Human seroprevalence of rift valley fever in the Timbuktu region, Mali, 2021

*Corresponding author: Issa DIARRA Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Human seroprevalence of rift valley fever in the Timbuktu region, Mali, 2021 Issa DIARRA 1, *, Abdourahamane TRAORE 1, Nazli AYHAN 2, Laurence THIRION 2, Mady CISSOKO 3, Seydou ARAMA 3, Moussa Hama SANKARE 3, Mouhamed THIERO 4, Bakary KOITA 5, Ami DIAKITE 1, Adama CISSE 2, Ousmane MAIGA 6 and Xavier de Lamballerie 2 1 Laboratoire de Biologie Moléculaire Appliquée (LBMA), Faculté de Pharmacie, Université des Sciences, des Techniques et des Technologies de Bamako (USTTB), Mali. 2 Unité des Virus Émergents (UVE: Aix-Marseille Univ – IRD 190 – Inserm 1207 – IHU Méditerranée Infection), Marseille, France. 3 Direction Régionale de la Santé de Tombouctou, Mali. 4 Hôpital NIANAKORO FOMBA de Ségou, Mali. 5 Infirmerie de Génie Militaire, Direction Centrale du Service de Santé des Armées, Bamako, Mali. 6 Universités des Sciences des Techniques et des Technologie de Bamako, Mali. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 266-273 Publication history: Received on 29 September 2025; revised on 05 November 2025; accepted on 08 November 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.2.0997 Abstract Rift Valley fever (RVF) is a zoonotic disease with a human mortality rate of up to 50%. Its prevalence in cattle in Timbuktu, in northern Mali, has been estimated at 10%. In this region, close contact between humans and animals increases the likelihood of exposure to the Rift Valley fever virus (RVFV). Despite this potential risk, no human epidemic has been reported in Timbuktu to date. To address this gap, we conducted a population-based study to estimate the seroprevalence of RVF in five districts of the Timbuktu region. Using the competitive ELISA technique, we found an overall human seroprevalence of RVF of 10.7% (95% CI: 8.8% - 12.8%) among 983 volunteers, closely like seroprevalence observed in cattle. These findings clearly demonstrated the circulation of the RVFV in the human population in all districts of the Timbuktu region and highlight the need to strengthen public health strategies to better prepare for potential future outbreaks. Keywords: Seroprevalence; Rift Valley Fever; Timbuktu; Mali 1. Introduction Rift Valley fever (RVF) is a zoonotic disease that mainly affects domestic and wild ruminants and can be transmitted to humans [1, 2]. The disease was first described in 1930s during an outbreak of enzootic hepatitis near Lake Naivasha in the Rift Valley in Kenya, where high mortality and numerous abortions (approximately 5,000 cases) were reported among ruminants [3]. The causative agent is RVF virus (RVFV), an RNA virus belonging to the Phenuiviridae family and the Phlebovirus genus, with the species identified as Phlebovirus riftense [4, 5]. Transmission of RVFV occurs through both mosquito-borne and non-mosquito-borne routed. non-vector transmission typically results from direct contact with infected animals or animal products (especially aborted foetus) – and is commonly associated with pastoral practices. Vector-borne transmission involves more than thirty mosquito species primarily from the genera Aedes, Anopheles, Culex and Mansonia. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 266-273 267 RVFV is responsible for recurrent outbreaks across Africa, causing severe impacts on public health, socioeconomic stability and growth, and food insecurity in particularly among livestock-dependent communities [6]. The early stages of an epizootic usually manifest as a wave of unexplained abortions, particularly in small ruminants. The disease is more severe in young animals, which present with acute hepatitis and haemorrhages, as well as variable but significant mortality. In humans, the incubation period is short (2 to 6 days) and most infections are asymptomatic. Symptoms are varied and non-specific, making clinical diagnosis difficult, particularly