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Objectives: This study assesses exposure to malaria vector mosquitos that is nonpreventable through use of nets, the contribution of outdoor and indoor biting towards residual vector exposure, and the risk factors for being bitten and for being infected with malaria parasites on Bioko Island, Equatorial Guinea., Methods: Human behavior and malaria infection data were collected from 13,735 randomly selected residents during cross-sectional surveys, concomitantly with entomological human landing catches, indoors and outdoors, in 20 locations on the Island. Self-reported time of going indoors, going to bed and whether using a net were analyzed to impute for each respondent the number of bites received outdoors and indoors during the night before the survey., Results: On average, each person received 2.7 (95% CI: 2.6-2.8) bites per night outdoors, 8.5 (8.3 to 8.7) bites indoors if not using a net, and 4.7 (4.5 to 4.8) bites indoors if using a net. Malaria infection was associated with more bites, regardless of whether received indoors or outdoors. Older age, male gender, not using a net, rural location, and going indoors later increased the risk of being bitten. The proportion of bites not averted by using a net was estimated as 66% (61 to 71)., Conclusions: A large proportion of biting, mostly indoors, may not be preventable by bednets. Tools targeting indoor biting should be prioritized in Bioko. Novel vector control tools are urgently needed to reduce overall exposure to mosquito bites., Competing Interests: Declarations of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Importation of malaria infections is a suspected driver of sustained malaria prevalence on areas of Bioko Island, Equatorial Guinea. Quantifying the impact of imported infections is difficult because of the dynamic nature of the disease and complexity of designing a randomized trial. We leverage a six-month travel moratorium in and out of Bioko Island during the initial COVID-19 pandemic response to evaluate the contribution of imported infections to malaria prevalence on Bioko Island. Using a difference in differences design and data from island wide household surveys conducted before (2019) and after (2020) the travel moratorium, we compare the change in prevalence between areas of low historical travel to those with high historical travel. Here, we report that in the absence of a travel moratorium, the prevalence of infection in high travel areas was expected to be 9% higher than observed, highlighting the importance of control measures that target imported infections., (© 2024. The Author(s).)

Background: Soil-transmitted helminth (STH) infection control remains a priority in endemic regions where local epidemiological data are needed for sustainable control strategies, particularly regarding population knowledge, attitudes, and practices (KAP). Our work assessed KAP toward STH infection and associated factors among residents of Bata district, Equatorial Guinea., Methods: A community-based cross-sectional study was conducted among 14 randomly selected communities in the Bata district. Eligible participants were interviewed face-to-face using a standardized questionnaire. Participants aged under 18 years were interviewed in the presence of their parents or legal guardians. For participants aged less than ten, a simplified version of the main questionnaire was developed focusing on children's practices toward STH and was administered to their parents or legal guardians., Results: A total of 399 participants were included in the present analysis. Among them, 58% responded to the main questionnaire. The mean (± SD) age of participants aged 10 and over was 37.5 (± 22.2) years, and 60% of them were females, while the mean (± SD) age of those aged less than ten was 5.0 (± 2.5) years. The respondents' overall knowledge, attitudes, and practices to STH were rated as bad (33%), very good (77%), and good (55%), respectively. Knowledge was significantly associated with education level (p = 0.04) with the knowledge level lower for participants with no formal education than for those with secondary/university education (β = -0.56, 95% CI: -1.00 - -0.12, p = 0.01); Appropriate attitudes level was significantly associated with occupation (p = 0.02) and education levels (p = 0.049) with the appropriate attitude level lower for students than for farmers/fishers (β = -1.24, 95% CI: -2.17--0.32, p = 0.01) and for primary-level participants than for those with secondary/university education (β = -0.68, 95% CI: -1.23--0.13, p = 0.02); while appropriate practice level were significantly associated with age (p = 0.01), occupation (p = 0.01), and education (p = 0.02), with the appropriate practices level increasing with age (β = 0.03, 95% CI: 0.005 - 0.05, p = 0.01) and lower in participants with no formal education than in those with secondary/university education (β = -1.19, -2.05 - -0.32, p = 0.007)., Conclusion: The present study revealed a lack of knowledge about STH in the study population, particularly regarding disease causes and transmission ways, highlighting the need for the implementation of integrated health education strategies, both at the community and school levels., (© 2024. The Author(s).)

Objective: To investigate the prevalence of single nucleotide polymorphisms (SNPs) of artemisinin resistance-related Pfubp1 and Pfap2mu genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea, so as to to provide baseline data for the formulation of malaria control strategies in Bioko Island., Methods: A total of 184 clinical blood samples were collected from patients with P. falciparum malaria in Bioko Island, Equatorial Guinea from 2018 to 2020, and genomic DNA was extracted. The Pfubp1 and Pfap2mu gene SNPs of P. falciparum were determined using a nested PCR assay and Sanger sequencing, and the gene sequences were aligned., Results: There were 159 wild-type P. falciparum isolates (88.83%) from Bioko Island, Equatorial Guinea, and 6 SNPs were identified in 20 Pfubp1 -mutant P. falciparum isolates (11.17%), in which 4 non-synonymous mutations were detected, including E1516G, K1520E, D1525E, E1528D. There was only one Pfubp1 gene mutation site in 19 Pfubp1 -mutant P. falciparum isolates (95.00%), in which non-synonymous mutations accounted for 68.42% (13/19). D1525E and E1528D were identified as major known epidemic mutation sites in the Pfubp1 gene associated with resistance to artemisinin-based combination therapies (ACTs). At amino acid position 1525, there were 178 wild-type P. falciparum isolates (99.44%) and 1 mutant isolate (0.56%), with such a mutation site identified in blood samples in 2018, and at amino acid position 1528, there were 167 wild-type P. falciparum isolates (93.30%) and 12 mutant isolates (6.70%). The proportions of wild-type P. falciparum isolates were 95.72% (134/140), 79.25% (126/159) and 95.83% (161/168) in the target amplification fragments of the three regions in the Pfap2mu gene ( Pfap2mu -inner1, Pfap2mu -inner2, Pfap2mu -inner3), respectively. There were 16 different SNPs identified in all successfully sequenced P. falciparum isolates, in which 7 non-synonymous mutations were detected, including S160N, K199T, A475V, S508G, I511M, L595F, and Y603H. There were 7 out of 43 Pfap2mu -mutant P. falciparum isolates (16.28%) that harbored only one gene mutation site, in which non-synonymous mutations accounted for 28.57% (2/7). For the known delayed clearance locus S160N associated with ACTs, there were 143 wild-type (89.94%) and 16 Pfap2mu -mutant P. falciparum isolates (10.06%)., Conclusions: Both Pfubp1 and Pfap2mu gene mutations were detected in P. falciparum isolates from Bioko Island, Equatorial Guinea from 2018 to 2020, with a low prevalence rate of Pfubp1 gene mutation and a high prevalence rate of Pfap2mu gene mutation. In addition, new mutation sites were identified in the Pfubp1 (E1504E and K1520E) and Pfap2mu genes (A475V and S508G).

Background: Tuberculosis (TB) is one of the leading causes of mortality from a single infectious disease agent. Equatorial Guinea is a country with high estimated TB incidence in 2021 (275 cases per 100,000 population) and low TB case detection (42%). Early diagnosis and prompt treatment are crucial for TB control. Failure to seek adequate health care increases the disease's transmission and leads to poor treatment outcome, the mortality, even for easily manageable conditions. Information regarding community management of TB and treatment-seeking patterns in Equatorial Guinea is rare. The aim of this study was to explore differences in TB health-seeking behaviour among urban and rural population TB cases in Equatorial Guinea and the factors associated with this behaviour., Methods: A national cross-sectional study of 770 household caregivers was conducted in 2020 in Equatorial Guinea using multistage stratified sampling. The 284 caregivers that reported having had a TB case in their family were included in this study. A practice index was created. Poisson regression with robust variance was performed with the practices index as dependent variable to assess the factors associated with the health-seeking behaviour., Results: Most of the cases (65%) have had good TB health-seeking practices. However, 23.2% of TB cases reported having abandoned treatment before 6 months. A higher probability of having good TB practices was observed with being women, aged and living in rural area. Those who were TB cases themselves have heard about TB on the radio, and had high knowledge about TB, hand also good practices., Conclusions: Disparities in tuberculosis health-seeking behaviour between rural and urban populations highlight the challenges existing in the fight against this infectious disease. The National Tuberculosis Control Program has to reinforce the health system needs to strengthen the follow-up of TB patients taking into account the population at risk of inappropriate TB behaviour., Trial Registration: Not applicable., (© 2023. The Author(s).)

