Spatial clustering of onchocerciasis in Bioko Island, Equatorial Guinea

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.S

Knowledge, Attitudes, and Stigma: The Perceptions of Tuberculosis in Equatorial Guinea

Tuberculosis remains one of the major causes of morbidity and mortality in Equatorial Guinea, with an estimated incidence of 280 per 100,000 inhabitants, an estimated mortality rate of 96 per 100,000 inhabitants, and a treatment non-adherence rate of 21.4%. This study aimed to identify the factors associated to TB-related knowledge, attitudes, and stigma in order to design community intervention strategies that could improve TB diagnostic and treatment adherence in Equatorial Guinea. A nationwide cross-sectional survey of 770 household caregivers was conducted in Equatorial Guinea about TB knowledge, attitudes, and practices. Knowledge, attitude, and stigma scores were calculated through correct answers and the median was used as cut-off. Associated factors were analyzed calculating prevalence ratio (PR) and a 95% confidence interval (95% CI) through Poisson regression with robust variance. The percentage of women was 53.0% and median age was 46 years (IQR: 33-60). The percentage of caregivers with high TB related knowledge was 34.9%, with a bad attitude (52.5%) and low stigma (40.4%). A greater probability of having good knowledge was observed in those 45 years old or less (PR: 1.3, 95% CI: 1.1-1.6), those with higher education level (PR: 1.4, 95% CI: 1.1-1.8) and higher wealth (PR: 1.4, 95% CI: 1.0-2.0), while sex (PR = 0.8, 95% CI: 0.6-0.9), religion (PR = 1.4, 95% CI: 1.0-1.8), and good knowledge (PR = 1.4, 95% CI: 1.2-1.7) were associated with good attitudes. Wage employment (PR = 95% CI: 1.2-1.4), feeling well informed (PR = 0.7, 95% CI: 0.6-0.8), having good TB knowledge (PR = 1.3, 95% CI: 1.1-1.7), and some sources of information were associated with having lower TB-related stigma. This study found that a high percentage of caregivers in Equatorial Guinea lack important knowledge about TB disease and have bad attitudes and high TB-related stigma. Given the epidemiological situation of TB in the country, it is urgent to improve TB knowledge and awareness among Equatorial Guinea's general population.This study was funded by Instituto de Salud Carlos III (PI18CIII/00023) and the Fundación Estatal Salud, Infancia y Bienestar Social (FCSAI).S

Characterization of the Plasmodium falciparum sarcoplasmic/endoplasmic reticulum Ca2+-ATPase gene in samples from Equatorial Guinea before implementation of artemisinin-based combination therapy

Plasmodium falciparum resistance to the primary drugs used for treatment of malaria has become the main obstacle to malaria control. Artemisinin combination therapies are the current treatment strategy, and it has been suggested that resistance to artemisinin derivatives may be related to mutations in the Plasmodium falciparum sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase ortholog of the mammalian sarco-endoplasmic reticulum Ca(2+) ATPase gene, known as the pfatp6 gene. Thus, the purpose of this study was to determine the prevalence of single-nucleotide polymorphisms (SNPs) in pfatp6. The presence of different SNPs was detected by polymerase chain reaction amplification of the pfatp6 gene, and then sequencing to identify all possible alleles of the gene. A total of 20 SNPs were detected, including eight SNPs that have not been previously described: K481R in Malabo; R801H on Annobon Island; and the synonymous SNPs a141t, c1788t, a2211g, t2739g, a2760c, and g2836a. The genotypic profile of pfatp6 in samples from Equatorial Guinea, may be a useful epidemiologic tool for monitoring local efficacy of artemisinin combination therapies.This study was supported by the Spanish Agency for International Development Cooperation and the Collaborative Research Network in Tropical Diseases.S

Failures in the case management of children with uncomplicated malaria in Bata district of Equatorial Guinea and associated factors

