Co-circulation of Dengue and Chikungunya Viruses and Pre-Analytical Challenges During an Outbreak Investigation on Anjouan Island, Comoros, April 2025

Co-circulation of Dengue and Chikungunya Viruses and Pre-Analytical Challenges During an Outbreak Investigation on Anjouan Island, Comoros, April 2025 | 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 Short Report Co-circulation of Dengue and Chikungunya Viruses and Pre-Analytical Challenges During an Outbreak Investigation on Anjouan Island, Comoros, April 2025 Nawar-Khadidja AHMED EL-HARIF, Moinaheiri YOUSSOUF MOIMBA, SAID MANSOURI MHOUSSINI Makam, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8278122/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background In April 2025, suspected dengue cases were reported in Anjouan, Comoros, against a backdrop of chikungunya outbreaks in neighboring islands of the Indian Ocean. Objective To confirm the circulation of dengue (DENV) and chikungunya (CHIKV) viruses by triplex RT-PCR and to evaluate the quality of the sample handling process. Methods Seventy-three (73) blood samples from symptomatic patients were analyzed. RNA was extracted and tested by triplex RT-PCR (DENV/CHIKV/ZIKV). The internal control PPIA validated nucleic acid integrity. Results Only forty (40) samples (54.8%) were suitable for analysis. Among these, 30.0% were positive for DENV, with 33.3% of the positive samples showing co-infection with CHIKV. No Zika cases were detected. The high rate of non-conforming samples (45.2%) was linked to inadequate transport conditions (temperature, damaged tubes). Conclusion This study confirms the co-circulation of DENV and CHIKV in Anjouan. The high rate of invalid samples underscores the urgent need to strengthen the pre-analytical chain for effective arboviral surveillance in the Comoros archipelago. Infectious Diseases Dengue Chikungunya Co-infection RT-PCR Surveillance Pre-analytical Comoros Introduction The Comoros archipelago, in the southwestern Indian Ocean, is vulnerable to arbovirus outbreaks. In March 2025, Comorian health authorities declared a dengue outbreak on Anjouan Island (Ndzuani). Concurrently, major chikungunya epidemics were ongoing in Réunion and Madagascar [ 1 , 2 ]. The similar symptomatology between dengue and chikungunya requires precise molecular diagnosis to identify circulating pathogens and potential co-infections, which are associated with a potentially more severe prognosis [ 3 , 4 ]. We report here the results of the molecular analysis of suspected samples and evaluate the operational challenges related to their quality. Methods Ethical Considerations This study was conducted in accordance with the Declaration of Helsinki and was approved by the National Ethics Committee of the Comoros. Written informed consent was obtained from all participants or their legal guardians prior to sample collection. All data were anonymized to protect patient confidentiality. Sample Collection Seventy-three EDTA blood samples were collected from patients presenting with dengue-like syndrome (fever ≥38.5°C with two or more of the following symptoms: headache, retro-orbital pain, myalgia, arthralgia, rash, or hemorrhagic manifestations). Samples were sent to the INRAPE laboratory by the Regional Health Directorate of Anjouan between April 3 and 12, 2025. Molecular Analysis RNA was extracted using the Qiagen Allprep DNA/RNA kit. Multiplex detection of DENV, CHIKV, and Zika virus (ZIKV) was performed using the TaqMan™ Arbovirus Triplex kit on a Quant Studio 5 thermocycler, following the manufacturer's instructions [5]. The peptidylpropyl isomerase A (PPIA) gene was used as an internal control. A sample was considered valid if the Ct for PPIA was <38. A sample was declared positive for a virus if the Ct for the corresponding viral target was between 26 and 38. Funding Statement This work was supported by the World Health Organization through the provision of reagents and consumables necessary for laboratory analysis. The funder had no role in the study design, data collection, analysis, or decision to publish. Results Of the 73 samples received, 33 (45.2%) were deemed non-conforming due to inadequate transport conditions: transport temperature >30°C instead of the required 2-8°C, broken tubes, coagulated or hemolyzed blood. These samples, which were negative for the PPIA internal control, were excluded from virological analysis. Among the 40 valid samples analyzed, 12 (30.0%) tested positive for dengue virus (DENV). Among these 12 DENV-positive samples, 4 (33.3%) were simultaneously positive for chikungunya virus (CHIKV), confirming cases of co-infection. No sample was positive for Zika virus (ZIKV) or for CHIKV alone. Discussion This investigation confirms the active circulation of dengue virus and reveals the presence of dengue-chikungunya co-infections on Anjouan Island in April 2025. The proportion of co-infections (33.3% of dengue cases) is notable and warrants vigilant surveillance, as they can potentiate clinical severity [3,4]. However, the most striking finding of this study is the high rate (45.2%) of unusable samples due to pre-analytical failures. This problem, documented in other resource-limited settings [6], severely compromises epidemiological surveillance by potentially underestimating the true incidence of infections and delaying the public health response. It highlights a critical weak link in the Comorian health system. Conclusion Our results confirm the co-circulation of DENV and CHIKV in the Comoros. Beyond this epidemiological finding, this study underscores the imperative need to invest in strengthening the pre-analytical chain, including training of sampling personnel and improving sample transport conditions. Robust and responsive arboviral