First laboratory-confirmed case of infection with mpox virus clade IIb lineage A.2.2 in the Republic of the Congo, co-circulation of clade Ia, Ib and clade IIb.

First laboratory-confirmed case of infection with mpox virus clade IIb lineage A.2.2 in the Republic of the Congo, co-circulation of clade Ia, Ib and clade IIb. | 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 First laboratory-confirmed case of infection with mpox virus clade IIb lineage A.2.2 in the Republic of the Congo, co-circulation of clade Ia, Ib and clade IIb. Félix Koukouikila-Koussounda, Claude Kwe Yinda, Pembe Issamou Mayengue, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7401723/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Apr, 2026 Read the published version in Nature Medicine → Version 1 posted You are reading this latest preprint version Abstract In 2022, mpox virus (MPXV) clade IIb emerged resulting in a global epidemic driven by human-to-human transmission mostly through sexual contact in the MSM population. To date, published data on the circulation of the MPXV clade IIb in the Central African region are absent. Here, we describe the first case of laboratory-confirmed mpox with MPXV clade IIb lineage A2.2 in Pointe-Noire, the second largest city of the Republic of the Congo (RoC). Whole genome phylogenetic analysis placed the MPXV in clade IIb, lineage A.2.2. currently emerging in West Africa, in particular Sierra Leone. The detection of clade IIb mpox, marks the third distinct MPXV clade and lineage co-circulating in the human population, together with clade Ia and clade Ib. This underscores the need for improved surveillance and diagnostic strategies to identify the respective clade and lineage circulating in the human population. Strengthening of the regional capacity for case detection, contact tracing, public health measures, and affordable vaccines are urgently needed to reduce the global risk for both clades I and Clade II MPXV. Biological sciences/Microbiology/Virology/Pox virus Health sciences/Diseases/Infectious diseases/Viral infection Health sciences/Health care/Diagnosis/Genetic testing Figures Figure 1 Figure 2 Introduction Mpox, is a viral zoonotic disease endemic in the Central and West African regions 1 . The disease is caused by two different clades of the mpox virus (MPXV), I and II, which are each further subdivided into subclades Ia and Ib, and IIa and IIb, respectively 2,3 . Historically, Clade I MPXV circulation was observed in Democratic Republic of the Congo (DRC), Republic of the Congo (RoC), Central African Republic, South Sudan, Gabon and Cameroon, whereas Clade 2 circulation was observed in Sierra Leone, Liberia, Ivory Coast, Ghana, Benin, Cameroon and Nigeria 4,5 . Cameroon has historically been the only country in which circulation of both Clade I and Clade II MPXV has been observed 6 . Clade IIb emerged early May 2022 causing a global epidemic of mpox that was declared for the first time a public health emergency of international concern (PHEIC) by the World Health Organization (WHO) on July 23, 2022 1 . The Clade IIb outbreak was characterized by rapid expansion of the disease in non-endemic countries especially in Europe and North America driven by sexual transmission among men who have sex with men (MSM) 4 . In August 2024, due to the rapid rising number of mpox cases caused by clade Ia and Ib in the DRC and neighboring countries 3,7 , the Africa Centers for Disease Control and Prevention (Africa CDC) has listed mpox as Public Health Emergency of Continental Security (PHECS). Subsequently, the WHO declared mpox a PHEIC for the second time since 2022 8,9 . Currently West Africa, in particular Sierra Leone is facing a major mpox Clade IIb outbreak of a novel clade IIb lineage A.2.2.1 virus derived from Nigerian A.2.2 viruses 10,11 . Similar to what has been observed for clade Ib, heterosexual contact transmision appears to be one of the drivers of this epidemic 12-15 . The RoC has experienced a dramatic increase in mpox cases over the last two years 16 . Historically Clade Ia has caused outbreaks primarily in the northern part of the country 17,18 , but several recent introductions of Clade Ib from Kinshasa, DRC were identified by us 2024 and 2025 19 . Both clade Ia and Ib, now display characteristics of sustained human to human transmission based on their apobec3 signatures in the genome 20 . Here we report the first detection of the introductions of clade IIb mpox in the RoC, highlighting the continuous evolving epidemiology of MPXV. Methods Case identification and sample collection The case is a 43-year-old male residing in Pointe-Noire which is the second largest city and the economic capital of the RoC. The patient reported a recent travel history to France and Ivory Coast. Pointe-Noire is divided into 6 health districts namely Lumumba, Mvou-Mvou, Tie-Tie, Loandjili, Ngoyo and Mongo-Mpoukou. Each health district includes several primary public health centers, one referral public hospital and private healthcare facilities. The patient was identified when visiting a private outpatient clinic in the sanitary district of Lumumba. He presented to the attending physician of the clinic on the morning of March 22, 2025 with complaints of fever, asthenia and skin rash in the genital area. This led the physician to initially suspect syphilis. After close physical examination of the patient, he noticed the presence of disseminated skin lesions on different parts