Longitudinal viral shedding and antibody response characteristics of men with acute infection of monkeypox virus: a prospective cohort study

Longitudinal viral shedding and antibody response characteristics of men with acute infection of monkeypox virus: a prospective cohort study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Longitudinal viral shedding and antibody response characteristics of men with acute infection of monkeypox virus: a prospective cohort study Yang Yang, Liuqing Yang, Shuo Song, Chenguang Shen, Siyu Niu, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3869240/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 May, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Background Elucidating the viral and antibody dynamics within the host and the viral dynamics of the environmental fomits is key for the successful public health measures against monkeypox virus (MPXV) infection. In this study, a total of 993 samples from multiple sites of patients and 1,633 samples from environmental fomits of 77 acute MPXV infections (HIV co-infection: N=42) were collected every two to three days and subjected for the detection of MPXV DNA, surface proteins specific antibody and neutralizing titers. Overall, skin lesions showed 100% positive rate of MPXV DNA, followed by rectal swab (88.16%), saliva (83.78%), oropharyngeal swab (78.95%), urine (55.26%)and plasma (31.17%). Positive rate of oropharyngeal swab decreased rapidly after 7 days post symptoms onset (d.p.o), while the rectal swab and saliva maintained close positive rates to the skin lesions. Viral dynamics were similar among skin lesions, saliva and oropharyngeal, withrapid increase to the peak at about 6 d.p.o and then decreased. While for the rectal swab, it peaked at the beginning of symptoms onset, and decreased rapidly with the disease progression. Totally, 52.66% (860/1633) of environmental fomits swabs were positive for MPXV DNA, with highest positive rate (69.89%) and mean viral loads (Ct values of 31.22) from the deposition area. Moreover, the proportion of Ct values between 15-30 maintained high for some fomits like the deposition area, floor and bedside cupboard during the disease progression.High seropositivity against A29L (100%) and H3L (94.74%) were detected, while the high correlation was only found between anti-A29L IgG titer and neutralizing titer. Notably, most of indexes were similar between HIV and Non-HIV participants, except the higher viral loads in the early stage and peak viral from the rectal swabs of the HIV participants. Our results contribute to an improved understanding of viral shedding and antibody response characteristics of acute Mpox. With the ongoing outbreak of Mpox worldwide, these data have profound implications for the diagnosis, treatment, prevention of transmission and development of vaccines for Mpox. Health sciences/Diseases/Infectious diseases/Viral infection Biological sciences/Microbiology/Infectious-disease diagnostics Monkeypox virus Viral shedding Neutralizing antibody Fomits Diagnostic accuracy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Mpox (formerly known as monkeypox) is a zoonotic illness caused by the monkeypox virus (MPXV), and the first case of human Mpox case was reported in 1970 in the Democratic Republic of the Congo 1 . Thereafter, sporadic cases caused by two distinct clades were confined to Central Africa (Clade 1) and West Africa (Clade 2) for decades until international transmission was first detected in the United States in 2003 2 . Since May 2022, another new lineage MPXV (Clade 2b) derived from West African clade with certain genetic changes caused a widespread epidemic worldwide, leading to the declaration of the Mpox outbreak a Public Health Emergency of International Concern (PHEIC) by World Health Organization (WHO) on July 23, 2022 3, 4 . The first imported Mpox case into mainland China was found in Sep, 2022, and no subsequent transmission occurred with the rapid diagnosis and isolation 5 . However, Mpox has been endemic in several cities of mainland China currently 6, 7, 8 . Animal-to-human transmissions were common in early sporadic human Mpox cases, while sustained human-to-human transmission become predominant among the widespread of MPXV 1 . Historically, the transmission of MPXV between humans has been thought to occur primarily through respiratory droplets 9, 10 . However, close contact or sexual contact with infectious sores or lesions on mucous membranes has been thought to be the primary mode of transmission during the 2022 outbreak 1, 10 . Recent studies have found that MPXV can also be detected in specimens from multiple sites of Mpox cases, including saliva, rectal swabs, blood, urine and semen 10, 11, 12, 13, 14, 15, 16, 17 , and skin lesions have been presumed to be the primary source of viral shedding and believed to be optimal for diagnosis 1 . However, one recent study has also found patients with negative MPXV DNA in skin lesions, while positive in other sites, highlighting the important roles of multi-site sampling in the diagnosis of Mpox 18 . Although some studies have compared the diagnostic accuracy of different sample sites, the evaluations with detailed temporal data are currently lacking. Moreover, asymptomatic and atypical MPXV infections which may serve as important source of transmission have been found 19, 20, 21, 22 , and the diagnostic accuracy of some alternative samples remains controversial 23 , posing challenges to laboratory diagnosis of Mpox. Studies have also set out to characterize the dynamics of MPXV shedding, while current understanding was restricted by either the limited number of participants and sampling frequency, short follow-up periods or biasing towards the Mpox patients with relatively mild disease 10, 12, 15, 24 . In addition, transmission of MPXV has also been shown to occur indirectly through contaminated fomites 1, 4, 12 . Therefore, systematic evaluation of the risk of this indirect transmission through environmental fomits is of great value to inform the disinfection and prevention strategies. Previous studies have also shown that MPXV DNA could be detected in different environmental fomits of Mpox patients, while application of the existing data has been hampered by limited number of samples and rooms, and lacking the longitudinal sampling in association with disease progression. Currently, there is no authorized Mpox specific vaccine available 25 . Vaccines against smallpox have been known to possess cross protective activity against Mpox, and the Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN, also known as JYNNEOS) has been approved by the US Food and Drug Administration (FDA) for pre-exposure vaccination against Mpox 4, 26 . One recent study has estimated that the vaccine effectiveness of MVA-BN against Mpox was 87% [95% confidence interval (CI): 84–90%] for one-dose vaccination and 89% (95% CI: 78–100%) for two-dose vaccination, based on pooled estimates during the 2022 Mpox outbreak 27 . Moreover, low levels of MPXV neutralizing antibodies were found after MVA-BN vaccination in healthy individuals 28 . Accordingly, it is in urgent need to develop Mpox specific vaccines able to effectively limit Mpox. Similar with other orthopoxviruses, MPXV possesses two diseases inducing infectious forms of the intracellular mature virion (IMV) and the extracellular enveloped virion (EEV) containing six surface proteins which were speculated to be the main neutralizing antibody-eliciting antigens 29 . However, little is known about the characteristics of surface proteins specific IgG responses following infection and the protective antigens, which is key for the design and development of MPXV specific vaccines. In this prospective cohort study, we systematically analyzed the longitudinal positive rate, viral shedding dynamics of different sample types and environmental fomits from acute MPXV infections in association with disease progression, and the antibody response characteristics against the surface proteins of MPXV. Methods Study design and participants We did a prospective cohort study on the longitudinal viral shedding and antibody response characteristics from men with acute infection of MPXV admitted to Shenzhen Third People's Hospital during June 9, 2023 to November, 5, 2023 (N = 77) (Shenzhen, Guangdong, China), and all the patients were followed-up to discharge. All the patients were living in the negative pressure isolation ward during hospitalization to reduce the possibility of further transmission. Clinical and epidemiological information were collected at the earliest time-point after admission. Each Ct value of quantitative real time PCR (qRT-PCR) from different sample types from the patients and the environmental fomites, and the specific IgG antibody against different MPXV surface proteins (including A29L, A35R, B6R, E8L, H3L and M1R) during hospitalization were detected and collected. For analyses, the participants were grouped based on the status of HIV infection (HIV, N = 42 and Non-HIV, N = 35). Moreover, all these specimens were further stratified into 3 groups based on the collection time related to the days post symptoms onset (d.p.o), including the 1 ~ 7 d.p.o, 8 ~ 14 d.p.o and 15 ~ 21 d.p.o groups. The study protocol was approved by the Ethics Committees of Shenzhen Third People’s Hospital (2021-030), and written informed consents were obtained. Sample collection and detection of the MPXV DNA Serial oropharyngeal swabs, saliva, rectal swab, skin lesions, urine and plasma samples were collected by professional nurse every two to three days. Samples from the fomits were also serially collected by the staffs in the Department of Healthcare-associated Infection Management every two days before the disinfection and clean. After the sample collection, the rooms were cleaned every 24 hours using 5000 ppm available chlorine sodium hypochlorite on all hard surfaces and floors, and 10000 ppm available chlorine sodium hypochlorite for the toilet, shower, wash basins as previously reported 30 . All the samples were subjected for the detection of MPXV DNAs using commercial kits (GeneoDX Co., Ltd.) based on qRT-PCR according to manufacturer’s instruction. The specimens were considered positive if the Ct value was < 40, and negative if the results were undetermined. The sequences of infected virus were determined by next-generation sequencing (BGI Co., Ltd.) and the genotypes were analyzed using the Nextclade ( https://clades.nextstrain.org/ ). Enzyme-linked immunosorbent assays (ELISAs) The MPXV surface proteins specific IgG antibodies in plasma specimens were detected using Enzyme-linked immunosorbent assays (ELISA) as previously reported and run in triplicate 31 . Briefly, microtiter plates (Sangon Biotech) were coated overnight at 4°C with 100 ng of each recombinant protein of MPXV, including A29L, H3L, B6R, A35R, E8L and M1R, which was expressed in E. coli expression system and purified by Ni-NTA. The plates were then washed twice with 1 × PBS (phosphate buffer saline) containing 0.1% v/v Tween-20 (PBST) and blocked with blocking solution (1 × PBS containing 5% w/v bovine serum albumin) overnight at 4°C. On the day of the experiment, the plates were washed once with PBST. The 3-fold serially diluted sera were added to the wells and incubated at 37°C for 60 min. Then the plates were washed five times using PBST and 100 µL horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody or HRP-conjugated goat anti-human IgM antibody solution (Sangon Biotech) was added to each plate, respectively, and incubated at 37°C for 60 min. Wash each plate with PBST five times, and 100 µL of tetramethylbenzidine (TMB) substrate (Sangon Biotech) was added to each plate at room temperature in the dark and reacted for 15 min. After the reaction, the plate was stopped with a 2M H 2 SO 4 solution. The absorbance was measured at 450 nm using a microplate reader. The ELISA titers were determined by endpoint dilution. Focus reduction neutralization test (FRNT) The neutralizing antibody (nAb) titers of the plasma samples against MPXV were