BACKGROUND: In Equatorial Guinea, malaria continues to be one of the main causes of morbidity and mortality among children. The National Therapeutic Guide established artesunate-amodiaquine (ASAQ) as first-line treatment for uncomplicated malaria, but compliance with this treatment is low. The aim of this study was to assess, for the first time, the performance of public healthcare workers in the diagnosis and treatment of uncomplicated malaria, their compliance with first-line Malaria National Therapeutic Guide and the associated factors. METHODS: A cross-sectional survey was conducted at the nine public health facilities in the Bata District of Equatorial Guinea to assess the management of uncomplicated malaria in children < 15 years of age. Bivariate and multivariate statistical analyses were used to determine the recommended treatment compliance and related factors. RESULTS: A total of 227 children with uncomplicated malaria were recorded from 9 public health facilities. Most of the treatments prescribed (83.3%) did not follow the first-line treatment recommended for uncomplicated malaria. The diagnosis was established with parasite confirmation in 182 cases (80.2%). After adjustment for other variables, children under 2 months of age, the use of parasite confirmation to the diagnosis of malaria and being familiar with the national therapeutic guide were significantly associated with the prescription of the first-line recommended treatment. Cases attended at the hospital or in a health facility with ASAQ in the pharmacy at the time of the study were also more likely to be prescribed with the recommended treatment, but with non-significant association after adjustment for other variables. CONCLUSIONS: This study identified the factors associated with the low compliance with the first-line treatment by the public healthcare facilities of Bata District of Equatorial Guinea. It seems necessary to improve case management of children with uncomplicated malaria; to reinforce the use of Malaria National Therapeutic Guide and to inform about the danger of using artemisinin monotherapy. Furthermore, it is crucial to provide recommended first-line treatment to the pharmacies of all public health facilities to ensure access to this treatment.This study was funded by the by the Agencia Española de Cooperación Internacional AECID2017/CTR/0900237 (http://www.aecid.es/ES) and the Tropical Diseases Research Network RD16CIII/003/001RICET (https://www.ricet.es/).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Caregivers' Malaria Knowledge, Beliefs and Attitudes, and Related Factors in the Bata District, Equatorial Guinea

OBJECTIVES: Adequate community knowledge about malaria is crucial in order to improve prevention by reducing exposure to the disease. Malaria is a major cause of morbidity and mortality among children of less than five years of age in Equatorial Guinea. However, information concerning the accuracy of community knowledge is insufficient. This study aimed at assessing the depth of caregivers' knowledge of malaria, their beliefs and attitudes about this disease, and their socioeconomic determinants in the Bata district of Equatorial Guinea. METHODOLOGY: A cross-sectional study was conducted in the district of Bata, involving 440 houses selected from 18 rural villages and 26 urban neighbourhoods. A combined "Malaria Knowledge Score" was generated based on caregivers' knowledge about transmission, symptoms, prevention, the treatment of children, and best place to seek treatment. Multivariate logistic regressions analyses were performed to assess those factors that are associated with knowledge about malaria. RESULTS: A total of 428 caregivers were interviewed; 255 (59.6%) and 173 (40.4%) lived in urban and rural areas respectively. Significant differences between rural and urban households were observed in caregivers' malaria knowledges and beliefs. Almost 42% of urban and 65% of rural caregivers were unaware as to how malaria is transmitted (OR = 2.69; 95% CI: 1.78-4.05). Together with rurality, the factors most significantly associated with the Malaria Knowledge were the level of education of the caregiver and the socioeconomic status of the household. CONCLUSIONS: Improvements in educational programs are needed to empower the most vulnerable households such that they can pro-actively implement malaria control measures. This could be achieved by a comprehensive communication strategy aimed at changing individual and community behaviours, and delivered by suitably trained community health workers and indoor residual spraying personnel.This study was funded by the Agencia Española de Cooperación Internacional (AECID), TREG1415/11, http://www.aecid.es/ES; and the Tropical Diseases Research Network (RICET), RD12/0018/0001, http://www.ricet.es/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Evaluation of LAMP for the diagnosis of Loa loa infection in dried blood spots compared to PCR-based assays and microscopy

Background: Loa loa is a filarial species found exclusively in West and Central Africa. Microscopy is the traditional diagnosis method for human loiasis. Several molecular methods have developed as an alternative approach for identification of L. loa filarial parasites. Objectives: The aim of this study was to evaluate a Loa-Loop-mediated isothermal amplification (LAMP) assay to diagnose loiasis disease on dried blood spots (DBS) samples, compared to microscopy, filaria-real time-polymerase chain reaction (PCR) and nested-Loa PCR. Methods: A total of 100 DBS samples and 100 blood smears were used for this study. DNA was extracted using saponin/Chelex method. DNA isolated was assayed by a Loa-LAMP assay in parallel to microscopy, filaria-real time PCR and nested-Loa PCR. The sensitivities and specificities of Loa-LAMP assay was computed comparing to each one of the reference methods. Findings: Loa-LAMP's sensitivity was more than 90% and specificity was nearly 100% when compared to molecular methods. On the other hand, sensitivity was decreased a bit when Loa-LAMP faced microscopy, but keeping the other statistical values high. Main conclusions: Loa-LAMP is an appropriate method for loiasis diagnosis in endemic areas. Though, it has disadvantages like the reagents' high price at the moment and not to be able to detect more filarial species at once.Financial support: Fundación Estatal, Salud, Infancia y Bienestar Social (Institute of Health Carlos III) and Cooperative Research Network on Tropical Diseases. T-HT-T is hired at the Malaria and Neglected Tropical Diseases Laboratory of National Centre of Tropical Medicine with a contract named ISCIII-SARA BORRELL CD17CIII/00018, financed by the Institute of Health Carlos III.S