The COVID-19 pandemic has not only strained healthcare systems in Africa but has also intensified the impact of emerging and re-emerging diseases. Specifically in Equatorial Guinea, mirroring the situation in other African countries, unique zoonotic outbreaks have occurred during this challenging period. One notable resurgence is Marburg virus disease (MVD), which has further burdened the already fragile healthcare system. The re-emergence of the Marburg virus amid the COVID-19 pandemic is believed to stem from a probable zoonotic spill-over, although the precise transmission routes remain uncertain. Given the gravity of the situation, addressing the existing challenges is paramount. Though the genome sequences from the current outbreak were not available for this study, we analyzed all the available whole genome sequences of this re-emerging pathogen to advocate for a shift towards active surveillance. This is essential to ensure the successful containment of any potential Marburg virus outbreak in Equatorial Guinea and the wider African context. This study, which presents an update on the phylodynamics and the genetic variability of MARV, further confirmed the existence of at least two distinct patterns of viral spread. One pattern demonstrates a slower but continuous and recurring virus circulation, while the other exhibits a faster yet limited and episodic spread. These results highlight the critical need to strengthen genomic surveillance in the region to effectively curb the pathogen's dissemination. Moreover, the study emphasizes the importance of prompt alert management, comprehensive case investigation and analysis, contact tracing, and active case searching. These steps are vital to support the healthcare system's response to this emerging health crisis. By implementing these strategies, we can better arm ourselves against the challenges posed by the resurgence of the Marburg virus and other infectious diseases.

A lack of HIV viral load (VL) and HIV drug resistance (HIVDR) monitoring in sub-Saharan Africa has led to an uncontrolled circulation of HIV-strains with drug resistance mutations (DRM), compromising antiretroviral therapy (ART). This study updates HIVDR data and HIV-1 variants in Equatorial Guinea (EG), providing the first data on children/adolescents in the country. From 2019−2020, 269 dried blood samples (DBS) were collected in Bata Regional Hospital (EG) from 187 adults (73 ART-naïve/114 ART-treated) and 82 children/adolescents (25 HIV-exposed-ART-naïve/57 ART-treated). HIV-1 infection was confirmed in Madrid by molecular/serological confirmatory tests and ART-failure by VL quantification. HIV-1 pol region was identified as transmitted/acquired DRM, predicted antiretroviral susceptibility (Stanfordv9.0) and HIV-1 variants (phylogeny). HIV infection was confirmed in 88.1% of the individuals and virological failure (VL > 1000 HIV-1-RNA copies/mL) in 84.2/88.9/61.9% of 169 ART-treated children/adolescents/adults. Among the 167 subjects with available data, 24.6% suffered a diagnostic delay. All 125 treated had experienced nucleoside retrotranscriptase inhibitors (NRTI); 95.2% were non-NRTI (NNRTI); 22.4% had experienced integrase inhibitors (INSTI); and 16% had experienced protease inhibitors (PI). At sampling, they had received 1 (37.6%), 2 (32%), 3 (24.8%) or 4 (5.6%) different ART-regimens. Among the 43 treated children−adolescents/37 adults with sequence, 62.8/64.9% carried viruses with major-DRM. Most harbored DRM to NNRTI (68.4/66.7%), NRTI (55.3/43.3%) or NRTI+NNRTI (50/33.3%). One adult and one child carried major-DRM to PI and none carried major-DRM to INSTI. Most participants were susceptible to INI and PI. DRM was absent in 36.2% of treated patients with VL > 1000 cp/mL, suggesting adherence failure. TDR prevalence in 59 ART-naïve adults was high (20.3%). One-half (53.9%) of the 141 subjects with pol sequence carried CRF02_AG. The observed high rate of ART-failure and transmitted/acquired HIVDR could compromise the 95-95-95-UNAIDS targets in EG. Routine VL and resistance monitoring implementation are mandatory for early detection of ART-failure and optimal rescue therapy selection ART regimens based on PI, and INSTI can improve HIV control in EG.

We present a comprehensive catalogue of the reptiles of Equatorial Guinea, consisting of 118 species belonging to 67 genera and 22 families. There are two species of Crocodylia, ten of Testudines and 106 of Squamata; this last taxon is represented by 62 species of snakes, two amphisbaenians and 42 lizards. Of these 118 species, seven are present only in Annobon, seven only in Bioko, 47 only in Río Muni, 53 occur both in Bioko and Río Muni (or Bioko, Río Muni and other islands), and four are sea turtles. Despite its high diversity, the level of endemism of Bioko is relatively low, with only four endemic species out of the 60 species reported for the island. In contrast, despite its low diversity, Annobon has the highest endemicity level, with five endemic species (and two introduced). No endemic species are known for the rest of the regions of Equatorial Guinea, which contain 100 species. We reveal several new country and species records, and point to some pending taxonomic questions to be addressed. Among the new species records, we highlight the presence of Cyclanorbis elegans, which represents the most meridional known population of the genus. Additional species are expected to be found in Equatorial Guinea as further field and taxonomic work is developed.

Artemisinin-based combination therapies (ACTs) resistance has emerged and could be diffusing in Africa. As an offshore island on the African continent, the island of Bioko in Equatorial Guinea is considered severely affected and resistant to drug-resistant Plasmodium falciparum malaria. However, the spatial and temporal distribution remain unclear. Molecular monitoring targeting the Pfcrt , Pfk13 , Pfpm2 , and Pfmdr1 genes was conducted to provide insight into the impact of current antimalarial drug resistance on the island. Furthermore, polymorphic characteristics, haplotype network, and the effect of natural selection of the Pfk13 gene were evaluated. A total of 152 Plasmodium falciparum samples (collected from 2017 to 2019) were analyzed for copy number variation of the Pfpm2 gene and Pfk13 , Pfcrt , and Pfmdr1 mutations. Statistical analysis of Pfk13 sequences was performed following different evolutionary models using 96 Bioko sequences and 1322 global sequences. The results showed that the prevalence of Pfk13, Pfcrt, and Pfmdr1 mutations was 73.68%, 78.29%, and 75.66%, respectively. Large proportions of isolates with multiple copies of Pfpm2 were observed (67.86%). In Bioko parasites, the genetic diversity of Pfk13 was low, and purifying selection was suggested by Tajima's D test (-1.644, P > 0.05) and the dN/dS test (-0.0004438, P > 0.05). The extended haplotype homozygosity analysis revealed that Pfk13 _K189T, although most frequent in Africa, has not yet conferred a selective advantage for parasitic survival. The results suggested that the implementation of continuous drug monitoring on Bioko Island is an essential measure. IMPORTANCE Malaria, one of the tropical parasitic diseases with a high transmission rate in Bioko Island, Equatorial Guinea, especially caused by P. falciparum is highly prevalent in this region and is commonly treated locally with ACTs. The declining antimalarial susceptibility of artemisinin-based drugs suggested that resistance to artemisinin and its derivatives is developing in P. falciparum. Copy number variants in Pfpm2 and genetic polymorphisms in Pfk13 , Pfcrt , and Pfmdr1 can be used as risk assessment indicators to track the development and spread of drug resistance. This study reported for the first time the molecular surveillance of Pfpm2, Pfcrt , Pfk13 , and Pfmdr1 genes in Bioko Island from 2017 to 2019 to assess the possible risk of local drug-resistant P. falciparum.

