Genomic Surveillance Enables the Identification of Co-infections With Multiple SARS-CoV-2 Lineages in Equatorial Guinea

COVID-19 disease caused by SARS-CoV-2 represents an ongoing global public health emergency. Rapid identification of emergence, evolution, and spread of SARS-CoV-2 variants of concern (VOC) would enable timely and tailored responses by public health decision-making bodies. Yet, global disparities in current SARS-CoV-2 genomic surveillance activities reveal serious geographical gaps. Here, we discuss the experiences and lessons learned from the SARS-CoV-2 monitoring and surveillance program at the Public Health Laboratory on Bioko Island, Equatorial Guinea that was implemented as part of the national COVID-19 response and monitoring activities. We report how three distinct SARS-CoV-2 variants have dominated the epidemiological situation in Equatorial Guinea since March 2020. In addition, a case of co-infection of two SARS-CoV-2 VOC, Beta and Delta, in a clinically asymptomatic and fully COVID-19 vaccinated man living in Equatorial Guinea is presented. To our knowledge, this is the first report of a person co-infected with Beta and Delta VOC globally. Rapid identification of co-infections is relevant since these might provide an opportunity for genetic recombination resulting in emergence of novel SARS-CoV-2 lineages with enhanced transmission or immune evasion potential.

A colorimetric test for the evaluation of the insecticide content of LLINs used on Bioko Island, Equatorial Guinea

Background Insecticide-treated nets and indoor residual spraying of insecticides are used as the vector control interventions in the fight against malaria. Measuring the actual amount of deposits of insecticides on bed nets and walls is essential for evaluating the quality and effectiveness of the intervention. A colorimetric “Test Kit” designed for use as a screening tool, able to detect the type II pyrethroids on fabrics and sprayed walls, was used for the first time to detect deltamethrin on long-lasting insecticidal nets (LLINs) deployed on Bioko Island, Equatorial Guinea. Methods LLINs were analysed using the colorimetric Test Kit performed in situ, which leads to the formation of an orange-red solution whose depth of colour indicates the amount of type II pyrethroid on the net. The kit results were validated by measuring the amount of extracted insecticide using high-performance liquid chromatography (HPLC) with diode array detection (DAD). Results Deltamethrin concentration was determined for 130 LLINs by HPLC–DAD. The deltamethrin concentration of these nets exhibited a significant decrease with the age of the net from 65 mg/m2 (< 12 months of use) to 31 mg/m2 (> 48 months; p < 0.001). Overall, 18% of the nets being used in households had < 15 mg/m2 of deltamethrin, thus falling into the “Fail” category as assessed by the colorimetric Test Kit. This was supported by determining the bio-efficacy of the nets using the WHO recommended cone bioassays. The Test Kit was field evaluated in situ and found to be rapid, accurate, and easy to use by people without laboratory training. The Test Kit was shown to have a reliable linear relationship between the depth of colour produced and deltamethrin concentration (R2 = 0.9135). Conclusion This study shows that this colorimetric test was a reliable method to assess the insecticidal content of LLINs under operational conditions. The Test Kit provides immediate results and offers a rapid, inexpensive, field-friendly alternative to the complicated and costly methods such as HPLC and WHO cone bioassays which also need specialist staff. Thus, enabling National Malaria Control Programmes to gain access to effective and affordable monitoring tools for use in situ.

Household food security access, and amidst Covid-19: a study case in Equatorial Guinea

Food is of primary importance for households. Equatorial Guinea (EG) has progressed much in income growth but less in living standard, which includes food security. To situating food–insecurity prevalence in EG, this paper researches household food security-access, and its link-cause to agriculture–considering the Covid-19– using the Household Hunger Scale (HHS). On the data, we surveyed 400 households, imparted in EG rural and urban areas of Bioko Island and the mainland. The results reveal a 2.4 mean for both rural and urban Bioko, placing households at moderate hunger level. On the mainland, rural scores 3.21 (the area most affected), yet falling in moderate hunger level; while the urban scores best 0.64 and stands in the food-safe zone. It derives that diet is rather monotonous and embrace–mainly– carbohydrates rice, bread, cassava, and plantain. A focus on boosting the productivity of staples is paramount for Food security and even national security.