of the body with vesicles (less than 15) and pustules on his limbs, trunk, back and buttocks. Diagnostic tests for syphilis, HIV, hepatitis B and hepatitis C were negative. Based on the clinical description the case was therefore referred to the disease control team of the Lumumba health district as a suspected mpox case. Skin lesion and blood samples were collected and transferred to the National Public Health Laboratory in Brazzaville for molecular diagnostics. The patient was isolated at home, given paracetamol and primalan for 5 days, and cyteal for the treatment of skin infection. The patients made a full recovery after 19 days. No transmission of the MPXV to family members or health care workers was observed. MPXV molecular detection Skin lesion and blood samples were inactivated in a class III biosafety cabinet and DNA was extracted using the High Pure PCR Template Preparation kit (Roche, Mannheim, Germany), following the manufacturer’s instructions. Extracted DNA was immediately kept at -20°C until MPXV DNA detection. Real-time PCR assays were performed using the XpertMpox cartridge (Cepheid, Sunnyvale, CA, USA) and RADI FAST Mpox detection kit (KH Medical Co.Ltd, South Korea) according to manufacturer’s instructions. The RADI FAST Mpox detection kit included specific primers for MPXV clade I and clade II targets. All assays were run on the QuantStudio 5 Real-Time PCR system (Applied Biosystems, foster City, CA, USA). Whole genome sequencing and bioinformatics analysis Nucleic acids extracted from the clinical samples were amplified using a mpox virus-specific primer set covering the MPXVgenome. 21 Amplification products were purified, then prepared for ONT sequencing using the Native Barcoding Kit 96 v14 (SQK-NBD114.96) according to the manufacturer's instructions. This method allows the addition of unique sequencing adapters and barcodes to each sample, without additional PCR, thus minimizing amplification bias. PCR products were then quantified using the Qubit instrument with the high-sensitivity QubitTM 1X dsDNA assay kit (ThermoFisher Scientific, Waltham, Mass, USA). Barcoded libraries were loaded onto an R10.4.1 flowcell and sequenced using an Oxford Nanopore GridION sequencer (Oxford Nanopore, Oxford, UK). Bioinformatics analysis A structured bioinformatics pipeline was implemented to analyze raw reads from Oxford Nanopore Technologies, following the steps below: Raw reads were subjected to a preliminary quality check using NanoPlot (v.1.44.1), to generate descriptive statistics (length distributions, quality scores) and identify any anomalies in the data. Residual adaptor sequences were automatically detected and removed using Porechop (v.0.2.4), optimizing read quality for subsequent steps. Rigorous filtering was applied using Filtlong (v.0.2.1), retaining only reads with a minimum length of 1500 bases, with an average quality score above 10 and representing the most reliable 80% of the initial set. The filtered reads were aligned against a reference sequence using Minimap2 (v.2.29-r1283) in map-ont mode, specifically optimized for long reads. The resulting alignments were then sorted and indexed with Samtools (v.1.21) for efficient exploitation. The consensus sequence was generated from the aligned reads using Medaka (v.2.0.1), a deep learning-based tool for correcting errors specific to ONT reads. The generated consensus sequence was submitted to Nextclade (https://clades.nextstrain.org/) for annotation, phylogenetic classification (clade assignment), and mutational event detection (substitutions, deletions, insertions). Phylogenetic analysis Preliminary overall phylogenetic tree was constructed using Nextclade including all sequences from RoC and Nextclade dataset "Mpox virus (All clades)”. Full-length genomes of historical clade IIb A.2.2 MPXV sequences were obtained from GISAID and Pathoplexus and used to construct a more detailed tree. Sequences were aligned using MAFFT (FFT-NS-1 algorithm) 22 , with the best model for distance estimates identified with the ModelFinder function 23 as the one with the lowest Bayesian information criterion (BIC). Maximum likelihood phylogenetic tree was constructed using IG-TREE2 24 and branch support was assessed using both ultrafast bootstrap approximation (ufBoot, 1000 replicates) 25 and SH-like approximate likelihood ratio test (SH-aLRT). The tree was visualized in FigTree ( http://tree .bio.ed.ac.uk/software/figtree/) and rooted using a clade IIb A.2 sequence. Bars indicate nucleotide substitutions per site. Ethics Specimen collection and laboratory confirmation were conducted under the passive national surveillance program. Written informed consent was obtained from the case without allowing publication of images. Ethical clearance was obtained from the Congolese Foundation for Medical Research Institutional Ethics Committee (Avis n°053/CEI/FCRM/2024). Results Laboratory results The initial diagnostic result for the suspect mpox case on the GeneExpert was mpox clade II positive. Cycle threshold (Ct) value of 22 for the skin lesion sample and a Ct-value of 34.8 for the blood sample and non-variola positive (generic assay to detect all non-variola orthopox viruses including mpox clade I and II) with a Ct-value of 21.7 for skin lesion sample and Ct value of 32.5 for the blood sample. In the second analysis using the RADI Fast Mpox detection kit, the result was positive for clade 2 with a Ct-value of 17 for the skin lesion sample