Introduction: In March 2021, a series of explosions shook a military base in Bata, Equatorial Guinea. As a response to government officials' request, the Israel Defense Forces Medical Corps (IDF-MC) deployed an emergency aid team that faced two major challenges: (1) understanding the scenario, the injury patterns, and the needs of the local medical system; and (2) minimizing the coronavirus disease 2019 (COVID-19) outbreak threats. This report describes the team design, the activities performed before and during the deployment, analyzes the pathology encountered, and shares lessons learned from the mission., Sources: Data were collected from the delegation protocols and IDF medical records. All activities of the Israeli delegation were coordinated with the local government., Observations: The local authorities reported that a total of 107 people were killed and more than 700 people were wounded. The team was the first international team to arrive at the scene and assisted the local medical teams to treat 231 patients in the three local hospitals and 213 patients in field clinics in the villages surrounding Bata. The COVID-19 pandemic influenced the operation of this mission, and caution measures were activated., Analysis: Unplanned explosions at munitions sites (UEMS) are a growing problem causing the medical teams to face unique challenges. By understanding the expected challenges, the team was reinforced with a plastic surgeon, portable ultrasound devices, a large amount and a variety of antibiotics, whole blood units, and freeze-dried plasma. Rehabilitation experts were needed in some cases in the week following the injury. An important key for the success of this kind of medical aid delegation is the collaboration with the local medical teams, which enhances patient care.

Background: Regular and comprehensive epidemiological surveys of the filarial nematodes Mansonella perstans and Loa loa in children, adolescents and adults living across Bioko Island, Equatorial Guinea are lacking. We aimed to demonstrate that blood retained on malaria rapid diagnostic tests, commonly deployed for malaria surveys, could be used as a source of nucleic acids for molecular based detection of M. perstans and L. loa. We wanted to determine the positivity rate and distribution of filarial nematodes across different age groups and geographical areas as well as to understand level of co-infections with malaria in an asymptomatic population., Methodology: M. perstans, L. loa and Plasmodium spp. parasites were monitored by qPCR in a cross-sectional study using DNA extracted from a subset malaria rapid diagnostic tests (mRDTs) collected during the annual malaria indicator survey conducted on Bioko Island in 2018., Principal Findings: We identified DNA specific for the two filarial nematodes investigated among 8.2% (263) of the 3214 RDTs screened. Positivity rates of M. perstans and L. loa were 6.6% and 1.5%, respectively. M. perstans infection were more prominent in male (10.5%) compared to female (3.9%) survey participants. M. perstans parasite density and positivity rate was higher among older people and the population living in rural areas. The socio-economic status of participants strongly influenced the infection rate with people belonging to the lowest socio-economic quintile more than 3 and 5 times more likely to be L. loa and M. perstans infected, respectively. No increased risk of being co-infected with Plasmodium spp. parasites was observed among the different age groups., Conclusions/significance: We found otherwise asymptomatic individuals were infected with M. perstans and L. loa. Our study demonstrates that employing mRDTs probed with blood for malaria testing represents a promising, future tool to preserve and ship NAs at room temperature to laboratories for molecular, high-throughput diagnosis and genotyping of blood-dwelling nematode filarial infections. Using this approach, asymptomatic populations can be reached and surveyed for infectious diseases beyond malaria., Competing Interests: The authors have declared that no competing interests exist.

Background: Surveillance programmes often use malaria rapid diagnostic tests (RDTs) to determine the proportion of the population carrying parasites in their peripheral blood to assess the malaria transmission intensity. Despite an increasing number of reports on false-negative and false-positive RDT results, there is a lack of systematic quality control activities for RDTs deployed in malaria surveillance programmes., Methods: The diagnostic performance of field-deployed RDTs used for malaria surveys was assessed by retrospective molecular analysis of the blood retained on the tests., Results: Of the 2865 RDTs that were collected in 2018 on Bioko Island and analysed in this study, 4.7% had a false-negative result. These false-negative RDTs were associated with low parasite density infections. In 16.6% of analysed samples, masked pfhrp2 and pfhrp3 gene deletions were identified, in which at least one Plasmodium falciparum strain carried a gene deletion. Among all positive RDTs analysed, 28.4% were tested negative by qPCR and therefore considered to be false-positive. Analysing the questionnaire data collected from the participants, this high proportion of false-positive RDTs could be explained by P. falciparum histidine rich protein 2 (PfHRP2) antigen persistence after recent malaria treatment., Conclusion: Malaria surveillance depending solely on RDTs needs well-integrated quality control procedures to assess the extent and impact of reduced sensitivity and specificity of RDTs on malaria control programmes., (© 2022. The Author(s).)

The need for tools that facilitate rapid detection and continuous monitoring of SARS-CoV-2 variants of concern (VOCs) is greater than ever, as these variants are more transmissible and therefore increase the pressure of COVID-19 on healthcare systems. To address this demand, we aimed at developing and evaluating a robust and fast diagnostic approach for the identification of SARS-CoV-2 VOC-associated spike gene mutations. Our diagnostic assays detect the E484K and N501Y single-nucleotide polymorphisms (SNPs) as well as a spike gene deletion (HV69/70) and can be run on standard laboratory equipment or on the portable rapid diagnostic technology platform peak PCR. The assays achieved excellent diagnostic performance when tested with RNA extracted from culture-derived SARS-CoV-2 VOC lineages and clinical samples collected in Equatorial Guinea, Central-West Africa. Simplicity of usage and the relatively low cost are advantages that make our approach well suitable for decentralized and rapid testing, especially in resource-limited settings.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2021. The Author(s).)

Objective: To investigate the genetic polymorphisms of Plasmodium falciparum multidrug resistance protein 1 ( PfMDR1 ), chloroquine resistance transporter ( PfCRT ) and Kelch 13 ( PfK13 ) genes in Bioko Island, Equatorial Guinea, so as to provide insights into the development of the malaria control strategy in local areas., Methods: A total of 85 peripheral blood samples were collected from patients with Plasmodium falciparum infections in Bioko Island, Equatorial Guinea in 2018 and 2019, and genomic DNA was extracted. The PfMDR1 , PfCRT and PfK13 genes were amplified using a nested PCR assay. The amplification products were sequenced, and the gene sequences were aligned., Results: There were no mutations associated with artemisinin resistance in PfK13 gene in Bioko Island, Equatorial Guinea, while drug-resistant mutations were detected in PfMDR1 and PfCRT genes, and the proportions of PfMDR1_N86Y, PfMDR1_Y184F and PfCRT_K76T mutations were 35.29% (30/85), 72.94% (62/85) and 24.71% (21/85), respectively., Conclusions: There are mutations in PfMDR1 , PfCRT and PfK13 genes in P. falciparum isolates from 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., (© 2021. The Author(s).)

Background: Prevalence of falciparum malaria on Bioko Island remains high despite sustained, intensive control. Progress may be hindered by high proportions of subpatent infections that are not detected by rapid diagnostic tests (RDT) but contribute to onward transmission, and by imported infections. Better understanding of the relationship between subpatent infections and RDT-detected infections, and whether this relationship is different from imported versus locally acquired infections, is imperative to better understand the sources of infection and mechanisms of transmission to tailor more effective interventions., Methods: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on a sub-set of samples from the 2015 Malaria Indicator Survey to identify subpatent infections. Households with RDT(+) individuals were matched 1:4 with households with no RDT(+) individuals. The association between living in a household with an RDT(+) individual and having a subpatent infection was evaluated using multivariate hierarchical logistic regression models with inverse probability weights for selection. To evaluate possible modification of the association by potential importation of the RDT(+) case, the analysis was repeated among strata of matched sets based on the reported eight-week travel history of the RDT(+) individual(s)., Results: There were 142 subpatent infections detected in 1,400 individuals (10.0%). The prevalence of subpatent infections was higher in households with versus without an RDT(+) individual (15.0 vs 9.1%). The adjusted prevalence odds of subpatent infection were 2.59-fold greater (95% CI: 1.31, 5.09) for those in a household with an RDT(+) individual compared to individuals in a household without RDT(+) individuals. When stratifying by travel history of the RDT(+) individual, the association between subpatent infections and RDT(+) infections was stronger in the strata in which the RDT(+) individual(s) had not recently travelled (adjusted prevalence odds ratio (aPOR) 2.95; 95% CI:1.17, 7.41), and attenuated in the strata in which recent travel was reported (aPOR 1.76; 95% CI: 0.54, 5.67)., Conclusions: There is clustering of subpatent infections around RDT(+) individual(s) when both imported and local infection are suspected. Future control strategies that aim to treat whole households in which an RDT(+) individual is found may target a substantial portion of infections that would otherwise not be detected., (© 2021. The Author(s).)