Population genetic analysis of the Plasmodium falciparum erythrocyte binding antigen-175 (EBA-175) gene in Equatorial Guinea

Background Plasmodium falciparum erythrocyte binding antigen-175 (PfEBA-175) is a candidate antigen for a blood-stage malaria vaccine, while various polymorphisms and dimorphism have prevented to development of effective vaccines based on this gene. This study aimed to investigate the dimorphism of PfEBA-175 on both the Bioko Island and continent of Equatorial Guinea, as well as the genetic polymorphism and natural selection of global PfEBA-175. Methods The allelic dimorphism of PfEBA-175 region II of 297 bloods samples from Equatorial Guinea in 2018 and 2019 were investigated by nested polymerase chain reaction and sequencing. Polymorphic characteristics and the effect of natural selection were analyzed using MEGA 7.0, DnaSP 6.0 and PopART programs. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 and Foldx program. Results Both Bioko Island and Bata district populations, the frequency of the F-fragment was higher than that of the C-fragment of PfEBA-175 gene. The PfEBA-175 of Bioko Island and Bata district isolates showed a high degree of genetic variability and heterogeneity, with π values of 0.00407 & 0.00411 and Hd values of 0.958 & 0.976 for nucleotide diversity, respectively. The values of Tajima's D of PfEBA-175 on Bata district and Bioko Island were 0.56395 and − 0.27018, respectively. Globally, PfEBA-175 isolates from Asia were more diverse than those from Africa and South America, and genetic differentiation quantified by the fixation index between Asian and South American countries populations was significant (FST > 0.15, P < 0.05). A total of 310 global isolates clustered in 92 haplotypes, and only one cluster contained isolates from three continents. The mutations A34T, K109E, D278Y, K301N, L305V and D329N were predicted as probably damaging. Conclusions This study demonstrated that the dimorphism of F-fragment PfEBA-175 was remarkably predominant in the study area. The distribution patterns and genetic diversity of PfEBA-175 in Equatorial Guinea isolates were similar another region isolates. And the levels of recombination events suggested that natural selection and intragenic recombination might be the main drivers of genetic diversity in global PfEBA-175. These results have important reference value for the development of blood-stage malaria vaccine based on this antigen.

Measuring the accuracy of gridded human population density surfaces: A case study in Bioko Island, Equatorial Guinea

Background Geospatial datasets of population are becoming more common in models used for health policy. Publicly-available maps of human population make a consistent picture from inconsistent census data, and the techniques they use to impute data makes each population map unique. Each mapping model explains its methods, but it can be difficult to know which map is appropriate for which policy work. High quality census datasets, where available, are a unique opportunity to characterize maps by comparing them with truth. Methods We use census data from a bed-net mass-distribution campaign on Bioko Island, Equatorial Guinea, conducted by the Bioko Island Malaria Elimination Program as a gold standard to evaluate LandScan (LS), WorldPop Constrained (WP-C) and WorldPop Unconstrained (WP-U), Gridded Population of the World (GPW), and the High-Resolution Settlement Layer (HRSL). Each layer is compared to the gold-standard using statistical measures to evaluate distribution, error, and bias. We investigated how map choice affects burden estimates from a malaria prevalence model. Results Specific population layers were able to match the gold-standard distribution at different population densities. LandScan was able to most accurately capture highly urban distribution, HRSL and WP-C matched best at all other lower population densities. GPW and WP-U performed poorly everywhere. Correctly capturing empty pixels is key, and smaller pixel sizes (100 m vs 1 km) improve this. Normalizing areas based on known district populations increased performance. The use of differing population layers in a malaria model showed a disparity in results around transition points between endemicity levels. Discussion The metrics in this paper, some of them novel in this context, characterize how these population maps differ from the gold standard census and from each other. We show that the metrics help understand the performance of a population map within a malaria model. The closest match to the census data would combine LandScan within urban areas and the HRSL for rural areas. Researchers should prefer particular maps if health calculations have a strong dependency on knowing where people are not, or if it is important to categorize variation in density within a city.