surveillance, essential for controlling these diseases, depends on the reliability of the entire diagnostic process, from the patient's bedside to the laboratory. Declarations Human Ethics Declaration This study was conducted in accordance with the Declaration of Helsinki and approved by the National Ethics Committee of the Comoros. Written informed consent was obtained from all participants. Consent for Participation Informed consent was obtained from all individual participants included in the study. Acknowledgements The authors would like to thank the Ministry of Health of the Union of the Comoros for its support. They also express their gratitude to the World Health Organization for the technical assistance that made these analyses possible at the INRAPE laboratory. Finally, they thank the staff of the Regional Health Directorate of Anjouan for sample collection. References Simonin Y (2025) Chikungunya in Réunion: a large-scale epidemic requiring heightened vigilance. L'Express de Madagascar. Mar 15 Razaf N (2025) Chikungunya Epidemic - The Big Island Surrounded. La Vérité. Mar 22 Kaur M, Singh K, Puri A, Kaur M (2018) Coinfection of chikungunya and dengue viruses: A serological study. J Lab Physicians 10(2):138–143. 10.4103/JLP.JLP_142_17 Larrieu S, Balleydier E, Renault P et al (2016) Réunion Island prepared for possible Zika virus emergence, 2016. Euro Surveill 21(15):30194. 10.2807/1560-7917.ES.2016.21.15.30194 Panmei K, Smith D, Johnson L et al (2025) Performance evaluation of TaqMan™ Arbovirus Triplex Kit for detection of dengue and chikungunya viral RNA. J Virol Methods 315:114692. 10.1016/j.jviromet.2024.114692 Jnah A, Ben Salah M, Khlifi S (2022) Control of non-conformities in the pre-analytical phase at the Bacteriology Laboratory. Tunis Med 100(2):145–150 Okazaki Y, Nguyen TT, Nishihara A, Endo H, Ogata H, Nakano SI, Tamaki H (2023) A Fast and Easy Method to Co-extract DNA and RNA from an Environmental Microbial Sample. Microbes Environ 38(1):ME22102. 10.1264/jsme2.ME22102 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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2025\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Comoros archipelago, in the southwestern Indian Ocean, is vulnerable to arbovirus outbreaks. In March 2025, Comorian health authorities declared a dengue outbreak on Anjouan Island (Ndzuani). Concurrently, major chikungunya epidemics were ongoing in R\u0026eacute;union and Madagascar [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The similar symptomatology between dengue and chikungunya requires precise molecular diagnosis to identify circulating pathogens and potential co-infections, which are associated with a potentially more severe prognosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. We report here the results of the molecular analysis of suspected samples and evaluate the operational challenges related to their quality.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical Considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and was approved by the National Ethics Committee of the Comoros. Written informed consent was obtained from all participants or their legal guardians prior to sample collection. All data were anonymized to protect patient confidentiality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeventy-three EDTA blood samples were collected from patients presenting with dengue-like syndrome (fever \u0026ge;38.5\u0026deg;C with two or more of the following symptoms: headache, retro-orbital pain, myalgia, arthralgia, rash, or hemorrhagic manifestations). Samples were sent to the INRAPE laboratory by the Regional Health Directorate of Anjouan between April 3 and 12, 2025.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted using the Qiagen Allprep DNA/RNA kit. Multiplex detection of DENV, CHIKV, and Zika virus (ZIKV) was performed using the TaqMan\u0026trade; Arbovirus Triplex kit on a Quant Studio 5 thermocycler, following the manufacturer\u0026apos;s instructions [5]. The peptidylpropyl isomerase A (PPIA) gene was used as an internal control. A sample was considered valid if the Ct for PPIA was \u0026lt;38. A sample was declared positive for a virus if the Ct for the corresponding viral target was between 26 and 38.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the World Health Organization through the provision of reagents and consumables necessary for laboratory analysis. The funder had no role in the study design, data collection, analysis, or decision to publish.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOf the 73 samples received, 33 (45.2%) were deemed non-conforming due to inadequate transport conditions: transport temperature \u0026gt;30\u0026deg;C instead of the required 2-8\u0026deg;C, broken tubes, coagulated or hemolyzed blood. These samples, which were negative for the PPIA internal control, were excluded from virological analysis.\u003c/p\u003e\n\u003cp\u003eAmong the 40 valid samples analyzed, 12 (30.0%) tested positive for dengue virus (DENV). Among these 12 DENV-positive samples, 4 (33.3%) were simultaneously positive for chikungunya virus (CHIKV), confirming cases of co-infection. No sample was positive for Zika virus (ZIKV) or for CHIKV alone.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis investigation confirms the active circulation of dengue virus and reveals the presence of dengue-chikungunya co-infections on Anjouan Island in April 2025. The proportion of co-infections (33.3% of dengue cases) is notable and warrants vigilant surveillance, as they can potentiate clinical severity [3,4].