BACKGROUND:This study aims to evaluate the effectiveness of an adapted methicillin-resistant Staphylococcus aureus (MRSA) decolonization program in an infirmary unit in Hong Kong that was inspired by successful interventions implemented in Orange County, California. METHODS:Nasal, skin, and rectal swabs were collected to assess MRSA colonization. Decolonization involved applying 10% povidone-iodine ointment to the anterior nares twice daily for five days every other week, along with twice weekly chlorhexidine gluconate (CHG) bathing for six months. Compliance with the application of povidone-iodine and CHG bathing techniques was monitored by measuring their respective levels in the anterior nares and on the skin. Air and environmental samples were collected and analyzed over time using linear regression. RESULTS:Among 60 patients in the infirmary unit (78% baseline MRSA carriers), overall MRSA colonization declined during the program, driven by significant reductions in skin colonization (65% to 29%, P < .001). Environmental contamination on high-touch patient-care equipment (bathing trolleys and slings) also significantly decreased over time (P < .001). These reductions coincided with the high-quality implementation of decolonization, evidenced by stable iodophor detection in nares during application weeks and sustained chlorhexidine levels on the skin, detectable 24 hours after bathing. In contrast, MRSA detection in air samples showed no significant change (P = .096), possibly due to dispersal by persistent carriers during care activities even as skin and environmental contamination declined. CONCLUSIONS:The adapted MRSA decolonization program was effective, significantly reducing overall MRSA colonization, especially at skin sites, while achieving high compliance with the protocol.

BACKGROUND:Early identification of patients with carbapenemase-producing Enterobacterales (CPE) colonization is crucial for infection control; however, microbiological testing may delay detection and be costly. Machine learning may enhance predictive analytics for timely identification of at-risk patients. METHODS:Four machine learning models: Decision Tree, Random Forest, Gradient Boosting, and XGBoost, were used to predict CPE colonization within 48 hours of admission using microbiological and demographic data. Model performance was assessed through sensitivity, specificity, and area under the receiver operating characteristic curve (AUROC). Uniform Manifold Approximation and Projection (UMAP) evaluated topological separability of CPE-positive cases and CPE-negative controls. RESULTS:From January 1, 2015 to December 31, 2024, 453,372 fecal specimens were submitted for CPE screening, with 194,917 (43.0%) collected within 48 hours of admission, comprising 3,328 CPE-positive cases (1.7%) and 191,589 CPE-negative controls. The Gradient Boosting classifier showed the best performance, achieving an AUROC of 0.598, sensitivity of 54.4%, and specificity of 59.1%. Demographic factors (age ≥ 75 and male sex), history of hospitalization, and known CPE colonization in the past year, and admission specialty (general medicine and general surgery) were consistently included in all models as top predictors. UMAP revealed significant overlap between CPE-positive and CPE-negative patients, indicating challenges in differentiating the risk profiles. CONCLUSIONS:This study highlights the complexities of using machine learning to predict CPE colonization within 48 hours of admission. The low AUROC values suggest that the models may not effectively predict CPE colonization at the patient level, potentially due to inherent rarity of events and overlapping risk profiles.

Three critically ill or fatal avian influenza A(H5N1) human infections have been reported in North America since November 2024. Notably, all were infected with genotype D1.1 instead of B3.13, the dominant genotype before November 2024. Here, we demonstrated that D1.1 could replicate to higher titers in human nasal and airway organoid–derived transwell monolayers from 6 donors. D1.1 exhibited a better binding to α2,3- and α2,6-linked sialic acid than B3.13. No significant differences in most inflammatory or antiviral cytokines/chemokines were observed. These observations suggest that D1.1 is better adapted to both the upper and lower human respiratory tract epithelium than B3.13.