Quantifying malaria acquired during travel and its role in malaria elimination on Bioko Island

Background Malaria elimination is the goal for Bioko Island, Equatorial Guinea. Intensive interventions implemented since 2004 have reduced prevalence, but progress has stalled in recent years. A challenge for elimination has been malaria infections in residents acquired during travel to mainland Equatorial Guinea. The present article quantifies how off-island contributes to remaining malaria prevalence on Bioko Island, and investigates the potential role of a pre-erythrocytic vaccine in making further progress towards elimination. Methods Malaria transmission on Bioko Island was simulated using a model calibrated based on data from the Malaria Indicator Surveys (MIS) from 2015 to 2018, including detailed travel histories and malaria positivity by rapid-diagnostic tests (RDTs), as well as geospatial estimates of malaria prevalence. Mosquito population density was adjusted to fit local transmission, conditional on importation rates under current levels of control and within-island mobility. The simulations were then used to evaluate the impact of two pre-erythrocytic vaccine distribution strategies: mass treat and vaccinate, and prophylactic vaccination for off-island travellers. Lastly, a sensitivity analysis was performed through an ensemble of simulations fit to the Bayesian joint posterior probability distribution of the geospatial prevalence estimates. Results The simulations suggest that in Malabo, an urban city containing 80% of the population, there are some pockets of residual transmission, but a large proportion of infections are acquired off-island by travellers to the mainland. Outside of Malabo, prevalence was mainly attributable to local transmission. The uncertainty in the local transmission vs. importation is lowest within Malabo and highest outside. Using a pre-erythrocytic vaccine to protect travellers would have larger benefits than using the vaccine to protect residents of Bioko Island from local transmission. In simulations, mass treatment and vaccination had short-lived benefits, as malaria prevalence returned to current levels as the vaccine’s efficacy waned. Prophylactic vaccination of travellers resulted in longer-lasting reductions in prevalence. These projections were robust to underlying uncertainty in prevalence estimates. Conclusions The modelled outcomes suggest that the volume of malaria cases imported from the mainland is a partial driver of continued endemic malaria on Bioko Island, and that continued elimination efforts on must account for human travel activity.

Incidence of Plasmodium falciparum malaria infection in 6-month to 45-year-olds on selected areas of Bioko Island, Equatorial Guinea

Background Extensive malaria control measures have been implemented on Bioko Island, Equatorial Guinea over the past 16 years, reducing parasite prevalence and malaria-related morbidity and mortality, but without achieving elimination. Malaria vaccines offer hope for reducing the burden to zero. Three phase 1/2 studies have been conducted successfully on Bioko Island to evaluate the safety and efficacy of whole Plasmodium falciparum (Pf) sporozoite (SPZ) malaria vaccines. A large, pivotal trial of the safety and efficacy of the radiation-attenuated Sanaria® PfSPZ Vaccine against P. falciparum is planned for 2022. This study assessed the incidence of malaria at the phase 3 study site and characterized the influence of socio-demographic factors on the burden of malaria to guide trial design. Methods A cohort of 240 randomly selected individuals aged 6 months to 45 years from selected areas of North Bioko Province, Bioko Island, was followed for 24 weeks after clearance of parasitaemia. Assessment of clinical presentation consistent with malaria and thick blood smears were performed every 2 weeks. Incidence of first and multiple malaria infections per person-time of follow-up was estimated, compared between age groups, and examined for associated socio-demographic risk factors. Results There were 58 malaria infection episodes observed during the follow up period, including 47 first and 11 repeat infections. The incidence of malaria was 0.25 [95% CI (0.19, 0.32)] and of first malaria was 0.23 [95% CI (0.17, 0.30)] per person per 24 weeks (0.22 in 6–59-month-olds, 0.26 in 5–17-year-olds, 0.20 in 18–45-year-olds). Incidence of first malaria with symptoms was 0.13 [95% CI (0.09, 0.19)] per person per 24 weeks (0.16 in 6–59-month-olds, 0.10 in 5–17-year-olds, 0.11 in 18–45-year-olds). Multivariate assessment showed that study area, gender, malaria positivity at screening, and household socioeconomic status independently predicted the observed incidence of malaria. Conclusion Despite intensive malaria control efforts on Bioko Island, local transmission remains and is spread evenly throughout age groups. These incidence rates indicate moderate malaria transmission which may be sufficient to support future larger trials of PfSPZ Vaccine. The long-term goal is to conduct mass vaccination programmes to halt transmission and eliminate P. falciparum malaria.