Evolution of pfhrp2 and pfhrp3 deletions in Equatorial Guinea between the pre- and post-RDT introduction.

BACKGROUND: Pfhrp2 and pfhrp3 deletions are threatening Plasmodium falciparum malaria diagnosis by rapid diagnostic tests (RDT) due to false negatives. This study assesses the changes in the frequencies of pfhrp2 and pfhrp3 deletions (pfhrp2Del and pfhrp3Del, respectively) and the genes in their flaking regions, before and after RDT introduction in Equatorial Guinea. METHODS: A total of 566 P. falciparum samples were genotyped to assess the presence of pfhrp2 and pfhrp3 deletions and their flanking genes. The specimens were collected 18 years apart from two provinces of Equatorial Guinea, North Bioko (Insular Region) and Litoral Province (Continental Region). Orthologs of pfhrp2 and pfhrp3 genes from other closely related species were used to compare sequencing data to assess pfhrp2 and pfhrp3 evolution. Additionally, population structure was studied using seven neutral microsatellites. RESULTS: This study found that pfhrp2Del and pfhrp3Del were present before the introduction of RDT; however, they increased in frequency after their use, reaching more than 15%. Haplotype networks suggested that pfhrp2Del and pfhrp3Del emerged multiple times. Exon 2 of pfhrp2 and pfhrp3 genes had high variability, but there were no significant changes in amino acid sequences. CONCLUSIONS: Baseline sampling before deploying interventions provides a valuable context to interpret changes in genetic markers linked to their efficacy, such as the dynamic of deletions affecting RDT efficacy.

Seasonal malaria chemoprevention in a context of high presumed sulfadoxine-pyrimethamine resistance: malaria morbidity and molecular drug resistance profiles in South Sudan.

BACKGROUND: Seasonal malaria chemoprevention (SMC) using sulfadoxine-pyrimethamine plus amodiaquine (SP-AQ), is a community-based malaria preventive strategy commonly used in the Sahel region of sub-Saharan Africa. However, to date it has not been implemented in East Africa due to high SP resistance levels. This paper is a report on the implementation of SMC outside of the Sahel in an environment with a high level of presumed SP-resistance: five cycles of SMC using SPAQ were administered to children 3-59 months during a period of high malaria transmission (July-December 2019) in 21 villages in South Sudan. METHODS: A population-based SMC coverage survey was combined with a longitudinal time series analysis of health facility and community health data measured after each SMC cycle. SMC campaign effectiveness was assessed by Poisson model. SPAQ molecular resistance markers were additionally analysed from dried blood spots from malaria confirmed patients. RESULTS: Incidence of uncomplicated malaria was reduced from 6.6 per 100 to an average of 3.2 per 100 after SMC administration (mean reduction: 53%) and incidence of severe malaria showed a reduction from 21 per 10,000 before SMC campaign to a mean of 3.3 per 10,000 after each cycle (mean reduction: 84%) in the target group when compared to before the SMC campaign. The most prevalent molecular haplotype associated with SP resistance was the IRNGE haplotype (quintuple mutant, with 51I/59R/108N mutation in pfdhfr + 437G/540E in pfdhps). In contrast, there was a low frequency of AQ resistance markers and haplotypes resistant to both drugs combined (< 2%). CONCLUSIONS: The SMC campaign was effective and could be used as an additional preventive tool in seasonal malaria settings outside of the Sahel, especially in areas where access to health care is unstable. Malaria case load reduction was observed despite the high level of resistance to SP.

Screening for K13-Propeller Mutations Associated with Artemisinin Resistance in Plasmodium falciparum in Yambio County (Western Equatoria State, South Sudan).

Artemisinin-combined treatments are the recommended first-line treatment of Plasmodium falciparum malaria, but they are being threatened by emerging artemisinin resistance. Mutations in pfk13 are the principal molecular marker for artemisinin resistance. This study characterizes the presence of mutations in pfk13 in P. falciparum in Western Equatoria State, South Sudan. We analyzed 468 samples from patients with symptomatic malaria and found 15 mutations (8 nonsynonymous and 7 synonymous). Each mutation appeared only once, and none were validated or candidate markers of artemisinin resistance. However, some mutations were in the same or following position of validated and candidate resistance markers, suggesting instability of the gene that could lead to resistance. The R561L nonsynonymous mutation was found in the same position as the R561H validated mutation. Moreover, the A578S mutation, which is widespread in Africa, was also reported in this study. We found a high diversity of other pfk13 mutations in low frequency. Therefore, routine molecular surveillance of resistance markers is highly recommended to promptly detect the emergence of resistance-related mutations and to limit their spread.

Updates on Malaria Epidemiology and Prevention Strategies.