detected using focus reduction neutralization test (FRNT) assay with clinical isolate of MPXV (hMpxV/China/SZ-SZTH41/2023, EPI_ISL_18213375) following the procedure as previously reported with modification 32 . All plasma samples were heat-inactivated at 56°C for 30 min before use. Serial dilutions of tested plasma samples were mixed with an equal volume monkeypox live virus (200 focus forming units) in 96-well microwell plates and incubated at 37°C for 1 h in the presence of 10% guinea pig serum (Beijing Bersee Technology Co., Ltd) as a source of complement. Mixtures were then transferred to 96-well plates seeded with 1.5 × 10 4 Vero E6 cells per well and incubated at 37°C for 18 h. Then the supernatant was removed and cells were fixed with 4% paraformaldehyde solution for 30 min, and permeabilized with 0.15% Triton X-100 for 10 min, followed by incubating with HRP-conjugated Vaccinia Virus Polyclonal Antibody (Invitrogen) for 2 h at room temperature. The reactions were developed with KPL TrueBlue Peroxidase substrates (Seracare Life Sciences Inc.). The numbers of MPXV foci were calculated using an EliSpot reader (Cellular Technology Ltd). Neutralizing antibody titers were calculated as a 50% inhibitory dose (ID 50 ) expressed as the dilution of plasma that resulted in a 50% reduction of the numbers of MPXV foci compared with virus control. Statistical analysis Statistical analyses were done as previously reported 33, 34 . The neutralizing titers of ID 50 were calculated using a five-parameter dose-response curve in Graphpad Prism. For descriptive analysis, data were presented as median (interquartile range, IQR) for continuous parameters and frequency (percentage) for categorical variables. Chi-squared and Fisher's exact tests were used to compare categorical variables, while for continuous variables the t-test was used for normal data and the Mann-Whitney U test for non-normal data. The Spearman rank correlation coefficient was used for linear correlation analysis between the ELISA titers and nAb titers. The kinetics of Ct values and IgG levels during hospitalization were calculated by the LOESS (locally estimated scatterplot smoothing) curve fitting polynomial regression using R version 4.1.0. P value less than 0·05 was considered statistically significant. Data availability The data supporting the findings of this study are available within the paper and the supplementary information files. Source data are provided with this paper. Results Baseline characteristics of the cohort During June 11, 2023 and November 11, 2023, a total of 139 laboratory confirmed Mpox patients were found in Shenzhen, China (Figure S1 ), and all the patients were men. Totally, 77 out of 81 hospitalized patients were enrolled in this study with a median age of 30 years (Table S1 ), and 42 patients were HIV-positive. Among the 77 patients, 72 patients were men who have sex with men (MSM) and 5 patients received smallpox vaccination. Skin lesions were the most common initial symptoms with 98.7% (76/77) positive rate, followed by some influenza-like symptoms including fever (71.43%), sore throat (61.04%), myalgia (35.06%). Lymphadenopathy and rectitis were found in 41.56% and 19.48% of the patients, respectively. There were no significant differences of the initial symptoms between HIV and Non-HIV participants, except a significantly higher prevalence of rectitis in the HIV participant (Table S1 ). The median days between symptoms onset to admission is 5, and median days hospitalization is 7. Fever clinic was the most common (66.23%) medical setting at first visit, followed by Department of Dermatology (9.09%), HIV clinic (7.79%), Department of Emergency (6.49%) and Anorectal surgery clinic (5.19%). The most common location of skin lesions were the Trunk and extremities (77.92%), genitals (62.34%), face (50.65%) and perianal region (37.66%), and a small portion on the hands and feet (12.99%). No participants received specific antiviral treatment for MPXV infection. MPXV genomes were sequenced from all the 77 participants and all the MPXV belonged to a newly defined lineage C.1 in the West African clade. Altogether, a total of 993 specimens from these patients were serially collected during hospitalization, including 166 oropharyngeal swabs, 154 saliva, 162 rectal swabs, 173 skin lesions, 156 urine and 182 plasma samples. Moreover, a total of 1633 environmental fomits swabs from 49 patients were also serially collected, including 89 Floor swabs, 125 Call button swabs, 124 Light switch swabs, 94 Television remote control swabs, 124 Bed handrail swabs, 125 Bedside cupboard swabs, 107 Chair (arm rest) swabs, 93 Door handle (patient room to bathroom) swabs, 93 Deposition area (Air conditioning air outlet) swabs, 125 Mobile phone swabs, 125 Clothes swabs, 125 Pillow swabs, 94 Toilet flush handle swabs, 95 Shower handle swabs and 95 Delivery window swabs (Table S2). These specimens were further stratified into 3 groups based on the collection time, including the 1 ~ 7 d.p.o, 8 ~ 14 d.p.o and 15 ~ 21 d.p.o groups (Table S1 and S2). Positive rate and dynamics of MPXV in multiple sites of Mpox patients Overall, about 78.95% (60/76), 83.78% (62/74), 88.16% (67/76), 100.00% (76/76), 55.26% (42/76) and 31.17% (24/76) participants showed positive detection of MPXV DNA in oropharyngeal swab, saliva, rectal swab, skin lesions, urine and plasma samples (Table 1 ), with no differences between the HIV and Non-HIV groups. For the three groups based on the collection date, all the skin lesions showed the highest positive rates, followed by rectal swab, saliva, oropharyngeal swab, urine and plasma. Of note, positive rates of 91.67%, 58.33% and 56.52% were found from the skin lesions, rectal swab and saliva samples in the 15 ~ 21 d.p.o group, respectively, while oropharyngeal swab only showed a positive rate of 25%. Moreover, only 2 plasma sample was positive for MPXV DNA in the 15 ~ 21 d.p.o group. Furthermore, we have also analyzed the positive rate of 143 paired samples of skin lesions, rectal swab, saliva and oropharyngeal swab. Generally, 139, 103, 101 and 70 positive samples were found for skin lesions, rectal swab, saliva and oropharyngeal swab, respectively (Fig. 1 ). In the 1 ~ 7 d.p.o group, the positive rates were similar among rectal swab, saliva and oropharyngeal swab. However, the positive rates of rectal swab and saliva were higher than oropharyngeal swab both in the 8 ~ 14 d.p.o and 15 ~ 21 d.p.o groups (Fig. 1 ). The viral loads (indicated as Ct values) were also analyzed, and the highest viral loads were found in the skin lesions, followed by rectal swab, saliva and oropharyngeal swab (Fig. 1 and Table 1 ). Moreover, there is no statistical differences on the positive rate and viral loads between HIV and Non-HIV groups, except a significantly higher positive rate of the urine during 15 ~ 21 d.p.o in the Non-HIV group and significantly higher viral loads in the rectal swab in the HIV group (Table 1 ). Table 1 Detection of MPXV DNA in multiple sites of Mpox cases during disease progression. Collection date Sample types Mpox patients Total HIV-positive (N = 42) HIV-negative (N = 35) p values Overall Positive rate (%, n/N) & Oropharyngeal 78.95 (60/76) 78.57 (33/42) 79.41 (27/34) > 0.9999 Saliva 83.78 (62/74) 87.18 (34/39) 80.00 (28/35) 0.5311 Rectal swab 88.16 (67/76) 92.86 (39/42) 82.35 (28/34) 0.2841 Skin lesions* 100.00 (76/76) 100.00 (42/42) 100.00 (34/34) > 0.9999 Urine 55.26 (42/76) 56.10 (23/41) 54.29 (19/35) > 0.9999 Plasma 31.17 (24/77) 38.10 (16/42) 22.86 (8/35) 0.1465 1 ~ 7 d.p.o Positive rate (%, n/N) Oropharyngeal 71.21 (47/66) 65.85 (27/41) 80.00 (20/25) 0.2703 Saliva 74.58 (44/59) 74.29 (26/35) 75.00 (18/24) > 0.9999 Rectal swab 75.41 (46/61) 78.38 (29/37) 70.83 (17/24) 0.5527 Skin lesions* 100.00 (74/74) 100.00 (49/49) 100.00 (25/25) > 0.9999 Urine 47.37 (27/57) 42.42 (14/33) 54.17 (13/24) 0.4295 Plasma 24.56 (14/57) 33.33 (11/33) 12.50 (3/24) 0.1183 Ct values (median; range) Oropharyngeal 33 (31–35) 33 (29.5–35) 32.9 (31–35) 0.2563 Saliva 30 (27–34) 30.5 (26.3–33.8) 29.3 (28.2–33.5) 0.7185 Rectal swab 26 (22–31) 24 (21–27) 33 (24–34) 0.0150 Skin lesions* 24 (22–26) 24 (23–26) 23 (21-24.5) 0.3717 Urine 35 (29.7–37) 34.9 (29.5–37) 36 (30.4–37) 0.7418 Plasma 34.8 (33.7–36.7) 34.5 (33.9–36.7) 35.1 (34.3–36.6) 0.7012 8 ~ 14 d.p.o Positive rate (%, n/N) Oropharyngeal 56.00 (42/75) 52.38 (22/42) 60.61 (20/33) 0.4936 Saliva 69.86(51/73) 76.92 (30/39) 61.76 (21/34) 0.2040 Rectal swab 72.73 (56/77) 76.74 (33/43) 67.65 (23/34) 0.4438 Skin lesions* 95.95 (71/74) 95.24 (40/42) 96.88 (31/32) > 0.9999 Urine 39.19 (29/74) 43.90 (18/41) 33.33 (11/33) 0.4731 Plasma 14.44 (13/90) 13.21 (7/53) 16.22 (6/37) 0.7649 Ct values (median; range) Oropharyngeal 33 (30 -35.15) 33.1 (30.1–35.2) 32.5 (29.5–35.3) 0.8400 Saliva 32 (28–35) 30.5 (27.25-36) 32 (31–34) 0.5962 Rectal swab 32.65 (26–35) 32 (26-35.8) 33 (28.5–35) 0.2525 Skin lesions* 26 (22–29) 26.5 (22-29.6) 26 (22.3–28.5) 0.7125 Urine 33 (30.4–34) 33 (30.8–35.4) 33 (29.35-34) 0.1999 Plasma 36.7 (35.7–37.1) 36.4 (35.3–36.7) 37 (36.3–37.4) 0.1353 15 ~ 21 d.p.o Positive rate (%, n/N) Oropharyngeal 25.00 (6/24) 26.67 (4/15) 22.22 (2/9) > 0.9999 Saliva 56.52 (13/23) 57.14 (8/14) 55.56 (5/9) > 0.9999 Rectal swab 58.33 (14/24) 60.00 (9/15) 55.56 (5/9) > 0.9999 Skin lesions* 91.67 (22/24) 93.33 (14/15) 88.89 (8/9) > 0.9999 Urine 39.13 (9/23) 20.00 (3/15) 75.00 (6/8) 0.0228 Plasma 5.71 (2/35) 10.53 (2/19) 0.00 (0/16) 0.4891 Ct values (median; range) Oropharyngeal 31.6 (28.8–33.6) 30.1 (26.7–32.7) 32.5 (31.8–33.3) 0.4456 Saliva 34 (27–36) 31.1 (26.5–36) 34 (34–37) 0.8951 Rectal swab 29.5 (27–36) 28 (27-30.2) 37 (30–38) 0.1088 Skin lesions* 28 (24.3–31.8) 28 (24.3–32.6) 28 (25.25–31.5) 0.3914 Urine 35 (29–36) 36 (31.4–37) 34.5 (30.3–35.8) 0.7896 Plasma 32.1 (31.9–32.4) 32.1 (31.9–32.4) NA NA & Individuals with positive detection of MPXV DNA in the indicated sample types. *Include swabs of lesion surface and exudate. NA: Not available. d.p.o: Days post symptoms onset. Then we analyzed the viral dynamics in different types of specimens from men with MPXV infection (Fig. 2 and Figure S2). For the skin lesions, viral loads slightly increased and reached the peak at about 6 d.p.o, and then slightly decreased to reach Ct values of 30 at about 17 d.p.o (Fig. 2 A). For the rectal swab, high viral loads were found at the at the beginning of symptoms onset, and decreased rapidly with the disease progression (Fig. 2 C). For the saliva and oropharyngeal swab, similar dynamics were found with the skin lesions, while the viral loads were much lower (Fig. 2 E and 2 G). When comparing between HIV and the Non-HIV groups, the dynamics and peak viral load were similar for the skin lesions, saliva, and oropharyngeal (Fig. 2 B, 2 D, 2 F, 2 H and Figure S2). Notably, much higher viral loads during the early phase of infection and peak viral loads were found in the rectal swabs from the HIV group (Fig. 2 E and Figure S2). Persistent detection of MPXV DNA on the environmental fomites from Mpox patients Totally, 52.66% (860/1633) of environmental fomits swabs were positive for MPXV DNA, with 62.92% (56/89) of the Floor swabs, 43.2% (54/125) of the Call button swabs, 37.1% (46/124) of the Light switch swabs, 44.68% (42/94) of the Television remote control swabs, 59.68% (74/124) of the Bed handrail swabs, 61.6% (77/125) of the Bedside cupboard swabs, 54.2% (58/107) Chair (arm rest) swabs, 45.16% (42/93) of the Door handle (patient room to bathroom) swabs, 69.89% (65/93) of the Deposition area (Air conditioning air outlet) swabs, 52.8% (66/125) of the Mobile phone swabs, 56.8% (71/125) of the Clothes swabs, 68% (85/125) of the Pillow swabs, 45.74% (43/94) of the Toilet flush handle swabs, 47.37% (45/95) of the Shower handle swabs and 37.89% (36/95) Delivery window swabs, respectively. The mean Ct values of these samples were 32.83, and Ct values showed significant differences among different swabs (anova, p = 0.008 ), among which Ct values from the deposition area is the lowest (Fig. 3 A and Table S3). By further dividing the samples according to their collection