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Introduction. Arboviruses and malaria pose a growing threat to public health, affecting not only the general population but also immunocompromised individuals and pregnant women. Individuals in vulnerable groups are at a higher risk of severe complications from the co-circulation and transmission of ZIKV, malaria, and FLAVI fever. In sub-Saharan countries, such as Nigeria, these mosquito-borne infections have clinical presentations that overlap with other diseases (dengue, West Nile virus, and Japanese encephalitis, chikungunya, and O’nyong o’nyong virus), making them a diagnostic challenge for clinicians in regions where they co-circulate. Vertical transmission can have a devastating impact on maternal health and fetal outcomes, including an increased risk of fetal loss and premature birth. Despite the global recognition of the burden of malaria and arboviruses, particularly ZIKV and other flaviviruses, there is limited data on their prevalence in Nigeria. In urban settings, where these diseases are endemic and share common biological, ecological, and economic factors, they may impact treatment outcomes and lead to epidemiological synergy. Hence, it is imperative to conduct sero-epidemiological and clinical studies to better understand the disease burden and hidden endemicity, thereby enabling improved prevention and clinical management. Method. Serum samples collected from outpatients between December 2020 and November 2021 in three regions of Nigeria were tested for the presence of IgG antibody seropositivity against ZIKV and FLAVI using immunoblot serological assay. Results. The overall cohort co-circulation antibody seropositivity of ZIKV, FLAVI and malaria was 24.0% (209/871). A total of 19.2% (167/871) of the study participants had ZIKV-seropositive antibodies and 6.2% (54/871) were FLAVI-seropositive, while 40.0% (348/871) of the subjects had malaria parasite antigens. Regional analysis revealed that participants from the southern region had the highest antibody seropositivity against ZIKV (21.7% (33/152)) and FLAVI (8.6% (13/152)), whereas those from the central region had a higher malaria parasite antigen (68.5% (287/419)). Conclusions. This study represents the largest comparative cross-sectional descriptive sero-epidemiological investigation of ZIKV-FLAVI and malaria cocirculation in Nigeria. The findings of this study revealed increased antibody seropositivity, hidden endemicity, and the burden of ZIKV, FLAVI, and malaria co-circulating in Nigeria.
A cross-sectional study was conducted at three university teaching hospital centers in Nigeria: namely, the Federal Medical Centre, Keffi, located in Nasarawa State; the Central Nigeria Abia State University Teaching Hospital, Aba, located in Abia State, Southern Nigeria; and the Baru-Diko Teaching Hospital, Kaduna, Kaduna State, located in Northern Nigeria (Figure 1). Arboviral and malaria study sites in Nigeria. The three states have a population of over 30 million inhabitants. Forty-five percent of the population live in urban areas (urban settlement in the context of the present study refers to high human population density and infrastructure of the built environment), 40% live in rural areas (open countryside with population densities of less than 500 people per square mile or places with fewer than 1500 people), and 15% live in slums or informal settlements (informal settlements within urban cities with inadequate housing, squalids, and miserable living conditions) [1]. The average annual temperature ranges from 21 °C to 37 °C, whereas in the interior lowlands, temperatures are generally above 27 °C. The mean annual precipitation is 1165.0 mm. It rains throughout southern Nigeria but much less so in the central and northern regions, with episodes of flooding and other environmental catastrophes. Most of the rainfall occurs between April and October, with minimal rainfall occurring between November and March. The main occupation of the inhabitants of the three regions is farming at both the commercial and subsistence levels. The study population comprised outpatients, including pregnant women enrolled for antenatal care and patients presenting with illness, at the rapid-access healthcare and antiretroviral (people living with AIDS) units of the hospitals between December 2020 and November 2021. These hospitals were purposefully selected to reflect the diversity of different cultures, religions, ethnicities, topographical and vegetation features, and human activities in the three geographical regions. The inclusion criteria were all outpatients within an age range of 0 months to 80 years who agreed to participate in the study and signed the consent form, including children, whose parents or guardians gave consent. The exclusion criteria were participants who were already undergoing treatment, those who refused to sign the consent form, and seriously ill hospitalized patients. A standardized questionnaire containing questions on demographics, medical history, vital signs and symptoms, clinical evaluation, hospitalization data, and a summary form was used to collect the information. All the research participants were examined for malaria-, ZIKV-, and FLAVI-related symptoms (fever, headaches, rashes, joint pain, conjunctivitis, and muscle discomfort) (Table 1). Before enrolment, participants were provided with protocol-specific information and had this clearly explained to them in English and their respective native languages. After enrolment, the participants signed an informed consent form. The participants who were unable to read or write were asked to verbally assent and give their thumbprint to indicate their willingness to participate. Signs and symptoms presented by ZIKV, FLAVI, and malaria monoinfected patients. The simple random sampling method was used to collect 871 samples from participants in the three regions. In total, 262 samples were collected from outpatients, 499 from HIV-positive patients, and 110 from blood banks. The sample size calculation (based on a 40% expected proportion of ZIKV and FLAVI in a total population of 500,000 patients with a 95% confidence interval and a p-value of 0.05) [17] indicated a minimum sample size of 384 serum samples, which was increased to 871 samples for subgroup analyses by region. All study participants provided 5 mL of venous blood, and a local clinical diagnostic laboratory technician collected the blood samples from the three blood banks. All participants were screened for malaria using an RDT specific for the parasite (SD BIOLINE Malaria Differential P.f/Pan Ag RDT (HRP II+ pLDH, Abbott, Mikrogen Diagnostik, Neuried, Germany). In brief, 5 µL of blood sample was transferred into the sample well using the appropriate device included in the kit, and five drops of lysis buffer were added to the buffer well. The results were read visually after 15–20 min. Screened samples were shipped on dry ice to the Institute of Virology Universitatsklinikum, Freiburg, where they were tested for the presence of human immunoglobulin G (IgG) antibodies using recomLine Tropical Fever for the presence of arboviral antibody serological marker IgG immunoblot (Mikrogen Diagnostik, Neuried, Germany) ZIKV NS1 and ZIKV Equad and flavivirus according to the manufacturer [18]. In brief, test strips were loaded with ZIKV and FLAVI antigens and incubated with diluted serum in a dish for 1 h. The strips were then washed three times. Peroxidase-conjugated anti-human antibodies (IgG-specific) were added, incubated for 45 min, and washed three times. Insoluble bands developed at the sites on the test strips occupied by antibodies 8 min after the addition of the coloring solution. Statistical analyses were performed using SPSS version 21. Descriptive statistics were employed for the analysis of the results, and 95% confidence intervals [CI] were used to identify the sociodemographic and behavioral characteristics of the study population. The results are presented in tables and figures. The chi-squared test was performed and deemed statistically significant at p ≤ 0.05.
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