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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. Graphical Abstract

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.

Background Malaria is endemic in Equatorial Guinea with stable transmission, and it remains a major cause of morbidity and mortality in children under 5 years of age. Adherence to artemisinin-based combination therapy (ACT) as a first-line treatment for uncomplicated malaria is critical to malaria control. Six years after the introduction of artesunate-amodiaquine (AS/AQ) therapy in Equatorial Guinea, adherence to the first-line treatment seems to be low in the Bata district. The factors associated with the choice of malaria treatment have not been studied previously in this area; therefore, this study aimed to analyse the preference and use of artemether as malaria treatment and its related factors in the Bata district of Equatorial Guinea. Methods In 2013, a cross-sectional study was conducted in the Bata district, which involved 428 households. Bivariate and multivariate statistical analyses were conducted to determine the relevance of socio-economic, geographical, and behavioural factors that played a role in the preference and use of artemether as malaria treatment. Results Artemether was considered the best treatment for malaria by 110 caregivers (26%), and was the antimalarial most administrated in the Bata district. It was prescribed to 117 children (27.34%); while, only 6.78% were administered AS/AQ. Caregivers living ≤ 3 km from the nearest health facility were almost two times more likely to consider artemether as the best treatment than those living farther away (95% CI 0.31–0.86). Caregivers with at least a secondary school education were 2.7 times more likely to consider artemether as the best treatment than those less educated. Children whose caregivers considered artemether the best treatment against malaria were five times more likely to be treated with artemether than children with caregivers who did not consider it the best (OR 5.07, 95% CI 2.93–8.78). In contrast, children that reported weakness as a symptom were less likely to be treated with artemether than those with other symptoms (OR 0.47, 95% CI 0.28–0.78). Conclusion Caregivers, public and private health staff, and drug sellers need to understand the importance of using ACT to treat uncomplicated malaria and the dangers of using artemisinin monotherapy.

BACKGROUND: Malaria in Equatorial Guinea remains a major public health problem. The country is a holo-endemic area with a year-round transmission pattern. In 2016, the prevalence of malaria was 12.09% and malaria caused 15% of deaths among children under 5 years. In the Continental Region, 95.2% of malaria infections were Plasmodium falciparum, 9.5% Plasmodium vivax, and eight cases mixed infection in 2011. The main strategy for malaria control is quick and accurate diagnosis followed by effective treatment. Early and accurate diagnosis of malaria is essential for both effective disease management and malaria surveillance. The quality of malaria diagnosis is important in all settings, as misdiagnosis can result in significant morbidity and mortality. Microscopy and RDTs are the primary choices for diagnosing malaria in the field. However, false-negative results may delay treatment and increase the number of persons capable of infecting mosquitoes in the community. The present study analysed the performance of microscopy and RDTs, the two main techniques used in Equatorial Guinea for the diagnosis of malaria, compared to semi-nested multiplex PCR (SnM-PCR). RESULTS: A total of 1724 samples tested by microscopy, RDT, and SnM-PCR were analysed. Among the negative samples detected by microscopy, 335 (19.4%) were false negatives. On the other hand, the negative samples detected by RDT, 128 (13.3%) were false negatives based on PCR. This finding is important, especially since it is a group of patients who did not receive antimalarial treatment. CONCLUSIONS: Owing to the high number of false negatives in microscopy, it is necessary to reinforce training in microscopy, the "Gold Standard" in endemic areas. A network of reference centres could potentially support ongoing diagnostic and control efforts made by malaria control programmes in the long term, as the National Centre of Tropical Medicine currently supports the National Programme against Malaria of Equatorial Guinea to perform all of the molecular studies necessary for disease control. Taking into account the results obtained with the RDTs, an exhaustive study of the deletion of the hrp2 gene must be done in EG to help choose the correct RDT for this area. This study (Project Reference PI14CIII/00064-TRPY 1282/15) was funded by the Institute of Health Carlos III, the Spanish Agency for International Development Cooperation (AECID) and the Network Biomedical Research on Tropical Diseases (RICET in Spanish): RD12/0018/0001. Sí