Purpose of Review: The objective of this review was to provide an update on recent malaria epidemiology, both globally and in non-endemic areas, to identify the current distribution and repercussions of genetically diverse Plasmodium species and summarize recently implemented intervention and prevention tools. Recent Findings: Notable changes in malaria epidemiology have occurred in recent years, with an increase in the number of total cases and deaths globally during 2020-2021, in part attributed to the COVID-19 pandemic. The emergence of artemisinin-resistant species in new areas and the expanding distribution of parasites harbouring deletions of the pfhrp2/3 genes have been concerning. New strategies to curb the burden of this infection, such as vaccination, have been implemented in certain endemic areas and their performance is currently being evaluated. Summary: Inadequate control of malaria in endemic regions may have an effect on imported malaria and measures to prevent re-establishment of transmission in malaria-free areas are essential. Enhanced surveillance and investigation of Plasmodium spp. genetic variations will contribute to the successful diagnosis and treatment of malaria in future. Novel strategies for an integrated One Health approach to malaria control should also be strengthened.

Prevalence of Schistosoma haematobium and Intestinal Helminth Infections among Nigerian School Children.

Schistosomiasis and soil-transmitted helminthiases (STH) are two parasitic diseases mainly affecting school children. The purpose of this study was to estimate the current prevalence and infection intensity, in addition to the associations of these infections with age and sex, in children aged 4-17 years living in Osun State, Nigeria. From each participant (250 children), one urine and one stool sample were taken for the study, for the microscopic detection of eggs or larvae in faeces by means of the Kato-Katz method and eggs in filtrated urine. The overall prevalence of urinary schistosomiasis was 15.20%, with light infection. The intestinal helminthic species identified (and their prevalence) were S. stercoralis (10.80%), S. mansoni (8%), A. lumbricoides (7.20%), hookworm (1.20%), and T. trichiura (0.4%), all of them being classified as light infections. Single infections (67.95%) are more frequent than multiple infections (32.05%). With this study, schistosomiasis and STH are still endemic in Osun State, but with a light to moderate prevalence and light infection intensity. Urinary infection was the most prevalent, with higher prevalence in children over 10 years. The >10 years age group had the highest prevalence for all of the intestinal helminths. There were no statistically significant associations between gender and age and urogenital or intestinal parasites.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions in Malaria-Hyperendemic Region, South Sudan.

Pfhrp2 and pfhrp3 gene deletions threaten the use of Plasmodium falciparum malaria rapid diagnostic tests globally. In South Sudan, deletion frequencies were 15.6% for pfhrp2, 20.0% for pfhrp3, and 7.5% for double deletions. Deletions were approximately twice as prevalent in monoclonal infections than in polyclonal infections.

Deletion patterns, genetic variability and protein structure of pfhrp2 and pfhrp3: implications for malaria rapid diagnostic test in Amhara region, Ethiopia.

BACKGROUND: Although rapid diagnostic tests (RDTs) play a key role in malaria-control strategies, their efficacy has been threatened by deletion and genetic variability of the genes pfhrp2/3. This study aims to characterize the deletion, genetic patterns and diversity of these genes and their implication for malaria RDT effectiveness, as well as their genetic evolution in the Amhara region of Ethiopia. METHODS: The study included 354 isolates from symptomatic patients from the Amhara region of Ethiopia who tested positive by microscopy. Exon 1-2 and exon 2 of genes pfhrp2 and -3 were amplified, and exon 2 was sequenced to analyse the genetic diversity, phylogenetic relationship and epitope availability. RESULTS: The deletion frequency in exon 1-2 and exon 2 was 22 and 4.6% for pfhrp2, and 68 and 18% for pfhrp3, respectively. Double deletion frequency for pfhrp2 and pfhrp3 was 1.4%. High genetic diversity, lack of clustering by phylogenetic analysis and evidence of positive selection suggested a diversifying selection for both genes. The amino-acid sequences, classified into different haplotypes, varied widely in terms of frequency of repeats, with novel amino-acid changes. Aminoacidic repetition type 2 and type 7 were the most frequent in all the sequences. The most frequent epitopes among protein sequences were those recognized by MAbs 3A4 and C1-13. CONCLUSION: Deletions and high amino acidic variation in pfhrp2 and pfhrp3 suggest their possible impact on RDT use in the Amhara region, and the high genetic diversity of these genes could be associated with a diversifying selection in Ethiopia. Surveillance of these genes is, therefore, essential to ensure the effectiveness of public health interventions in this region.

Comparison of three PCR-based methods to detect Loa loa and Mansonella perstans in long-term frozen storage dried blood spots.

