猴痘透過精液的病毒複製和傳染性

資料來源：http://www.thelancet.com/infection Vol 22 November 2022 / 財團法人台灣紅絲帶基金會編譯

  我們懷著極大的興趣閱讀了 Daniele Lapa 及其同事提出的研究結果，顯示成功從感染患者的精液中分離出猴痘病毒 DNA。作者認為，猴痘可能有生殖器宿主，因為精液樣本中的病毒持續脫落，即使在病毒拷貝數較低的情況下也是如此。這些發現可能顯示，當前的猴痘爆發主要通過性傳播傳播，特別是在各種報告估計大多數猴痘病例是在與男性發生性關係的男性中報告的之後。了解傳播方式有助於制定適當的介入措施，以降低當前疫情的強度。

  精液中存在猴痘 DNA 可能是由於局部生殖器複製或尿液、血液或生殖器損傷的被動擴散。然而，這一事件的確切機制在文獻中仍有爭議。儘管 Lapa 及其同事報告說，由於他們的樣本中沒有病毒 DNA，因此不太可能來自其他來源（血液和尿液）的交叉污染，但由於某些觀點，這一發現應謹慎解釋。首先，該發現基於從單個患者獲得的結果。因此，無法從本報告中得出適當的結論。此外，Noe 及其同事在使用 VeroE6 細胞系培養兩名猴痘患者的猴痘病毒精液樣本時，沒有顯示出生長。

  其次，之前的調查已經在猴痘患者的血液和尿液樣本中檢測到猴痘病毒DNA。例如，Thornhill 及其同事報告了從總共 528 名猴痘患者中採集的 7% 的血液樣本和 3% 的尿液樣本中的猴痘病毒 DNA 陽性 PCR 結果。儘管這些比率微不足道，但應予以考慮，特別是因為在其他相關調查中進一步報告了陽性血液和尿液樣本。在這些樣本中檢測到病毒碎片可能顯示這些顆粒可能會交叉污染精液。儘管作者在他們的患者中排除了這種可能性，但樣本量仍然是一個主要限制。第三，病毒顆粒的交叉污染也可能來自生殖器病變（例如脫落的上皮細胞）。然而，作者並未排除這種可能性，因為他們的病變樣本僅取自頭部傷口。根據作者和其他研究的證據，皮膚病變的病毒脫落間隔最長，病毒濃度最高。例如，Thornhill 及其同事報告說，與其他樣本相比，從皮膚和肛門生殖器區域獲得的樣本的 PCR 陽性結果最高（97%）。此外，Tarín-Vicente 及其同事報告說，他們的猴痘人群中 99% 的皮膚拭子和 78% 的肛門拭子呈陽性。這些發現顯示這些病變有可能引起精液交叉污染。但是，Lapa 及其同事也沒有具體說明這一點。

  此外，在精液中檢測到猴痘病毒並不足以顯示其性傳播，因為先前關於引起病毒血症並可以在精液中檢測到的其他病毒的研究的證據並未顯示它們的性傳播。在男性生殖系統中檢測到病毒顆粒通常是繼發於病毒血症，主要是在發生局部或全身炎症時，因為血液睾丸屏障容易感染病毒。無論其複制能力如何，病毒也可能透過管道持續存在，因為睾丸可能是病毒免疫的優勢部位。因此，我們建議仔細解釋當前的證據，直到其他調查證實了調查結果。

作者回復

我們感謝 Abdullah Reda 及其同事對我們工作的評論。我們同意，在精液中檢測猴痘病毒基因組的來源可能來自其他體液的被動擴散或生殖器病變的樣本污染，這一可能性值得仔細考慮，也基於以前對其他人類病毒的經驗。然而，在我們的案例中，一些發現使得這種可能性不太可能發生。有利於從血液擴散到生殖道的一種可能機制是由於睾丸炎等炎症條件導致的血液屏障通透性增加。現有證據顯示，天花期間的睾丸炎極為罕見，患者的生殖道炎症被排除在外。此外，正如我們在評論中指出的，我們發現尿液的猴痘病毒 PCR 檢測結果為陰性。此外，這種 PCR 測試的循環閾值比在同一時間範圍內收集的外周血樣本中的精液要高得多，因此精液不太可能被這些液體污染。最後，為了避免陰莖上唯一的生殖器損傷處感染猴痘病毒，我們要求在採集樣本前清潔手和陰莖，並明確說明在採集精液樣本期間排除接觸或損傷磨損。

在針對猴痘病毒在不同身體部位分佈的幾項研究中，外源性污染是精液樣本中存在猴痘病毒的唯一或主要原因，也被認為不太可能。在一個大型猴痘病例係列中，77 名患者中有 58 名（75%）的精液中含有猴痘病毒 DNA，這支持了將其歸為單純污染的常見發現。對其他病毒的研究也強調了從精液中分離出具有複制能力的病毒是困難的或零星的。傳染性玆卡病毒是從 78 個精液樣本中的 3 個（4%）中分離出的，其病毒 RNA 可檢測到，因此顯示沒有病毒分離至少部分歸因於技術限制，而不是精液中沒有病毒。