and Ct-value of 33 for the blood sample. Passive surveillance data of mpox in Republic of the Congo January – August 2025. Fifty-six mpox cases from January 2025 to August 2025 were identified in RoC using the above-described molecular diagnostic and sequencing pipelines. The samples were obtained from cases from 7 of the 12 administrative departments of the RoC namely, Brazzaville, Pointe-Noire, Likouala, Cuvette, Kouilou, Plateaux and Niari. During this time 16 cases of clade Ia, 32 cases of clade Ib, and the two introductions of clade IIb were identified. Epidemiological data suggest that during this period in 2025 several lineages of clade Ia, as well as clade Ib are co-circulating with the major administrative (Brazzaville) and economic (Pointe-Noire) hubs being the hot spots of the sustained human-to-human transmission of mpox in RoC (Figure 1). Phylogenetic analysis Whole-genome sequences were obtained from an mpox-positive specimen, with a genome length of 197,200 base pairs. Phylogenetic analysis identified this sequence as MPXV clade IIb, lineage A.2.2 (Figure 2). The Congolese sequence clustered with recent lineage A.2.2 sequences from Nigeria, Togo, the United States of America (USA), and Australia, forming a clade that branches into the new G.1 cluster, which is composed mostly of sequences from Sierra Leone. Discussion Since 2022, molecular diagnostics for MPXV has been established at the NPHL, Brazzaville, RoC allowing for the implementation of a passive mpox surveillance system in the country. In 2023, next generation sequencing capacity was added, and the passive surveillance system allows for molecular diagnostics of mpox suspect cases, clade and lineage defining qRT-PCR assays and the ability to perform molecular epidemiological studies using genomic mpox data. Our mpox surveillance successfully identified the co-circulation of multiple Clade Ia lineages in RoC in 2025, identified the introduction of Clade Ib from the Democratic Republic of the Congo on two independent occasions in 2024 and now identified the first identified introduction of Clade IIb. Phylogenetic analysis placed the clade IIb MPXV into the lineage A.2.2. MPXV Clade IIb has been circulating cryptically for nearly a decade in Nigeria. The ongoing circulation of Clade IIb in Nigeria, including distinct sub lineages as the A.2.2., have resulted the recently identified regional outbreaks in Sierra Leone, as well as travel related cases outside Africa, including German, USA and Australia. The rapid spread in West Africa, introduction into Central Africa and travel related cases outside Africa bears resemblance to the rapid global spread of the global mpox outbreak in 2022 as the A2 lineage also originates from Nigeria and has spread throughout West Africa and has been detected in Australia, Europe and the USA. In the phylogenetic tree, our clade IIb sequence was of the A.2.2. lineage and clustered with sequences of MPXV isolates from West Africa, USA, Germany and Australia all collected in 2025. Together with the recent travel history to France and Ivory Coast, his suggests that the mpox patient infected with clade IIb MPXV likely contracted the infection while traveling in West Africa or Europe. The rapid rise of clade IIb lineage 2.2 highlights the vulnerability of the region for undetected circulation of mpox. It also underscores significant gaps in regional preparedness, in particular the availability of diagnostics and the ability to conduct molecular epidemiology. Our patient was not able to provide comprehensive contact-tracing information to health workers, showing the complexity of tracking imported cases. This work presents epidemiological and phylogenetic characteristics of the first laboratory-confirmed mpox case with MPXV clade IIb, lineage A.2.2 in Pointe-Noire, RoC. The detection of this first case of clade IIb in this region, highlights the growing complexity of mpox epidemiology in an historically endemic region. In 2025, cases of Clade Ia, Ib, and now IIb have been detected in RoC. The current genetic and epidemiological data from RoC suggest that each of these MPXV clades and sublineages harbors distinct human to human characteristics based on apobec 3 signatures and epidemiological characteristics. 16,19,26 . Strengthening of the regional capacity for case detection, contact tracing, public health measures, and affordable vaccines are needed to implement interventions to reduce the global risk for both Clade I and Clade II MPXV. 27 Declarations Specimen collection and laboratory confirmation were conducted under the passive national surveillance program. Written informed consent was obtained from the case for publishing without allowing publication of images. Ethical clearance was obtained from the Congolese Foundation for Medical Research Institutional Ethics Committee (Avis n°053/CEI/FCRM/2024). Author contributions FKK, DAEB, RGE and FRN conceived and designed the study; FKK, DAEB, RGE, GDI, VRBA, LHL, and ISO processed samples and generated the data; CKY, VJM, PIM, LNM, IJL, JELD and FRN accessed and verified all the data; FKK, DAEB, FRN and CKY curated, analyzed and interpreted data; FKK, DAEB, CKY, VJM, and RGE wrote the first draft of the manuscript; ISO, JMK, AGN, PIM, EML, NPMD, AFA, CKY, VJM and FRN revised the manuscript. All authors provided critical revision of the manuscript. All authors had full access to all the data of the study and had final responsibility for the decision