OBJECTIVES: Loa loa and Mansonella perstans are two very common filarial species in Africa. Although microscopy is the traditional diagnostic method for human filariasis, several polymerase chain reaction (PCR) methods have emerged as an alternative approach for identifying filarial parasites. The aim of this study is to compare three molecular methods and decide which is the most suitable for diagnosing human loiasis and mansonellosis in non-endemic regions using dried blood spot (DBS) as a medium for sample collection and storage. METHODS: A total of 100 DBS samples, with their corresponding thin and thick blood smears, were selected for this study. Microscopy was used as the reference method to diagnose and calculate the microfilaraemia. Filarial DNA was extracted using the saponin/Chelex method and the DNA isolated was assayed by Filaria-real time-PCR, filaria-nested PCR, and cytochrome oxidase I PCR. All PCR products were subsequently purified and sequenced. The statistical values for each molecular test were calculated and compared. RESULTS: Overall, 64 samples were identified as negative by all tests and a further 36 samples were positive by at least one of the methods used. The sensitivity and specificity were similar for the different molecular methods, all of which demonstrated good agreement with microscopy. CONCLUSIONS: Based on this study, and from a practical point of view (single and short amplification round), the optimal technique for diagnosing filarial infection in non-endemic regions is filaria-real time-PCR, which presents high sensitivity and specificity and is also able to detect a wide range of human filariae.

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.

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.

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.

Therapeutic efficacy of artesunate-amodiaquine and artemether-lumefantrine and polymorphism in Plasmodium falciparum kelch13-propeller gene in Equatorial Guinea.

BACKGROUND: Artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) are the currently recommended first- and second-line therapies for uncomplicated Plasmodium falciparum infections in Equatorial Guinea. This study was designed to evaluate the efficacy of these artemisinin-based combinations and detect mutations in P. falciparum kelch13-propeller domain gene (Pfkelch13). METHODS: A single-arm prospective study evaluating the efficacy of ASAQ and AL at three sites: Malabo, Bata and Ebebiyin was conducted between August 2017 and July 2018. Febrile children aged six months to 10 years with confirmed uncomplicated P. falciparum infection and other inclusion criteria were sequentially enrolled first in ASAQ and then in AL at each site, and followed up for 28 days. Clinical and parasitological parameters were assessed. The primary endpoint was PCR-adjusted adequate clinical and parasitological response (ACPR). Samples on day-0 were analysed for mutations in Pfkelch13 gene. RESULTS: A total 264 and 226 patients were enrolled in the ASAQ and AL treatment groups, respectively. Based on per-protocol analysis, PCR-adjusted cure rates of 98.6% to 100% and 92.4% to 100% were observed in patients treated with ASAQ and AL, respectively. All study children in both treatment groups were free of parasitaemia by day-3. Of the 476 samples with interpretable results, only three samples carried non-synonymous Pfkelch13 mutations (E433D and A578S), and none of them is the known markers associated with artemisinin resistance. CONCLUSION: The study confirmed high efficacy of ASAQ and AL for the treatment of uncomplicated falciparum infections as well as the absence of delayed parasite clearance and Pfkelch13 mutations associated with artemisinin resistance. Continued monitoring of the efficacy of these artemisinin-based combinations, at least every two years, along with molecular markers associated with artemisinin and partner drug resistance is imperative to inform national malaria treatment policy and detect resistant parasites early. Trial registration ACTRN12617000456358, Registered 28 March 2017; http://www.anzctr.org.au/trial/MyTrial.aspx.

Impact of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions on malaria control worldwide: a systematic review and meta-analysis.

BACKGROUND: Deletion of pfhrp2 and/or pfhrp3 genes cause false negatives in malaria rapid diagnostic test (RDT) and threating malaria control strategies. This systematic review aims to assess the main methodological aspects in the study of pfhrp2 and pfhrp3 gene deletions and its global epidemiological status, with special focus on their distribution in Africa; and its possible impact in RDT. METHODS: The systematic review was conducted by examining the principal issues of study design and methodological workflow of studies addressing pfhrp2 deletion. Meta-analysis was applied to represent reported prevalences of pfhrp2 and pfhrp3 single and double deletion in the World Health Organization (WHO) region. Pooled-prevalence of deletions was calculated using DerSimonnian-Laird random effect model. Then, in-deep analysis focused on Africa was performed to assess possible variables related with these deletions. Finally, the impact of these deletions in RDT results was analysed combining reported information about RDT sensitivity and deletion prevalences. RESULTS: 49 articles were included for the systematic review and 37 for the meta-analysis, 13 of them placed in Africa. Study design differs significantly, especially in terms of population sample and information reported, resulting in high heterogeneity between studies that difficulties comparisons and merged conclusions. Reported prevalences vary widely in all the WHO regions, significantly higher deletion were reported in South-Central America, following by Africa and Asia. Pfhrp3 deletion is more prevalent (43% in South-Central America; 3% in Africa; and 1% in Asia) than pfhrp2 deletion (18% in South-Central America; 4% in Africa; and 3% in Asia) worldwide. In Africa, there were not found differences in deletion prevalence by geographical or population origin of samples. The prevalence of deletion among false negatives ranged from 0 to 100% in Africa, but in Asia and South-Central America was only up to 90% and 48%, respectively, showing substantial relation between deletions and false negatives. CONCLUSION: The concerning prevalence of pfhrp2, pfhrp3 and pfhrp2/3 gene deletions, as its possible implications in malaria control, highlights the importance of regular and systematic surveillance of these deletions. This review has also outlined that a standardized methodology could play a key role to ensure comparability between studies to get global conclusions.

