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.

Plasmodium falciparum Genetic Diversity in Continental Equatorial Guinea before and after Introduction of Artemisinin-Based Combination Therapy.

Efforts to control malaria may affect malaria parasite genetic variability and drug resistance, the latter of which is associated with genetic events that promote mechanisms to escape drug action. The worldwide spread of drug resistance has been a major obstacle to controlling Plasmodium falciparum malaria, and thus the study of the origin and spread of associated mutations may provide some insights into the prevention of its emergence. This study reports an analysis of P. falciparum genetic diversity, focusing on antimalarial resistance-associated molecular markers in two socioeconomically different villages in mainland Equatorial Guinea. The present study took place 8 years after a previous one, allowing the analysis of results before and after the introduction of an artemisinin-based combination therapy (ACT), i.e., artesunate plus amodiaquine. Genetic diversity was assessed by analysis of the Pfmsp2 gene and neutral microsatellite loci. Pfdhps and Pfdhfr alleles associated with sulfadoxine-pyrimethamine (SP) resistance and flanking microsatellite loci were investigated, and the prevalences of drug resistance-associated point mutations of the Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes were estimated. Further, to monitor the use of ACT, we provide the baseline prevalences of K13 propeller mutations and Pfmdr1 copy numbers. After 8 years, noticeable differences occurred in the distribution of genotypes conferring resistance to chloroquine and SP, and the spread of mutated genotypes differed according to the setting. Regarding artemisinin resistance, although mutations reported as being linked to artemisinin resistance were not present at the time, several single nucleotide polymorphisms (SNPs) were observed in the K13 gene, suggesting that closer monitoring should be maintained to prevent the possible spread of artemisinin resistance in Africa.

Molecular evidence of a Trypanosoma brucei gambiense sylvatic cycle in the human african trypanosomiasis foci of Equatorial Guinea.

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.

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

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

Screening of Trypanosoma brucei gambiense in domestic livestock and tsetse flies from an insular endemic focus (Luba, Equatorial Guinea).

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.

An alternative approach to detect Trypanosoma in Glossina (Diptera, Glossinidae) without dissection.

BACKGROUND: Determining if a tsetse fly is infected by trypanosomes and thus potentially able to transmit trypanosome-related human and animal diseases is an extremely laborious and time-consuming task to perform, especially under field conditions. In this study we tested a possible alternative approach that uses the entire insect vector for DNA extraction and PCR analysis to detect and identify Trypanosoma spp. in field collected tsetse flies. METHODOLOGY: DNA extraction was performed using a method originally developed for tick DNA extraction followed by PCR detection and identification of Trypanosoma spp. RESULTS: Two out of 62 flies captured in Equatorial Guinea carried DNA of T. brucei s.l. and Trypanosoma vivax. T. congolense forest, T. congolense savannah and T. congolense Kilifi were not detected. CONCLUSIONS: The approach we employed allowed the molecular detection and species identification of trypanosomes using the whole vector body for DNA extraction. Although the approach does not give direct information on tsetse infectivity, it provides valuable information about trypanosome species circulating in a tsetse fly vector population. The method allows an effective processing of a large number of field captured tsetse in a central laboratory.

Spatial and temporal variability of the Glossina palpalis palpalis population in the Mbini focus (Equatorial Guinea).

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.

Predicted distribution and movement of Glossina palpalis palpalis (Diptera: Glossinidae) in the wet and dry seasons in the Kogo trypanosomiasis focus (Equatorial Guinea).

The aim of this study was to predict the distribution and movement of populations of the tsetse fly, Glossina palpalis palpalis (Diptera: Glossinidae), in the wet and dry seasons and to analyze the impact of the use of mono-pyramidal traps on fly populations in the Kogo focus in 2004 and 2005. Three Glossina species are present in Kogo: Glossina palpalis palpalis, major HAT vector in West-Central Africa, Glossina caliginea, and Glossina tabaniformis. The apparent density (AD) of G. p. palpalis clearly fell from 1.23 tsetse/trap/day in July 2004 to 0.27 in December 2005. A significant reduction in the mean AD for this species was noted between seasons and years. The diversity of Glossina species was relatively low at all the sampling points; G. p. palpalis clearly predominated over the other species and significantly dropped as a consequence of control activities. The predictive models generated for the seasonal AD showed notable differences not only in the density but in the distribution of the G. p. palpalis population between the rainy and dry season. The mono-pyramidal traps have proven to be an effective instrument for reducing the density of the tsetse fly populations, although given that the Kogo trypanosomiasis focus extends from the southern Equatorial Guinea to northern Gabon, interventions need to be planned on a larger scale, involving both countries, to guarantee the long-term success of control.