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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 Mass drug administration (MDA) can rapidly reduce the burden of Plasmodium falciparum (Pf). However, concerns remain about its contribution to select for antimalarial drug resistance. Methods We used Sanger sequencing and real-time PCR to determine the proportion of molecular markers associated with antimalarial resistance (k13, pfpm2, pfmdr1 and pfcrt) in Pf isolates collected before (n = 99) and after (n = 112) the implementation of two monthly MDA rounds with dihydroartemisinin–piperaquine (DHAp) for two consecutive years in Magude district of Southern Mozambique. Results None of the k13 polymorphisms associated with artemisinin resistance were observed in the Pf isolates analyzed. The proportion of Pf isolates with multiple copies of pfpm2, an amplification associated with piperaquine resistance, was similar in pre- (4.9%) and post-MDA groups (3.4%; p = 1.000). No statistically significant differences were observed between pre- and post-MDA groups in the proportion of Pf isolates neither with mutations in pfcrt and pfmdr1 genes, nor with the carriage of pfmdr1 multiple copies (p>0.05). Conclusions This study does not show any evidence of increased frequency of molecular makers of antimalarial resistance after MDA with DHAp in southern Mozambique where markers of antimalarial resistance were absent or low at the beginning of the intervention.

International audience; Background: Tick-borne diseases caused by Anaplasma species put serious constraints on the health and production of domestic cattle in tropical and sub-tropical regions. After recovering from a primary infection, cattle typically become persistent carriers of pathogens and play a critical role in the epidemiology of the disease, acting as reservoirs of the Anaplasma spp. Methods: In this study a duplex PCR assay was used for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle using two primer pairs targeting msp4 and msp2 genes, respectively. We used this method to analyze DNA preparations derived from 328 blood cattle samples that were collected from 80 farms distributed among Tunisia's four bioclimatic zones. Results: The prevalence of the A. marginale infection (24.7 %) was significantly higher and more widespread (in all bioclimatic areas) than that of A. phagocytophilum (0.6 %), which was found in a mixed infection with A. marginale. Conclusions: The duplex PCR assay used proved to be a rapid, specific and inexpensive mean for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle blood. It allowed us to report the identification of A. phagocytophilum for the first time in cattle in Tunisia and confirm the presence of A. marginale in cattle from several geographical areas of the country. Further epidemiological studies undertaken using this assay will help improve the surveillance of the associated diseases in the regions where they are endemic

BACKGROUND: Onchocerciasis or "river blindness" is a chronic parasitic disease caused by the filarial worm Onchocerca volvulus, transmitted through infected blackflies (Simulium spp.). Bioko Island (Equatorial Guinea) used to show a high endemicity for onchocerciasis. During the last years, the disease control programmes using different larvicides and ivermectin administration have considerably reduced the prevalence and intensity of infection. Based on this new epidemiological scenario, in the present work we aimed to assess the impact of the strategies applied against onchocerciasis in Bioko Island by an evaluation of IgG4 antibodies specific for recombinant Ov-16 in ELISA. METHODS: A cross-sectional study was conducted in Bioko Island from mid-January to mid-February, 2014. Twenty communities were randomly selected from rural and urban settings. A total of 140 households were chosen. In every selected household, all individuals aged 5 years and above were recruited; 544 study participants agreed to be part of this work. No previous data on onchocerciasis seroprevalence in the selected communities were available. Blood samples were collected and used in an "ELISA in-house" prepared with recombinant Ov-16, expressed and further purified. IgG4 antibodies specific for recombinant Ov-16 were evaluated by ELISA in all of the participants. RESULTS: Based on the Ov-16 ELISA, the onchocerciasis seroprevalence was 7.9 %, mainly concentrated in rural settings; samples from community Catedral Ela Nguema (# 16) were missed during the field work. Among the rural setups, communities Inasa Maule (# 7), Ruiché (# 20) and Barrios Adyacentes Riaba (# 14), had the highest seropositivity percentages (29.2, 26.9 and 23.8 %, respectively). With respect to the urban settings, we did not find any positive case in communities Manzana Casa Bola (# 3), Colas Sesgas (# 6), Getesa (# 8), Moka Bioko (# 9), Impecsa (# 10), Baney Zona Baja (# 12) and Santo Tomás de Aquino (# 1). No onchocerciasis seropositive samples were found in 10-year-old individuals or younger. The IgG4 positive titles increased in older participants. CONCLUSIONS: A significant decline in onchocerciasis prevalence was observed in Bioko Island after years of disease-vector control and CDTI strategy. The seroprevalence increased with age, mainly in rural settings that could be due to previous exposure of population to the filarial parasite, eliminated by the control programmes introduced against onchocerciasis. A new Ov-16 serological evaluation with a larger sample size of children below 10 years of age is required to demonstrate the interruption of transmission of O. volvulus in the human population of Bioko Island (Equatorial Guinea) according to the WHO criteria. We would like to thank the National Program for Control of Onchocerciasis and other Filariasis in Equatorial Guinea for supporting us to obtain the information on which this study is based. We are grateful to the study participants for volunteering to participate in the study and the data collectors for performing field work. Our gratitude to the Unit of Serological Diagnosis, Department of Parasitology, Centro Nacional de Microbiología, and the Spanish Red Cross for providing some control samples. Thanks are also due to Diana Gomez-Barroso, from the National Centre of Epidemiology (ISCIII) for her help with the mapping. Sí

