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Background: Identification of local Plasmodium vivax transmission foci and its hidden reservoirs are crucial to eliminating residual vivax malaria transmission. This study assessed whether reactive case detection (RCD) could better identify P. vivax cases and infection incidences in Arjo-Didessa, Southwestern Ethiopia., Methods: A RCD survey was conducted from November 2019 to October 2021 in Arjo-Didessa and the surrounding vicinity in southwestern Ethiopia. RCD was performed at 0, 30, and 60 days following reports of P. vivax infections by health facilities to detect further cases and potential transmission networks. Household members of the index case and neighbours living within 200 m of the index household were screened for P. vivax. Households 200-500 m away are considered controls and were also screened for P. vivax. Plasmodium vivax was detected by microscopy, rapid diagnostic testing (RDT), and quantitative polymerase chain reaction (qPCR). Risk factors associated with vivax malaria were analysed using generalized estimating equations (GEE)., Results: A total of 3303 blood samples were collected from the index (n = 427), neighbouring (n = 1626), and control (n = 1240) household in the three rounds of follow-up visits for malaria infection, the overall positivity rate of P. vivax malaria was 1.6% (95% CI 1.2-2.2%), 1.9% (95% CI 1.5-2.4), and 3.9% (95% CI 3.2-4.6%) by microscopy, RDT, and qPCR, respectively. Microscopy and RDT detected 41.5% (54 of 130) and 49.1% (64 of 130) of the qPCR-confirmed P. vivax cases, respectively. Of qPCR-positive samples, 77.7% of the total P. vivax infections circulated in the index and neighbouring households, while control households accounted for 23.3% of the infections. Of the P. vivax infections detected 81.0% (95% CI 72.9-87.1%) were asymptomatic. In this study, P. vivax infection incidence was higher in index case households (53.8 cases per 1000 person-months) and (44.0 cases per 1000 person-months) in neighbouring households compared to the control households (25.1 cases per 1000 person-months) with statistical difference (p = 0.02). In index case households, children < 5 years and school-age children were at higher risk of P. vivax infection (AOR: 6.3, 95% CI: 2.24-18.02, p = 0.001 and AOR: 2.7, 95% CI: 1.10-6.64, p = 0.029)., Conclusions: This study found clustering of asymptomatic and sub-microscopic P. vivax infections in the index case household and their neighbours using RCD and molecular methods. Children under 5 years and of school age were more likely to have P. vivax infection in index households. Thus, tailored RCD approaches and targeted interventions for interrupting residual P. vivax transmission networks are needed to eliminate P. vivax malaria in low transmission settings., Competing Interests: Declarations: Ethics approval and consent to participate The protocol was reviewed and approved by the National Research Ethics Review Committee (NRERC) of Ethiopia (Ref. no.: 10/31/2018) and Jimma University Institute of Health, Institutional Review Board (Reference No. IHRPGD/362/2). Permission was obtained from the relevant Buno Bedele and East Wollega health offices, and Arjo-Didessa Sugar Factory, Oromia regional state, Ethiopia. A written consent was obtained from the study participants and the parents and guardians of children. All malaria-positive index cases and positive study participants identified by microscopic examination of blood film and Rapid Diagnostic tests (RDT) at PCD clinics were treated as per the national malaria treatment guideline for free by health professionals. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Malaria prevention and control strategies have been hampered by urbanization and the spread of Anopheles stephensi. The spread of this vector into Africa further complicates the already complex malaria situation, that could put about 126 million Africans at risk of infection. Hence, this study aimed to assess the determinants of urban malaria, focusing on the role of urbanization and the distribution of An. stephensi in Eastern Ethiopia., Methods: A matched case control study was conducted among febrile urban residents of Dire Dawa (malaria positive as cases and negative as a control). A capillary blood sample was collected for parasite identification using microscopic examination and an interviewer administered questionnaire was used to collect additional data. Centers for Disease Control and Prevention miniature light traps (CDC-LT) and Prokopack aspirator were used to collect adult mosquito vectors from the selected cases and control houses to identify the mosquito vector species. Then, the data were exported to STATA for analysis. Conditional logistic regression was done to identify determinants, and principal component Analysis (PCA) was done for some independent variables., Results: This study enrolled 132 cases and 264 controls from urban setting only. Of the 132 cases, 90 cases were positive for Plasmodium falciparum, 34 were positive for Plasmodium vivax and 8 had mixed infections. All cases and controls were similar with regard to their respective age and sex. Travel history (AOR: 13.1, 95% CI 2.8-61.4), presence of eves and holes on walls (AOR: 2.84, 95% CI 1.5-5.5), history of malaria diagnosis (AOR: 2.4, 95% CI 1.1-5.3), owning any livestock (AOR: 7.5, 95% CI 2.4-22.8), presence of stagnant water in the area (AOR: 3.2, 95% CI 1.7-6.1), sleeping under bed net the previous night (AOR: 0.21, 95% CI 0.1-0.6) and knowledge on malaria and its prevention (AOR: 2.2, 95% CI 1.2-4.1) were determinants of urban malaria infection. About 34 adult Anopheles mosquitoes were collected and identified from those selected cases and control houses and 27 of them were identified as An. stephensi., Conclusion: Among the cases, the dominant species were P. falciparum. This study identified travel history, house condition, past infection, livestock ownership, stagnant water, bed net use, and malaria knowledge as determinants of infection. This study also found the dominance of the presence of An. stephensi among the collected mosquito vectors. This suggests that the spread of An. stephensi may be impacting malaria infection in the study area. Hence, strengthening urban-targeted malaria interventions should be enhanced to prevent and control further urban malaria infection and spread., (© 2024. The Author(s).)

