Viral replication and infectivity of monkeypox through semen - Authors' reply.

We thank Abdullah Reda and colleagues for their comments on our work. 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. Existing evidence shows that orchitis during smallpox was exceedingly rare, and inflammation in the genital tract was excluded in the patient. Moreover, as pointed out in our Comment, we found that monkeypox virus PCR test for urine was negative. Furthermore, this PCR test had a much higher cycle threshold than semen in peripheral blood samples collected within the same timeframe, thus making it unlikely that semen was contaminated by these fluids. Finally, to avoid monkeypox virus contamination from the only genital lesion located on the penis, we required the hands and penis to be cleaned before sample collection and clear instructions were given for excluding contact or lesion abrasion during the collection of the semen sample.

That contamination by exogenous sources represents the only or the major cause for the presence of monkeypox virus in semen samples is also deemed unlikely in several studies addressing the monkeypox virus distribution in different body sites. In a large case series of monkeypox,2, 3, 4 58 (75%) of 77 patients had monkeypox virus DNA in their semen, supporting that it is a too frequent finding for relegating it to mere contamination. Studies of other viruses also highlight the difficult or sporadic isolation of replication-competent viruses from semen. Infectious Zika virus was isolated from 3 (4%) of 78 semen samples with detectable viral RNA, thus suggesting that the absence of viral isolation could be at least in part attributed to technical limitations rather than the absence of virus in the seminal fluid.

Notably, we have achieved monkeypox virus isolation by culturing semen from a second patient with monkeypox who was followed up at the National Institute for Infectious Diseases ‘Lazzaro Spallanzani’, with a quantification cycle value of 22·7. We agree that epidemiological and laboratory data from large cohorts are needed to clarify the potential role played by semen in monkeypox virus transmission, and that a more in-depth analysis of seminal tropism of monkeypox virus should be performed to assess whether viral particles or DNA are associated to the cellular fractions (ie, seminal leukocytes, exfoliated epithelial cells, or sperm cells) or to seminal plasma. However, it is worthy to mention that, when it comes to veterinary poxviruses, evidence already exist for semen-driven transmission.

We declare no competing interests. The authors acknowledge the contribution of the INMI Monkeypox Study Group (appendix). DL and FC contributed equally.