Knowledge and practices regarding malaria and the National Treatment Guidelines among public health workers in Equatorial Guinea.

BACKGROUND: In 2018, an estimated 228 million cases of malaria occurred worldwide. Countries are far from having achieved reasonable levels of national protocol compliance among health workers. Lack of awareness of treatment protocols and treatment resistance by prescribers threatens to undermine progress when it comes to reducing the prevalence of this disease. This study sought to evaluate the degree of knowledge and practices regarding malaria diagnosis and treatment amongst prescribers working at the public health facilities of Bata, Equatorial Guinea. METHODS: A cross-sectional survey was conducted in October-December 2017 amongst all public health professionals who attended patients under the age of 15 years, with suspected malaria in the Bata District of Equatorial Guinea. Practitioners were asked about their practices and knowledge of malaria and the National Malaria Treatment Guidelines. A bivariate analysis and a logistic regression model were used to determine factors associated with their knowledge. RESULTS: Among the 44 practitioners interviewed, 59.1% worked at a Health Centre and 40.9% at the District Hospital of Bata. Important differences in knowledge and practices between hospital and health centre workers were found. Clinical diagnosis was more frequently by practitioners at the health centres (p = 0.059), while microscopy confirmation was more frequent at regional hospital (100%). Intramuscular artemether was the anti-malarial most administrated at the health centres (50.0%), while artemether-lumefantrine was the treatment most used at the regional hospital (66.7%). Most practitioners working at public health facilities (63.6%) have a low level of knowledge regarding the National Malaria Treatment Guidelines. While knowledge regarding malaria, the National Malaria Treatment Guidelines and treatment resistances is low, it was higher amongst hospital workers than amongst practitioners at health centres. CONCLUSIONS: It is essential to reinforce practitioners' knowledge, treatment and diagnosis practices and use of the National Malaria Treatment Guidelines in order to improve malaria case management and disease control in the region. A specific malaria training programme ensuring ongoing updates training is necessary in order to ensure that greater experience does not entail obsolete knowledge and, consequently, inadequate diagnosis and treatment practices.

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.

First evidence of the deletion in the pfhrp2 and pfhrp3 genes in Plasmodium falciparum from Equatorial Guinea.

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.

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.

Comparison of three diagnostic methods (microscopy, RDT, and PCR) for the detection of malaria parasites in representative samples from Equatorial Guinea.

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.

Differentially expressed microRNAs in experimental cerebral malaria and their involvement in endocytosis, adherens junctions, FoxO and TGF-ß signalling pathways.

Cerebral malaria (CM) is the most severe manifestation of infection with Plasmodium, however its pathogenesis is still not completely understood. microRNA (miRNA) have been an area of focus in infectious disease research, due to their ability to affect normal biological processes, and have been shown to play roles in various viral, bacterial and parasitic infections, including malaria. The expression of miRNA was studied following infection of CBA mice with either Plasmodium berghei ANKA (causing CM), or Plasmodium yoelii (causing severe but non-cerebral malaria (NCM)). Using microarray analysis, miRNA expression was compared in the brains of non-infected (NI), NCM and CM mice. Six miRNA were significantly dysregulated between NCM and CM mice, and four of these, miR-19a-3p, miR-19b-3p, miR-142-3p and miR-223-3p, were further validated by qPCR assays. These miRNA are significantly involved in several pathways relevant to CM, including the TGF-ß and endocytosis pathways. Dysregulation of these miRNA during CM specifically compared with NCM suggests that these miRNA, through their regulation of downstream targets, may be vitally involved in the neurological syndrome. Our data implies that, at least in the mouse model, miRNA may play a regulatory role in CM pathogenesis.

Malaria determining risk factors at the household level in two rural villages of mainland Equatorial Guinea.

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.