to submit for publication. Acknowledgements We thank all the health workers who were involved in identification the case, epidemiological information, collection of specimens and transfer of samples to the NPHL. We express our gratitude to Africa CDC, WHO Congo and CDC Atlanta for continuous support. Disclosure statement The authors declare no competing interest. Funding This work received no external funding and was fully supported by the National Public Health Laboratory. CKY and VJM were supported by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH authors were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. Data availability statement Whole genome sequences of the MPXV generated in this study are available at GISAID XX - XX References WHO. Multi-country monkeypox outbreak in non-endemic countries. World Health Organization2022. Ulaeto D, Agafonov A, Burchfield J, et al. New nomenclature for mpox (monkeypox) and monkeypox virus clades. Lancet Infect Dis 2023;23(3):273-275. 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Felix Koukouikila-Koussounda PIM, Golmard Reiche Elenga, Ghislain Dzeret Indolo, Yanne Vanessa Thiécesse Mavoungou, Reize Vishnou Ampiri, Lucette Nathalie Macosso, Igor Judicaël Louzolo, Valchy Bel-Beby Miegakanda, Jean Medar Kankou, Eric Maurice Leroy, Nicole Prisca Makaya Dangui, Dachel Aymard Eyenet Boussam, Claude Kwe Yinda, Vincent Jacobus J. Munster, Fabien Roch Niama. Molecular detection of mpox virus in clinical samples from suspected cases identified through a passive surveillance program in the Republic of the Congo, 2022-2024: positivity rate and characteristics of laboratory-confirmed versus negative cases. Lancet Microbe 2025;in press. Ndembi N, Folayan MO, Komakech A, et al. Evolving Epidemiology of Mpox in Africa in 2024. N Engl J Med 2025;392(7):666-676. DOI: 10.1056/NEJMoa2411368. Additional Declarations There is NO Competing Interest. 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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-7401723","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":505371007,"identity":"2efbb9dd-99e7-4c0e-9d64-6e12eafe78db","order_by":0,"name":"Félix Koukouikila-Koussounda","email":"","orcid":"","institution":"Laboratoire National de Santé Publique (LNSP)","correspondingAuthor":false,"prefix":"","firstName":"Félix","middleName":"","lastName":"Koukouikila-Koussounda","suffix":""},{"id":505371008,"identity":"231901cf-6cef-4821-a7f0-fd6cb876aa4b","order_by":1,"name":"Claude Kwe Yinda","email":"","orcid":"","institution":"National Institute of 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(LNSP)","correspondingAuthor":false,"prefix":"","firstName":"Isaac","middleName":"Samuel","lastName":"Onyakouang","suffix":""},{"id":505371018,"identity":"e350eec9-2840-4e4c-bd5b-c021b4c9b57f","order_by":11,"name":"Jordy Exaucé Lyelet Demboux","email":"","orcid":"","institution":"Laboratoire National de Santé Publique (LNSP)","correspondingAuthor":false,"prefix":"","firstName":"Jordy","middleName":"Exaucé Lyelet","lastName":"Demboux","suffix":""},{"id":505371019,"identity":"4cc2f316-eef5-4c57-b872-fe9b6ff70ea3","order_by":12,"name":"Jean-Médard Kankou","email":"","orcid":"","institution":"Laboratoire National de Santé Publique (LNSP)","correspondingAuthor":false,"prefix":"","firstName":"Jean-Médard","middleName":"","lastName":"Kankou","suffix":""},{"id":505371020,"identity":"adeecd75-5f10-4ee4-a0ec-b9671f0bb70a","order_by":13,"name":"Aristide Gilbert Nianga","email":"","orcid":"","institution":"Directorate of Epidemiology and Disease Control, Ministry of Health and Population","correspondingAuthor":false,"prefix":"","firstName":"Aristide","middleName":"Gilbert","lastName":"Nianga","suffix":""},{"id":505371021,"identity":"b563c571-7a38-4df5-aa46-0f865f4b6e7a","order_by":14,"name":"Avelin Aghokeng","email":"","orcid":"","institution":"Research Institute for Development (RID), MIVEGEC","correspondingAuthor":false,"prefix":"","firstName":"Avelin","middleName":"","lastName":"Aghokeng","suffix":""},{"id":505371006,"identity":"0019c3c6-cb01-4736-88bf-b1defce74641","order_by":15,"name":"Vincent Munster","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACZjS+HAMzD1FaDCCcAwwMxoS1MKBpSWxgIKDF4DjvwQ8fd/xh0J2Re/jzh4p76dvZeQ8wfqk4jFvLYb5kyZlnDBjMbuSlSRw4U5y7s5kvgVnmDG4tks08Zsy8bSAtOWYMB9sScjcc5jFglmxLw6/lL0SL8YeD/xLSDQhp4WcGamGEaDGQONiQkADSwvixzQafFmPJ3jZjHrMzb8wkzhxLMAQ57DDDGdxa2PjPGH742SYnZ3Yc6LCKmgR5g/NnDB/+qJDAqQUGUOPiMHGxiQwYf5CsZRSMglEwCoYxAACc6k4ILOJRtAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2288-3196","institution":"National Institute of Allergy and Infectious Diseases","correspondingAuthor":true,"prefix":"","firstName":"Vincent","middleName":"","lastName":"Munster","suffix":""},{"id":505371022,"identity":"acafb528-68e3-409b-9d33-5f737eb87ffa","order_by":16,"name":"Fabien Roch Niama","email":"","orcid":"","institution":"Laboratoire National de Santé Publique (LNSP)","correspondingAuthor":false,"prefix":"","firstName":"Fabien","middleName":"Roch","lastName":"Niama","suffix":""}],"badges":[],"createdAt":"2025-08-18 16:46:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7401723/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7401723/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41591-026-04256-2","type":"published","date":"2026-04-03T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89993550,"identity":"0a09dad5-1e29-4fe5-b1b6-13a10fa38ab4","added_by":"auto","created_at":"2025-08-27 07:45:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":97953,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the Republic of the Congo (RoC) showing departments with mpox-positive cases in 2015. Pie size represents the number of mpox cases, and colors indicate the number of cases per subclade.