BACKGROUND: The emergence of drug resistance in Plasmodium falciparum has been a major contributor to the global burden of malaria. Drug resistance complicates treatment, and it is one of the most important problems in malaria control. This study assessed the level of mutations in P. falciparum genes, pfdhfr, pfdhps, pfmdr1, and pfcrt, related to resistance to different anti-malarial drugs, in the Continental Region of Equatorial Guinea, after 8 years of implementing artesunate combination therapies as the first-line treatment. RESULTS: A triple mutant of pfdhfr (51I/59R/108N), which conferred resistance to sulfadoxine/pyrimethamine (SP), was found in 78% of samples from rural settings; its frequency was significantly different between urban and rural settings (p = 0.007). The 164L mutation was detected for the first time in this area, in rural settings (1.4%). We also identified three classes of previously described mutants and their frequencies: the partially resistant (pfdhfr 51I/59R/108N + pfdhps 437G), found at 54% (95% CI 47.75-60.25); the fully resistant (pfdhfr 51I/59R/108N + pfdhps 437G/540E), found at 28% (95% CI 7.07-14.93); and the super resistant (pfdhfr 51I/59R/108N + pfdhps 437G/540E/581G), found at 6% (95% CI 0.48-4.32). A double mutation in pfmdr1 (86Y + 1246Y) was detected at 2% (95% CI 0.24-3.76) frequency, distributed in both urban and rural samples. A combination of single mutations in the pfmdr1 and pfcrt genes (86Y + 76T), which was related to resistance to chloroquine and amodiaquine, was detected in 22% (95% CI 16.8-27.2) of samples from the area. CONCLUSIONS: The high level of mutations detected in P. falciparum genes related to SP resistance could be linked to the unsuccessful withdrawal of SP treatment in this area. Drug resistance can reduce the efficacy of intermittent prophylactic treatment with SP for children under 5 years old and for pregnant women. Although a high number of mutations was detected, the efficacy of the first-line treatment, artemisinin/amodiaquine, was not affected. To avoid increases in the numbers, occurrence, and spread of mutations, and to protect the population, the Ministry of Health should ensure that health centres and hospitals are supplied with appropriate first-line treatments for malaria. We would like to thank the National Malaria Control Programme and Ministry of Health and Ministry of Health and Social Welfare of Equatorial Guinea for their assistance. We would also like to thank the Network of Tropical Diseases Research Centres (Red de Investigación Cooperativa en Enfermedades Tropicales/ RICET-: RD12/0018/0001). This work has been done under the project: PI14CIII/00064-TRPY 1282/15 and was funded by the Institute of Health Carlos III. Sí

BACKGROUND: Malaria remains a major cause of morbidity and mortality in children under 5 years of age in Equatorial Guinea. Early appropriate treatment can reduce progression of the illness to severe stages, thus reducing of mortality, morbidity and onward transmission. The factors that contribute to malaria treatment delay have not been studied previously in Equatorial Guinea. The objective of this study was to assess the determinants of delay in seeking malaria treatment for children in the Bata district, in mainland Equatorial Guinea. METHODOLOGY: A cross-sectional study was conducted in Bata district, in 2013, which involved 428 houses in 18 rural villages and 26 urban neighbourhoods. Household caregivers were identified in each house and asked about their knowledge of malaria and about the management of the last reported malaria episode in a child 15 years and younger under their care. Bivariate and multivariate statistical analyses were conducted to determine the relevance of socio-economic, geographical and behavioural factors on delays in care-seeking behaviour. RESULTS: Nearly half of the children sought treatment at least 24 h after the onset of the symptoms. The median delay in seeking care was 2.8 days. Children from households with the highest socio-economic status were less likely to be delayed in seeking care than those from households with the lowest socio-economic status (OR 0.37, 95% CI 0.19-0.72). Children that first received treatment at home, mainly paracetamol, were more than twice more likely to be delayed for seeking care, than children who did not first receive treatment at home (OR 2.36, 95% CI 1.45-3.83). Children living in a distance >3 km from the nearest health facility were almost two times more likely to be delayed in seeking care than those living closer to a facility but with non significant association once adjusted for other variables (OR 1.75, 95% CI 0.88-3.47). CONCLUSION: To decrease malaria morbidity and mortality in Bata district, efforts should be addressed to reduce household delays in seeking care. It is necessary to provide free access to effective malaria diagnosis and treatment, to reinforce malaria management at community level through community health workers and drug sellers and to increase awareness on the severity of malaria, the importance of early diagnosis and appropriate treatment. The authors thank the study participants for volunteering, the data collectors for conducting the fieldwork and the Bata Nursing School, and the MoHSW of Equatorial Guinea. We also thank S. Walter and P. Berzosa for their useful comments. The corresponding author’s affiliation centre belongs to the Spanish Network on Tropical Diseases Research (RICET in Spanish): RD12/0018/0001. Sí