time, we found no statistical differences among the 1 ~ 7 d.p.o, 8 ~ 14 d.p.o and 15 ~ 21 d.p.o groups (Table S3). Further analysis showed that the proportion of Ct values between 15–30 were highest for deposition area (21.35%), followed by floor (18.05%), bedside cupboard (14.37%) and then the pillow (11.72%) (Fig. 3 B). Moreover, the proportion of samples with Ct values between 15–30 increased with the disease progression for most environmental fomites (Fig. 3 C). Antibody characteristics of men with acute MPXV infection Plasma samples were also serially collected from these 67 participants and subjected for the test of MPXV specific IgG antibody response, including A29L, A35R, B6R, E8L, H3L and M1R proteins. About 94.02%, 16.42%, 41.79%, 35.82%, 80.6% and 2.99% individuals showed positive IgG response against A29L, A35R, B6R, E8L, H3L and M1R, respectively, with no differences between HIV and Non-HIV groups (Fig. 4 A). Then we further analyzed the anti-A29L and anti-H3L IgG in detail. The positive rate for anti-A29L and anti-H3L IgG during 1 ~ 7 d.p.o could reach 86.36 and 79.55, and increased to 100% and 94.74% during 15 ~ 21 d.p.o, respectively (Fig. 4 B and 4 C). The endpoint titers of anti-A29L IgG increased obviously along with the disease progression and reached the peak at about 12 d.p.o, while no increase was found for anti-H3L IgG titers (Fig. 4 E). The dynamics of anti-A29L and anti-H3L IgG titers were similar between HIV and Non-HIV groups (Fig. 4 D and 4 E). Moreover, ID 50 of 67 plasma samples against authentic MPXV were detected using FRNT assay, and neutralizing activity was detected in 97.92% (47/48) of the samples collected after 7 d.p.o. The Spearman rank correlation coefficient analyses showed a high correlation between ID 50 and anti-A29L IgG titer, while not between ID 50 and anti-H3L IgG titer (Fig. 4 F and 4 G). Discussion The qRT-PCR assay has been regarded as the gold standard and the most common method for the diagnosis of MPXV infection 23 . Viral shedding patterns varied in different site of viral infections 10, 35 , and diagnostic accuracy of different sample types also varied in association with disease progression 36 . In our study, the specimens were further stratified into 1~7 d.p.o, 8~14 d.p.o and 15~21 d.p.o groups based on the collection time, covering different stages of acute viral infections. Consistent with previous studies 10, 12, 17 , skin lesions possessed the highest sensitivity for the diagnosis of MPXV infection during the whole infection course, followed by rectal swabs, saliva and then the oropharyngeal swab during disease progression. (Table 1 and Figure 1). It is worth noting that the positive rate of oropharyngeal swab is similar to that of rectal swabs and saliva during 1~7 d.p.o, while decreased dramatically and showed much lower positive rate between 8~21 d.p.o, which might explain the controversial results about the diagnostic accuracy of oropharyngeal swab 23 . The positive rates of rectal swabs and saliva maintain similar during disease progression and close to the positive rates of skin lesions, indicating that these two types of specimens could serve as excellent alternatives for the diagnosis of MPXV infection, especially for the asymptomatic and atypical infections when skin lesions are not available. In addition, serological assessment of MPXV infection could be a complementary method for the diagnosis of Mpox, and useful in some other settings including identification of self-attenuated infection, assessing population seroprevalence to determine asymptomatic 37 . Our result from the serological response to the six main neutralizing-eliciting antigens of MPXV following infection demonstrated a notably high seropositivity rate of A29L specific IgG, then the H3L specific IgG (Figure 4). Furthermore, the seropositivity of A29L and H3L-specific IgG within 7 d.p.o could reach about 80%, suggesting two prominent serological markers for the early diagnosis of MPXV infection. Human-to-human transmission of MPXV has been shown to occur through direct contact with skin lesions, body fluids, or respiratory droplets from infected humans 1, 9, 12 , therefore, viral load and viral shedding dynamics of different sample types have important implications on transmissibility, treatment and public policy 33 . In our study, we enrolled a total of 77 patients (42 patients were HIV-positive), and a total of 993 different types of specimens from these patients were serially collected during hospitalization for the analyses of viral dynamics. Consistent with previous studies, skin lesion showed the highest viral loads, followed by anal swabs, saliva, and oropharyngeal swabs, and the viral loads in urine and plasma were low 12 . These high positive rate and viral loads in skin lesion and rectal swabs are in line with previous findings that direct contact with infectious sores or lesions on mucous membranes has been the primary transmission mode of transmission during the 2022 outbreak 1, 38, 39, 40, 41, 42 . Moreover, high occurrence of genital and perianal lesions further support that close contact during sex might be the dominant form of transmission in the current outbreak as previously reported 41 . Meanwhile, the high positive rate of MPXV in saliva and oropharyngeal swabs the transmission of MPXV through breathing and kissing as previously reported 12, 15 . Furthermore, our viral kinetic analyses revealed that the viral load in skin lesions peaked at around day 6 d.p.o, followed by a gradual decline, with the Ct values > 30 at approximately 17 d.p.o. Similar kinetics were found for the saliva, and oropharyngeal swabs, with much lower viral loads when compared with skin lesion (Figure 1, Figure 2 and Figure S1). Notably, our results from rectal swabs showed the highest viral loads following symptoms onset, and then rapidly decreased with the Ct values > 30 at approximately 7 d.p.o (Figure 2). Moreover, HIV patients had much higher peak viral loads compared to non-HIV patients. These results indicated that highest risk of transmission through skin lesion, saliva and breathing occurred at about 6 d.p.o, while immediately post symptoms onset for contact with the rectal mucosa, especially for MSM with HIV. Therefore, such populations with high risk of transmission should be specially targeted by public health messaging. Environmental contamination in the living spaces of Mpox patients have been confirmed and viable MPXV has also been successfully recovered, suggesting a risk of onward transmission to close contacts or members of the general public present in these locations 30, 43 . In our study, we collected a total of 1633 environmental fomits swabs from 49 patients covering different stages of acute infection. Our results further confirmed the extensive environmental contamination during disease progression, with no statistical differences of Ct values among the 1~7 d.p.o, 8~14 d.p.o and 15~21 d.p.o groups (Table S3). Notably, a substantial proportion samples with Ct values between 15-30 were found, with the highest for deposition area, followed by floor and bedside cupboard (Figure 1B). Moreover, the proportion of samples with Ct values between 15-30 were higher in 8~14 d.p.o and 15~21 d.p.o groups in most cases (Figure 1C). These results suggest the potentially high prevalence of infection-competent MPXV around living spaces of Mpox patients and the high risk of transmission to close contact. Although our study was based on the specialist health-care environment, while the large scale and longitudinal sampling provide us solid evidence about the environmental contamination in the living spaces of Mpox patients, which could be widely applied to other spaces and settings where Mpox patients stay prolonged periods. Therefore, it is of great importance for the surface cleaning protocols, the use of appropriate personal protective equipment (PPE), and robust doffing procedures to avoid potential onward transmission to family members and healthcare staff 30, 44 . Until recently, researchers have developed several mRNA vaccines and sub-unit vaccines based on different set of antigens 31, 45, 46, 47, 48, 49, 50, 51 . Several surface proteins of MPXV, including M1R, E8L, H3L, A29L on the intracellular mature virion, A35R, and B6R proteins on the extracellular enveloped virion, has been found to be potential vaccine targets for MPVX 29 . However, which protein could serve as the main antigen to induce strong neutralizing immunity against MPXV remained controversial in these studies, which might associate with the differences in the design, modification, and composition of antigens, the experimental settings, and the used animal models 52 . Moreover, nearly all the evaluation of the neutralizing and protection efficiency were done with VACV, while the intrinsic differences between VACV and MPXV might influence the application of obtained results. Our results from the Mpox patients showed that the A29L specific IgG raised rapidly after symptoms onset, and the titers of A29L specific IgG were highly correlated with the neutralizing titers. These data suggest A29L is an essential antigen component to induce neutralizing antibody during vaccine development. Moreover, the similar seropositivity rates of the surface proteins between HIV and Non-HIV participants provides valuable information on the immunogenicity of MPXV antigens in immunocompromised populations. Previously, all MPXV infections have been thought to be symptomatic and serve as the main source of transmission through close contact 20, 53 . Studies have shown that the secondary attack rate (SAR) of MPXV was estimated to be 10% for unvaccinated household contacts Mpox cases with the Congo Basin monkeypox clade, and even lower for the Western African clade accounting for the current endemic 54 . Therefore, outbreak of Mpox in the general population can be quickly contained and tends toward extinction in the absence of repeated animal-to-human transmission, evidenced by several outbreaks in endemic regions non-endemic countries 55, 56, 57 . Similarly, the first confirmed Mpox case in Shenzhen was found on June 11, 2023 7 , then Shenzhen has experienced a mini-wave of MPXV infection between until August 10, 2023, followed by sporadic cases (Figure S1). Of note, fever clinic was the most common (66.23%) medical setting at first visit for the case series, and it is easy to be ignored about the possibility of Mpox if the doctor lacks such experience. Cases with initial symptom of influenza-like symptoms were found in our cohort and other studies 22 . Moreover, etiological and serological evidences of asymptomatic MPXV infection and individuals without recognized symptoms have been reported 20, 21 . Although asymptomatic carriership was thought to play a negligible role in the spread of orthopox viruses, undiagnosed MPXV infections may play a much more significant role in the transmission of MPXV due to the dense sexual network of MSM men 20 , as studies have shown that the basic reproduction number (R0) for MPXV greatly exceeds 1 among networks of MSM compared to R0 levels below 1 in non-MSM sexual networks 58, 59 . There are limitations about our study. Firstly, we only detected the DNA of MPXV, and virus isolation assays were not undertaken during the analyses. Secondly, all the enrolled patients discharged within 21 d.p.o, and the viral clearance were not observed for most of them. Therefore, we could not analyze the time for viral clearance. In conclusion, our results contribute to an improved understanding of the sample selection for higher laboratory diagnosis accuracy at different stages of infection, the viral shedding kinetics of multiple sites from Mpox patients, the transmission risk of fomits during disease progression, and the antibody response characteristics against the main neutralizing antibody-eliciting antigens of MPXV. With the ongoing outbreak of Mpox worldwide, these data have profound implications for the diagnosis, treatment, prevention of transmission and development of vaccines for Mpox. Declarations Contributors YY, YL and HL conceived and designed the study. YY, CS and LG contributed to the analysis and interpretation of data. LY, FW, YX and TH enrolled of patients and collection of samples. SS, SN, YP, SZ, JC, LZ and YC carried out the experiment. LY, JL and SC collected the clinical data. YY and SS drafted the article. YY, LY, SS, CS and SN have accessed and verified all the data reported in this study. All authors reviewed and revised the manuscript and approved the final version. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. Declaration of interests The authors have declared that no competing interests exist. Data sharing The data supporting the findings of this study are available in the manuscript or appendix. Further requests might require ethical approval and should contact the corresponding authors. Acknowledgments This work was supported by grants National Science and Technology Major Project (2021YFC0863300), National Natural Science Foundation of China (32170939), and the Shenzhen High-level Hospital Construction Fund (23250G1001 and XKJS-CRGRK-004). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3869240","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267413128,"identity":"ffd61a45-839a-4b98-9441-ea61921906e1","order_by":0,"name":"Yang 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B-D: Comparative positive rates and viral loads among paired samples of skin lesions, rectal swab, saliva and oropharyngeal swabs during 1~7 d.p.o (B), 8~14 d.p.o (C) and 15~21 d.p.o (D). Ct values between different sample types were analyzed using Wilcoxon signed-rank test, and \u003cem\u003ep\u003c/em\u003e values less than 0.05 were considered statistically significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3869240/v1/9d370e9f21e9e7762e945dde.png"},{"id":50384754,"identity":"a84518ed-116c-4e6e-8b67-2d89c6b6875c","added_by":"auto","created_at":"2024-01-30 17:31:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1020059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViral shedding dynamics of MPXV in different site of Mpox patients. \u003c/strong\u003eA, C, E and G show the overall viral shedding dynamics of MPXV in skin lesions (A), rectal swab (C), saliva (E) and oropharyngeal swabs (G) from Mpox patients. B, D, F and H show the comparative viral shedding dynamics of MPXV in skin lesions(B), rectal swab (D), saliva(F) and oropharyngeal swabs (H) from Mpox patientswith (HIV) or without HIV (Non-HIV). The kinetics of Ct values during hospitalization were calculated by the LOESS (locally estimated scatterplot smoothing) curve fitting polynomial regression using R.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3869240/v1/9f033dce2ae7779662ce7c3e.png"},{"id":50384753,"identity":"741f98e7-e189-4925-8683-54d99792de9a","added_by":"auto","created_at":"2024-01-30 17:31:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":485046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of the Ct values of MPXV DNA on different environmental fomits from Mpox patients in association with disease progression. \u003c/strong\u003eA: Comparison of the Ct values of MPXV DNA on different environmental fomits from Mpox patients. B: Overall distribution of the Ct values of MPXV DNA on different environmental fomits from Mpox patients. C: Distribution of the Ct values of MPXV DNA on different environmental fomits from Mpox patients in association with disease progression.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3869240/v1/07c5ba9a13d2ce04dce24aa2.png"},{"id":50384755,"identity":"78a78eb6-64e2-42fb-8b3c-3ee24510e77e","added_by":"auto","created_at":"2024-01-30 17:31:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":696541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMPXV specific antibody characteristics of acute infections. \u003c/strong\u003eA: Overall seropositivity of the specific IgG response against different surface proteins of MPXV, including A29L, A35R, B6R, E8L, H3L and M1R proteins. B and C: Comparative seropositivity of the specific IgG response against A29L (B) and H3L (C) in HIV and Non-HIV participants. D and E: Dynamics A29L (D) and H3L (E) specific IgG endpoint titers. F and G: The linear correlation analyses between the A29L (F) and H3L (G) specific IgG endpointtiters and nAb titers.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3869240/v1/f13a3860c712e78ac5444901.png"},{"id":57250408,"identity":"d1de769a-3858-49ff-ac21-2263c5c904b9","added_by":"auto","created_at":"2024-05-28 07:08:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4339593,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3869240/v1/4fe6e52a-8713-4902-8a20-280a3daf10b5.pdf"},{"id":50384756,"identity":"4f56b6f2-a9d3-4b70-a775-b479a79680e2","added_by":"auto","created_at":"2024-01-30 17:31:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":775803,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementarymaterials24.01.14.docx","url":"https://assets-eu.researchsquare.com/files/rs-3869240/v1/64a4fea3d03256fb0c2dc17b.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Longitudinal viral shedding and antibody response characteristics of men with acute infection of monkeypox virus: a prospective cohort study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMpox (formerly known as monkeypox) is a zoonotic illness caused by the monkeypox\u003c/p\u003e \u003cp\u003evirus (MPXV), and the first case of human Mpox case was reported in 1970 in the Democratic Republic of the Congo\u003csup\u003e1\u003c/sup\u003e. Thereafter, sporadic cases caused by two distinct clades were confined to Central Africa (Clade 1) and West Africa (Clade 2) for decades until international transmission was first detected in the United States in 2003\u003csup\u003e2\u003c/sup\u003e. Since May 2022, another new lineage MPXV (Clade 2b) derived from West African clade with certain genetic changes caused a widespread epidemic worldwide, leading to the declaration of the Mpox outbreak a Public Health Emergency of International Concern (PHEIC) by World Health Organization (WHO) on July 23, 2022\u003csup\u003e3, 4\u003c/sup\u003e. The first imported Mpox case into mainland China was found in Sep, 2022, and no subsequent transmission occurred with the rapid diagnosis and isolation\u003csup\u003e5\u003c/sup\u003e. However, Mpox has been endemic in several cities of mainland China currently\u003csup\u003e6, 7, 8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnimal-to-human transmissions were common in early sporadic human Mpox cases, while sustained human-to-human transmission become predominant among the widespread of MPXV\u003csup\u003e1\u003c/sup\u003e. Historically, the transmission of MPXV between humans has been thought to occur primarily through respiratory droplets\u003csup\u003e9, 10\u003c/sup\u003e. However, close contact or sexual contact with infectious sores or lesions on mucous membranes has been thought to be the primary mode of transmission during the 2022 outbreak\u003csup\u003e1, 10\u003c/sup\u003e. Recent studies have found that MPXV can also be detected in specimens from multiple sites of Mpox cases, including saliva, rectal swabs, blood, urine and semen\u003csup\u003e10, 11, 12, 13, 14, 15, 16, 17\u003c/sup\u003e, and skin lesions have been presumed to be the primary source of viral shedding and believed to be optimal for diagnosis\u003csup\u003e1\u003c/sup\u003e. However, one recent study has also found patients with negative MPXV DNA in skin lesions, while positive in other sites, highlighting the important roles of multi-site sampling in the diagnosis of Mpox\u003csup\u003e18\u003c/sup\u003e. Although some studies have compared the diagnostic accuracy of different sample sites, the evaluations with detailed temporal data are currently lacking. Moreover, asymptomatic and atypical MPXV infections which may serve as important source of transmission have been found\u003csup\u003e19, 20, 21, 22\u003c/sup\u003e, and the diagnostic accuracy of some alternative samples remains controversial\u003csup\u003e23\u003c/sup\u003e, posing challenges to laboratory diagnosis of Mpox. Studies have also set out to characterize the dynamics of MPXV shedding, while current understanding was restricted by either the limited number of participants and sampling frequency, short follow-up periods or biasing towards the Mpox patients with relatively mild disease\u003csup\u003e10, 12, 15, 24\u003c/sup\u003e. In addition, transmission of MPXV has also been shown to occur indirectly through contaminated fomites\u003csup\u003e1, 4, 12\u003c/sup\u003e. Therefore, systematic evaluation of the risk of this indirect transmission through environmental fomits is of great value to inform the disinfection and prevention strategies. Previous studies have also shown that MPXV DNA could be detected in different environmental fomits of Mpox patients, while application of the existing data has been hampered by limited number of samples and rooms, and lacking the longitudinal sampling in association with disease progression.\u003c/p\u003e \u003cp\u003eCurrently, there is no authorized Mpox specific vaccine available\u003csup\u003e25\u003c/sup\u003e. Vaccines against smallpox have been known to possess cross protective activity against Mpox, and the Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN, also known as JYNNEOS) has been approved by the US Food and Drug Administration (FDA) for pre-exposure vaccination against Mpox\u003csup\u003e4, 26\u003c/sup\u003e. One recent study has estimated that the vaccine effectiveness of MVA-BN against Mpox was 87% [95% confidence interval (CI): 84\u0026ndash;90%] for one-dose vaccination and 89% (95% CI: 78\u0026ndash;100%) for two-dose vaccination, based on pooled estimates during the 2022 Mpox outbreak\u003csup\u003e27\u003c/sup\u003e. Moreover, low levels of MPXV neutralizing antibodies were found after MVA-BN vaccination in healthy individuals\u003csup\u003e28\u003c/sup\u003e. Accordingly, it is in urgent need to develop Mpox specific vaccines able to effectively limit Mpox. Similar with other orthopoxviruses, MPXV possesses two diseases inducing infectious forms of the intracellular mature virion (IMV) and the extracellular enveloped virion (EEV) containing six surface proteins which were speculated to be the main neutralizing antibody-eliciting antigens\u003csup\u003e29\u003c/sup\u003e. However, little is known about the characteristics of surface proteins specific IgG responses following infection and the protective antigens, which is key for the design and development of MPXV specific vaccines.\u003c/p\u003e \u003cp\u003eIn this prospective cohort study, we systematically analyzed the longitudinal positive rate, viral shedding dynamics of different sample types and environmental fomits from acute MPXV infections in association with disease progression, and the antibody response characteristics against the surface proteins of MPXV.