Background: Thrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described., Methods: 153 blood spot samples from Bioko malaria patients were collected during 2016-2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools., Results: A total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN-dS (6.2231, p < 0.05) hinted at natural selection of PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p < 0.05). 667 Asian isolates clustered in 136 haplotypes and 739 African isolates clustered in 528 haplotypes by network analysis. The mutations I116T, L221I, Y128F, G228V and P299S were predicted as probably damaging by PolyPhen online service, while mutations L49V, R285G, R285S, P299S and K421N would lead to a significant increase of free energy difference (ΔΔG > 1) indicated a destabilization of protein structure., Conclusions: Evidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.

Plasmodium falciparum sporozoite (PfSPZ) Vaccine (radiation-attenuated, aseptic, purified, cryopreserved PfSPZ) and PfSPZ-CVac (infectious, aseptic, purified, cryopreserved PfSPZ administered to subjects taking weekly chloroquine chemoprophylaxis) have shown vaccine efficacies (VEs) of 100% against homologous controlled human malaria infection (CHMI) in nonimmune adults. Plasmodium falciparum sporozoite-CVac has never been assessed against CHMI in African vaccinees. We assessed the safety, immunogenicity, and VE against homologous CHMI of three doses of 2.7 × 10 6 PfSPZ of PfSPZ Vaccine at 8-week intervals and three doses of 1.0 × 10 5 PfSPZ of PfSPZ-CVac at 4-week intervals with each arm randomized, double-blind, placebo-controlled, and conducted in parallel. There were no differences in solicited adverse events between vaccinees and normal saline controls, or between PfSPZ Vaccine and PfSPZ-CVac recipients during the 6 days after administration of investigational product. However, from days 7-13, PfSPZ-CVac recipients had significantly more AEs, probably because of Pf parasitemia. Antibody responses were 2.9 times higher in PfSPZ Vaccine recipients than PfSPZ-CVac recipients at time of CHMI. Vaccine efficacy at a median of 14 weeks after last PfSPZ-CVac dose was 55% (8 of 13, P = 0.051) and at a median of 15 weeks after last PfSPZ Vaccine dose was 27% (5 of 15, P = 0.32). The higher VE in PfSPZ-CVac recipients of 55% with a 27-fold lower dose was likely a result of later stage parasite maturation in the liver, leading to induction of cellular immunity against a greater quantity and broader array of antigens.

Providing medical care for participants in clinical trials in resource-limited settings can be challenging and costly. Evaluation and treatment of a young man who developed cervical lymphadenopathy during a malaria vaccine trial in Equatorial Guinea required concerted efforts of a multinational, multidisciplinary team. Once a diagnosis of diffuse large B-cell lymphoma was made, the patient was taken to India to receive immunochemotherapy. This case demonstrates how high-quality medical care was provided for a serious illness that occurred during a trial that was conducted in a setting in which positron emission tomography for diagnostic staging, an oncologist for supervision of treatment, and an optimal therapeutic intervention were not available. Clinical researchers should anticipate the occurrence of medical conditions among study subjects, clearly delineate the extent to which health care will be provided, and set aside funds commensurate with those commitments.

Fifteen years of investment in malaria control on Bioko Island, Equatorial Guinea (EG), dramatically reduced malaria-associated morbidity and mortality, but the impact has plateaued. To progress toward elimination, EG is investing in the development of a malaria vaccine. We assessed the unique public-private partnership that has had such a significant impact on malaria on Bioko Island and now added a major effort on malaria vaccine development. As part of a $79M commitment, the EG government (75%) and three American energy companies (25%) have invested since 2012 greater than $55M in the Equatoguinean Malaria Vaccine Initiative (EGMVI) to support clinical development of Sanaria ® PfSPZ vaccines (Sanaria Inc., Rockville, MD). In turn, the vaccine development program is building human capital and physical capacity. The EGMVI established regulatory and ethical oversight to ensure compliance with the International Conference on Harmonization and Good Clinical Practices for the first importation of investigational product, ethical approval, and conduct of a clinical trial in Equatoguinean history. The EGMVI has completed three vaccine trials in EG, two vaccine trials in Tanzania, and a malaria incidence study, and initiated preparations for a 2,100-volunteer clinical trial. Personnel are training for advanced degrees abroad and have been trained in Good Clinical Practices and protocol-specific methods. A new facility has established the foundation for a national research institute. Biomedical research and development within this visionary, ambitious public-private partnership is fostering major improvements in EG. The EGMVI plans to use a PfSPZ Vaccine alongside standard malaria control interventions to eliminate Pf malaria from Bioko, becoming a potential model for elimination campaigns elsewhere.

At the beginning of 2019, a sudden surge of malaria cases was observed in the district of Riaba, Bioko Island. Between January and April, confirmed malaria cases increased 3.8-fold compared to the same period in 2018. Concurrently, anopheline human biting rate (HBR) increased 2.1-fold. During the outbreak, 82.2% of the district population was tested for malaria with a rapid diagnostic test; 37.2% of those tested had a detectable infection and were treated according to national guidelines. Vector control interventions, including indoor residual spraying and larval source management were scaled-up. After the interventions, the number of confirmed cases decreased by 70% and the overall parasite prevalence in the communities by 43.8%. Observed prevalence in a follow up malaria indicator survey, however, was significantly higher than elsewhere on the island, and higher than in previous years. There was no significant reduction in HBR, which remained high for the rest of the year. The surge was attributed to various factors, chiefly increased rainfall and a large number of anthropogenic anopheline breeding sites created by construction works. This case study highlights the need for sustained vector control interventions and multi-sector participation, particularly in malaria control and elimination settings with persistently high local malaria receptivity.

Objective: To explore whether the climate has played a role in the COVID-19 outbreak, we compared virus lethality in countries closer to the Equator with others. Lethality in European territories and in territories of some nations with a non-temperate climate was also compared., Materials and Methods: Lethality was calculated as the rate of deaths in a determinate moment from the outbreak of the pandemic out of the total of identified positives for COVID-19 in a given area/nation, based on the COVID-John Hopkins University website. Lethality of countries located within the 5th parallels North/South on 6 April and 6 May 2020, was compared with that of all the other countries. Lethality in the European areas of The Netherlands, France and the United Kingdom was also compared to the territories of the same nations in areas with a non-temperate climate., Results: A lower lethality rate of COVID-19 was found in Equatorial countries both on April 6 (OR=0.72 CI 95% 0.66-0.80) and on May 6 (OR=0.48, CI 95% 0.47-0.51), with a strengthening over time of the protective effect. A trend of higher risk in European vs. non-temperate areas was found on April 6, but a clear difference was evident one month later: France (OR=0.13, CI 95% 0.10-0.18), The Netherlands (OR=0.5, CI 95% 0.3-0.9) and the UK (OR=0.2, CI 95% 0.01-0.51). This result does not seem to be totally related to the differences in age distribution of different sites., Conclusions: The study does not seem to exclude that the lethality of COVID-19 may be climate sensitive. Future studies will have to confirm these clues, due to potential confounding factors, such as pollution, population age, and exposure to malaria.

The use of malaria rapid diagnostic tests (RDTs) as a source for nucleic acids that can be analyzed via nucleic acid amplification techniques has several advantages, including minimal amounts of blood, sample collection, simplified storage and shipping conditions at room temperature. We have systematically developed and extensively evaluated a procedure to extract total nucleic acids from used malaria RDTs. The co-extraction of DNA and RNA molecules from small volumes of dried blood retained on the RDTs allows detection and quantification of P. falciparum parasites from asymptomatic patients with parasite densities as low as 1 Pf/µL blood using reverse transcription quantitative PCR. Based on the extraction protocol we have developed the ENAR (Extraction of Nucleic Acids from RDTs) approach; a complete workflow for large-scale molecular malaria surveillance. Using RDTs collected during a malaria indicator survey we demonstrated that ENAR provides a powerful tool to analyze nucleic acids from thousands of RDTs in a standardized and high-throughput manner. We found several, known and new, non-synonymous single nucleotide polymorphisms in the propeller region of the kelch 13 gene among isolates circulating on Bioko Island, Equatorial Guinea.

