date accessed: July 28, 2022Google Scholar The vast majority of cases have been reported in Europe and other non-endemic countries, mostly diagnosed in young men, self-identifying as men who have sex with men (MSM). Monkeypox virus transmission might occur through close contact of mucosa or non-intact skin with infectious material, or large respiratory droplets during prolonged face-to-face contact.5European Centre for Disease Prevention and ControlRisk assessment: monkeypox multi-country outbreak.https://www.ecdc.europa.eu/en/publications-data/risk-assessment-monkeypox-multi-country-outbreakDate: May 23, 2022Date accessed: July 22, 2022Google Scholar Whether monkeypox virus can be sexually transmitted via genital fluids remains under investigation. Monkeypox virus transmission during sexual intercourse has been documented in the UK in two men with no travel history to endemic countries and evidenced by the temporal association of symptoms with sexual contact and the location of primary lesion sites matching those of sexual contact.6Heskin J Belfield A Milne C et al.Transmission of monkeypox virus through sexual contact—a novel route of infection.J Infect. 2022; (published online June 1.)https://doi.org/10.1016/j.jinf.2022.05.028Summary Full Text Full Text PDF Google Scholar Viral DNA detection in semen samples has been reported in three cases in Italy and subsequently in two patients with monkeypox in Germany.7Antinori A Mazzotta V Vita S et al.Epidemiological, clinical and virological characteristics of four cases of monkeypox support transmission through sexual contact, Italy, May 2022.Euro Surveill. 2022; 272200421Crossref PubMed Scopus (36) Google Scholar, 8Noe S Zange S Seilmaier M et al.Clinical and virological features of first human monkeypox cases in Germany.Infection. 2022; (published online July 11.)https://doi.org/10.1007/s15010-022-01874-zCrossref Scopus (7) Google Scholar Furthermore, monkeypox DNA was detected in the seminal fluid of 29 (91%) of 32 people affected by monkeypox in a large case series on the 2022 global outbreak.9Thornhill JP Barkati S Walmsley S et al.Monkeypox virus infection in humans across 16 countries—April–June 2022.N Engl J Med. 2022; (published online July 21.)https://doi.org/10.1056/NEJMoa2207323Crossref PubMed Google Scholar However, to date, no evidence is available on the infectiousness of monkeypox virus in semen. Therefore, we investigated viral shedding in longitudinal semen samples collected 5–19 days after symptom onset from one confirmed monkeypox virus case diagnosed at the National Institute for Infectious Diseases ‘Lazzaro Spallanzani’ (Rome, Italy; appendix p 1). The patient was a 39-year-old man, who travelled in Austria during the first 2 weeks of May, 2022. He self-identified as an MSM and sex worker and reported condomless sexual intercourse with several male partners during the previous month. The patient was HIV-infected, treated with dolutegravir and lamivudine, with viral suppression and immune recovery, and reported a history of sexually transmitted infections. He was admitted to the hospital 5 days after symptom onset. His symptoms included fever, followed by the appearance of clustered itchy papular lesions in the anal region and single lesions on the head, thorax, legs, arms, hand, and penis. The patient reported one dose of smallpox vaccination during childhood, more than 30 years earlier. He did not receive any current treatment for monkeypox virus infection. Monkeypox virus infection was confirmed by real-time PCR on a skin lesion swab (quantification cycle [Cq] 18·9) and scab (Cq 21·4) collected on day 5 after symptom onset. The virus was successfully isolated in vitro from a swab of a skin lesion on the head (appendix p 2). Plasma, urine, and semen samples were longitudinally collected to monitor the duration of viral shedding (table). Monkeypox virus DNA was detected in plasma collected on day 8 after symptom onset only. Urine samples were negative. Monkeypox virus DNA was detected in all semen samples tested during the period of observation (Cq range 27·8–40·6). Semen collected on day 6 after symptom onset was inoculated in Vero E6 cells (ATCC; Manassas VA, USA). Clear cytopathic effect was observed 48 h after the inoculum and monkeypox virus replication was confirmed by real-time PCR on DNA purified from cell growth medium collected after 48 h, 72 h, and 96 h. Notably, on day 6 after symptom onset, anti-monkeypox virus IgG antibodies (1:80) were detected by immunofluorescence assay in the patient's serum but IgM was not detected. A serum sample on day 8 after symptom onset was positive for both IgG (1:320) and IgM (1:20). This early detection of specific IgG might be related to a possible cross-reaction from childhood smallpox vaccination. However, the fast generation of IgG compared with IgM might not exclude unconventional antibody kinetics, or the presence of low IgM levels during the acute phase of infection, with a titre near the detection limit of the method. Overall, our findings support that prolonged shedding of monkeypox virus DNA can occur in the semen of infected patients for weeks after symptoms onset, and show that semen collected in the acute phase of infection (day 6 after symptom onset) might contain a replication-competent virus and represent a potential source of infection. Viral particles in semen might derive from passive diffusion from blood, urine, or genital lesions (eg, exfoliated epithelial cells) or local genital replication.10Le Tortorec A Matusali G Mahé D et al.From ancient to emerging infections: the odyssey of viruses in the male genital tract.Physiol Rev. 2020; 100: 1349-1414Crossref PubMed Scopus (37) Google Scholar Whether infectious monkeypox virus found in semen could be associated with seminal cells or if viral replication occurs in the genital tract remains to be established. In the case discussed