Colorimetric and Real-Time Loop-Mediated Isothermal Amplification (LAMP) for Detection of Loa loa DNA in Human Blood Samples

Loiasis, caused by the filarial nematode Loa loa, is endemic in Central and West Africa. Loa loa has been associated with severe adverse reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. Diagnosis of loiasis still depends on microscopy in blood samples, but this is not effective for large-scale surveys. New diagnostics methods for loiasis are urgently needed. Previously, we developed a colorimetric high-sensitive and species-specific LAMP for Loa loa DNA detection. Here, we evaluate it in a set of 100 field-collected clinical samples stored as dried blood spots. In addition, Loa loa-LAMP was also evaluated in real-time testing and compared with microscopy and a specific PCR/nested PCR. A simple saponin/Chelex-based method was used to extract DNA. Colorimetric and real-time LAMP assays detected more samples with microscopy-confirmed Loa loa and Loa loa/Mansonella perstans mixed infections than PCR/nested-PCR. Samples with the highest Loa loa microfilariae counts were amplified faster in real-time LAMP assays. Our Loa loa-LAMP could be a promising molecular tool for the easy, rapid and accurate screening of patients for loiasis in endemic areas with low-resource settings. The real-time testing (feasible in a handheld device) could be very useful to rule out high-microfilariae loads in infected patients.This research was funded by the Institute of Health Carlos III, ISCIII, Spain (www.isciii.es), grants: RICET RD16/0027/0018 (A.M.), RD16/0027/0000 (A.B.), FCSAI-ISCIII (P.N.) and PI19/01727 (P.F.-S.), European Union co-financing by FEDER (Fondo Europeo de Desarrollo Regional) ‘Una manera de hacer Europa’. We also acknowledge support by the Predoctoral Fellowship Program of Junta de Castilla y León co-financing by Fondo Social Europeo (BDNS (Identif.): 422058 and BDNS (Identif.): 487971), by the ISCIII-Sara Borrell contract CD17CIII/00018 financed by the Institute of Health Carlos III and Predoctoral Fellowship Program of University of Salamanca, and co-financing by Santander Bank.S

Temporal evolution of the resistance genotypes of Plasmodium falciparum in isolates from Equatorial Guinea during 20 years (1999 to 2019)

Background: Malaria is one of the deadliest diseases in the world, particularly in Africa. As such, resistance to anti-malarial drugs is one of the most important problems in terms of global malaria control. This study assesses the evolution of the different resistance markers over time and the possible influence of interventions and treatment changes that have been made in Equatorial Guinea. Methods: A total of 1223 biological samples obtained in the period 1999 to 2019 were included in the study. Screening for mutations in the pfdhfr, pfdhps, pfmdr1, and pfcrt genes was carried out by nested PCR and restriction-fragment length polymorphisms (RFLPs), and the study of pfk13 genes was carried out by nested PCR, followed by sequencing to determine the presence of mutations. Results: The partially and fully resistant haplotypes (pfdhfr + pfdhps) were found to increase over time. Moreover, in 2019, the fully resistant haplotype was found to be increasing, although its super-resistant counterpart remains much less prevalent. A continued decline in pfmdr1 and pfcrt gene mutations over time was also found. The number of mutations detected in pfk13 has increased since 2008, when artemisinin-based combination therapy (ACT) were first introduced, with more mutations being observed in 2019, with two synonymous and five non-synonymous mutations being detected, although these are not related to resistance to ACT. In addition, the non-synonymous A578S mutation, which is the most frequent on the African continent, was detected in 2013, although not in the following years. Conclusions: Withdrawal of the use of chloroquine (CQ) as a treatment in Equatorial Guinea has been shown to be effective over time, as wild-type parasite populations outnumber mutant populations. The upward trend observed in sulfadoxine-pyrimethamine (SP) resistance markers suggest its misuse, either alone or in combination with artesunate (AS) or amodiaquine (AQ), in some areas of the country, as was found in a previous study conducted by this group, which allows selective pressure from SP to continue. Single nucleotide polymorphisms (SNPs) 540E and 581G do not exceed the limit of 50 and 10%, respectively, thus meaning that SP is still effective as an intermittent preventive treatment (IPT) in this country. As for the pfk13 gene, no mutations have been detected in relation to resistance to ACT. However, in 2019 there is a greater accumulation of non-synonymous mutations compared to years prior to 2008.The projects where the samples were taken were funded by Spanish Agency for International Cooperation and Development (AECID), ISCIII, Cooperative Research Network on Tropical Diseases (RICET) and by the Strategic Action in Health (Acción Estratégica en Salud) of the Institute of Health Carlos III (Madrid, Spain), project No. TRPY111/2018 (PI17CIII/0016).S