in the early stages of infection [7]. RVFV has a broad geographic distribution, with documented outbreaks and seroprevalence studies in countries neighbouring Mali, highlighting the regional risk of transmission and critical need for cross-border surveillance and preparedness [7–12]. Prior studies on emerging pathogens in Mali have focused on mammalian reservoirs (Dromedaries, cattle, sheep, goat and mice), which are considered indicator species for the circulation of these viruses [13–16]. Data indicating a 10% infection rate in cattle cohabiting near human populations in northern Mali villages [13] strongly suggests frequent zoonotic spillover potential, increasing the likelihood of human exposure to RVFV and subsequent disease development. [13]. Nevertheless, despite increased recognition of cattle RVF in Timbuktu, there is a crucial gap in human health data. To date no human outbreaks have been detected and the prevalence of the disease in humans is unknown. Consequently, to better understand the risk of human RVFV infection in Northern Mali, we conducted a cross-sectional serologic survey of inhabitants of five Health Districts within Timbuktu region (Timbuktu, Niafunke, Dire, Goundam and Gourma-Rharous) and determined the seroprevalence of the RVF in the region. 2. Materials and methods 2.1. Ethical and Professional statements The research protocol was approved by the ethic committees of the University of Sciences, Techniques and Technologies of Bamako (USTTB) through Letter No. 2021/282/USTTB dated October 28, 2021. Before we conducted this study, permission was granted from regional health professionals as well as from village elders and chiefs. Consent was obtained from all participants aged 18 years and older prior to their involvement in the study. For participants aged 6 months to 17 years, consent was obtained from a parent or legal guardian before any study-related procedures were initiated. The study protocol was explained to both the parents and children aged 11 years and older. Both the parent/guardian and the child (if aged 11 or older) signed or provided a fingerprint on the assent/consent form, as appropriate. For children aged 2 to 11 years, the decision to participate in the study was made solely by the parents or legal guardians. For children aged 12 to 14 years, participation was based on a joint decision between the child and their parent or guardian. Both the child (aged 12–14 years) and their parent were informed that participation was voluntary and that they could withdraw from the study at any time without any consequences or risk of harm. 2.2. Study design This was a cross-sectional, prospective study conducted from November 10 to 16, 2021, across the five health districts of the Timbuktu region (figure 1). According to the updated 2009 General Population and Housing Census, the Timbuktu region had an estimated population of 856,609 inhabitants in 2021, distributed as follows in the 5 regional Health Districts: Dire – 147,302; Goundam – 198,675; Niafunke – 250,871; Gourma-Rharous – 151,632; and Timbuktu – 108,129. Based on findings from previous investigations for arboviruses, including a Zika virus seroprevalence of 12 % in Mali [17], the minimum sample size required for statistical significance was calculated. Using a 95 % confidence level, a standard normal deviate (Z) of 1.96, a margin of error of 5 %, and accounting for an anticipated 5 % loss to follow-up, the estimated sample size was 930 participants. The study included male and female volunteers aged over six months, drawn from the general population across the five health districts of the Timbuktu region: Timbuktu, Niafunké, Diré, Goundam, and Gourma-Rharous. Participants in the study were required to meet the following criteria: no fever, be over six months of age, provide informed consent, and have resided in the locality for at least six months prior to enrolment. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 266-273 268 Figure 1 Study sites for assessment of RVFV seroprevalence in humans, Timbuktu, Mali, 2021. The map indicates the 5 Health Districts of Timbuktu region 2.3. Sample Collection and storage After obtaining assent or consent, a nurse or a biologist from the ward collected approximately 2 mL of blood into a serum-separating tube (SST). Following coagulation, the samples were centrifuged at 2,900 rpm for 10 minutes to separate the serum. The recovered serum was stored at a temperature below –15 °C before being transported to the Applied Molecular Biology Laboratory (LBMA) at the Faculty of Sciences and Techniques (FST) in Bamako, where the samples were kept frozen until analysis. 2.4. Sample processing Laboratory, serological analyses were conducted by the National Reference Center (NRC) for Arboviruses in Marseille, part of the Emerging Viruses Unit (UVE) at Aix-Marseille University. A commercially available competitive ELISA (ID Screen® Rift Valley Fever Competition multispecies -ID vet, Innovative Diagnostics, France) was used for the detection and quantification of IgG antibodies specific to the nucleocapsid RVFV, following the manufacturer’s instructions. Briefly, this ELISA is based on a competitive binding principle. The wells are pre-coated with recombinant RVF virus nucleoprotein. If the human serum sample contains anti-nucleoprotein antibodies, these will compete with a labeled anti-nucleoprotein conjugate (HRP-conjugated) for binding to the coated antigen. Statistical Analysis Data were collected using a structured questionnaire, entered into Microsoft Excel, and analyzed with IBM SPSS Statistics version 25. Pearson’s Chi-square test was used to compare proportions between categorical variables, while Analysis of Variance (ANOVA) was applied to compare means among groups. A p-value less than 0.05 was considered statistically significant. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 266-273 269 3. Results • Enrolment - A total of 983 participants were enrolled in this study: Timbuktu 519 (52.8%) Diré 199 (20.2%) Goundam 125 (12.7%) Niafunké 77 (7.8%) Gourma-Rharous 63 (6.4%). Both oral and written consent was obtained from all participants before their inclusion in the study. In cases where a participant was illiterate, informed consent was obtained in the presence of a literate witness mostly local teacher chosen by the participant or a trusted community member designated by the community. Individuals who have not provided informed consent or assent, those currently participating in a clinical vaccination trial, those who had received a blood transfusion within the past six months, or were undergoing immunosuppressive therapy were excluded from the study. • Socio-demographic results – Within the study population, the sex ratio was ~3.0 (740 women to 243 men), The median age was 27 years, with an age range from 1 to 88 years. The median age was 29 years for men and 26 years for women. The age range for men was 1 to 88 years, and for women, 2 to 76 years. • Seroprevalence rate – The overall seroprevalence of RVF in 2021 was 10.7% [95% CI: 0.8% - 12.8%]. • Sex: seroprevalence was significantly higher in men compared to women, with rates of 19.0% [95% CI: 14.2% - 24.4%] and 8.0% [95% CI: 6.1% - 10,1%] respectively (Pearson’s χ² test, p < 0.001; see Table 1). Table 1 RVF seroprevalence according to sex in the Timbuktu region, 2021 RVF seroprevalence Negative N (%) Doubtful N (%) Positive N (%) Total N Sex Male 3 (1,2) 194 (79,8) 46 (19,0) 243 Female 7 (0,9) 674 (91,1) 59 (8,0) 740 Total 10 (1,0) 868 (88,3) 105 (10,7) 983 • Age: seroprevalence significantly increased with age, rising from less than 2% below the age of 15 to ca. 20% in those over the age of 45 (Pearson’s χ² test, p < 0.001, see Table 2). We also observed a significant increase of the seroprevalence among volunteers over the age of 30 (born before the 1980s). Table 2 RVF seroprevalence according to age groups in the Timbuktu region, 2021 Years RVF