herein, the isolation of live replication-competent monkeypox virus from semen, and prolonged viral DNA shedding, even at low viral copies, might hint at a possible genital reservoir. No monkeypox virus DNA was detected in urine and blood samples, suggesting absence of semen cross-contamination from other potential sources. Our patient was an HIV-infected, viro-immunological responder, thus we cannot entirely exclude the possibility of an effect of HIV-associated chronic immune dysregulation on prolonged monkeypox virus shedding in semen. At the time of writing, we detected monkeypox virus DNA in semen samples from 11 (79%) of 14 patients, and live and replication-competent virus was isolated from the positive seminal fluid (Cq 22·7) of a second patient with HIV.TableTimeline of monkeypox virus DNA detection in plasma, urine, and semen samples with increasing days from symptom onsetDay 5Day 6Day 7Day 8Day 9Day 10Day 11Day 13Day 14Day 15Day 16Day 17Day 19PlasmaNANANAPositive (34·5)NANegativeNANegativeNANegativeNegativeNegativeNegativeUrineNANANegativeNANegativeNANegativeNegativeNANegativeNANANegativeSemenPositive (28·0)Positive (29·3)Positive (27·8)NANANANANAPositive (34·3)Positive (35·6)NAPositive (38·7)Positive (40·6)Rash or skin lesionPositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositiveNegativeQuantification cycle values are indicated in brackets after positive results. The cutoff cycle threshold is 45, thresholds of 42 or higher are retested for confirmation. Negative indicates no detection of monkeypox virus DNA or absence of rash or skin lesions. Positive indicates detection of monkeypox virus DNA or presence of rash or skin lesions. NA=not available. Open table in a new tab Quantification cycle values are indicated in brackets after positive results. The cutoff cycle threshold is 45, thresholds of 42 or higher are retested for confirmation. Negative indicates no detection of monkeypox virus DNA or absence of rash or skin lesions. Positive indicates detection of monkeypox virus DNA or presence of rash or skin lesions. NA=not available. In our opinion, the case discussed herein supports that transmission of monkeypox virus during sexual activity might be a viable and recognised route, especially in the current 2022 outbreak of disease. Further studies on the viral tropism for the genital tract and data on the frequency of virus detection and duration of replicative monkeypox virus shedding in the seminal fluid, also in patients who do not have HIV, are crucial to better understand the viral pathogenesis and the potential role of semen-driven transmission in the spreading of monkeypox infection and disease burden. We declare no competing interests. DL and FC contributed equally to this work. AA and FM contributed equally to this work. The patient provided written informed consent to the use of their data and clinical samples for the research purposes of the present study, for which we thank them. We thank the nursing and laboratory staff for their crucial contributions in collecting and processing samples. This work was supported by the Italian Ministry of Health (Ricerca Corrente—line 1 and 2) and the European Commission Horizon 2020 (European-Virus-Archive-GLOBAL—871029). The INMI Monkeypox Study Group includes Isabella Abbate, Alessandro Agresta, Alessandra Amendola, Andrea Antinori, Francesco Baldini, Tommaso Ascoli Bartoli, Alessia Beccacece, Rita Bellagamba, Giulia Berno, Aurora Bettini, Nazario Bevilacqua, Licia Bordi, Marta Camici, Fabrizio Carletti, Angela Corpolongo, Stefania Cicalini, Francesca Colavita, Alessandra D'Abramo, Gabriella De Carli, Federico De Zottis, Lavinia Fabeni, Francesca Faraglia, Federica Forbici, Concetta Maria Fusco, Roberta Gagliardini, Anna Rosa Garbuglia, Saba Gebremeskel, Maria Letizia Giancola, Emanuela Giombini, Enrico Girardi, Giulia Gramigna, Elisabetta Grilli, Susanna Grisetti, Cesare Ernesto Maria Gruber, Eleonora Lalle, Simone Lanini, Daniele Lapa, Gaetano Maffongelli, Fabrizio Maggi, Alessandra Marani, Andrea Mariano, Ilaria Mastrorosa, Giulia Matusali, Silvia Meschi, Valentina Mazzotta, Claudia Minosse, Klizia Mizzoni, Martina Moccione, Annalisa Mondi, Vanessa Mondillo, Nicoletta Orchi, Sandrine Ottou, Carmela Pinnetti, Silvia Pittalis, Vincenzo Puro, Silvia Rosati, Gabriella Rozera, Martina Rueca, Laura Scorzolini, Eliana Specchiarello, Francesco Vaia, Francesco Vairo, Beatrice Valli, Alessandra Vergori, and Serena Vita. Download .pdf (.44 MB) Help with pdf files Supplementary appendix Viral replication and infectivity of monkeypox through semen - Authors' replyWe thank Abdullah Reda and colleagues for their comments on our work.1 We agree that the possibility that sources for the detection of monkeypox virus genomes in the semen could derive from passive diffusion from other body fluids or specimen contamination from genital lesions deserves careful consideration, based also on previous experience with other human viruses. However, several findings make this possibility unlikely in our case. A possible mechanism favouring diffusion from the blood to the genital tract is increased blood barrier permeability due to inflammatory conditions such as orchitis. Full-Text PDF Viral replication and infectivity of monkeypox through semenWith great interest, we read the findings presented by Daniele Lapa and colleagues,1 showing the successful isolation of monkeypox viral DNA from the seminal fluid of an infected patient. The authors suggested that monkeypox might have a genital reservoir because of the persistent viral shedding in seminal samples, even at low viral copies. These findings could indicate that the current monkeypox outbreak predominantly spreads through sexual transmission, especially after the various reports that estimated that most monkeypox cases were reported among individuals who identify as men who have sex with men. Full-Text PDF