Objective: To investigate the drug-resistant gene polymorphisms in Plasmodium falciparum imported from Equatorial Guinea to Shandong Province., Methods: From 2015 to 2016, blood samples were collected from imported P. falciparum malaria patients returning from Equatorial Guinea to Shandong Province, and genome DNA of the malaria parasite was extracted. The drug-resistant Pfcrt , Pfmdr1 , Pfdhfr , Pfdhps , and K13 genes of P. falciparum were amplified using a PCR assay, followed by DNA sequencing, and the sequences were aligned., Results: The target fragments of all 5 drug-resistant genes of P. falciparum were successfully amplified and sequenced. There were 72.8%, 18.6%, and 8.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfcrt gene, respectively, and all mutant haplotypes were CVI ET (the underline indicates the mutation site). There were 20.0%, 61.4% and 18.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfmdr1 gene, respectively, and the mutant haplotypes mainly included YF and NF (the underlines indicate the mutation sites). There were 1.4%, 98.6%, and 0 of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfdhfr gene, respectively, and A IRN I was the predominant mutant haplotype (the underline indicates the mutation site). There were 1.4%, 94.3%, and 4.3% of P. falci parum parasites carrying the wild-, mutant-, and mixed-type Pfdhps gene, respectively, and S G KAA was the predominant mutant haplotype (the underline indicates the mutation site). The complete drug-resistant IRNGE genotype consisted of 8.6% of the Pfdhfr and Pfdhps genes, and the K13 gene A578S mutation occurred in 1.4% of the parasite samples., Conclusions: There are mutations in the Pfcrt , Pfmdr1 , Pfdhfr , Pfdhps , and K13 genes of P. falciparum imported from Equatorial Guinea to Shandong Province, with a low frequency in the Pfcrt gene mutation and a high frequency in the Pfmdr1 , Pfdhfr , and Pfdhps gene mutations, and the K13 gene A578S mutation is detected in the parasite samples.

Background: The World Health Organization (WHO) recommends rapid diagnostic tests (RDTs) as a good alternative malaria-diagnosis method in remote parts of sub-Saharan Africa. The majority of commercial RDTs currently available detect the Plasmodium falciparum protein histidine-rich protein 2 (PfHRP2). There have also been recent reports of pfhrp2 gene deletions being found in parasites collected from several African countries. The WHO has concluded that lacking the pfhrp2 gene must be monitored in Africa. The purpose of the study was to analyse why the samples that were positive by PCR were negative by RDTs and, therefore, to determine whether there have been deletions in the pfhrp2 and/or pfhrp3 genes., Methods: Malaria NM-PCR was carried out on all the samples collected in the field. A group of 128 samples was positive by PCR but negative by RDT; these samples were classified as RDT false-negatives. PCR was carried out for exon2 of pfhrp2 and pfhrp3 genes to detect the presence or absence of these two genes. Frequencies with 95% confidence intervals (CIs) were used for prevalence estimates. Associations were assessed by the Chi square test or Fisher´s exact test. The level of significance was set at p ≤ 0.05. Statistical analyses were performed using the software package SPSSv.15.0., Results: After PCR, 81 samples were identified (4.7%, 95% CI 3.8-5.8) which had deletion in both genes, pfhrp2 and pfhrp3. Overall, however, 11 samples (0.6%, 95% CI 0.36-1.14) had deletion only in pfhrp2 but not in pfhrp3, and 15 (0.9%, 95% CI 0.6-1.5) presented with deletion only in pfhrp3 but not in pfhrp2. Considering the pfhrp2 gene separately, within the total of 1724 samples, 92 (5.3%, 95% CI 4.37-6.5) had evidence of deletion., Conclusion: The present study provides the first evidence of deletion in the pfhrp2 and pfhrp3 genes in P. falciparum isolates from Equatorial Guinea. However, larger studies across different regions within the country and across different seasonal profiles are needed to determine the full extent of pfhrp2 and pfhrp3 deletion. It is strongly recommended to implement an active surveillance programme in order to detect any increases in pfhrp2 and pfhrp3 deletion frequencies.

Objective: To evaluate the nutritional, clinical and immunological status of children at HIV diagnosis in the continental region of Equatorial Guinea., Methods: Children <18 years diagnosed with HIV between 2009 and 2017 were included. Clinical, immunological and nutritional data were collected. Weight-for-height, weight-for-age and height-for-age Z-scores were calculated using WHO Child Growth Standards. The population was assessed in two equal periods (2009-2013 and 2014-2017) from the time of diagnosis., Results: A total of 213 children were diagnosed with HIV (49.3% males), median age 3.8 years (IQR: 1.5-8.2). A total of 121 cases (56.8%) were at WHO clinical stage III, and 53 (24.9%) were at WHO clinical stage IV. CDC immunological stage II was diagnosed in 51/130 (39.2%) and CDC stage III in 44/130 (33.8%). About 56.2% of children were underweight (weight-for-age <-2 SD); 20.1% moderately and 36.1% severely so. About 27.6% of children were wasted (weight-for-height <-2 SD); 11.9% moderately and 15.7% severely so. About 56.3% of children were stunted (height-for-age <-2 SD); 20.7% moderately and 35.6% severely so. The prevalence of wasting was higher in children ≤5 years than in children >5 years (36.4% vs. 19.1%, P = 0.026). In the second period, the prevalence of moderate-severe immunodeficiency decreased (87.2% to 67.0%, P = 0.018), without significant differences in the other nutritional or clinical data. Severe underweight was a risk factor for moderate-severe immunodeficiency (aOR: 4 [95% CI: 1.4-11.4], P = 0.010)., Conclusions: We highlight a high proportion of malnutrition at the time of HIV diagnosis in Guinea´s paediatric population. Early diagnosis of HIV infection is a priority, achievable by training Guinea´s physicians to suspect HIV early, introducing HIV molecular diagnostic techniques and ensuring intensive nutritional treatment., (© 2019 John Wiley & Sons Ltd.)

Aims: Little data have been published so far on the epidemiological aspects of hepatitis D virus (HDV) infection in immigrant populations and even poorer is the information on the virological, phylogenetic, and clinical aspects of this infection in these populations. This review article, aimed primarily at physicians caring for immigrants, summarizes the information available on HDV infection and analyzes data on this topic concerning the immigrant populations., Methods and Results: The prevalence of HDV infection in HBsAg-positive immigrants varies according to the country of origin. For example, in immigrants from sub-Saharan Africa, this prevalence is higher in those born in Equatorial Guinea (24.4%) than those from other African countries (10.3%). The epidemiological impact of HDV infection linked to migratory flows is a function of the different endemicity between countries of origin and countries in which a new existence has been established. This impact is high when immigrants from areas endemic to HDV infection (eg, Equatorial Guinea) settle in areas of low endemicity (eg, Germany or England, with a prevalence of around 4%), while the impact is lesser or nonexistent if the migratory flows are directed toward countries with intermediate endemicity (eg, Italy and Greece, with a prevalence of around 10%)., Conclusion: This impact of immigration on HDV epidemiology can be strong when HDV endemicity is high in the country of origin and low in the host country and slight when immigrants move to high or medium endemic countries., (© 2019 Wiley Periodicals, Inc.)

Over the past decade, insecticide resistance to malaria vectors has been identified in 71 malaria endemic countries. This has posed a major global health challenge in the fight against malaria, with declining rates of indoor residual spraying coverage attributed to pyrethroid-resistance. As part of its vector control monitoring strategies, the Bioko Island Malaria Control Project (BIMCP) in Equatorial Guinea conducted routine insecticide resistance bioassays using the WHO's standard susceptibility tests from 2013 to 2018. During the same period, the frequency of the target-site knockdown resistance allele (kdr) in the local vector population was also determined via PCR for detection of the L1014 F mutation. Biochemical analysis for metabolic resistance was also conducted in 2015. From 2016-2017, Fludora™ fusion, a formulated combination of clothianidin (a neonicotinoid) and deltamethrin (a pyrethroid) was evaluated for 9 months on Bioko Island, using the WHO's standard test procedure for determining residual effectiveness of insecticides on sprayed surfaces. In 2016, the mortality rate of the vectors on 0.05% deltamethrin was as low as 38%. The frequency of the West African form of knockdown resistance allele, L1014 F, in the vector population was as high as 80%, and metabolic resistance analysis indicated high upregulated cytochrome P450 s. However, the residual effectiveness of Fludora™ fusion recorded mortalities above 80% after 72 h of exposure for 8 months. Although both target-site knockdown resistance and metabolic resistance to pyrethroids were implicated in the local malaria vector population, Fludora™ fusion was effective under field conditions in controlling the resistant vectors for a period of 8 months on wooden surfaces on Bioko Island and represents a valuable addition to IRS programs, especially in regions with high levels of pyrethroid resistance., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Malaria burden on Bioko Island has decreased significantly over the past 15 years. The impact of interventions on malaria prevalence, however, has recently stalled. Here, we use data from island-wide, annual malaria indicator surveys to investigate human movement patterns and their relationship to Plasmodium falciparum prevalence. Using geostatistical and mathematical modelling, we find that off-island travel is more prevalent in and around the capital, Malabo. The odds of malaria infection among off-island travelers are significantly higher than the rest of the population. We estimate that malaria importation rates are high enough to explain malaria prevalence in much of Malabo and its surroundings, and that local transmission is highest along the West Coast of the island. Despite uncertainty, these estimates of residual transmission and importation serve as a basis for evaluating progress towards elimination and for efficiently allocating resources as Bioko makes the transition from control to elimination.