BACKGROUND:Candidozyma auris has emerged as a global health threat. While air dispersal of C. auris has been reported, its role in non-outbreak settings remains underexplored. METHODS:We implemented proactive infection control measures, including active surveillance, early isolation of cases, contact tracing, and environmental sampling of air grilles, air handling units (AHUs), and commonly shared items to assess C. auris contamination rates using culture and polymerase chain reaction. Patient and environmental isolates were selected for whole-genome sequencing (WGS). RESULTS:Between 1 January 2023 and 31 December 2025, we collected 11,458 specimens from 6310 patients (8999 hospitalization episodes). Of these, 0.4% (25/6310) were colonized with C. auris, with 84% (21/25) diagnosed through active surveillance. Contact tracing of 1499 patients identified four secondary cases, resulting in a transmission rate of 0.3% (4/1499). Among 1854 environmental samples, C. auris was identified in 16.7% (309/1854) of samples, with higher rates in non-air grille sources than air grilles related items (17.5%, 288/1646 vs 10.1%, 21/208; P = 0.007). Contamination rates were significantly higher in items within isolation areas than those outside (33.1%, 209/631 vs 6.0%, 44/730; P < 0.001). For a patient isolated in a side room, C. auris contamination of air return grilles inside the room and supply grilles outside the room showed temporal and spatial association. WGS confirmed clonal relatedness between the patient isolate and air grille isolates. CONCLUSIONS:Our study demonstrates air dispersal of C. auris in non-outbreak settings, highlighting the need for innovative infection control measures.

BACKGROUND:There are few treatment options for patients with chronic hepatitis E unresponsive to ribavirin. Drug repurposing is required to identify new treatments. Molnupiravir, a nucleoside analogue, is approved for the treatment of coronavirus disease 2019 (COVID-19). This study evaluated the activity of molnupiravir against hepatitis E virus (HEV) in cell culture and animal models. METHODS:Cytotoxicity and antiviral efficacy of molnupiravir, ribavirin, and sofosbuvir were investigated using infectious cDNA clones and wild-type HEV isolates in PLC/PRF/5 cells and primary rat hepatocytes. Immunosuppressed rats were infected with HEV and treated with molnupiravir and ribavirin. The effectiveness of molnupiravir in clearing HEV in serum, feces, and liver tissue was compared with that of untreated and ribavirin-treated animals. Mutations arising in virus populations during treatment were assessed using next-generation sequencing. RESULTS:The antiviral effect of molnupiravir was comparable to that of ribavirin and superior to that of sofosbuvir against HEV strains in vitro, with decreased HEV RNA in supernatant (p<0.05) and cytoplasmic viral protein expression. No additive effect with sofosbuvir was observed. Rats (n=14 per group) treated with 400 mg/kg/d molnupiravir cleared viremia within 4 weeks of treatment, and 9/14 of these animals also cleared viral shedding in stool. Mean viremia and fecal viral loads were reduced compared with untreated and ribavirin-treated animals (p≤0.005). Partial effectiveness was apparent at the lower 250 mg/kg/d molnupiravir dose. Molnupiravir-treated rats had improved liver histology compared with control animals. Frequent transition mutations were observed in HEV from molnupiravir-treated animals. CONCLUSIONS:Molnupiravir limits HEV infection in cell culture and animal models. Molnupiravir could be an alternative for ribavirin-refractory chronic hepatitis E.

H9N2 is currently the second most common avian influenza A virus subtype infecting humans. Monitoring viral phenotypic and genotypic adaptation to humans is crucial for risk assessment. Here, we compared the replication of an H9N2 human isolate collected in 2024 (A/HK/2346/2024) to a human isolate collected in 1999 (A/HK/1073/1999). In Madin Darby canine kidney (MDCK) cells, A/HK/2346/2024 and A/HK/1073/1999 replicated to 8 and 5 log10 plaque-forming units (PFU) per ml, respectively. In both human nasal and lung organoids, A/HK/2346/2024 replicated to 6 log10 PFU/ml, but A/HK/1073/1999 failed to replicate in either organoid. The infection rates of both ciliated and non-ciliated cells and the ratios of infected 2,6/2,3 cells were higher for A/HK/2346/2024 than A/HK/1073/1999. Apart from the mammalian adaptive substitutions that were present in the nasopharyngeal specimen collected on day 1 post-symptom onset (pso) (HA-D183N/D190 T/Q192R/Q226L; NA-del62-64; PB2-A588V/K702R; PB1-I368V; PA-K356R/S409N; M1-R95K), the mammalian-adaptive substitution PB2-D253N emerged de novo on day 7 pso. Analysis of all human (n = 96) and avian influenza (n = 14,762) H9N2 deposited at GISAID showed the dominance of several human-adaptive substitutions in H9N2 strains collected from humans in recent years. In summary, we demonstrated that a recent H9N2 virus is more adapted to humans, and is able to replicate to high titres in both upper and lower human respiratory tract which may confer higher person-to-person transmissibility and virulence. Our study underscores the importance of human organoid-based phenotypic monitoring and inter/intrahost genotypic monitoring for assessing the zoonotic risk of avian influenza viruses.