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7401723/v1/d76174ab2b4ba8c97348b842.png"},{"id":89993552,"identity":"b41e8f06-8f34-4db6-b7bf-82ce251be076","added_by":"auto","created_at":"2025-08-27 07:45:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":490441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Phylogenetic analysis of MPXV nucleotide sequences of all clades and lineages, with the contemporary MPXV genomes of clade Ia and Ib, and Clade IIb obtained from the surveillance in the Republic of the Congo depicted as red circles in the phylogenetic tree. \u003cstrong\u003eB\u003c/strong\u003e. Close-up tree of clade IIb, lineage A.2, with the colors of the circles representing the origin of the MPXV genome, with clade IIb obtained from the surveillance in the Republic of the Congo depicted as red circle in the phylogenetic tree. The tree was visualized in FigTree (\u003ca href=\"http://tree.bio.ed.ac.uk/software/figtree/\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/a\u003e). Scale bar indicates nucleotide substitutions per site.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7401723/v1/8c0bd330635428da5f430177.png"},{"id":106138322,"identity":"76a9cd5a-596d-4981-87c6-8f33332b2ee1","added_by":"auto","created_at":"2026-04-04 07:10:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1170064,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7401723/v1/6fbd3094-80ad-4fc8-ba5c-301eeb1a04c1.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"First laboratory-confirmed case of infection with mpox virus clade IIb lineage A.2.2 in the Republic of the Congo, co-circulation of clade Ia, Ib and clade IIb.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMpox, is a viral zoonotic disease endemic in the Central and West African regions\u003csup\u003e1\u003c/sup\u003e. The disease is caused by two different clades of the mpox virus (MPXV), I and II, which are each further subdivided into subclades \u0026nbsp;Ia and Ib, and IIa and IIb, respectively\u003csup\u003e2,3\u003c/sup\u003e. Historically, Clade I MPXV circulation was observed in Democratic Republic of the Congo (DRC), Republic of the Congo (RoC), Central African Republic, South Sudan, Gabon and Cameroon, whereas Clade 2 circulation was observed in Sierra Leone, Liberia, Ivory Coast, Ghana, Benin, Cameroon and Nigeria\u003csup\u003e4,5\u003c/sup\u003e. Cameroon has historically been the only country in which circulation of both Clade I and Clade II MPXV has been observed\u003csup\u003e6\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClade IIb emerged early May 2022 causing a global epidemic of mpox that was declared for the first time a public health emergency of international concern (PHEIC) by the World Health Organization (WHO) on July 23, 2022\u003csup\u003e1\u003c/sup\u003e. The Clade IIb outbreak was characterized by rapid expansion of the disease in non-endemic countries especially in Europe and North America driven by sexual transmission among men who have sex with men (MSM)\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn August 2024, due to the rapid rising number of mpox cases caused by clade Ia and Ib in the DRC and neighboring countries\u003csup\u003e3,7\u003c/sup\u003e, the Africa Centers for Disease Control and Prevention (Africa CDC) has listed mpox as Public Health Emergency of Continental Security (PHECS). Subsequently, the WHO declared mpox a PHEIC for the second time since 2022\u003csup\u003e8,9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCurrently West Africa, in particular Sierra Leone is facing a major mpox Clade IIb outbreak of a novel clade IIb lineage A.2.2.1 virus derived from Nigerian A.2.2 viruses\u003csup\u003e10,11\u003c/sup\u003e. Similar to what has been observed for clade Ib, heterosexual contact transmision appears to be one of the drivers of this epidemic\u003csup\u003e12-15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe RoC has experienced a dramatic increase in mpox cases over the last two years\u003csup\u003e16\u003c/sup\u003e. Historically Clade Ia has caused outbreaks primarily in the northern part of the country\u003csup\u003e17,18\u003c/sup\u003e, but several recent introductions of Clade Ib from Kinshasa, DRC were identified by us 2024 and 2025\u003csup\u003e19\u003c/sup\u003e. Both clade Ia and Ib, now display characteristics of sustained human to human transmission based on their apobec3 signatures in the genome\u003csup\u003e20\u003c/sup\u003e. Here we report the first detection of the introductions of clade IIb mpox in the RoC, highlighting the continuous evolving epidemiology of MPXV.