HIV diagnosis in Equatorial Guinea. Keys to reduce the diagnostic and therapeutic delay.

BACKGROUND: In Equatorial Guinea, only 54 % of people living with HIV know their HIV status. There are no confirmatory or molecular diagnostic techniques for early diagnosis or monitoring of infection in the country. Rapid diagnostic tests can induce false-positive diagnoses if used as a confirmatory technique. Our study aimed to identify the challenges of early HIV diagnosis in Equatorial Guinea by analyzing the rate of false positive diagnoses, diagnostic and therapeutic delays, and treatment failures among those on antiretroviral therapy. METHODS: From 2019-2022, dried blood from 341 children, adolescents and adults diagnosed in Equatorial Guinea as HIV-positive by rapid diagnostic testing, and from 54 HIV-exposed infants were collected in Bata and sent to Madrid to confirm HIV-infection by molecular (Xpert HIV-1Qual, Cepheid) and/or serological confirmatory assays (Geenius-HIV-1/2, BioRad). HIV diagnostic delay (CD4 <350cells/mm3), advanced disease at diagnosis (CD4 <200cells/mm3) and antiretroviral treatment delay and failure (viraemia >1,000RNA-HIV-1-copies/ml) were also studied after viral quantification (XpertVL HIV-1, Cepheid). RESULTS: False-positive diagnoses were identified in 5 % of analysed samples. HIV infection was confirmed in 90.5 % of previously diagnosed patients in Equatorial Guinea and 3.7 % of HIV-exposed children undiagnosed in the field. Two-thirds of each new HIV patient had delayed diagnosis, and one-third had advanced disease. Treatment delay occurred in 28.3 % of patients, being around four times more likely in adolescents/adults than children. More than half (56 %) of 232 treated patients presented treatment failure, being significantly higher in children/adolescents than in adults (82.9 %/90 % vs. 45.6 %, p < 0.001). CONCLUSION: We identified some challenges of early HIV diagnosis in Equatorial Guinea, revealing a high rate of false positive diagnoses, diagnostic/treatment delays, and treatment failures that need to be addressed. The implementation of more accurate rapid diagnostic techniques and confirmatory tests, along with improving access to care, treatment, awareness, and screening, would contribute to controlling the spread of HIV in the country.

Rural-Urban Inequities in Tuberculosis-Related Practices in Equatorial Guinea.

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.

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.

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.

High Drug Resistance Levels Compromise the Control of HIV Infection in Pediatric and Adult Populations in Bata, Equatorial Guinea.

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.

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.

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.

Interruption of onchocerciasis transmission in Bioko Island: Accelerating the movement from control to elimination in Equatorial Guinea.

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.

The use and preference of artemether as a first-choice treatment for malaria: results from a cross-sectional survey in the Bata district, Equatorial Guinea.

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.

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.

Prevalence of anemia and associated factors in children living in urban and rural settings from Bata District, Equatorial Guinea, 2013.

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.

Spatial clustering and risk factors of malaria infections in Bata district, 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.

Profile of molecular mutations in pfdhfr, pfdhps, pfmdr1, and pfcrt genes of Plasmodium falciparum related to resistance to different anti-malarial drugs in the Bata District (Equatorial Guinea).

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.