BACKGROUND & AIMS:Rocahepevirus ratti genotype 1 (rat hepatitis E virus; rHEV) is an emerging hepatitis agent that is genetically distinct from conventional human-infecting HEV. Zoonotic assessment of rHEV has been hampered by a lack of human-derived strain sequences and subtyping methods. We aimed to expand the dataset of human-derived rHEV genomes, develop a robust subtyping method, and investigate potential markers of human adaptation. METHODS:Human rHEV cases were detected in Hong Kong by population-wide PCR-based hepatitis surveillance. rHEV genomes were obtained using Sanger sequencing. New sequences were aligned with publicly available rHEV genomes to infer clade and subtype-level cut-offs based on maximum likelihood patristic distances. rHEV codon usage biases and selection pressures were measured, stratified by host, gene, and phylogenetic parameters. Bayesian phylogeny was used to estimate time to most recent common ancestor. RESULTS:Complete or partial rHEV sequences were obtained from 22 human rHEV cases identified in Hong Kong from 2017-2024. Distance-based subtyping of this enhanced dataset revealed two rHEV clades and six subtypes. Human-derived rHEV sequences clustered across both clades and four subtypes, indicating genotype-wide zoonotic potential. An open-source web application was created to classify sequences using this subtyping framework. Significantly lower codon usage bias was identified in ORF1 of human-derived rHEV strains. rHEV strains were well adapted to human codon usage patterns irrespective of the derived host. We observed significant selection relaxation in ORF2 of human-derived rHEV (k = 0.19; p = 5.48E-04), indicating higher tolerance to accumulation of non-synonymous mutations. The Hong Kong rHEV supercluster emerged c.30-50 years ago, decades before the first human case. CONCLUSIONS:rHEV shows genotype-wide potential for human spillover infection across its phylogeny. Strengthened global surveillance and sequencing in both humans and rodents are essential to assess and manage this emerging threat. IMPACT AND IMPLICATIONS:Rat hepatitis E virus (rHEV) is an emerging cause of viral hepatitis in humans, but a comprehensive zoonotic assessment is hampered by a lack of sequences and systematic subtyping methods. Using a new, extended dataset of human-derived rHEV strains, we found a wide phylogenetic distribution of human-derived rHEV globally. Regardless of derived host, rHEV codon adaptation was similar for humans, indicating that all rHEV strains should be assumed to have zoonotic potential. Selection analyses revealed that human-derived sequences may be more tolerant to the accumulation of mutations at ORF2, which encodes the viral capsid. Some human-derived strains exhibited even lower codon usage bias, suggesting enhanced potential for replication and persistence across multiple hosts. These results are relevant to clinicians and policymakers as they prove the importance of instituting rHEV surveillance in humans and rodents.

Neuraminidase inhibitors (NAIs), such as oseltamivir and zanamivir, serve as the primary treatment for influenza virus infection. NAI-resistant influenza A(H1N1) strains were widespread during the 2008–09 influenza season, especially in Japan, where 100% of the strains were resistant to oseltamivir.1 However, after the NAI-susceptible 2009 pandemic subtype (A[H1N1]pdm09) replaced the previous seasonal A(H1N1) subtype in 2009, the incidence of A(H1N1)pdm09 with reduced inhibition (10-fold to 100-fold) or highly reduced inhibition (>100-fold) was only approximately 1% in the 2019–20 influenza season.