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and participants\u003c/h2\u003e \u003cp\u003eWe did a prospective cohort study on the longitudinal viral shedding and antibody response characteristics from men with acute infection of MPXV admitted to Shenzhen Third People's Hospital during June 9, 2023 to November, 5, 2023 (N\u0026thinsp;=\u0026thinsp;77) (Shenzhen, Guangdong, China), and all the patients were followed-up to discharge. All the patients were living in the negative pressure isolation ward during hospitalization to reduce the possibility of further transmission. Clinical and epidemiological information were collected at the earliest time-point after admission. Each Ct value of quantitative real time PCR (qRT-PCR) from different sample types from the patients and the environmental fomites, and the specific IgG antibody against different MPXV surface proteins (including A29L, A35R, B6R, E8L, H3L and M1R) during hospitalization were detected and collected. For analyses, the participants were grouped based on the status of HIV infection (HIV, N\u0026thinsp;=\u0026thinsp;42 and Non-HIV, N\u0026thinsp;=\u0026thinsp;35). Moreover, all these specimens were further stratified into 3 groups based on the collection time related to the days post symptoms onset (d.p.o), including the 1\u0026thinsp;~\u0026thinsp;7 d.p.o, 8\u0026thinsp;~\u0026thinsp;14 d.p.o and 15\u0026thinsp;~\u0026thinsp;21 d.p.o groups. The study protocol was approved by the Ethics Committees of Shenzhen Third People\u0026rsquo;s Hospital (2021-030), and written informed consents were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and detection of the MPXV DNA\u003c/h2\u003e \u003cp\u003eSerial oropharyngeal swabs, saliva, rectal swab, skin lesions, urine and plasma samples were collected by professional nurse every two to three days. Samples from the fomits were also serially collected by the staffs in the Department of Healthcare-associated Infection Management every two days before the disinfection and clean. After the sample collection, the rooms were cleaned every 24 hours using 5000 ppm available chlorine sodium hypochlorite on all hard surfaces and floors, and 10000 ppm available chlorine sodium hypochlorite for the toilet, shower, wash basins as previously reported\u003csup\u003e30\u003c/sup\u003e. All the samples were subjected for the detection of MPXV DNAs using commercial kits (GeneoDX Co., Ltd.) based on qRT-PCR according to manufacturer\u0026rsquo;s instruction. The specimens were considered positive if the Ct value was \u0026lt;\u0026thinsp;40, and negative if the results were undetermined. The sequences of infected virus were determined by next-generation sequencing (BGI Co., Ltd.) and the genotypes were analyzed using the Nextclade (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://clades.nextstrain.org/\u003c/span\u003e\u003cspan address=\"https://clades.nextstrain.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assays (ELISAs)\u003c/h2\u003e \u003cp\u003eThe MPXV surface proteins specific IgG antibodies in plasma specimens were detected using Enzyme-linked immunosorbent assays (ELISA) as previously reported and run in triplicate\u003csup\u003e31\u003c/sup\u003e. Briefly, microtiter plates (Sangon Biotech) were coated overnight at 4\u0026deg;C with 100 ng of each recombinant protein of MPXV, including A29L, H3L, B6R, A35R, E8L and M1R, which was expressed in E. coli expression system and purified by Ni-NTA. The plates were then washed twice with 1 \u0026times; PBS (phosphate buffer saline) containing 0.1% v/v Tween-20 (PBST) and blocked with blocking solution (1 \u0026times; PBS containing 5% w/v bovine serum albumin) overnight at 4\u0026deg;C. On the day of the experiment, the plates were washed once with PBST. The 3-fold serially diluted sera were added to the wells and incubated at 37\u0026deg;C for 60 min. Then the plates were washed five times using PBST and 100 \u0026micro;L horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody or HRP-conjugated goat anti-human IgM antibody solution (Sangon Biotech) was added to each plate, respectively, and incubated at 37\u0026deg;C for 60 min. Wash each plate with PBST five times, and 100 \u0026micro;L of tetramethylbenzidine (TMB) substrate (Sangon Biotech) was added to each plate at room temperature in the dark and reacted for 15 min. After the reaction, the plate was stopped with a 2M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution. The absorbance was measured at 450 nm using a microplate reader. The ELISA titers were determined by endpoint dilution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFocus reduction neutralization test (FRNT)\u003c/h2\u003e \u003cp\u003eThe neutralizing antibody (nAb) titers of the plasma samples against MPXV were detected using focus reduction neutralization test (FRNT) assay with clinical isolate of MPXV (hMpxV/China/SZ-SZTH41/2023, EPI_ISL_18213375) following the procedure as previously reported with modification\u003csup\u003e32\u003c/sup\u003e. All plasma samples were heat-inactivated at 56\u0026deg;C for 30 min before use. Serial dilutions of tested plasma samples were mixed with an equal volume monkeypox live virus (200 focus forming units) in 96-well microwell plates and incubated at 37\u0026deg;C for 1 h in the presence of 10% guinea pig serum (Beijing Bersee Technology Co., Ltd) as a source of complement. Mixtures were then transferred to 96-well plates seeded with 1.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e Vero E6 cells per well and incubated at 37\u0026deg;C for 18 h. Then the supernatant was removed and cells were fixed with 4% paraformaldehyde solution for 30 min, and permeabilized with 0.15% Triton X-100 for 10 min, followed by incubating with HRP-conjugated Vaccinia Virus Polyclonal Antibody (Invitrogen) for 2 h at room temperature. The reactions were developed with KPL TrueBlue Peroxidase substrates (Seracare Life Sciences Inc.). The numbers of MPXV foci were calculated using an EliSpot reader (Cellular Technology Ltd). Neutralizing antibody titers were calculated as a 50% inhibitory dose (ID\u003csub\u003e50\u003c/sub\u003e) expressed as the dilution of plasma that resulted in a 50% reduction of the numbers of MPXV foci compared with virus control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were done as previously reported\u003csup\u003e33, 34\u003c/sup\u003e. The neutralizing titers of ID\u003csub\u003e50\u003c/sub\u003e were calculated using a five-parameter dose-response curve in Graphpad Prism. For descriptive analysis, data were presented as median (interquartile range, IQR) for continuous parameters and frequency (percentage) for categorical variables. Chi-squared and Fisher's exact tests were used to compare categorical variables, while for continuous variables the t-test was used for normal data and the Mann-Whitney U test for non-normal data. The Spearman rank correlation coefficient was used for linear correlation analysis between the ELISA titers and nAb titers. The kinetics of Ct values and IgG levels during hospitalization were calculated by the LOESS (locally estimated scatterplot smoothing) curve fitting polynomial regression using R version 4.1.0. \u003cem\u003eP\u003c/em\u003e value less than 0\u0026middot;05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data supporting the findings of this study are available within the paper and the supplementary information files. Source data are provided with this paper.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBaseline characteristics of the cohort\u003c/h2\u003e \u003cp\u003eDuring June 11, 2023 and November 11, 2023, a total of 139 laboratory confirmed Mpox patients were found in Shenzhen, China (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and all the patients were men. Totally, 77 out of 81 hospitalized patients were enrolled in this study with a median age of 30 years (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and 42 patients were HIV-positive. Among the 77 patients, 72 patients were men who have sex with men (MSM) and 5 patients received smallpox vaccination. Skin lesions were the most common initial symptoms with 98.7% (76/77) positive rate, followed by some influenza-like symptoms including fever (71.43%), sore throat (61.04%), myalgia (35.06%). Lymphadenopathy and rectitis were found in 41.56% and 19.48% of the patients, respectively. There were no significant differences of the initial symptoms between HIV and Non-HIV participants, except a significantly higher prevalence of rectitis in the HIV participant (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The median days between symptoms onset to admission is 5, and median days hospitalization is 7. Fever clinic was the most common (66.23%) medical setting at first visit, followed by Department of Dermatology (9.09%), HIV clinic (7.79%), Department of Emergency (6.49%) and Anorectal surgery clinic (5.19%). The most common location of skin lesions were the Trunk and extremities (77.92%), genitals (62.34%), face (50.65%) and perianal region (37.66%), and a small portion on the hands and feet (12.99%). No participants received specific antiviral treatment for MPXV infection. MPXV genomes were sequenced from all the 77 participants and all the MPXV belonged to a newly defined lineage C.1 in the West African clade.\u003c/p\u003e \u003cp\u003eAltogether, a total of 993 specimens from these patients were serially collected during hospitalization, including 166 oropharyngeal swabs, 154 saliva, 162 rectal swabs, 173 skin lesions, 156 urine and 182 plasma samples. Moreover, a total of 1633 environmental fomits swabs from 49 patients were also serially collected, including 89 Floor swabs, 125 Call button swabs, 124 Light switch swabs, 94 Television remote control swabs, 124 Bed handrail swabs, 125 Bedside cupboard swabs, 107 Chair (arm rest) swabs, 93 Door handle (patient room to bathroom) swabs, 93 Deposition area (Air conditioning air outlet) swabs, 125 Mobile phone swabs, 125 Clothes swabs, 125 Pillow swabs, 94 Toilet flush handle swabs, 95 Shower handle swabs and 95 Delivery window swabs (Table S2). These specimens were further stratified into 3 groups based on the collection time, including the 1\u0026thinsp;~\u0026thinsp;7 d.p.o, 8\u0026thinsp;~\u0026thinsp;14 d.p.o and 15\u0026thinsp;~\u0026thinsp;21 d.p.o groups (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePositive rate and dynamics of MPXV in multiple sites of Mpox patients\u003c/h2\u003e \u003cp\u003eOverall, about 78.95% (60/76), 83.78% (62/74), 88.16% (67/76), 100.00% (76/76), 55.26% (42/76) and 31.17% (24/76) participants showed positive detection of MPXV DNA in oropharyngeal swab, saliva, rectal swab, skin lesions, urine and plasma samples (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with no differences between the HIV and Non-HIV groups. For the three groups based on the collection date, all the skin lesions showed the highest positive rates, followed by rectal swab, saliva, oropharyngeal swab, urine and plasma. Of note, positive rates of 91.67%, 58.33% and 56.52% were found from the skin lesions, rectal swab and saliva samples in the 15\u0026thinsp;~\u0026thinsp;21 d.p.o group, respectively, while oropharyngeal swab only showed a positive rate of 25%. Moreover, only 2 plasma sample was positive for MPXV DNA in the 15\u0026thinsp;~\u0026thinsp;21 d.p.o group. Furthermore, we have also analyzed the positive rate of 143 paired samples of skin lesions, rectal swab, saliva and oropharyngeal swab. Generally, 139, 103, 101 and 70 positive samples were found for skin lesions, rectal swab, saliva and oropharyngeal swab, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the 1\u0026thinsp;~\u0026thinsp;7 d.p.o group, the positive rates were similar among rectal swab, saliva and oropharyngeal swab. However, the positive rates of rectal swab and saliva were higher than oropharyngeal swab both in the 8\u0026thinsp;~\u0026thinsp;14 d.p.o and 15\u0026thinsp;~\u0026thinsp;21 d.p.o groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The viral loads (indicated as Ct values) were also analyzed, and the highest viral loads were found in the skin lesions, followed by rectal swab, saliva and oropharyngeal swab (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, there is no statistical differences on the positive rate and viral loads between HIV and Non-HIV groups, except a significantly higher positive rate of the urine during 15\u0026thinsp;~\u0026thinsp;21 d.p.o in the Non-HIV group and significantly higher viral loads in the rectal swab in the HIV group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetection of MPXV DNA in multiple sites of Mpox cases during disease progression.