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.00765, Gambiense trypanosomiasis is considered an anthroponotic disease. Consequently, control programs are generally aimed at stopping transmission of Trypanosoma brucei gambiense (T. b. gambiense) by detecting and treating human cases. However, the persistence of numerous foci despite efforts to eliminate this disease questions this strategy as unique tool to pursue the eradication. The role of animals as a reservoir of T. b. gambiense is still controversial, but could partly explain maintenance of the infection at hypo-endemic levels. In the present study, we evaluated the presence of T. b. gambiense in wild animals in Equatorial Guinea. The infection rate ranged from 0.8% in the insular focus of Luba to more than 12% in Mbini, a focus with a constant trickle of human cases. The parasite was detected in a wide range of animal species including four species never described previously as putative reservoirs. Our study comes to reinforce the hypothesis that animals may play a role in the persistence of T. b. gambiense transmission, being particularly relevant in low transmission settings. Under these conditions the integration of sustained vector control and medical interventions should be considered to achieve the elimination of gambiense trypanosomiasis., We thank the hunter teams of Equatorial Guinea and the staff of the National Sleeping Sickness Control Programme. We wanted to extend our thanks to the authorities of the Ministry of Health and Social Welfare of Equatorial Guinea and the Spanish Agency for International Development Cooperation (AECID). We are grateful to Dr. Vincent Jamonneau (IRD) for providing valuable comments of the manuscript. J-MB is supported by a Miguel Servet Fellowship (CP09/00300) and funded by “Fondo de Investigación Sanitaria”(FIS) PI10/01128. AB and MN are granted by ISCIII -Subdirección General de Redes y Centros de InvestigaciónCooperativa (RICET) RD12/0018/0001 and RD12/0018/0015, respectively.