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCase identification and sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe case is a 43-year-old male residing in Pointe-Noire which is the second largest city and the economic capital of the RoC. The patient reported a recent travel history to France and Ivory Coast. Pointe-Noire is divided into 6 health districts namely Lumumba, Mvou-Mvou, Tie-Tie, Loandjili, Ngoyo and Mongo-Mpoukou. Each health district includes several primary public health centers, one referral public hospital and private healthcare facilities. The patient was identified when visiting a private outpatient clinic in the sanitary district of Lumumba. He presented to the attending physician of the clinic on the morning of March 22, 2025 with complaints of fever, asthenia and skin rash in the genital area. This led the physician to initially suspect syphilis. After close physical examination of the patient, he noticed the presence of disseminated skin lesions on different parts of the body with vesicles (less than 15) \u0026nbsp;and pustules on his limbs, trunk, back and buttocks. Diagnostic tests for syphilis, HIV, hepatitis B and hepatitis C were negative. Based on the clinical description the case was therefore referred to the disease control team of the Lumumba health district as a suspected mpox case.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSkin lesion and blood samples were collected and transferred to the National Public Health Laboratory in Brazzaville for molecular diagnostics. The patient was isolated at home, given paracetamol and primalan for 5 days, and cyteal for the treatment of skin infection. The patients made a full recovery after 19 days. No transmission of the MPXV to family members or health care workers was observed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMPXV molecular detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSkin lesion and blood samples were inactivated in a class III biosafety cabinet and DNA was extracted using the High Pure PCR Template Preparation kit (Roche, Mannheim, Germany), following the manufacturer’s instructions. Extracted DNA was immediately kept at -20°C until MPXV DNA detection. Real-time PCR assays were performed using the XpertMpox cartridge (Cepheid, Sunnyvale, CA, USA) and RADI FAST Mpox detection kit (KH Medical Co.Ltd, South Korea) according to manufacturer’s instructions. The RADI FAST Mpox detection kit included specific primers for MPXV clade I and clade II targets. All assays were run on the QuantStudio 5 Real-Time PCR system (Applied Biosystems, foster City, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole genome sequencing and bioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNucleic acids extracted from the clinical samples were amplified using a mpox virus-specific primer set covering the MPXVgenome.\u003csup\u003e21\u003c/sup\u003e Amplification products were purified, then prepared for ONT sequencing using the Native Barcoding Kit 96 v14 (SQK-NBD114.96) according to the manufacturer's instructions. This method allows the addition of unique sequencing adapters and barcodes to each sample, without additional PCR, thus minimizing amplification bias. PCR products were then quantified using the Qubit instrument with the high-sensitivity QubitTM 1X dsDNA assay kit (ThermoFisher Scientific, Waltham, Mass, USA). Barcoded libraries were loaded onto an R10.4.1 flowcell and sequenced using an Oxford Nanopore GridION sequencer (Oxford Nanopore, Oxford, UK).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA structured bioinformatics pipeline was implemented to analyze raw reads from Oxford Nanopore Technologies, following the steps below: Raw reads were subjected to a preliminary quality check using NanoPlot (v.1.44.1), to generate descriptive statistics (length distributions, quality scores) and identify any anomalies in the data. Residual adaptor sequences were automatically detected and removed using Porechop (v.0.2.4), optimizing read quality for subsequent steps. Rigorous filtering was applied using Filtlong (v.0.2.1), retaining only reads with a minimum length of 1500 bases, with an average quality score above 10 and representing the most reliable 80% of the initial set. The filtered reads were aligned against a reference sequence using Minimap2 (v.2.29-r1283) in map-ont mode, specifically optimized for long reads. The resulting alignments were then sorted and indexed with Samtools (v.1.21) for efficient exploitation. The consensus sequence was generated from the aligned reads using Medaka (v.2.0.1), a deep learning-based tool for correcting errors specific to ONT reads. The generated consensus sequence was submitted to Nextclade (https://clades.nextstrain.org/) for annotation, phylogenetic classification (clade assignment), and mutational event detection (substitutions, deletions, insertions).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreliminary overall phylogenetic tree was constructed using Nextclade including all sequences from RoC and Nextclade dataset \"Mpox virus (All clades)”. Full-length genomes of historical clade IIb A.2.2 MPXV sequences were obtained from GISAID and Pathoplexus and used to construct a more detailed tree. Sequences were aligned using MAFFT (FFT-NS-1 algorithm) \u003csup\u003e22\u003c/sup\u003e, with the best model for distance estimates identified with the ModelFinder function \u003csup\u003e23\u003c/sup\u003e as the one with the lowest Bayesian information criterion (BIC). Maximum likelihood phylogenetic tree was constructed using IG-TREE2 \u003csup\u003e24\u003c/sup\u003e and branch support was assessed using both ultrafast bootstrap approximation (ufBoot, 1000 replicates) \u003csup\u003e25\u003c/sup\u003e and SH-like approximate likelihood ratio test (SH-aLRT). The tree was visualized in FigTree (\u003ca href=\"http://tree\"\u003ehttp://tree\u003c/a\u003e.bio.ed.ac.uk/software/figtree/) and rooted using a clade IIb A.2 sequence. Bars indicate nucleotide substitutions per site.