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCollection date\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample types\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eMpox patients\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHIV-positive (N\u0026thinsp;=\u0026thinsp;42)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHIV-negative (N\u0026thinsp;=\u0026thinsp;35)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep values\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverall\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive rate (%, n/N)\u003c/b\u003e\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOropharyngeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.95 (60/76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.57 (33/42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79.41 (27/34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaliva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83.78 (62/74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.18 (34/39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.00 (28/35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5311\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRectal swab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.16 (67/76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.86 (39/42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82.35 (28/34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2841\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSkin lesions*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.00 (76/76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.00 (42/42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.00 (34/34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.26 (42/76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.10 (23/41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54.29 (19/35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.17 (24/77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.10 (16/42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.86 (8/35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1465\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u0026thinsp;~\u0026thinsp;7 d.p.o\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive rate (%, n/N)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOropharyngeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.21 (47/66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.85 (27/41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.00 (20/25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2703\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaliva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.58 (44/59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.29 (26/35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.00 (18/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRectal swab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.41 (46/61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.38 (29/37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e70.83 (17/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5527\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSkin lesions*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.00 (74/74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.00 (49/49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.00 (25/25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.37 (27/57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.42 (14/33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54.17 (13/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4295\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.56 (14/57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.33 (11/33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.50 (3/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1183\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCt values (median; range)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOropharyngeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33 (31\u0026ndash;35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33 (29.5\u0026ndash;35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.9 (31\u0026ndash;35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2563\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaliva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (27\u0026ndash;34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.5 (26.3\u0026ndash;33.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.3 (28.2\u0026ndash;33.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7185\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRectal swab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (22\u0026ndash;31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 (21\u0026ndash;27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33 (24\u0026ndash;34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSkin lesions*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 (22\u0026ndash;26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 (23\u0026ndash;26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23 (21-24.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.3717\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35 (29.7\u0026ndash;37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.9 (29.5\u0026ndash;37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36 (30.4\u0026ndash;37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7418\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.8 (33.7\u0026ndash;36.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.5 (33.9\u0026ndash;36.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.1 (34.3\u0026ndash;36.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u0026thinsp;~\u0026thinsp;14 d.p.o\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive rate (%, n/N)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOropharyngeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.00 (42/75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.38 (22/42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.61 (20/33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4936\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaliva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.86(51/73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.92 (30/39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e61.76 (21/34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2040\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRectal swab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.73 (56/77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.74 (33/43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.65 (23/34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4438\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSkin lesions*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.95 (71/74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.24 (40/42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96.88 (31/32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.19 (29/74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.90 (18/41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33.33 (11/33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4731\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.44 (13/90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.21 (7/53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.22 (6/37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7649\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCt values (median; range)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOropharyngeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33 (30 -35.15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.1 (30.1\u0026ndash;35.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.5 (29.5\u0026ndash;35.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.8400\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaliva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32 (28\u0026ndash;35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.5 (27.25-36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32 (31\u0026ndash;34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5962\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRectal swab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.65 (26\u0026ndash;35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32 (26-35.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33 (28.5\u0026ndash;35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2525\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSkin lesions*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (22\u0026ndash;29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.5 (22-29.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26 (22.3\u0026ndash;28.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7125\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33 (30.4\u0026ndash;34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33 (30.8\u0026ndash;35.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33 (29.35-34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.7 (35.7\u0026ndash;37.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.4 (35.3\u0026ndash;36.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37 (36.3\u0026ndash;37.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1353\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15\u0026thinsp;~\u0026thinsp;21 d.p.o\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive rate (%, n/N)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOropharyngeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.00 (6/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.67 (4/15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.22 (2/9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaliva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.52 (13/23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.14 (8/14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55.56 (5/9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRectal swab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.33 (14/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.00 (9/15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55.56 (5/9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSkin lesions*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.67 (22/24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93.33 (14/15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88.89 (8/9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.13 (9/23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.00 (3/15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.00 (6/8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0228\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.71 (2/35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.53 (2/19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00 (0/16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4891\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCt values (median; range)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOropharyngeal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.6 (28.8\u0026ndash;33.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.1 (26.7\u0026ndash;32.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.5 (31.8\u0026ndash;33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4456\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaliva\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34 (27\u0026ndash;36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.1 (26.5\u0026ndash;36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34 (34\u0026ndash;37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.8951\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRectal swab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.5 (27\u0026ndash;36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28 (27-30.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37 (30\u0026ndash;38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1088\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSkin lesions*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28 (24.3\u0026ndash;31.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28 (24.3\u0026ndash;32.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28 (25.25\u0026ndash;31.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.3914\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35 (29\u0026ndash;36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36 (31.4\u0026ndash;37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.5 (30.3\u0026ndash;35.