Background Sleeping sickness is spread over 36 Sub-Saharan African countries. In West and Central Africa, the disease is caused by Trypanosoma brucei gambiense, which produces a chronic clinical manifestation. The Luba focus (Bioko Island, Equatorial Guinea) has not reported autochthonous sleeping sickness cases since 1995, but given the complexity of the epidemiological cycle, the elimination of the parasite in the environment is difficult to categorically ensure. Methodology/Principal Findings The aim of this work is to assess, by a molecular approach (Polymerase Chain Reaction, PCR), the possible permanence of T. b. gambiense in the vector (Glossina spp.) and domestic fauna in order to improve our understanding of the epidemiological situation of the disease in an isolated focus considered to be under control. The results obtained show the absence of the parasite in peridomestic livestock but its presence, although at very low rate, in the vector. On the other hand, interesting entomological data highlight that an elevated concentration of tsetse flies was observed in two out of the ten villages considered to be in the focus. Conclusions These findings demonstrate that even in conditions of apparent control, a complete parasite clearance is difficult to achieve. Further investigations must be focused on animal reservoirs which could allow the parasites to persist without leading to human cases. In Luba, where domestic livestock are scarcer than other foci in mainland Equatorial Guinea, the epidemiological significance of wild fauna should be assessed to establish their role in the maintenance of the infection., Author Summary Sleeping sickness is a neglected disease with an important impact on public health of many countries of Sub-Saharan Africa. It is transmitted by tsetse fly bites (the vector) and mainly affects remote and rural populations. The chronic form, caused by Trypanosoma brucei gambiense, includes almost 90% of reported cases, and it is often misdiagnosed or lately detected after months or years of infection. Many efforts have been carried out to control the disease and interesting advances have been achieved. Although elimination is considered possible, there is an urgent need to understand the disease dynamic, especially in foci with very low rate (or absent) of infection for a long time. We performed a parasite screening in tsetse flies and livestock from Luba focus (Equatorial Guinea), considered to be “controlled” since 1995 (no human cases for fifteen years). The obtained results demonstrate that T. b. gambiense still remains in the environment and entomological data reveal high population density of the vector in some localities. This finding suggests that other intervention ways focused on control of vector populations, combined with the detection of human cases, could be necessary to achieve the total elimination of the parasite in hypoendemic foci.

BACKGROUND: Malaria has traditionally been a major endemic disease in Equatorial Guinea. Although parasitaemia prevalence on the insular region has been substantially reduced by vector control in the past few years, the prevalence in the mainland remains over 50% in children younger than five years. The aim of this study is to investigate the risk factors for parasitaemia and treatment seeking behaviour for febrile illness at country level, in order to provide evidence that will reinforce the EG National Malaria Control Programme. METHODS: The study was a cross-sectional survey of children 0 to 5 years old, using a multistaged, stratified, cluster-selected sample at the national level. It included a socio-demographic, health and dietary questionnaires, anthropometric measurements, and thick and thin blood smears to determine the Plasmodium infection. A multivariate logistic regression model was used to determine risk factors for parasitaemia, taking into account the cluster design. RESULTS: The overall prevalence of parasitemia was 50.9%; it was higher in rural (58.8%) compared to urban areas (44.0%, p = 0.06). Age was positively associated with parasitemia (p < 0.0001). In rural areas, risk factors included longer distance to health facilities (p = 0.01) and a low proportion of households with access to protected water in the community (p = 0.02). Having had an episode of cough in the 15 days prior to the survey was inversely related to parasitemia (p = 0.04). In urban areas, the risk factors were stunting (p = 0.005), not having taken colostrum (p = 0.01), and that someone in the household slept under a bed net (p = 0.002); maternal antimalarial medication intake during pregnancy (p = 0.003) and the household socio-economic status (p = 0.0002) were negatively associated with parasitemia. Only 55% of children with fever were taken outside their homes for care, and treatment seeking behaviour differed substantially between rural and urban populations. CONCLUSION: Results suggest that a national programme to fight malaria in Equatorial Guinea should take into account the differences between rural and urban communities in relation to risk factors for parasitaemia and treatment seeking behaviour, integrate nutrition programmes, incorporate campaigns on the importance of early treatment, and target appropriately for bed nets to reach the under-fives. The study was funded by the Instituto de Salud Carlos III (ISCIII), the Spanish Research Network of Tropical Diseases (RICET), and the Spanish Agency for International Cooperation (AECI). Sí