值得注意的是，我們透過培養來自國家傳染病研究所“Lazzaro Spallanzani”的第二名猴痘患者的精液，實現了猴痘病毒的分離，量化週期值為 22∙7。我們同意需要來自大型世代的流行病學和實驗室數據來闡明精液在猴痘病毒傳播中的潛在作用，並且應該對猴痘病毒的精液嗜性進行更深入的分析，以評估病毒顆粒或 DNA 是否與細胞部分相關（即精液白細胞、脫落的上皮細胞或精細胞）或精漿相關。然而，值得一提的是，在獸類痘病毒方面，已經存在精液驅動傳播的證據。

我們聲明沒有競爭利益。

Abdullah Reda, Ranjit Sah, Alfonso J Rodriguez-Morales, *Jaffer Shah jaffer.shah@drexel.edu 

埃及開羅，愛資哈爾大學醫學院， (AR)；尼泊爾加德滿都，特里布萬大學教學醫院，醫學研究所（RS）；哥倫比亞佩雷拉 ， Grupo de Investigación Biomedicina，醫學院，Fundación Universitaria Autónoma de las Américas (AJR-M)；秘魯利馬大學臨床流行病學和生物統計學碩士（AJR-M）；美國紐約，紐約州衛生處 (JS)

Viral replication and infectivity of monkeypox through semen

http://www.thelancet.com/infection Vol 22 November 2022

  With great interest, we read the findings presented by Daniele Lapa and colleagues, 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. Understanding the mode of transmission could allow for the development of proper interventional approaches to reduce the intensity of the current outbreak.

  Monkeypox DNA presence in the seminal fluids might be due to local genital replication or passive diffusion from urine, blood, or genital lesions. However, the exact mechanism of this event remains controversial in the literature. Although Lapa and colleagues reported that crosscontamination from other sources (blood and urine) is unlikely due to the absence of viral DNA in their specimens, this finding should be interpreted with caution due to some points. First, the finding is based on the results obtained from a single patient. Therefore, an appropriate conclusion is not attainable from this report. Moreover, Noe and colleagues showed no growth when culturing the monkeypox virus seminal samples of two patients with monkeypox using VeroE6 cell lines. 

  Second, previous investigations have detected monkeypox viral DNA in the blood and urine samples of patients with monkeypox. For example, Thornhill and colleagues reported monkeypox viral DNA positive PCR results in 7% of blood samples and 3% of urine samples taken from a total of 528 patients with monkeypox. Although these rates are meager, they should be considered, especially because positive  blood and urine samples were further reported in other relevant investigations. Detecting viral shreds in these samples might suggest potential semen cross contamination by these particles. Although the authors excluded this possibility in their patient, the sample size is still a major limitation. Third, cross-contamination of viral particles might also occur from genital lesions (eg, exfoliated epithelial cells). However, the authors did not exclude this possibility because their lesion samples were obtained from the head only. According to the evidence from the authors and other studies, skin lesions have the most extended viral shedding intervals and highest viral concentrations. For example, Thornhill and colleagues4 reported that samples obtained from skin and anogenital regions had the highest positive PCR results (97%) when compared with other samples. Moreover, Tarín-Vicente and colleagues reported that 99% of skin swabs and 78% of anal swabs were positive in their monkeypox population. These findings indicate the potential ability of these lesions to induce cross-contamination with seminal fluids. However, this was not also specified by Lapa and colleagues. 

  Furthermore, monkeypox viral detection in semen is not sufficient to indicate its sexual transmission since evidence from previous studies on other viruses that caused viremia and could be detected in semen did not indicate their sexual transmission Detecting viral particles within the male reproductive system is commonly secondary to viraemia because the blood–testis barrier is liable to viruses, mainly when local or systemic inflammation occurs. Viral persistence through the tract is also likely, irrespective of its ability to replicate because the testes can be an immunologically favored site for the virus. Accordingly, we suggest that the current evidence be carefully interpreted until other investigations confirm the findings. 

Authors’ reply 

We 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. 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,1 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, 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.

Abdullah Reda, Ranjit Sah, Alfonso J Rodriguez-Morales, *Jaffer Shah jaffer.shah@drexel.edu Faculty of Medicine, Al-Azhar University, Cairo, Egypt (AR); Tribhuvan University Teaching Hospital, Institute of Medicine, Kathmandu, Nepal (RS); Grupo de Investigación Biomedicina, Faculty of Medicine, Fundación Universitaria Autónoma de las Américas, Pereira, Colombia (AJR-M); Master of Clinical Epidemiology and Biostatistics, Universidad Científica del Sur, Lima, Peru (AJR-M); New York State Department of Health, New York, NY 10013, USA (JS)