Glossina palpalis palpalis populations from Equatorial Guinea belong to distinct allopatric clades.

BACKGROUND: Luba is one of the four historical foci of Human African Trypanosomiasis (HAT) on Bioko Island, in Equatorial Guinea. Although no human cases have been detected since 1995, T. b. gambiense was recently observed in the vector Glossina palpalis palpalis. The existence of cryptic species within this vector taxon has been previously suggested, although no data are available regarding the evolutionary history of tsetse flies populations in Bioko. METHODS: A phylogenetic analysis of 60 G. p. palpalis from Luba was performed sequencing three mitochondrial (COI, ND2 and 16S) and one nuclear (rDNA-ITS1) DNA markers. Phylogeny reconstruction was performed by Distance Based, Maximum Likelihood and Bayesian Inference methods. RESULTS: The COI and ND2 mitochondrial genes were concatenated and revealed 10 closely related haplotypes with a dominant one found in 61.1% of the flies. The sequence homology of the other 9 haplotypes compared to the former ranged from 99.6 to 99.9%. Phylogenetic analysis clearly clustered all island samples with flies coming from the Western African Clade (WAC), and separated from the flies belonging to the Central Africa Clade (CAC), including samples from Mbini and Kogo, two foci of mainland Equatorial Guinea. Consistent with mitochondrial data, analysis of the microsatellite motif present in the ITS1 sequence exhibited two closely related genotypes, clearly divergent from the genotypes previously identified in Mbini and Kogo. CONCLUSIONS: We report herein that tsetse flies populations circulating in Equatorial Guinea are composed of two allopatric subspecies, one insular and the other continental. The presence of these two G. p. palpalis cryptic taxa in Equatorial Guinea should be taken into account to accurately manage vector control strategy, in a country where trypanosomiasis transmission is controlled but not definitively eliminated yet

Circulation of SARS-CoV-2 and co-infection with Plasmodium falciparum in Equatorial Guinea

The impact of COVID-19 in Africa has been a big concern since the beginning of the pandemic. However, low incidence of COVID-19 case severity and mortality has been reported in many African countries, although data are highly heterogeneous and, in some regions, like Sub-Saharan Africa, very scarce. Many of these regions are also the cradle of endemic infectious diseases like malaria. The aim of this study was to determine the prevalence of SARS-CoV-2, the diversity and origin of circulating variants as well as the frequency of co-infections with malaria in Equatorial Guinea. For this purpose, we conducted antigen diagnostic tests for SARS-CoV-2, and microscopy examinations for malaria of 1,556 volunteers at six health centres in Bioko and Bata from June to October 2021. Nasopharyngeal swab samples were also taken for molecular detection of SARS-COV-2 by RT-qPCR and whole genome viral sequencing. We report 3.0% of SARS-CoV-2 and 24.4% of malaria prevalence over the sampling in Equatorial Guinea. SARS-CoV-2 cases were found at a similar frequency in all age groups, whereas the age groups most frequently affected by malaria were children (36.8% [95% CI 30.9-42.7]) and teenagers (34.7% [95% CI 29.5-39.9]). We found six cases of confirmed co-infection of malaria and SARS-CoV-2 distributed among all age groups, representing a 0.4% frequency of co-infection in the whole sampled population. Interestingly, the majority of malaria and SARS-CoV-2 co-infections were mild. We obtained the genome sequences of 43 SARS-CoV-2 isolates, most of which belong to the lineage Delta (AY.43) and that according to our pandemic-scale phylogenies were introduced from Europe in multiple occasions (7 transmission groups and 17 unique introductions). This study is relevant in providing first-time estimates of the actual prevalence of SARS-CoV-2 in this malaria-endemic country, with the identification of circulating variants, their origin, and the occurrence of SARS-CoV-2 and malaria co-infection.We would like to thank all volunteers who participated in this study and the local authorities and communities in Equatorial Guinea for their support. We also thank the IPBLN and IBV core facilities for their support to project activitiesN