Background: Malaria can be transmitted by blood transfusion from human to human and it is responsible for the majority of transfusion-transmitted infectious diseases worldwide. In sub-Saharan Africa, it had been estimated that almost a quarter of blood donations contain malaria parasites. Since rapid diagnostic tests and thick blood smear microscopy lack sensitivity for low density parasitaemia, particularly in asymptomatic adults, the most reliable method to assess the problem of transfusion-transmitted malaria are nucleic acid-based molecular approaches such as quantitative polymerase chain reaction. The study was undertaken to determine the prevalence of sub-microscopic malaria parasite infection among blood donors in Malabo, Equatorial Guinea., Methods: Between July and August 2017, a total of 200 individual blood samples from blood donors at the Malabo Blood Bank were collected and screened by rapid diagnostic tests and thick blood smear microscopy. Retrospectively, the same samples were analysed for the presence of undetected, low-density malaria parasites using quantitative polymerase chain reaction., Results: In comparison to 6.5% (13/200) by rapid diagnostic test and 2.0% (4/200) by microscopy, the proportion of Plasmodium falciparum positive blood donations analysed by quantitative polymerase chain reaction was significantly higher (26%, 52/200). Densities of P. falciparum positive blood donations were ranging from 0.06 to 3707.0 parasites/µL with 79.6% below 100 parasites/µL and therefore not detectable by non-molecular malaria diagnostic tests. qPCR based species identification revealed that P. falciparum was the dominating species responsible for 88.1% (52/59) of positive blood donations, followed by Plasmodium malariae (15.3%, 9/59) and Plasmodium ovale (3.4%, 2/59)., Conclusions: This study confirms that in malaria endemic settings, sub-patent malaria infections among blood donors are prevalent. In blood collected from healthy donors living in Malabo, P. falciparum, P. malariae and P. ovale parasites were identified. Currently widely used malaria diagnostic tools have missed more than 75% of P. falciparum containing blood donations, demonstrating the value of quantitative polymerase chain reaction to reliably detect low density P. falciparum infections. Since the availability of molecular diagnostic methods in malaria endemic countries is still limited, the blood recipients living in malaria endemic countries should be treated following WHO recommendations.

Background: Malaria is still a serious public health problem on Bioko Island (Equatorial Guinea), although the number of annual cases has been greatly reduced since 2004 through the Bioko Island Malaria Control Project (BIMCP). A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate the genetic diversity of Plasmodium falciparum populations and multiplicity of infection (MOI) on Bioko Island 7 years after BIMCP., Methods: A total of 181 patients with uncomplicated P. falciparum malaria diagnosed with microscopy were collected from Bioko Island from January 2011 to December 2014. Parasite DNA was extracted using chelex-100 and species were identified using a real-time PCR followed by high-resolution melting. Plasmodium falciparum msp1 and msp2 allelic families were determined using nested PCR., Results: Three msp1 alleles (K1, MAD20, and RO33) and two msp2 alleles (FC27 and 3D7) were analysed in all samples. In msp1, the MAD20 allelic family was predominant with 96.69% (175/178) followed respectively by the K1 allelic family with 96.07% (171/178) and R033 allelic family with 70.78% (126/178). In msp2, the FC27 allelic family was the most frequently detected with 97.69% (169/173) compared to 3D7 with 72.25% (125/173). Twenty-six different alleles were observed in msp1 with 9 alleles for K1, 9 alleles for MAD20 and 8 alleles for R033. In msp2, 25 individual alleles were detected with 5 alleles for FC27 and 20 alleles for 3D7. The overall MOI was 5.51 with respectively 3.5 and 2.01 for msp1 and msp2. A significant increase in overall MOI was correlated with the age group of the patients (P = 0.026) or parasite densities (P = 0.04)., Conclusions: The present data showed high genetic diversity and MOI values among the P. falciparum population in the study, reflecting both the high endemic level and malaria transmission on Bioko Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area.

Introduction: Onchocerciasis is a chronic neglected tropical disease caused by the filarial nematode Onchocerca volvulus, which is endemic in Equatorial Guinea. The aim was to estimate the current spatial distribution of onchocerciasis, and its related factors, in Bioko Island after several years of mass drug administration and vector control activities, by using GIS technics., Methodology: The survey was carried out within the framework of a wider research project entitled "Strengthening the National Programme for Control of Onchocerciasis and other Filariasis in Equatorial Guinea". A structured questionnaire was designed to cover basic socio-demographic information and risk factors for onchocerciasis and the coordinates of household. the hydrographic network data to calculate the positive onchocerciasis rate was used. Poisson generalized linear model was used to explore the association between onchocerciasis and the following covariates: distance to the river, preventive practices, water source and household´s main source of income. Two different cluster analysis methods were used: Getis-Ord Gi statistic and SaTScan™ purely spatial statistic estimator., Results: The risk of onchocerciasis was higher for those who drank water from external sources (RR 25.3) than for those who drank home tap water (RR 8.0). The clusters with z-score higher were located at the east of the island. For 5 km and 1 km distances, one significant cluster in the east was detected (RR 5.91 and RR 7.15)., Conclusion: No environmental factors related with onchocerciasis were found, including proximity to rivers. This could be partially explained by the fact that the vector was eliminated in 2005., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2018 Diana Gómez-Barroso, Laura Moya, Zaida Herrador, Belén García, Justino Nguema, Policarpo Ncogo, Pilar Aparicio, Agustín Benito.)

Background: Portuguese-speaking countries (PSC) share the influence of the Portuguese culture but have socioeconomic development patterns that differ from that of Portugal., Objective: To describe trends in cardiovascular disease (CVD) morbidity and mortality in the PSC between 1990 and 2016, stratified by sex, and their association with the respective sociodemographic indexes (SDI)., Methods: This study used the Global Burden of Disease (GBD) 2016 data and methodology. Data collection followed international standards for death certification, through information systems on vital statistics and mortality surveillance, surveys, and hospital registries. Techniques were used to standardize causes of death by the direct method, as were corrections for underreporting of deaths and garbage codes. To determine the number of deaths due to each cause, the CODEm (Cause of Death Ensemble Model) algorithm was applied. Disability-adjusted life years (DALYs) and SDI (income per capita, educational attainment and total fertility rate) were estimated for each country. A p-value <0.05 was considered significant., Results: There are large differences, mainly related to socioeconomic conditions, in the relative impact of CVD burden in PSC. Among CVD, ischemic heart disease was the leading cause of death in all PSC in 2016, except for Mozambique and Sao Tome and Principe, where cerebrovascular diseases have supplanted it. The most relevant attributable risk factors for CVD among all PSC are hypertension and dietary factors., Conclusion: Collaboration among PSC may allow successful experiences in combating CVD to be shared between those countries.