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7896\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlasma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.1 (31.9\u0026ndash;32.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.1 (31.9\u0026ndash;32.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e\u0026amp;\u003c/sup\u003eIndividuals with positive detection of MPXV DNA in the indicated sample types.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*Include swabs of lesion surface and exudate.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eNA: Not available.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003ed.p.o: Days post symptoms onset.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen we analyzed the viral dynamics in different types of specimens from men with MPXV infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figure S2). For the skin lesions, viral loads slightly increased and reached the peak at about 6 d.p.o, and then slightly decreased to reach Ct values of 30 at about 17 d.p.o (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). For the rectal swab, high viral loads were found at the at the beginning of symptoms onset, and decreased rapidly with the disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). For the saliva and oropharyngeal swab, similar dynamics were found with the skin lesions, while the viral loads were much lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). When comparing between HIV and the Non-HIV groups, the dynamics and peak viral load were similar for the skin lesions, saliva, and oropharyngeal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and Figure S2). Notably, much higher viral loads during the early phase of infection and peak viral loads were found in the rectal swabs from the HIV group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and Figure S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePersistent detection of MPXV DNA on the environmental fomites from Mpox patients\u003c/h2\u003e \u003cp\u003eTotally, 52.66% (860/1633) of environmental fomits swabs were positive for MPXV DNA, with 62.92% (56/89) of the Floor swabs, 43.2% (54/125) of the Call button swabs, 37.1% (46/124) of the Light switch swabs, 44.68% (42/94) of the Television remote control swabs, 59.68% (74/124) of the Bed handrail swabs, 61.6% (77/125) of the Bedside cupboard swabs, 54.2% (58/107) Chair (arm rest) swabs, 45.16% (42/93) of the Door handle (patient room to bathroom) swabs, 69.89% (65/93) of the Deposition area (Air conditioning air outlet) swabs, 52.8% (66/125) of the Mobile phone swabs, 56.8% (71/125) of the Clothes swabs, 68% (85/125) of the Pillow swabs, 45.74% (43/94) of the Toilet flush handle swabs, 47.37% (45/95) of the Shower handle swabs and 37.89% (36/95) Delivery window swabs, respectively. The mean Ct values of these samples were 32.83, and Ct values showed significant differences among different swabs (anova, \u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.008\u003c/em\u003e), among which Ct values from the deposition area is the lowest (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Table S3). By further dividing the samples according to their collection time, we found no statistical differences among the 1\u0026thinsp;~\u0026thinsp;7 d.p.o, 8\u0026thinsp;~\u0026thinsp;14 d.p.o and 15\u0026thinsp;~\u0026thinsp;21 d.p.o groups (Table S3). Further analysis showed that the proportion of Ct values between 15\u0026ndash;30 were highest for deposition area (21.35%), followed by floor (18.05%), bedside cupboard (14.37%) and then the pillow (11.72%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Moreover, the proportion of samples with Ct values between 15\u0026ndash;30 increased with the disease progression for most environmental fomites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAntibody characteristics of men with acute MPXV infection\u003c/h2\u003e \u003cp\u003ePlasma samples were also serially collected from these 67 participants and subjected for the test of MPXV specific IgG antibody response, including A29L, A35R, B6R, E8L, H3L and M1R proteins. About 94.02%, 16.42%, 41.79%, 35.82%, 80.6% and 2.99% individuals showed positive IgG response against A29L, A35R, B6R, E8L, H3L and M1R, respectively, with no differences between HIV and Non-HIV groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Then we further analyzed the anti-A29L and anti-H3L IgG in detail. The positive rate for anti-A29L and anti-H3L IgG during 1\u0026thinsp;~\u0026thinsp;7 d.p.o could reach 86.36 and 79.55, and increased to 100% and 94.74% during 15\u0026thinsp;~\u0026thinsp;21 d.p.o, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The endpoint titers of anti-A29L IgG increased obviously along with the disease progression and reached the peak at about 12 d.p.o, while no increase was found for anti-H3L IgG titers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The dynamics of anti-A29L and anti-H3L IgG titers were similar between HIV and Non-HIV groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Moreover, ID\u003csub\u003e50\u003c/sub\u003e of 67 plasma samples against authentic MPXV were detected using FRNT assay, and neutralizing activity was detected in 97.92% (47/48) of the samples collected after 7 d.p.o. The Spearman rank correlation coefficient analyses showed a high correlation between ID\u003csub\u003e50\u003c/sub\u003e and anti-A29L IgG titer, while not between ID\u003csub\u003e50\u003c/sub\u003e and anti-H3L IgG titer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe qRT-PCR assay has been regarded as the gold standard and the most common method for the diagnosis of MPXV infection\u003csup\u003e23\u003c/sup\u003e. Viral shedding patterns varied in different site of viral infections\u003csup\u003e10, 35\u003c/sup\u003e, and diagnostic accuracy of different sample types also varied in association with disease progression\u003csup\u003e36\u003c/sup\u003e.\u0026nbsp;In our study, the\u0026nbsp;specimens were further stratified into 1~7 d.p.o, 8~14 d.p.o and 15~21 d.p.o groups based on the collection time, covering different stages of acute viral infections. Consistent with previous studies\u003csup\u003e10, 12, 17\u003c/sup\u003e,\u0026nbsp;skin lesions possessed the highest sensitivity for the diagnosis of MPXV infection during the whole infection course, followed by rectal swabs, saliva and then the oropharyngeal swab during disease progression.\u0026nbsp;(Table 1 and Figure 1). It is worth noting that the positive rate of\u0026nbsp;oropharyngeal swab is similar to that of rectal swabs and saliva during\u0026nbsp;1~7 d.p.o, while decreased dramatically and showed much lower positive rate between\u0026nbsp;8~21 d.p.o, which might explain the\u0026nbsp;controversial results about the diagnostic accuracy of oropharyngeal swab\u003csup\u003e23\u003c/sup\u003e. The positive rates of rectal swabs and saliva maintain similar during disease progression and close to the positive rates of skin lesions, indicating that these two types of specimens could serve as excellent alternatives for the diagnosis of MPXV infection, especially for the asymptomatic and atypical infections when skin lesions are not available. In addition, serological assessment of MPXV infection could be a complementary method for the diagnosis of Mpox, and useful in some other settings including identification of self-attenuated infection, assessing population seroprevalence to determine asymptomatic\u003csup\u003e37\u003c/sup\u003e. Our result from the serological response to the six main neutralizing-eliciting antigens of MPXV following infection demonstrated a notably high seropositivity rate of A29L specific IgG, then the H3L specific IgG (Figure 4). Furthermore, the seropositivity of A29L and H3L-specific IgG within 7 d.p.o could reach about 80%, suggesting two prominent serological markers for the early diagnosis of MPXV infection.\u003c/p\u003e\n\u003cp\u003eHuman-to-human transmission of MPXV has been shown to occur through direct contact with skin lesions, body fluids, or respiratory droplets from infected humans\u003csup\u003e1, 9, 12\u003c/sup\u003e, therefore, viral load and viral shedding dynamics of different sample types have important implications on transmissibility, treatment and public policy\u003csup\u003e33\u003c/sup\u003e.\u0026nbsp;In our study, we enrolled a total of 77 patients (42 patients were HIV-positive), and a total of 993 different types of specimens from these patients were serially collected during hospitalization for the analyses of viral dynamics. Consistent with previous studies, skin\u0026nbsp;lesion\u0026nbsp;showed the highest viral loads, followed by anal swabs, saliva, and oropharyngeal swabs, and the viral loads in urine and plasma were low\u003csup\u003e12\u003c/sup\u003e. These high positive rate and viral loads in skin lesion and rectal swabs are in line with previous findings that direct contact with infectious sores or lesions on mucous membranes has been the primary transmission mode of transmission during the 2022 outbreak\u003csup\u003e1, 38, 39, 40, 41, 42\u003c/sup\u003e. Moreover, high occurrence of genital and perianal lesions further support that close contact during sex might be the dominant form of transmission in the current outbreak as previously reported\u003csup\u003e41\u003c/sup\u003e. Meanwhile, the high positive rate of MPXV in saliva and oropharyngeal swabs the transmission of MPXV through breathing and kissing as previously reported\u003csup\u003e12, 15\u003c/sup\u003e. Furthermore, our viral kinetic analyses revealed that the viral load in skin lesions peaked at around day 6 d.p.o, followed by a gradual decline, with the Ct values \u0026gt; 30 at approximately 17 d.p.o. Similar kinetics were found for the saliva, and oropharyngeal swabs, with much lower viral loads when compared with skin lesion (Figure 1, Figure 2 and Figure S1). Notably, our results from rectal swabs showed the highest viral loads following symptoms onset, and then rapidly decreased with the Ct values \u0026gt; 30 at approximately 7 d.p.o (Figure 2). Moreover, HIV patients had much higher peak viral loads compared to non-HIV patients. These results indicated that highest risk of transmission through skin lesion, saliva and breathing occurred at about 6 d.p.o, while immediately post symptoms onset for contact with the rectal mucosa, especially for MSM with HIV. Therefore, such populations with high risk of transmission should be specially targeted by public health messaging.\u003c/p\u003e\n\u003cp\u003eEnvironmental contamination in the living spaces of Mpox patients have been confirmed and viable MPXV has also been successfully recovered, suggesting a risk of onward transmission to close contacts or members of the general public present in these locations\u003csup\u003e30, 43\u003c/sup\u003e. In our study, we collected a total of\u0026nbsp;1633 environmental fomits swabs from 49 patients covering different stages of acute infection. Our results further confirmed the extensive environmental contamination during disease progression, with no statistical differences of Ct values among the 1~7 d.p.o, 8~14 d.p.o and 15~21 d.p.o groups (Table S3). Notably, a substantial proportion samples with Ct values between 15-30 were found, with the highest for deposition area, followed by floor and bedside cupboard (Figure 1B). Moreover, the proportion of samples with Ct values between 15-30 were higher in 8~14 d.p.o and 15~21 d.p.o groups in most cases (Figure 1C). These results suggest the potentially high prevalence of infection-competent MPXV around\u0026nbsp;living spaces of Mpox patients and\u0026nbsp;the high risk of transmission to close contact.\u0026nbsp;Although our study was based on the specialist health-care environment, while the large scale and\u0026nbsp;longitudinal\u0026nbsp;sampling provide us solid evidence about the environmental contamination in the living spaces of Mpox patients, which could be widely applied to other spaces and settings where Mpox patients stay prolonged periods.