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecimen collection and laboratory confirmation were conducted under the passive national surveillance program. Written informed consent was obtained from the case without allowing publication of images. Ethical clearance was obtained from the Congolese Foundation for Medical Research Institutional Ethics Committee (Avis n°053/CEI/FCRM/2024).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLaboratory results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial diagnostic result for the suspect mpox case on the GeneExpert was mpox clade II positive. Cycle threshold (Ct) value of 22 for the skin lesion sample and a Ct-value of 34.8 for the blood sample and non-variola positive (generic assay to detect all non-variola orthopox viruses including mpox clade I and II) with a Ct-value of 21.7 for skin lesion sample and Ct value of 32.5 for the blood sample. In the second analysis using the RADI Fast Mpox detection kit, the result was positive for clade 2 with a Ct-value of 17 for the skin lesion sample and Ct-value of 33 for the blood sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePassive surveillance data of mpox in Republic of the Congo January – August 2025.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFifty-six mpox cases from January 2025 to August 2025 were identified in RoC using the above-described molecular diagnostic and sequencing pipelines. The samples were obtained from cases from 7 of the 12 administrative departments of the RoC namely, Brazzaville, Pointe-Noire, Likouala, Cuvette, Kouilou, Plateaux and Niari. During this time 16 cases of clade Ia, 32 cases of clade Ib, and the two introductions of clade IIb were identified. Epidemiological data suggest that during this period in 2025 several lineages of clade Ia, as well as clade Ib are co-circulating with the major administrative (Brazzaville) and economic (Pointe-Noire) hubs being the hot spots of the sustained human-to-human transmission of mpox in RoC (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-genome sequences were obtained from an mpox-positive specimen, with a genome length of 197,200 base pairs. Phylogenetic analysis identified this sequence as MPXV clade IIb, lineage A.2.2 (Figure 2). The Congolese sequence clustered with recent lineage A.2.2 sequences from Nigeria, Togo, the United States of America (USA), and Australia, forming a clade that branches into the new G.1 cluster, which is composed mostly of sequences from Sierra Leone.\u003c/p\u003e\n"},{"header":"Discussion","content":"\n\u003cp\u003eSince 2022, molecular diagnostics for MPXV has been established at the NPHL, Brazzaville, RoC allowing for the implementation of a passive mpox surveillance system in the country. In 2023, next generation sequencing capacity was added, and the passive surveillance system allows for molecular diagnostics of mpox suspect cases, clade and lineage defining qRT-PCR assays and the ability to perform molecular epidemiological studies using genomic mpox data.\u003c/p\u003e\n\u003cp\u003eOur mpox surveillance successfully identified the co-circulation of multiple Clade Ia lineages in RoC in 2025, identified the introduction of Clade Ib from the Democratic Republic of the Congo on two independent occasions in 2024 and now identified the first identified introduction of Clade IIb.\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis placed the clade IIb MPXV into the lineage A.2.2. MPXV Clade IIb has been circulating cryptically for nearly a decade in Nigeria. The ongoing circulation of Clade IIb in Nigeria, including distinct sub lineages as the A.2.2., have resulted the recently identified regional outbreaks in Sierra Leone, as well as travel related cases outside Africa, including German, USA and Australia. The rapid spread in West Africa, introduction into Central Africa and travel related cases outside Africa bears resemblance to the rapid global spread of the global mpox outbreak in 2022 as the A2 lineage also originates from Nigeria and has spread throughout West Africa and has been detected in Australia, Europe and the USA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the phylogenetic tree, our clade IIb sequence was of the A.2.2. lineage and clustered with sequences of MPXV isolates from West Africa, USA, Germany and Australia all collected in 2025. Together with the recent travel history to France and Ivory Coast, his suggests that the mpox patient infected with clade IIb MPXV likely contracted the infection while traveling in West Africa or Europe. The rapid rise of clade IIb lineage 2.2 highlights the vulnerability of the region for undetected circulation of mpox. It also underscores significant gaps in regional preparedness, in particular the availability of diagnostics and the ability to conduct molecular epidemiology. \u0026nbsp;Our patient was not able to provide comprehensive contact-tracing information to health workers, showing the complexity of tracking imported cases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work presents epidemiological and phylogenetic characteristics of the first laboratory-confirmed mpox case with MPXV clade IIb, lineage A.2.2 in Pointe-Noire, RoC. The detection of this first case of clade IIb in this region, highlights the growing complexity of mpox epidemiology in an historically endemic region. In 2025, cases of Clade Ia, Ib, and now IIb have been detected in RoC. The current genetic and epidemiological data from RoC suggest that each of these MPXV clades and sublineages harbors distinct human to human characteristics based on apobec 3 signatures and epidemiological characteristics.