Background: After the introduction of an artemisinin-based combination therapy, the reduction of prevalence of malaria infections has shown a remarkable progress during the last decade. However due to the lack of a consistent malaria control programme and socioeconomic inequalities, Plasmodium infection is still one of the major cause of disease in Equatorial Guinea, namely in the rural communities. This study explored the associated risk factors of malaria transmission at the microeconomic level (households) in two rural villages of mainland Equatorial Guinea., Methods: This survey involved 232 individuals living in 69 households located in two rural villages, Ngonamanga and Miyobo, of coastal and interior of Equatorial Guinea, respectively. Malaria prevalence was measured by PCR and parasitaemia level by optical microscopy; household socioeconomic status (SES) was measured based on house characteristics using a 2-step cluster analysis. Logistic regression analysis was performed to investigate the relationship of a diverse set of independent variables on being diagnosed with malaria and on showing high levels of parasitaemia., Results: The prevalence of Plasmodium spp. infection was 69%, with 80% of households having at least one parasitaemic member. The majority of houses have eaves (80%), walls of clay/wood (90%) and zinc roof (99%) and only 10% of them have basic sanitation facilities. The studied areas showed reduced rates of indoor residual spraying coverage (9%), and long-lasting insecticide-treated net ownership (35%), with none of these preventive tools showing any significant effects on malaria risk in these areas. Neither the risk of malaria infection (PCR positive result) or the development of high parasitaemia did show association with SES., Conclusions: This study has contributed to reinforce the importance of living conditions associated to a high risk of malaria infection and vulnerability to develop high parasitaemia. This study also contributes to future malaria control interventions to be implemented in mainland Equatorial Guinea or in other countries with similar environmental conditions.

Background: Onchocerciasis, also known as river blindness, is a parasitic disease. More than 99 percent of all cases occur in Africa. Bioko Island (Equatorial Guinea) is the only island endemic for onchocerciasis in the world. Since 2005, when vector Simulium yahense was eliminated, there have not been any reported cases of infection. This study aimed to demonstrate that updated WHO criteria for stopping mass drug administration (MDA) have been met., Methodology/principal Findings: A cross-sectional study was conducted from September 2016 to January 2017. Participants were 5- to 9-year-old school children. Onchocerciasis/lymphatic Filariasis (LF, only in endemic districts) rapid diagnostic tests (RDTs) were performed. Blood spots were collected from RDT positive children and 10 percent of the RDT negatives to determine Ov16 and Wb123 IgG4 antibodies through enzyme-linked immunosorbent assay (ELISA). Skin snips were collected from RDT positives. Filarial detection was performed by PCR in positives and indeterminate sera. Black fly collection was carried out in traditional breeding sites. A total of 7,052 children, ranging from 5 to 9 years of age, were included in the study. Four children (0.06%) were Ov16 IgG4 RDT positives, but negative by ELISA Ov16, while 6 RDT negative children tested positive by ELISA. A total of 1,230 children from the Riaba and Baney districts were tested for LF. One child was Wb123 RDT positive (0.08%), but ELISA negative, while 3 RDT negative children were positive by Wb123 ELISA. All positive samples were negative by PCR for onchocerciasis and LF (in blood spot and skin snip). All fly collections and larval prospections in the traditional catching and prospection sites were negative., Conclusions/significance: WHO criteria have been met, therefore MDA in Bioko Island can be stopped. Three years of post-treatment surveillance should be implemented to identify any new occurrences of exposure or infection.

Since 2004, indoor residual spraying (IRS) and long-lasting insecticide-impregnated bednets (LLINs) have reduced the malaria parasite prevalence in children on Bioko Island, Equatorial Guinea, from 45% to 12%. After target site-based (knockdown resistance; kdr ) pyrethroid resistance was detected in 2004 in Anopheles coluzzii (formerly known as the M form of the Anopheles gambiae complex), the carbamate bendiocarb was introduced. Subsequent analysis showed that kdr alone was not operationally significant, so pyrethroid-based IRS was successfully reintroduced in 2012. In 2007 and 2014-2015, mass distribution of new pyrethroid LLINs was undertaken to increase the net coverage levels. The combined selection pressure of IRS and LLINs resulted in an increase in the frequency of pyrethroid resistance in 2015. In addition to a significant increase in kd r frequency, an additional metabolic pyrethroid resistance mechanism had been selected. Increased metabolism of the pyrethroid deltamethrin was linked with up-regulation of the cytochrome P450 CYP9K1. The increase in resistance prompted a reversion to bendiocarb IRS in 2016 to avoid a resurgence of malaria, in line with the national Malaria Control Program plan., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Human filariae are vector-borne parasites and the causative agents of various diseases, including human onchocerciasis and lymphatic filariasis. Onchocerciasis causes a spectrum of cutaneous and ophthalmologic manifestations (including blindness) and has long been a major public health problem in Bioko Island (Equatorial Guinea). Bioko Island has been included in the WHO's Onchocerciasis Control Program since 1987. In Bioko Island, the specificity and sensitivity of clinical Onchocerca volvulus diagnosis is key. The objective of this work was to update onchocerciasis elimination progress in Bioko Island, after 18 years of mass ivermectin intervention, and the general filariasis situation through a rapid and accurate molecular method. A cross-sectional study was conducted in Bioko Island from mid-January to mid-February 2014. A total of 543 subjects were included in the study. Whole blood and one skin snip (from lumbar regions) were analysed with a real time PCR assay. Two other skin biopsies were analysed by an expert microscopist. All positive samples were confirmed by sequencing. Traditional microscopic examination of the skin biopsies failed to detect any microfilariae. However, 11 (2.03%) infections were detected using PCR assay, including one O. volvulus, two Mansonella streptocerca, seven Mansonella perstans and one Loa loa infections. PCR assays in blood detected 52 filariae-positive individuals (9.6%) which harboured M. perstans or L. loa. The low prevalence of O. volvulus confirms the success of the Onchocerciasis Control Programme and suggests that Mass Drug Administration in Bioko Island can be interrupted in the near future. The very high prevalence of M. perstans found in skin snips assays raises doubts about the reliability of microscope-based diagnosis of O. volvulus infections., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Whilst there have been substantial reductions in malaria transmission over the past decade, in many countries in West and Central Africa the malaria burden remains high. Monitoring and evaluation of malaria transmission trends and intervention strategies are key elements for malaria control programmes. This study uses a time series of annual malaria indicator surveys to track the progress of malaria control in Bioko Island, Equatorial Guinea, over a 13 year period of intensive interventions. Malaria infection and haemoglobin were measured annually in children (1 to 14 years) in cross-sectional household surveys from 2004 to 2016 in 18 sentinel sites across the island. Trends in transmission patterns were assessed and the impact of the vector control interventions (net use and spray coverage) was evaluated., Results: Between 2004 and 2016 approximately 106,500 individual tests for parasitaemia were conducted using rapid diagnostic tests. Although spray coverage remained relatively high (> 70%) over the time period, reported net usage was generally below 40%. Parasite prevalence reduced from 43.3 to 10.5% between 2004 and 2016. The prevalence of moderate to severe anaemia in children aged 1-5 years reduced from 14.9 to 1.6%. Impact in individual sites ranged from 57 to 100% reductions in parasite prevalence between 2004 and 2016. Sleeping under a net and living in a house that had been sprayed in the past 12 months were independently protective against infection (OR = 0.69 [95%CI 0.61-0.80] and OR = 0.87 [95% CI 0.78-0.97], respectively), whilst recent travel to the mainland increased the odds of infection nearly fourfold (OR = 3.94 [95%CI 2.79-5.56])., Conclusion: Island-wide interventions have resulted in a substantial reduction in malaria transmission on Bioko Island. This unique time series of 13 consecutive annual malaria indicator surveys clearly demonstrates the long-term effectiveness of the sustained use of two vector control interventions, indoor residual spraying and LLINs, and the value of comprehensive and sustained surveillance. Despite considerable success in reducing the burden on the island, malaria is still endemic, with populations in some areas remaining at high risk of infection.

Equatorial Guinea (EG) is a country in Central Africa with typical tropical weather. Sheep are an important source of food in EG, but the absence of information regarding infectious diseases that affect the native flocks of small ruminants is a concern. The country is currently implementing several new practices in the sheep industry associated with the importation of sheep from other countries. This study aimed to evaluate the seroprevalence of bacterial infections that are important to the sheep industry in EG sheep flocks. The detection of specific antibodies for the four agents studied was performed using enzyme-linked immunosorbent assay in 1,002 serum samples from EG sheep. The results showed a true prevalence of 13.37% for antibodies against Corynebacterium pseudotuberculosis, 0.59% for Brucella ovis, 19.89% for Chlamydophila abortus and 0.79% for Mycoplasma agalactiae in animals from production flocks. Among a group of 35 samples from isolated native animals, 47.56% were seropositive for antibodies against C. pseudotuberculosis, 42.84% for B. ovis, 54.28% for C. abortus and 11.35% for M. agalactiae. These results comprise the first report of the prevalence of infectious diseases in sheep in EG. They highlight the importance of adopting adequate measures to prevent infection by bacteria endemic to EG native flocks during the development of the sheep industry in the country.