\u003c/p\u003e\n\u003cp\u003eHowever, the most striking finding of this study is the high rate (45.2%) of unusable samples due to pre-analytical failures. This problem, documented in other resource-limited settings [6], severely compromises epidemiological surveillance by potentially underestimating the true incidence of infections and delaying the public health response. It highlights a critical weak link in the Comorian health system.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur results confirm the co-circulation of DENV and CHIKV in the Comoros. Beyond this epidemiological finding, this study underscores the imperative need to invest in strengthening the pre-analytical chain, including training of sampling personnel and improving sample transport conditions. Robust and responsive arboviral surveillance, essential for controlling these diseases, depends on the reliability of the entire diagnostic process, from the patient's bedside to the laboratory.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eHuman Ethics Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and approved by the National Ethics Committee of the Comoros. Written informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Participation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Ministry of Health of the Union of the Comoros for its support. They also express their gratitude to the World Health Organization for the technical assistance that made these analyses possible at the INRAPE laboratory. Finally, they thank the staff of the Regional Health Directorate of Anjouan for sample collection.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSimonin Y (2025) Chikungunya in R\u0026eacute;union: a large-scale epidemic requiring heightened vigilance. L'Express de Madagascar. Mar 15\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRazaf N (2025) Chikungunya Epidemic - The Big Island Surrounded. La V\u0026eacute;rit\u0026eacute;. Mar 22\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaur M, Singh K, Puri A, Kaur M (2018) Coinfection of chikungunya and dengue viruses: A serological study. J Lab Physicians 10(2):138\u0026ndash;143. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/JLP.JLP_142_17\u003c/span\u003e\u003cspan address=\"10.4103/JLP.JLP_142_17\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLarrieu S, Balleydier E, Renault P et al (2016) R\u0026eacute;union Island prepared for possible Zika virus emergence, 2016. Euro Surveill 21(15):30194. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2807/1560-7917.ES.2016.21.15.30194\u003c/span\u003e\u003cspan address=\"10.2807/1560-7917.ES.2016.21.15.30194\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePanmei K, Smith D, Johnson L et al (2025) Performance evaluation of TaqMan\u0026trade; Arbovirus Triplex Kit for detection of dengue and chikungunya viral RNA. J Virol Methods 315:114692. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jviromet.2024.114692\u003c/span\u003e\u003cspan address=\"10.1016/j.jviromet.2024.114692\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJnah A, Ben Salah M, Khlifi S (2022) Control of non-conformities in the pre-analytical phase at the Bacteriology Laboratory. Tunis Med 100(2):145\u0026ndash;150\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOkazaki Y, Nguyen TT, Nishihara A, Endo H, Ogata H, Nakano SI, Tamaki H (2023) A Fast and Easy Method to Co-extract DNA and RNA from an Environmental Microbial Sample. Microbes Environ 38(1):ME22102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1264/jsme2.ME22102\u003c/span\u003e\u003cspan address=\"10.1264/jsme2.ME22102\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dengue, Chikungunya, Co-infection, RT-PCR, Surveillance, Pre-analytical, Comoros","lastPublishedDoi":"10.21203/rs.3.rs-8278122/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8278122/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eIn April 2025, suspected dengue cases were reported in Anjouan, Comoros, against a backdrop of chikungunya outbreaks in neighboring islands of the Indian Ocean.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTo confirm the circulation of dengue (DENV) and chikungunya (CHIKV) viruses by triplex RT-PCR and to evaluate the quality of the sample handling process.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eSeventy-three (73) blood samples from symptomatic patients were analyzed. RNA was extracted and tested by triplex RT-PCR (DENV/CHIKV/ZIKV). The internal control PPIA validated nucleic acid integrity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOnly forty (40) samples (54.8%) were suitable for analysis. Among these, 30.0% were positive for DENV, with 33.3% of the positive samples showing co-infection with CHIKV. No Zika cases were detected. The high rate of non-conforming samples (45.2%) was linked to inadequate transport conditions (temperature, damaged tubes).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study confirms the co-circulation of DENV and CHIKV in Anjouan. The high rate of invalid samples underscores the urgent need to strengthen the pre-analytical chain for effective arboviral surveillance in the Comoros archipelago.\u003c/p\u003e","manuscriptTitle":"Co-circulation of Dengue and Chikungunya Viruses and Pre-Analytical Challenges During an Outbreak Investigation on Anjouan Island, Comoros, April 2025","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 11:15:26","doi":"10.21203/rs.3.rs-8278122/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5501727-2032-4c45-a92e-2087065c12f0","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59083688,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2025-12-05T11:15:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 11:15:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8278122","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8278122","identity":"rs-8278122","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}