Background De novo amino acid substitutions (DNS) frequently emerge among immunocompromised patients with chronic SARS-CoV-2 infection. While previous studies have reported these DNS, their significance has not been systematically studied. Methods We performed a review of DNS that emerged during chronic SARS-CoV-2 infection. We searched PubMed until June 2023 using the keywords “(SARS-CoV-2 or COVID-19) and (mutation or sequencing) and ((prolonged infection) or (chronic infection) or (long term))”. We included patients with chronic SARS-CoV-2 infection who had SARS-CoV-2 sequencing performed for at least 3 time points over at least 60 days. We also included 4 additional SARS-CoV-2 patients with chronic infection of our hospital not reported previously. We determined recurrent DNS that has appeared in multiple patients and determined the significance of these mutations among epidemiologically-significant variants. Findings A total of 34 cases were analyzed, including 30 that were published previously and 4 from our hospital. Twenty two DNS appeared in ≥3 patients, with 14 (64%) belonging to lineage-defining mutations (LDMs) of epidemiologically-significant variants and 10 (45%) emerging among chronically-infected patients before the appearance of the corresponding variant. Notably, nsp9-T35I substitution (Orf1a T4175I) emerged in all three patients with BA.2.2 infection in 2022 before the appearance of Variants of Interest that carry nsp9-T35I as LDM (EG.5 and BA.2.86/JN.1). Structural analysis suggests that nsp9-T35I substitution may affect nsp9-nsp12 interaction, which could be critical for the function of the replication and transcription complex. Interpretation DNS that emerges recurrently in different chronically-infected patients may be used as a marker for potential epidemiologically-significant variants. Funding Theme-Based Research Scheme [T11/709/21-N] of the Research Grants Council (See acknowledgements for full list).

Background: While airborne transmission of rhinovirus is recognized in indoor settings, its role in hospital transmission remains unclear. Methods: We investigated an outbreak of rhinovirus in a pediatric intensive care unit (PICU) to assess air dispersal. We collected clinical, environmental, and air samples, and staff's surgical masks for viral load and phylogenetic analysis. Hand hygiene compliance and the number of air changes per hour in the PICU were measured. A case-control analysis was performed to identify nosocomial rhinovirus risk factors. Results: Between March 31, 2023, and April 2, 2023, three patients acquired rhinovirus in a cubicle (air changes per hour: 14) of 12 -bed PICU. A portable aircleaning unit was placed promptly. Air samples (72,000 L in 6 hours) from the cohort area, and outer surfaces of staff's masks (n = 8), were rhinovirus RNAnegative. Hand hygiene compliance showed no significant differences (31/34, 91.2% vs 33/37, 89.2%, P = 1) before and during outbreak. Only 1 environmental sample (3.8%) was positive (1.86 x 10 3 copies/mL). Case-control and next-generation sequencing analysis implicated an infected staff member as the source. Conclusions: Our findings suggest that air dispersal of rhinovirus was not documented in the well-ventilated PICU during the outbreak. Further research is needed to better understand the dynamics of rhinovirus transmission in health care settings. (c) 2023 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Circovirus human is a new viral species that includes the human circovirus (HCirV), which has been linked to hepatitis in immunocompromised persons. We investigated prevalence of HCirV infection in 278 patients with hepatitis and 184 asymptomatic persons using real-time PCR and sequencing assays. HCirV viremia and sequences were found in 8 (2.9%) hepatitis patients and no asymptomatic patients. Alternate causes of hepatitis (hepatitis E and cholangitis) were clearly identifiable in 2 HCirV-infected patients. HCirV could not be ruled out as a contributor to hepatitis in the remaining 6 patients, 4 of whom were immunocompromised. Persistent infections were documented in 3 patients, but only 1 had relapsing hepatitis. One HCirV patient displayed symptoms of an infectious mononucleosis-like syndrome. Isolates clustered with known HCirV strains from France and China. HCirV-derived virus-like particles bound to PLC/PRF/5 and Hep-G2 human hepatoma cells but not to lung epithelial cells, indicating hepatic tropism.

SARS-CoV-2 Omicron variant has evolved into sublineages. Here, we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However, there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population.