Anemia in children under 5 years of age is a global public health problem. According to the World Health Organization the current rate of anemia among preschool aged children in Equatorial Guinea is 66%. No information is available above this age. The cross-sectional Prevamal Survey was conducted in 2013 aimed at providing baseline data on malaria prevalence in children aged 2 months-15 years old. Sampling was carried out with the use of a multistage, stratified cluster strategy in the district of Bata, Equatorial Guinea. The χ2 test and adjusted Poisson regression models were applied to assess the association between social-demographic and economic factors, malaria and anemia. A total of 1436 children were tested, out of which 1,421 children (99%) were tested for anemia. Over 85% were anemic; out of them, 284 (24%), 815 (67%) and 111 (9%) children had mild, moderate and severe anemia, respectively. Severe anemia was more frequent among children aged 2-12 months old and those living in rural sites. About 47% tested positive for malaria via a rapid diagnostic test (RDT). This rate was significantly higher in rural villages (66%; p<0.001). The prevalence of anemia and malaria was higher in rural settings (p<0.001). On the other hand, anemia in urban areas displayed a heterogeneity and complexity that differed from the rural environment: in urban neighbourhoods, children with concomitant malaria infection were more likely to be anemic (adjusted prevalence rate (aPR):1.19; CI 95%: 1.12-1.28). Moreover, the prevalence of anemia was higher in children aged above 13 months compared to younger children (p<0.005). Belonging to the poorest wealth tertile were positively (aPR: 1.14, 95% CI: 1.05-1.24) and children' parents being employees (aPR: 0.86, 95% CI: 0.76-0.96) or self-employed (aPR: 0.86, 95% CI: 0.76-0.97) vs. working in agriculture and/or fishing negatively associated with anemia among urban children. This marked urban-rural variation indicates the importance of targeting specific areas or districts. Strategies aimed at reducing malaria are clearly paramount in this country. Prevention and treatment of other factors associated with the etiology of anemia (e.g., iron deficiency) are also likely necessary to combat the burden of anemia in Equatorial Guinea.

Background: The transmission of malaria is intense in the majority of the countries of sub-Saharan Africa, particularly in those that are located along the Equatorial strip. The present study aimed to describe the current distribution of malaria prevalence among children and its environment-related factors as well as to detect malaria spatial clusters in the district of Bata, in Equatorial Guinea., Methods: From June to August 2013 a representative cross-sectional survey using a multistage, stratified, cluster-selected sample was carried out of children in urban and rural areas of Bata District. All children were tested for malaria using rapid diagnostic tests (RDTs). Results were linked to each household by global position system data. Two cluster analysis methods were used: hot spot analysis using the Getis-Ord Gi statistic, and the SaTScan™ spatial statistic estimates, based on the assumption of a Poisson distribution to detect spatial clusters. In addition, univariate associations and Poisson regression model were used to explore the association between malaria prevalence at household level with different environmental factors., Results: A total of 1416 children aged 2 months to 15 years living in 417 households were included in this study. Malaria prevalence by RDTs was 47.53%, being highest in the age group 6-15 years (63.24%, p < 0.001). Those children living in rural areas were there malaria risk is greater (65.81%) (p < 0.001). Malaria prevalence was higher in those houses located <1 km from a river and <3 km to a forest (IRR: 1.31; 95% CI 1.13-1.51 and IRR: 1.44; 95% CI 1.25-1.66, respectively). Poisson regression analysis also showed a decrease in malaria prevalence with altitude (IRR: 0.73; 95% CI 0.62-0.86). A significant cluster inland of the district, in rural areas has been found., Conclusions: This study reveals a high prevalence of RDT-based malaria among children in Bata district. Those households situated in inland rural areas, near to a river, a green area and/or at low altitude were a risk factor for malaria. Spatial tools can help policy makers to promote new recommendations for malaria control.

Malaria and HIV are two of the most severe public health problems in Africa. However, epidemiological data on Bioko Island is scarce. To investigate the prevalence of malaria and HIV infections and assess association of malaria and HIV infections and possible confounding factors, we performed a cross-sectional survey of people of malaria-endemic Bioko Island, Equatorial Guinea. A cross-sectional study of 1 526 subjects was carried out to determine the prevalence of malaria and HIV infection in Malabo region hospital on Bioko Island. Questionnaires were administered and venous blood samples were drawn for malaria parasites and HIV detection. The prevalence of participants infected with malaria and HIV in this area were 13.8% and 6.6% respectively. The average prevalence of co-infection for malaria and HIV was 0.92%. HIV-infection was significantly associated with the age and gender. Malaria infections were significantly associated with the age. This study showed that the prevalence of HIV and malaria on Bioko Island was higher than expected, although the co-infection prevalence of malaria and HIV was low. The results also indicated that malaria and HIV infections lead to more public health risk to youngsters and women.

Objective: Molecular epidemiology techniques in tuberculosis (TB) can identify high-risk strains that are actively transmitted. We aimed to implement a novel strategy to optimize the identification and control of multidrug-resistant (MDR) TB in a specific population., Methods: We developed a strain-specific PCR tailored from whole genome sequencing (WGS) data to track a specific MDR prevalent strain in Equatorial Guinea (EG-MDR)., Results: The PCR was applied prospectively on remnants of GeneXpert reaction mixtures owing to the lack of culture facilities in Equatorial Guinea. In 147 (93%) of 158 cases, we were able to differentiate between infection by the EG-MDR strain or by any other strain and found that 44% of all rifampicin-resistant TB cases were infected by EG-MDR. We also analysed 93 isolates obtained from Equatorial Guinea 15 years ago, before MDR-TB had become the problem it is today. We found that two of the scarce historical MDR cases were infected by EG-MDR. WGS revealed low variability-six single nucleotide polymorphisms acquired by this strain over 15 years-likely because of the lack in the country of a specific program to treat MDR-TB., Conclusions: Our novel strategy, which integrated WGS analysis and strain-specific PCRs, represents a low-cost, rapid and transferable strategy that allowed a prospective efficient survey and fast historical analysis of MDR-TB in a population., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objectives: This is the first study describing drug resistance mutations (DRM) and HIV-1 variants among infected pregnant women in Equatorial Guinea (GQ), a country with high (6.2%) and increasing HIV prevalence., Methods: Dried blood spots (DBS) were collected from November 2012 to December 2013 from 69 HIV-1 infected women participating in a prevention of mother-to-child transmission program in the Hospital Regional of Bata and Primary Health Care Centre María Rafols, Bata, GQ. The transmitted (TDR) or acquired (ADR) antiretroviral drug resistance mutations at partial pol sequence among naive or antiretroviral therapy (ART)-exposed women were defined following WHO or IAS USA 2015 lists, respectively. HIV-1 variants were identified by phylogenetic analyses., Results: A total of 38 of 69 HIV-1 specimens were successfully amplified and sequenced. Thirty (79%) belonged to ART-experienced women: 15 exposed to nucleoside reverse transcriptase inhibitors (NRTI) monotherapy, and 15 to combined ART (cART) as first regimen including two NRTI and one non-NRTI (NNRTI) or one protease inhibitor (PI). The TDR rate was only found for PI (3.4%). The ADR rate was 37.5% for NNRTI, 8.7% for NRTI and absent for PI or NRTI+NNRTI. HIV-1 group M non-B variants caused most (97.4%) infections, mainly (78.9%) recombinants: CRF02&#95;AG (55.2%), CRF22&#95;A101 (10.5%), subtype C (10.5%), unique recombinants (5.3%), and A3, D, F2, G, CRF06&#95;cpx and CRF11&#95;cpx (2.6% each)., Conclusions: The high rate of ADR to retrotranscriptase inhibitors (mainly to NNRTIs) observed among pretreated pregnant women reinforces the importance of systematic DRM monitoring in GQ to reduce HIV-1 resistance transmission and to optimize first and second-line ART regimens when DRM are present., Competing Interests: The authors have declared that no competing interests exist.