Monkeypox infection among men who have sex with men: PCR testing on seminal fluids.

Dear Editor,

in this journal recently J Heskin et al. described how sexual transmission is becoming predominant among new monkeypox (MPX) cases, Awan UA et al. identified as critical to achieve infection control raising public knowledge of risk factors and informing of the strategies they may take to decrease their exposure to the virus, , and we provided an example of an atypical MPX proctitis following sexual intercourse.

In this case-series we presented data on MPX virus detection on seminal fluids to corroborate the hypothesis of sexual transmission.

Overall, 36 men who have sex with men (MSM) diagnosed with MPX infection at the Infectious Diseases Unit of San Raffaele Scientific Institute, Milan, Italy, were tested on seminal fluids.

Real-time (RT) PCR (RealStar® Orthopoxvirus PCR Kit 1.0 – altona DIAGNOTICS) targeting variola virus and non-variola Orthopoxvirus species (cowpox, monkeypox, raccoonpox, camelpox, vaccinia virus) was used to detect non-variola DNA on swabs, serum, plasma, seminal fluids and urines and a specific RT PCR targeting Monkeypox virus DNA (Liferiver - SHANGHAI ZJ BIO-TECH CO., LTD) subsequently confirmed MPX infections.

All individuals had a positive oropharyngeal, cutaneous, genital or rectal MPX swab and were at different time points tested for MPX also on seminal fluids and urines.

All reported high-risk sexual behaviors in the 3 months before diagnosis, with unprotected sexual intercourse with >10 partners. 15/36 (42%) were people living with HIV (PLWH) and 15/36 (42%) were HIV pre-exposure prophylaxis (PrEP) users.

Among 22/36 (61%) individuals MPX virus was detected also in seminal fluids, whereas in 8/36 (22%) in urines. Median (interquartile) cycle thresholds (CT) of MPX were 34 (29–36.5) in seminal fluids and 34 (33–36) in blood.

Individuals with positive seminal fluids tested positive for MPX on plasma or serum in 17/22 (77%) of cases, whilst 7/14 (50%) of those with negative seminal fluids tested positive on plasma or serum. Full details on virologic data are presented in Table 1 .

Table 1

Virologic data on different specimens of monkeypox (MPX) virus.

Cases	MPX swab§	Seminal fluids	Urines	Serum/Plasma
Case 1	+	+	+	+
Case 2	+	–	–	–
Case 3	+	+	+	+
Case 4	+	+	–	+
Case 5	+	–	–	–
Case 6	+	+	–	+
Case 7	+	–	–	–
Case 8	+	+	–	+
Case 9	+	–	–	–
Case 10	+	–	–	+
Case 11	+	+	–	+
Case 12	+	–	–	–
Case 13	+	–	–	+
Case 14	+	+	–	+
Case 15	+	–	–	–
Case 16	+	–	–	+
Case 17	+	+	–	+
Case 18	+	+	–	–
Case 19	+	+	–	+
Case 20	+	–	–	+
Case 21	+	+	+	–
Case 22	+	–	–	–
Case 23	+	+	+	+
Case 24	+	+	+	+
Case 25	+	–	–	+
Case 26	+	+	+	–
Case 27	+	+	–	+
Case 28	+	+	–	+
Case 29	+	–	–	+
Case 30	+	+	–	+
Case 31	+	+	–	+
Case 32	+	+	–	–
Case 33	+	+	–	+
Case 34	+	+	–	–
Case 35	+	–	+	+
Case 36	+	+	+	+

: “MPX -swab” refers to either cutaneous, rectal, oropharyngeal or genital swabs.

Genital lesions were present among 10/22 (45%) of MSM with positive seminal fluids and 3/14 (21%) with negative. Among individuals without genital lesions, 18/23 (78%) had rectal lesions, 20/23 (87%) cutaneous lesions and 15/23 (65%) both: 10/18 (55%), 10/20 (50%), 8/15 (53%), respectively, tested positive on seminal fluids for MPX.

Overall, only one individual with positive seminal fluids had a concurrent sexually transmitted infection (STI), whilst three with negative seminal fluids had a concurrent one.

Individuals’ characteristics among those who tested positive or negative for MPX on seminal fluids are presented in Table 2 .

Table 2

Individuals’ characteristics among those who tested positive or negative for MPX on seminal fluids.

Characteristics	Overall (n = 36)	Positive seminal fluids (n = 22)	Negative seminal fluids (n = 14)	p-value
Age (IQR)	41.5 (31.25–35.5)	37.5 (35–45)	35 (31–37)	0.303 a
HIV infection	15 (42%)	7 (32%)	8 (57%)	0.175 b
PrEP users	15 (42%)	9 (41%)	6 (43%)	1 b
Number of sexual partners10–2020–30>30	20 (55%)10 (28%)6 (17%)	15 (68%)6 (27%)4 (18%)	5 (36%)4 (28%)2 (14%)	0.947 b
Type of intercourse:ReceptiveInsertiveBoth	4 (11%)4 (11%)26 (72%)	2 (9%)2 (9%)18 (82%)	2 (15%)2 (15%)10 (71%)	0.975 b
Previous STIs	36 (100%)	22 (100%)	14 (100%)	1 b
Concurrent STIs	4 (11%)	1 (4%)	3 (21%)	0.277 b
Presence of genital lesions	13 (36%)	10 (45%)	3 (21%)	0.175b
Presence of rectal lesions°	18 (50%)	10 (45%)	8 (57%)	0.730 b
Presence of cutaneous lesions°	20 (55%)	10 (45%)	10 (71%)	0.176 b
Positive serum or plasmaCT (median, IQR)	24 (66%)34 (33–36)	17 (77%)33.5 (30–36)	7 (50%)34.5 (34–36)	0.147 b0.263 a

: by Mann-Whitney test;.

: by chi-square or Fisher's exact test.

: without genital lesions

Abbreviations: STI: sexually transmitted infection; IQR: interquartile; CT: cycle threshold.

In this case-series positive seminal fluids were frequent among MPX cases, in line with other previously reported preliminary results. A higher proportion of MSM tested positive on seminal fluids than urines, suggesting that MPX testing on seminal fluids rather than urines could be possibly included in clinical assessment of individuals.

We observed that also individuals who presented without genital lesions or who did not engage in insertive intercourse had positive seminal fluids. Most MSM with positive seminal fluids tested positive for MPX also on serum or plasma. Given these two factors, we hypothesize that MPX presence in seminal fluids follows bloodstream dissemination of the virus.

Furthermore, concurrent STIs seemed to not have an effect on the positivity rate of seminal fluids.

A key question remains the duration of viral shedding in seminal fluids following clinical healing of MPX lesions, especially among those living with an immune impairment, such as PLWH. Moreover, the significance of detecting MPX DNA on seminal fluids and urines is still uncertain, as to this day no data are available regarding viral culture on seminal fluids. However, we believe that these data, in our high-risk population, corroborate the hypothesis of sexual transmission.

Contributorship statement

S.N., A.R.R. and E.B. visited the individuals and contributed to writing the article. C.C and D.C. visited the individuals and contributed to the reviewing of the article. A.C. coordinated clinical activities and contributed to the reviewing of the article. D.M. and A.R. coordinated virologic activities and performed PCR tests for MPX. All authors have read and agreed to the published version of the manuscript.

Declaration of Competing Interest

None.