BACKGROUND:The duration and magnitude of SARS-CoV-2 air dispersal during nosocomial outbreaks remain uncertain. This study evaluates the impact of mobile modular high-efficiency particulate air filter units (MMHUs) on SARS-CoV-2 air dispersal. METHODS:We investigated a nosocomial COVID-19 outbreak in an infirmary unit. The viral load (VL) of SARS-CoV-2 in air samples was correlated with the VL in nasopharyngeal swabs with or without MMHU. These samples underwent whole-genome sequencing and phylogenetic analysis. RESULTS:Upon outbreak declaration (August 2, 2024, day 0), 44 (69.8%) of 63 patients acquired COVID-19 in Ward 2B (19 male) and 2C (25 female) by day 4. The VL of SARS-CoV-2 remained detectable in air until day 11 (2B) and day 20 (2C). The VL of air samples was significantly correlated with the VL in nasopharyngeal swabs collected on days 5, 7, 10, and 13 in 2C (r = -0.975, P = .004). Using MMHU, the mean daily ratio of SARS-CoV-2 RNA (copies/1,000 L of air/patient) in 2B was 5 times lower than in 2C from days 5 to 10. Whole-genome sequencing revealed all 41 tested strains belonged to the Omicron variant, KP.3.1.1, phylogenetically related to the prevailing community strains. CONCLUSIONS:Using MMHU mitigates the duration and magnitude of SARS-CoV-2 air dispersal during nosocomial outbreak.

Background The spread of emerging SARS-CoV-2 immune escape sublineages, especially JN.1 and KP.2, has resulted in new waves of COVID-19 globally. The evolving memory B cell responses elicited by the parental Omicron variants to subvariants with substantial antigenic drift remain incompletely investigated. Methods Using the single B cell antibody cloning technology, we isolated single memory B cells, delineated the B cell receptor repertoire and conducted the pseudovirus-based assay for recovered neutralizing antibodies (NAb) screening. We analyzed the cryo-EM structures of top broadly NAbs (bnAbs) and evaluated their in vivo efficacy (golden Syrian hamster model). Findings By investigating the evolution of human B cell immunity, we discovered a new panel of bnAbs arising from vaccinees after Omicron BA.2/BA.5 breakthrough infections. Two lead bnAbs neutralized major Omicron subvariants including JN.1 and KP.2 with IC50 values less than 10 ng/mL, representing ultrapotent receptor binding domain (RBD)-specific class I bnAbs. They belonged to the IGHV3-53/3-66 clonotypes instead of evolving from the pre-existing vaccine-induced IGHV1-58/IGKV3-20 bnAb ZCB11. Despite sequence diversity, they targeted previously unrecognized, highly conserved conformational epitopes in the receptor binding motif (RBM) for ultrapotent ACE2 blockade. The lead bnAb ZCP3B4 not only protected the lungs of hamsters intranasally challenged with BA.5.2, BQ.1.1 and XBB.1.5 but also prevented their contact transmission. Interpretation Our findings demonstrated that class I bnAbs have evolved an ultrapotent mode of action protecting against highly transmissible and broad Omicron escape variants, and their epitopes are potential targets for novel bnAbs and vaccine development. Funding A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

The emergence of SARS-CoV-2 mutations poses significant challenges to diagnostic tests, as these mutations can reduce the sensitivity of commonly used RT-PCR assays. Therefore, there is a need to design diagnostic assays with multiple targets to enhance sensitivity. In this study, we identified a novel diagnostic target, the nsp10 gene, using nanopore sequencing. Firstly, we determined the analytical sensitivity and specificity of our COVID-19-nsp10 assay. The COVID-19-nsp10 assay had a limit of detection of 74 copies/mL (95% confidence interval: 48–299 copies/mL) and did not show cross-reactivity with other respiratory viruses. Next, we determined the diagnostic performance of the COVID-19-nsp10 assay using 261 respiratory specimens, including 147 SARS-CoV-2-positive specimens belonging to the ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using a LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were found to be 100%. The median Cp values for the LightMix E-gene RT-PCR and our COVID-19-nsp10 RT-PCR were 22.48 (range: 12.95–36.60) and 25.94 (range 16.37–36.87), respectively. The Cp values of the COVID-19-nsp10 RT-PCR assay correlated well with those of the LightMix E-gene RT-PCR assay (Spearman’s ρ = 0.968; p < 0.0001). In conclusion, nsp10 is a suitable target for a SARS-CoV-2 RT-PCR assay.