seroprevalence Negative N (%) Doubtful N (%) Positive N (%) Total N Age groups 1-15 151 (98.1) 0 (0.0) 3 (1.9) 154 16-30 408 (92.7) 1 (0.2) 31 (7.0) 440 31-45 173 (81.2) 3 (1.4) 37 (17.4) 213 +45 136 (77.3) 6 (3.4) 34 (19.3) 176 Total 868 (88.3) 10 (1.0) 105 (10.7) 983 • Health District seroprevalence: In 2021, the overall seroprevalence of RVF in the Timbuktu region was 10.7% [95% CI: 0.8% - 12.8%]. The highest seroprevalence rates were recorded in the health districts of GourmaRharous (33.3% [ 95% CI/ 22.0% - 46.3%]) and Dire (29.6% [95% CI: 23.4% - 36.5%]), while the lowest was observed in Goundam (2.4% [95% CI: 0.5% - 6.9%]), denoting very different exposure according to the different zones (Table 3). World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 266-273 270 Table 3 RVF seroprevalence according to health districts in the Timbuktu region, 2021 RVF seroprevalence Negative N (%) Doubtful N (%) Positive N (%) Total N Health Districts Gourma-Rharous 41 (65,1) 1 (1,6) 21 (33,3) 63 Dire 137 (68,8) 3 (1,5) 59 (29,7) 199 Niafunke 70 (90,9) 2 (2,6) 5 (6,5) 77 Timbuktu 499 (96,1) 3 (0,6) 17 (3,3) 519 Goundam 121 (96,8) 1 (0,8) 3 (2,4) 125 Total 868 (88,3) 10 (1,0) 105 (10,7) 983 4. Discussion The objective of this study was to estimate the seroprevalence of Rift Valley Fever (RVF) in the human population of the Timbuktu region. The findings provide evidence of RVFV circulation in the area, with a seroprevalence rate of 10.7%. Notably, this seroprevalence in humans closely mirrors that reported in animals (10%) within the same region, as documented by Subudhi et al. [13]. The seroprevalence of RVF in humans varies considerably across regions and is strongly influenced by occupational exposure. Reported rates include 4.5% among agro-pastoral and pastoral communities in northern Tanzania, 0.7% among rural adults in Kenya, and 2.8% in the Northern KwaZulu-Natal Province of South Africa. In Saudi Arabia, approximately 9% of local personnel working in close contact with animals tested seropositive using the RVF competition ELISA. Notably, a cross-sectional study conducted near Lake Malawi in Tanzania reported a seroprevalence as high as 29.3% [18]. The seroprevalence of RVF was significantly higher in men (19.0%) compared to women (8.0%), with a Pearson p-value of 0.001. A similar gender disparity was reported in Tanzania by Emmanuel Senyael Swai et al., who observed seroprevalence rates of 5.3% in men and 1.5% in women [19]. Seropositivity was significantly associated with increasing age in our study, reaching up to 19.3% among individuals over 45 years old. This increase was observed in both men and women (see supplementary tables 2 and 2). This trend indicates that RVF seroprevalence increases with age, a pattern similarly observed by Cook et al. in the Lake Victoria Basin, western Kenya [20], and by Heinrich et al. in the Mbeya region of Tanzania [21], that suggesting cumulative lifetime exposure is a key determinant of serostatus. By grouping the volunteers into 10-year age categories, we observe that the seroprevalence of RVF is virtually zero among individuals aged 1–10 years (supplementary table 3). This indicates that transmission is non-vectorial in this age group likely occurring through contact with livestock. Furthermore, the age distribution reveals a sudden increase in seroprevalence starting in the 31–40 age group (individuals born between 1981 and 1990). This pattern suggests that an RVF epidemic likely occurred during that decade, of which mosquitoes played a primary role in transmission. This inference is corroborated by historical records. An epidemic (epizootic) of RFV was declared in 1987 in the Rosso region, a locality situated between Senegal and Mauritania. This epidemic occurred after the construction of a barrage on the Senegal River for agricultural irrigation [22]. In addition, several RVF outbreaks (which affected both humans and animals) occurred in Mauritania, notably in 2010, 2015, 2020 and 2022 [1]. We propose that these recurrent epidemics in Mauritania have had a direct epidemiological impact on the Timbuktu region. This cross-border transmission is likely facilitated by the transhumance (seasonal migration of livestock and herders) routes connecting Mauritania with the Timbuktu localities, which also explains the increasing seroprevalence of RFV with age in our study. This age-related increase suggests repeated natural exposure to the RVF virus within the Timbuktu population. 