BACKGROUND: Human African Trypanosomiasis is a vector-borne parasitic disease. The geographical distribution of the disease is linked to the spatial distribution of the tsetse fly. As part of a control campaign using traps, the spatial and temporal variability is analysed of the glossina populations present in the Mbini sleeping sickness foci (Equatorial Guinea). RESULTS: A significant drop in the annual mean of the G. p. palpalis apparent density was noted from 2004 to 2005, although seasonal differences were not observed. The apparent density (AD) of G. p. palpalis varies significantly from one biotope to another. The fish dryers turned out to be zones with the greatest vector density, although the AD of G. p. palpalis fell significantly in all locations from 2004 to 2005. CONCLUSION: Despite the tsetse fly density being relatively low in fish dryers and jetties, the population working in those zones would be more exposed to infection. The mono-pyramidal traps in the Mbini focus have been proven to be a useful tool to control G. p. palpalis, even though the activity on the banks of the River Wele needs to be intensified. The application of spatial analysis techniques and geographical information systems are very useful tools to discriminate zones with high and low apparent density of G. p. palpalis, probably associated with different potential risk of sleeping sickness transmission. We would like to thank the National Sleeping Sickness Control Program, the Republic of Equatorial Guinea's Ministry of Health and Social Welfare, and the Institute of Forestry Promotion and Development (INDEPROF) for its technical support during the study. This study was funded by the Spanish International Cooperation Agency (AECI) and the Instituto de Salud Carlos III within the Network of Tropical Diseases Research Centers (RICET-RD06/0021). Sí

Background Malaria transmission varies from one country to another and there are also local differences in time and space. An important variable when explaining the variability in transmission is the breeding behaviour of the different vector species and the availability of breeding sites. The aim of this study was to determine the geographical variability of certain entomological parameters: human biting rate (HBR), sporozoitic index (SI) for Plasmodium falciparum and entomological inoculation rate (EIR). Methods The study was carried out in a small village in the mainland region of Equatorial Guinea. Adult mosquitoes were collected by CDC light traps. Polymerase Chain Reaction was employed to identify the species within the Anopheles gambiae complex and to detect P. falciparum sporozoites. The geographical position of all the dwellings in the village were taken using a global positioning system receiver unit. Data relating to the dwelling, occupants, use of bednets and the mosquitoes collection data were used to generate a geographical information system (GIS). This GIS allowed the minimum distance of the dwellings to the closest water point (potential breeding sites) to be determined. Results A total of 1,173 anophelines were caught: 279 A. gambiae s.l. (217 A. gambiae s.s. and one Anopheles melas), 777 Anopheles moucheti and 117 Anopheles carnevalei. A. moucheti proved to be the main vector species and was responsible for 52.38 [95% IC: 33.7–71] night infective bites during this period. The highest SI was found in A. carnevalei (24%), even though the HBR was the lowest for this species. A significant association was found between the distance from the dwellings to the closest water point (River Ntem or secondary streams) and the total HBR. Conclusion A clear association has been observed between the distance to potential breeding sites and the variability in the anopheline density, while the other parameters measured do not seem to condition this spatial variability. The application of GIS to the study of vector-transmitted diseases considerably improves the management of the information obtained from field surveys and facilitates the study of the distribution patterns of the vector species.

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania., Author Summary Leishmaniasis, one of the most neglected tropical diseases, affects over 2 million people each year. Visceral leishmaniasis (VL), also known as Kala-azar, is caused by the protozoan parasites Leishmania donovani and Leishmania infantum and is fatal if left untreated. Because existing treatments are often ineffective due to parasite resistance and/or toxicity new drugs are urgently needed. Leishmaniasis is transmitted to humans by the bite of an infected sandfly. In the insect vector, parasites exist as flagellated forms—promastigotes, which infect macrophage cells of the human host, where they differentiate to round forms known as amastigotes. Amastigotes and promastigotes are substantially different from a molecular perspective. Drug discovery for leishmaniasis has traditionally been complicated by the unavailability of validated drug targets and of relevant drug assays. Whole cell-based assays have been widely used, as they bypass the need for a validated target. However, they use the insect form of the parasite; indeed, the human form, the intracellular amastigote, is difficult to obtain in the laboratory in quantities compatible with drug screening. We describe here the technical advances that made it possible to adapt the intracellular amastigote form of L. donovani to a drug assay compatible with high-throughput screening.