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Background: How often mpox causes asymptomatic infections, particularly among persons who have received the Modified Vaccinia Ankara (MVA) vaccine, is unknown., Methods: We performed mpox polymerase chain reaction testing on rectal and pharyngeal specimens collected from symptomatic and asymptomatic patients at a sexual health clinic in Seattle, WA, between May 2022 and May 2023. Analyses evaluated the prevalence of asymptomatic or subclinical infection and, among persons with polymerase chain reaction-positive tests, the association of MVA vaccination status with the symptomatic infection., Results: The study population included 1663 persons tested for mpox during 2353 clinic visits. Ninety-three percent of study participants were cisgender men and 96% were men who have sex with men. A total of 198 symptomatic patients (30%) had a first mpox-positive test during 664 visits. Eighteen patients (1.1%) tested during 1689 visits had asymptomatic or subclinical mpox based on a positive rectal or pharyngeal test done in the absence of testing done because of clinical suspicion for mpox. Fourteen (78%) of 18 persons with asymptomatic/subclinical mpox and 53 (26%) of 198 persons with symptomatic mpox had received at least 1 dose of the MVA vaccine ( P < 0.0001). Controlling for calendar month, study subjects who received 1 and 2 doses of MVA vaccine were 4.4 (95% confidence interval, 1.3-15) and 11.9 (3.6-40) times more likely to have asymptomatic versus symptomatic mpox, respectively, than persons who were unvaccinated., Conclusions: Asymptomatic mpox is uncommon. Modified Vaccinia Ankara vaccination is associated with an asymptomatic/subclinical infection among persons with mpox., Competing Interests: Conflict of Interest and Sources of Funding: M.R.G., and O.O.S. P.S., have received research support from Hologic and SpeeDx. C.C. has been a paid consultant to Roche Diagnostics. M.R. has received research support from Nabriva. J.C.D. has received research support from Hologic and Mayne Pharmaceuticals. A.L.G. has done contract laboratory testing for Abbott, Cepheid, Novavax, Pfizer, Janssen, and Hologic, and has received research support from Gilead. E.J.C., E.N.K., M.M., J.P.G., and A.B. have no conflicts of interest. None of the listed conflicts of interest relate to mpox or the work presented in this manuscript., (Copyright © 2024 American Sexually Transmitted Diseases Association. All rights reserved.)

Background: In spring of 2022, an outbreak of monkeypox (mpox) spread worldwide. Here, we describe performance characteristics of monkeypox virus (MPXV)-specific and pan-orthopoxvirus qPCR assays for clinical use., Methods: We validated probe-based qPCR assays targeting MPXV-specific loci F3L and G2R (genes MPXVgp052/OPG065 and MPXVgp002 and gp190/OPG002, respectively) and a pan-orthopoxvirus assay targeting the E9L locus (MPXVgp057/OPG071). Clinical samples and synthetic controls were extracted using the Roche MP96 or Promega Maxwell 48 instrument. qPCR was performed on the AB7500 thermocycler. Synthetic control DNA and high concentration clinical samples were quantified by droplet PCR. Cross-reactivity was evaluated for camelpox and cowpox genomic DNA, vaccinia culture supernatant, and HSV- and VZV-positive clinical specimens. We also tested the performance of the F3L assay using dry swabs, Aptima vaginal and rectal swabs, nasopharyngeal, rectal, and oral swabs, cerebrospinal fluid, plasma, serum, whole blood, breastmilk, urine, saliva, and semen., Results: The MPXV-F3L assay is reproducible at a limit of detection (LoD) of 65.6 copies/mL of viral DNA in viral transport medium/universal transport medium (VTM/UTM), or 3.3 copies/PCR reaction. No cross-reactivity with herpesviruses or other poxviruses was observed. MPXV-F3L detects MPXV DNA in alternative specimen types, with an LoD ranging between 260-1000 copies/mL, or 5.7-10 copies/PCR reaction. In clinical swab VTM specimens, MPXV-F3L and MPXV-G2R assays outperformed OPXV-E9L by an average of 2.4 and 2.8 Cts, respectively. MPXV-G2R outperformed MPXV-F3L by 0.4 Cts, consistent with presence of two copies of G2R present in labile inverted terminal repeats (ITRs) of MPXV genome., Conclusions: MPXV is readily detected by qPCR using three clinically validated assays., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ALG reports contract testing from Abbott, Cepheid, Novavax, Pfizer, Janssen and Hologic and research support from Gilead and Merck, outside of the described work. Dr Kachikis reported serving as a research consultant for Pfizer and GlaxoSmithKline on maternal immunization-related projects in 2020 and as an unpaid consultant for GlaxoSmithKline in 2022 outside the submitted work. Dr Kachikis reported receiving grant support from Merck and Pfizer outside the submitted work., (Copyright © 2022 Elsevier B.V. All rights reserved.)