There are only eight approved small molecule antiviral drugs for treating COVID-19. Among them, four are nucleotide analogues (remdesivir, JT001, molnupiravir, and azvudine), while the other four are protease inhibitors (nirmatrelvir, ensitrelvir, leritrelvir, and simnotrelvir-ritonavir). Antiviral resistance, unfavourable drug-drug interaction, and toxicity have been reported in previous studies. Thus there is a dearth of new treatment options for SARS-CoV-2. In this work, a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity. One compound, designated 172, demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern. Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease (3CLpro) by binding to an allosteric site and reduces 3CLpro dimerization. A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro. . In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice. Overall, this study identified an alternative druggable site on the SARS-CoV-2 3CLpro, proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.

Chronic kidney disease (CKD) patients are at higher risk of severe COVID-19. Humoral and cellular immunity from prior infection or vaccination are important for protection, but the neutralizing antibody (nAb) response against SARS-CoV-2 variants is impaired. We investigated the variant-specific nAb and T cell immunity among CKD patients. Adult CKD patients were recruited between August and October 2022. nAb against the SARS-CoV-2 (ancestral strains and four Omicron sublineages) and T cell response were measured using the live virus neutralization assay and interferon-gamma release assay (IGRA). The correlation between nAb/T-cell response and subsequent infection after recruitment were also determined. Among the 88 recruited patients, 95.5% had prior infection or had completed the primary vaccine series. However, only 77.3% had detectable nAb against at least one SARS-CoV-2 strains, 59.1% tested positive in IGRA, and 52.3% had detectable nAb and tested positive in the IGRA. The nAb geometic mean titers (GMTs) against XBB.1, BA.5 and BA.2.3.20 were significantly lower than those against BA.2 and ancestral strain. Prior SARS-CoV-2 infection was associated with elevated nAb and T cell response. More kidney transplant recipients (KTRs) showed absent nAb and T cell response (36.8% vs. 10.1%), despite a higher prevalence of vaccine booster in this population (94.7% vs. 50.7%). Lower levels of nAb titer and T cell response were significantly associated with subsequent infection. A considerable proportion of CKD patients, especially KTRs, showed absence of humoral and cellular protective immunity against SARS-CoV-2. Strategies to improve immunogenicity in this population are urgently needed.

Abstract We report the first imported case of human monkeypox in Hong Kong, manifesting as infectious mononucleosis-like syndrome. Viral load in deep throat saliva was comparable to vesicle swabs collected from face, arm, and back of this patient, suggesting deep throat saliva as an alternative specimen for early diagnosis of monkeypox.

Background Nirmatrelvir-ritonavir (Paxlovid) and ensitrelvir are 3-chymotrypsin-like cysteine protease (3CLpro) inhibitors which have been approved for the treatment of COVID-19 in 2021 and 2022, respectively. Previous studies have identified 3CLpro mutations that are associated with reduced susceptibility to these antivirals. The aim of the current study was to estimate the global prevalence of 3CLpro inhibitor-resistant SARS-CoV-2 strains.Methods We compiled a list of 3CLpro mutations which have been associated with nirmatrelvir or ensitrelvir resis-tance based on either viral replication or 3CLpro activity assays, and determined their prevalence among 13.4 million sequences deposited in GISAID as of December 14, 2022, about 1 year after the approval of nirmatrelvir-ritonavir. We analyzed the prevalence for different time periods, SARS-CoV-2 lineages and geographical locations.Findings Overall, 0.5% (67,095/13,446,588) of the sequences contained at least one mutation that was shown to affect the inhibitory activity of nirmatrelvir or ensitrelvir on viral replication or 3CLpro activity. We did not observe any increasing trend of resistance after the widespread clinical use of nirmatrelvir-ritonavir. G15S (2070 per million) and T21I (1386 per million) were the most prevalent mutations, and these mutations were dominant in some SARS-CoV-2 lineages. E166V and S144E, previously shown to affect the inhibitory activity of nirmatrelvir on viral replication or protease activity by > 100-folds, were found in <1 per million sequences. Interpretation Our data suggest that 3CLpro inhibitor resistance is currently rare. However, continuous global genotypic and phenotypic surveillance would be crucial in the early detection of resistant mutants. Copyright (c) 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).