5. Conclusion The high seroprevalence rate we document highlights the need for increased surveillance for RVFV in Mali. Further work is needed to support these findings, including human, animal and entomological surveillance in other regions of Mali. These findings confirm that exposure to RVFV is occurring in Timbuktu. Continued research into the specific epidemiological features of RVFV is essential to determine whether the infection pattern in Mali represents a state of World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 266-273 271 endemism, intermittent emergence, or a risk for major epidemics. This knowledge is crucial for developing targeted public health and veterinary preparedness strategies. Compliance with ethical standards Acknowledgments We thank the study district health officers and the study population for their cooperation. We are grateful to LBMA et UVE personnel for invaluable technical contribution. We would also like to thank Mr Mathias Amagoron DOLO for the cartographic representation of the study sites and Professor Ousmane KOITA for supervising the study in the field. Disclosure of conflict of interest No conflicting interest of the authors for publication. Statement of ethical approval The research protocol was approved by the ethic committees of the University of Sciences, Techniques and Technologies of Bamako (USTTB) through Letter No. 2021/282/USTTB dated October 28, 2021. Statement of informed consent Informed consent was obtained from all individual participants included in the study. Funding This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme under ZIKAlliance Grant Agreement no. 734548 and the European Virus Archive EVAg; European UnionHorizon 2020 programme under grant agreement no. 653316. References [1] Tinto B, Quellec J, Cêtre-Sossah C, et al. Rift Valley fever in West Africa: A zoonotic disease with multiple socioeconomic consequences. One Health; 17. Epub ahead of print 1 December 2023. DOI: 10.1016/j.onehlt.2023.100583. [2] Lumley S, Horton DL, Hernandez-Triana LLM, et al. Rift valley fever virus: Strategies for maintenance, survival and vertical transmission in mosquitoes. Journal of General Virology 2017; 98: 875–887. [3] Wright D, Kortekaas J, Bowden TA, et al. Rift valley fever: Biology and epidemiology. Journal of General Virology 2019; 100: 1187–1199. [4] Ikegami T. Molecular biology and genetic diversity of Rift Valley fever virus. Antiviral Res 2012; 95: 293–310. [5] Fatima I, Ahmad S, Alamri MA, et al. Discovery of Rift Valley fever virus natural pan-inhibitors by targeting its multiple key proteins through computational approaches. Sci Rep; 12. Epub ahead of print 1 December 2022. DOI: 10.1038/s41598-022-13267-1. [6] Muvunyi CM, Ngabonziza JCS, Siddig EE, et al. Rift Valley Fever in Rwanda Is Urging for Enhancing Global Health Security Through Multisectoral One Health Strategy. Microorganisms; 13. Epub ahead of print 1 January 2025. DOI: 10.3390/MICROORGANISMS13010091. [7] Ould El Mamy AB, Baba MO, Barry Y, et al. Unexpected rift valley fever outbreak, Northern Mauritania. Emerg Infect Dis 2011; 17: 1894–1896. [8] Lagare A, Fall G, Ibrahim A, et al. First occurrence of Rift Valley fever outbreak in Niger, 2016. Vet Med Sci 2019; 5: 70–78. [9] Kanouté YB, Gragnon BG, Schindler C, et al. Epidemiology of brucellosis, Q Fever and Rift Valley Fever at the human and livestock interface in northern Côte d’Ivoire. Acta Trop 2017; 165: 66–75. [10] Trabelsi MK, Hachid A, Derrar F, et al. Serological evidence of Rift Valley fever viral infection among camels imported into Southern Algeria. Comp Immunol Microbiol Infect Dis; 100. Epub ahead of print 1 September 2023. DOI: 10.1016/j.cimid.2023.102035. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 266-273 272 [11] Durand B, Modou M Lo, Tran A, et al. Rift Valley fever in northern Senegal: A modelling approach to analyse the processes underlying virus circulation recurrence. PLoS Negl Trop Dis 2020; 14: e0008009. [12] Barry Y, Elbara A, Bollahi MA, et al. Rift Valley fever, Mauritania, 2020: Lessons from a one health approach. One Health; 15. Epub ahead of print 1 December 2022. DOI: 10.1016/j.onehlt.2022.100413. [13] Subudhi S, Dakouo M, Sloan A, et al. Seroprevalence of rift valley fever virus antibodies in cattle in Mali, 20052014. American Journal of Tropical Medicine and Hygiene 2018; 98: 872–874. [14] Maiga O, Sas MA, Rosenke K, et al. Serosurvey of Crimean-Congo hemorrhagic fever virus in Cattle, Mali, West Africa. American Journal of Tropical Medicine and Hygiene 2017; 96: 1341–1345. [15] Falzarano D, Kamissoko B, de Wit E, et al. Dromedary camels in northern Mali have high seropositivity to MERSCoV. One Health 2017; 3: 41–43. [16] Safronetz D, Strong JE, Feldmann F, et al. A recently isolated Lassa virus from Mali demonstrates atypical clinical disease manifestations and decreased virulence in cynomolgus macaques. Journal of Infectious Diseases 2013; 207: 1316–1327. [17] Diarra I, Nurtop E, Sangaré AK, et al. Zika virus circulation in Mali. Emerg Infect Dis 2020; 26: 945–952. [18] Heinrich N, Saathoff E, Weller N, et al. High seroprevalence of Rift Valley fever and evidence for endemic circulation in Mbeya region, Tanzania, in a cross-sectional study. PLoS Negl Trop Dis; 6. Epub ahead of print March 2012. DOI: 10.1371/JOURNAL.PNTD.0001557. [19] Swai ES, Schoonman L. A survey of zoonotic diseases in trade cattle slaughtered at Tanga city abattoir: a cause of public health concern. Asian Pac J Trop Biomed 2012; 2: 55. [20] Cook EAJ, Grossi-Soyster EN, de Glanville WA, et al. The sero-epidemiology of Rift Valley fever in people in the Lake Victoria Basin of western Kenya. PLoS Negl Trop Dis; 11. Epub ahead of print 7 July 2017. DOI: 10.1371/JOURNAL.PNTD.0005731. [21] Heinrich N, Saathoff E, Weller N, et al. High seroprevalence of Rift Valley fever and evidence for endemic circulation in Mbeya region, Tanzania, in a cross-sectional study. PLoS Negl Trop Dis; 6. Epub ahead of print March 2012. DOI: 10.1371/JOURNAL.PNTD.0001557. [22] Saluzzo JF, Digoutte JP, Chartier C, et al. FOCUS OF RIFT VALLEY FEVER VIRUS TRANSMISSION IN SOUTHERN MAURITANIA. The Lancet 1987; 329: 504. Appendix Supplementary table 1: RVF seroprevalence according to age groups in males, Timbuktu region, 2021. Years RVF seroprevalence Negative N (%) Doubtful N (%) Positive N (%) Total N Age groups 1-15 51 (98.1) 0 (0.0) 1 (1.9) 52 16-30 65 (80.2) 0 (0.0) 16 (19.8) 81 31-45 37 (68.5) 1 (1.9) 16 (29.6) 54 +45 41 (73.2) 2 (3.6) 13 (23.2) 66 Total 194 (79.8) 3 (1.2) 46 (18.9) 243 Pearson’s χ² test, p = 0.004 World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 266-273 273 Supplementary table 2: RVF seroprevalence according to age groups in females, Timbuktu region, 2021. Years RVF seroprevalence Negative N (%) Doubtful N (%) Positive N (%) Total N Age groups 1-15 100 (98.0) 0 (0.0) 2 (2.0) 102 16-30 344 (95.5) 1 (0.3) 15 (4.2) 359 31-45 136 (85.5) 2 (1.3) 21 (13.2) 159 +45 95 (79.2) 4 (3.3) 21 (17.5) 120 Total 674 (91.1) 7 (0.9) 69 (8.0) 740 Pearson’s χ² test, p < 0.000 Supplementary table 3: Age Specific Seroprevalence of Rift Valley Fever by Health District in the Timbuktu Region. 1 - 10 years 11 - 20 years 21 - 30 years 31 - 40 years 41 - 50 years 51 - 60 years 60+ years Gourmarharous 0.0 11.1 31.6 21.4 55.6 80.0 33.3 Dire 0.0 18.8 19.4 34.2 50.0 46.7 44.4 Nianfunke 0.0 0.0 5.3 26.7 0.0 0.0 0.0 Timbuktu 1.8 2.0 2.2 1.4 11.8 5.9 5.6 Goundam 0.0 0.0 0.0 8.0 0.0 25.0 0.0