\u003csup\u003e16,19,26\u003c/sup\u003e. Strengthening of the regional capacity for case detection, contact tracing, public health measures, and affordable vaccines are needed to implement interventions to reduce the global risk for both Clade I and Clade II MPXV.\u003csup\u003e27\u003c/sup\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eSpecimen collection and laboratory confirmation were conducted under the passive national surveillance program. Written informed consent was obtained from the case for publishing without allowing publication of images. Ethical clearance was obtained from the Congolese Foundation for Medical Research Institutional Ethics Committee (Avis n\u0026deg;053/CEI/FCRM/2024).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFKK, DAEB, RGE and FRN conceived and designed the study; FKK, DAEB, RGE, GDI, VRBA, LHL, and ISO processed samples and generated the data; CKY, VJM, PIM, LNM, IJL, JELD and FRN accessed and verified all the data; FKK, DAEB, FRN and CKY curated, analyzed and interpreted data; FKK, DAEB, CKY, VJM, and RGE wrote the first draft of the manuscript; ISO, JMK, AGN, PIM, EML, NPMD, \u0026nbsp;AFA, CKY, VJM and FRN revised the manuscript. All authors provided critical revision of the manuscript. All authors had full access to all the data of the study and had final responsibility for the decision to submit for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the health workers who were involved in identification the case, epidemiological information, collection of specimens and transfer of samples to the NPHL. We express our gratitude to Africa CDC, WHO Congo and CDC Atlanta for continuous support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work received no external funding and was fully supported by the National Public Health Laboratory. CKY and VJM were supported by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH authors were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole genome sequences of the MPXV generated in this study are available at GISAID XX - XX\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO. Multi-country monkeypox outbreak in non-endemic countries. World Health Organization2022.\u003c/li\u003e\n\u003cli\u003eUlaeto D, Agafonov A, Burchfield J, et al. New nomenclature for mpox (monkeypox) and monkeypox virus clades. Lancet Infect Dis 2023;23(3):273-275. 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DOI: 10.1056/NEJMoa2411368.\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":"","lastPublishedDoi":"10.21203/rs.3.rs-7401723/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7401723/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"In 2022, mpox virus (MPXV) clade IIb emerged resulting in a global epidemic driven by human-to-human transmission mostly through sexual contact in the MSM population. To date, published data on the circulation of the MPXV clade IIb in the Central African region are absent. Here, we describe the first case of laboratory-confirmed mpox with MPXV clade IIb lineage A2.2 in Pointe-Noire, the second largest city of the Republic of the Congo (RoC). Whole genome phylogenetic analysis placed the MPXV in clade IIb, lineage A.2.2. currently emerging in West Africa, in particular Sierra Leone. The detection of clade IIb mpox, marks the third distinct MPXV clade and lineage co-circulating in the human population, together with clade Ia and clade Ib. This underscores the need for improved surveillance and diagnostic strategies to identify the respective clade and lineage circulating in the human population. Strengthening of the regional capacity for case detection, contact tracing, public health measures, and affordable vaccines are urgently needed to reduce the global risk for both clades I and Clade II MPXV.","manuscriptTitle":"First laboratory-confirmed case of infection with mpox virus clade IIb lineage A.2.2 in the Republic of the Congo, co-circulation of clade Ia, Ib and clade IIb.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 07:44:58","doi":"10.21203/rs.3.rs-7401723/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nm","sideBox":"Learn more about [Nature Medicine](http://www.nature.com/nm/)","snPcode":"","submissionUrl":"","title":"Nature Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"16d53016-484f-443f-b4a6-ea83b359393d","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53678415,"name":"Biological sciences/Microbiology/Virology/Pox virus"},{"id":53678416,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":53678417,"name":"Health sciences/Health care/Diagnosis/Genetic testing"}],"tags":[],"updatedAt":"2026-04-04T07:10:03+00:00","versionOfRecord":{"articleIdentity":"rs-7401723","link":"https://doi.org/10.1038/s41591-026-04256-2","journal":{"identity":"nature-medicine","isVorOnly":false,"title":"Nature Medicine"},"publishedOn":"2026-04-03 04:00:00","publishedOnDateReadable":"April 3rd, 2026"},"versionCreatedAt":"2025-08-27 07:44:58","video":"","vorDoi":"10.1038/s41591-026-04256-2","vorDoiUrl":"https://doi.org/10.1038/s41591-026-04256-2","workflowStages":[]},"version":"v1","identity":"rs-7401723","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7401723","identity":"rs-7401723","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}