\u0026nbsp;Therefore, it is of great\u0026nbsp;importance for the surface cleaning protocols, the use of appropriate personal protective equipment (PPE), and robust doffing procedures to avoid potential onward transmission to family members and healthcare staff\u003csup\u003e30, 44\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUntil recently, researchers have developed several mRNA vaccines and sub-unit vaccines based on different set of antigens\u003csup\u003e31, 45, 46, 47, 48, 49, 50, 51\u003c/sup\u003e. Several surface proteins of MPXV, including M1R, E8L, H3L, A29L on the intracellular mature virion, A35R, and B6R proteins on the extracellular enveloped virion, has been found to be potential vaccine targets for MPVX\u003csup\u003e29\u003c/sup\u003e. However, which protein could serve as the main antigen to induce strong neutralizing immunity against MPXV remained controversial in these studies, which might associate with the differences in the design, modification, and composition of antigens, the experimental settings, and the used animal models\u003csup\u003e52\u003c/sup\u003e. Moreover, nearly all the evaluation of the neutralizing and protection efficiency were done with VACV, while the intrinsic differences between VACV and MPXV might influence the application of obtained results. Our results from the Mpox patients showed that the A29L specific IgG raised rapidly after symptoms onset, and the titers of A29L specific IgG were highly correlated with the neutralizing titers. These data suggest A29L is an essential antigen component to induce neutralizing antibody during vaccine development.\u0026nbsp;Moreover, the similar seropositivity rates of the surface proteins between HIV and Non-HIV participants provides valuable information on the immunogenicity of MPXV antigens in immunocompromised populations.\u003c/p\u003e\n\u003cp\u003ePreviously, all MPXV infections have been thought to be symptomatic and serve as the main source of transmission through close contact\u003csup\u003e20, 53\u003c/sup\u003e. Studies have shown that the secondary attack rate (SAR) of MPXV was estimated to be 10% for unvaccinated household contacts Mpox cases with the Congo Basin monkeypox clade, and even lower for the Western African clade accounting for the current endemic\u003csup\u003e54\u003c/sup\u003e. Therefore, outbreak of Mpox in the general population can be quickly contained and tends toward extinction in the absence of repeated animal-to-human transmission, evidenced by several outbreaks in endemic regions non-endemic countries\u003csup\u003e55, 56, 57\u003c/sup\u003e. Similarly, the first confirmed Mpox case in Shenzhen was found on June 11, 2023\u003csup\u003e7\u003c/sup\u003e, then Shenzhen has experienced a mini-wave of MPXV infection between until August 10, 2023, followed by sporadic cases (Figure S1). Of note, fever clinic was the most common (66.23%) medical setting at first visit for the case series, and it is easy to be ignored about the possibility of Mpox if the doctor lacks such experience.\u0026nbsp;Cases with initial symptom of\u0026nbsp;influenza-like symptoms\u0026nbsp;were found in our cohort and other studies\u003csup\u003e22\u003c/sup\u003e. Moreover, etiological and serological evidences of asymptomatic MPXV infection and individuals without recognized symptoms have been reported\u003csup\u003e20, 21\u003c/sup\u003e. Although asymptomatic carriership was thought to play a negligible role in the spread of orthopox viruses, undiagnosed MPXV infections may play a much more significant role in the transmission of MPXV due to the dense sexual network of MSM men\u003csup\u003e20\u003c/sup\u003e, as studies have shown that the basic reproduction number (R0) for MPXV greatly exceeds 1 among networks of MSM compared to R0 levels below 1 in non-MSM sexual networks\u003csup\u003e58, 59\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThere are limitations about our study. Firstly, we only detected the DNA of MPXV, and virus isolation assays were not undertaken during the analyses. Secondly, all the enrolled patients discharged within 21 d.p.o, and the viral clearance were not observed for most of them. Therefore, we could not analyze the time for viral clearance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, our results contribute to an improved understanding of the sample selection for higher laboratory diagnosis accuracy at different stages of infection, the viral shedding kinetics of multiple sites from Mpox patients, the transmission risk of fomits during disease progression, and the antibody response characteristics against the main neutralizing antibody-eliciting antigens of MPXV.\u0026nbsp;With the ongoing outbreak of Mpox worldwide, these data have profound implications for the diagnosis, treatment, prevention of transmission and development of vaccines for Mpox.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContributors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYY, YL and HL\u0026nbsp;conceived and designed the study.\u0026nbsp;YY, CS and LG contributed to the analysis and interpretation of data. LY, FW, YX and TH enrolled of patients and collection of samples. SS, SN, YP, SZ, JC, LZ and YC carried out the experiment. LY, JL and SC collected the clinical data. YY and SS drafted the article. YY, LY, SS, CS and SN have accessed and verified all the data reported in this study. All authors reviewed and revised the manuscript and approved the final version. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData sharing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available in the manuscript or appendix. Further requests might require ethical approval and should contact the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants\u0026nbsp;National Science and Technology Major Project (2021YFC0863300), National Natural Science Foundation of China (32170939), and the Shenzhen High-level Hospital Construction Fund (23250G1001 and XKJS-CRGRK-004).\u0026nbsp;The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMitj\u0026agrave; O\u003cem\u003e, et al.\u003c/em\u003e Monkeypox. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e401\u003c/strong\u003e, 60-74 (2023).\u003c/li\u003e\n \u003cli\u003eReed KD\u003cem\u003e, et al.\u003c/em\u003e The detection of monkeypox in humans in the Western Hemisphere. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e350\u003c/strong\u003e, 342-350 (2004).\u003c/li\u003e\n \u003cli\u003eIsidro J\u003cem\u003e, et al.\u003c/em\u003e Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 1569-1572 (2022).\u003c/li\u003e\n \u003cli\u003eLum FM\u003cem\u003e, et al.\u003c/em\u003e Monkeypox: disease epidemiology, host immunity and clinical interventions. \u003cem\u003eNat Rev Immunol\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 597-613 (2022).\u003c/li\u003e\n \u003cli\u003eZhao H\u003cem\u003e, et al.\u003c/em\u003e The First Imported Case of Monkeypox in the Mainland of China - Chongqing Municipality, China, September 16, 2022. \u003cem\u003eChina CDC Wkly\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 853-854 (2022).\u003c/li\u003e\n \u003cli\u003eDou X\u003cem\u003e, et al.\u003c/em\u003e Clinical, epidemiological, and virological features of Mpox in Beijing, China - May 31-June 21, 2023. \u003cem\u003eEmerg Microbes Infect\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2254407 (2023).\u003c/li\u003e\n \u003cli\u003eWan J\u003cem\u003e, et al.\u003c/em\u003e Investigation into the epidemiology, genetic characteristics, and clinical manifestations of the first monkeypox outbreak in Shenzhen, China. \u003cem\u003eBiosafety and Health\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 259-265 (2023).\u003c/li\u003e\n \u003cli\u003eXu T, Zhang L. 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Stochastic model for interhuman spread of monkeypox. \u003cem\u003eAm J Epidemiol\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 1082-1092 (1987).\u003c/li\u003e\n \u003cli\u003eElsayed S, Bondy L, Hanage WP. Monkeypox Virus Infections in Humans. \u003cem\u003eClin Microbiol Rev\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, e0009222 (2022).\u003c/li\u003e\n \u003cli\u003eEndo A\u003cem\u003e, et al.\u003c/em\u003e Heavy-tailed sexual contact networks and monkeypox epidemiology in the global outbreak, 2022. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e378\u003c/strong\u003e, 90-94 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Monkeypox virus, Viral shedding, Neutralizing antibody, Fomits, Diagnostic accuracy","lastPublishedDoi":"10.21203/rs.3.rs-3869240/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3869240/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElucidating the viral and antibody dynamics within the host and the viral dynamics of the environmental fomits is key for the successful public health measures against monkeypox virus (MPXV) infection. In this study, a total of 993 samples from multiple sites of patients and 1,633 samples from environmental fomits of 77 acute MPXV infections (HIV co-infection: N=42) were collected every two to three days and subjected for the detection of MPXV DNA, surface proteins specific antibody and neutralizing titers. Overall, skin lesions showed 100% positive rate of MPXV DNA, followed by rectal swab (88.16%), saliva (83.78%), oropharyngeal swab (78.95%), urine (55.26%)and plasma (31.17%). Positive rate of oropharyngeal swab decreased rapidly after 7 days post symptoms onset (d.p.o), while the rectal swab and saliva maintained close positive rates to the skin lesions. Viral dynamics were similar among skin lesions, saliva and oropharyngeal, withrapid increase to the peak at about 6 d.p.o and then decreased. While for the rectal swab, it peaked at the beginning of symptoms onset, and decreased rapidly with the disease progression. Totally, 52.66% (860/1633) of environmental fomits swabs were positive for MPXV DNA, with highest positive rate (69.89%) and mean viral loads (Ct values of 31.22) from the deposition area. Moreover, the proportion of Ct values between 15-30 maintained high for some fomits like the deposition area, floor and bedside cupboard during the disease progression.High seropositivity against A29L (100%) and H3L (94.74%) were detected, while the high correlation was only found between anti-A29L IgG titer and neutralizing titer. Notably, most of indexes were similar between HIV and Non-HIV participants, except the higher viral loads in the early stage and peak viral from the rectal swabs of the HIV participants. Our results contribute to an improved understanding of viral shedding and antibody response characteristics of acute Mpox. With the ongoing outbreak of Mpox worldwide, these data have profound implications for the diagnosis, treatment, prevention of transmission and development of vaccines for Mpox.\u003c/p\u003e","manuscriptTitle":"Longitudinal viral shedding and antibody response characteristics of men with acute infection of monkeypox virus: a prospective cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-30 17:31:48","doi":"10.21203/rs.3.rs-3869240/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c69e7ded-bd74-4afd-8c8b-7b7c2aca240d","owner":[],"postedDate":"January 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28172252,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":28172253,"name":"Biological sciences/Microbiology/Infectious-disease diagnostics"}],"tags":[],"updatedAt":"2024-05-28T07:08:10+00:00","versionOfRecord":{"articleIdentity":"rs-3869240","link":"https://doi.org/10.1038/s41467-024-48754-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-05-27 04:00:00","publishedOnDateReadable":"May 27th, 2024"},"versionCreatedAt":"2024-01-30 17:31:48","video":"","vorDoi":"10.1038/s41467-024-48754-8","vorDoiUrl":"https://doi.org/10.1038/s41467-024-48754-8","workflowStages":[]},"version":"v1","identity":"rs-3869240","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3869240","identity":"rs-3869240","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}