Background: Malaria is a major disease burden in Ethiopia. Migration can influence malaria transmission dynamics, with individuals relocating from malaria-free highland regions to malarious lowlands potentially facing elevated risks of contracting malaria. Migrants may find it difficult to protect themselves against malaria and have limited access to diagnosis or treatment. Settlers in gold mining sites are one type of migrant and are often neglected in malaria research yet may have particularly high malaria risk. This study was a malaria prevalence survey among settlers in a new gold mining settlement in the highly malarious Gambella Region, Ethiopia., Methods: n = 590 people were surveyed for demographic information and their knowledge and practices of malaria. Participants were tested for malaria using rapid diagnostic tests and microscopy. Using logistic regressions, the influence of demographic characteristics on malaria infections and bed net access were analysed. A sub-sample of participants was interviewed to comprehend settlement living conditions and healthcare accessibility., Results: The overall prevalence of Plasmodium falciparum was 37.5% (CI 32.4-42.3%). Young children were most likely to have malaria, with individuals aged 15-24 having 67% lower odds (aOR: 0.33; CI 0.13-0.86) of infection compared to those aged 1-4 years old. Meanwhile, those age 25-plus had 75% decreased odds of malaria infection (aOR 0.25; CI 0.10-0.65). Individuals with bed nets had ~ 50% decreased odds of testing positive for falciparum malaria than those reporting having no bed net (aOR: 0.47; CI 0.22-0.97). Individuals who relocated from low elevation with high malaria test positivity rate areas were more prone to testing positive for malaria, as were those residing in densely populated households with multiple malaria cases. Conversely, individuals from higher elevations with low malaria test positivity rates, and those living in households with 5-10 occupants and < 2 malaria infections, were more likely to possess bed nets., Conclusions: This gold mining settlement provides an example of an oft-neglected atypical community where malaria is a significant, but under-addressed, health problem. Within this community, future interventions focused on distributing bed nets, particularly to larger households and those with children, have great potential to alleviate the malaria burden. Efforts should also be made to provide affordable, and accessible, early diagnosis and treatment., (© 2024. The Author(s).)

Background: To interrupt residual malaria transmission and achieve successful elimination of Plasmodium falciparum in low-transmission settings, the World Health Organization (WHO) recommends the administration of a single dose of 0.25 mg/kg (or 15 mg/kg for adults) primaquine (PQ) combined with artemisinin-based combination therapy (ACT), without glucose-6-phosphate dehydrogenase (G6PD) testing. However, due to the risk of haemolysis in patients with G6PD deficiency (G6PDd), PQ use is uncommon. Thus, this study aimed to assess the safety of a single low dose of PQ administered to patients with G6PD deficiency., Methods: An observational cohort study was conducted with patients treated for uncomplicated P. falciparum malaria with either single-dose PQ (0.25 mg/kg) (SLD PQ) + ACT or ACT alone. Microscopy-confirmed uncomplicated P. falciparum malaria patients visiting public health facilities in Arjo Didessa, Southwest Ethiopia, were enrolled in the study from September 2019 to November 2022. Patients with uncomplicated P. falciparum malaria were followed up for 28 days through clinical and laboratory diagnosis, such as measurements of G6PD levels and haemoglobin (Hb) concentrations. G6PD levels were measured by a quantiative CareSTART™ POCT S1 biosensor machine. Patient interviews were also conducted, and the type and frequency of clinical complaints were recorded. Hb data were taken on days (D) 7, 14, 21, and 28 following treatment with SLD-PQ + ACT or ACT alone., Results: A total of 249 patients with uncomplicated P. falciparum malaria were enrolled in this study. Of these, 83 (33.3%) patients received ACT alone, and 166 (66.7%) received ACT combined with SLD-PQ treatment. The median age of the patients was 20 (IQR 28-15) years. G6PD deficiency was found in 17 (6.8%) patients, 14 males and 3 females. There were 6 (7.2%) and 11 (6.6%) phenotypic G6PD-deficient patients in the ACT alone and ACT + SLD-PQ arms, respectively. The mean Hb levels in patients treated with ACT + SLD-PQ were reduced by an average of 0.45 g/dl (95% CI = 0.39 to 0.52) in the posttreatment phase (D7) compared to a reduction of 0.30 g/dl (95% CI = 0.14 to - 0.47) in patients treated with ACT alone (P = 0.157). A greater mean Hb reduction was observed on day 7 in the G6PDd ACT + SLD-PQ group (- 0.60 g/dL) than in the G6PDd ACT alone group (- 0.48 g/dL); however, there was no statistically significant difference (P = 0.465). Overall, D14 losses were 0.10 g/dl (95% CI = - 0.00 to 0.20) and 0.05 g/dl (95% CI = - 0.123 to 0.22) in patients with and without SLD-PQ, respectively (P = 0.412)., Conclusions: This study's findings indicate that using SLD-PQ in combination with ACT is safe for uncomplicated P. falciparum malaria regardless of the patient's G6PD status in Ethiopian settings. Caution should be taken in extrapolating this finding in other settings with diverse G6DP phenotypes., (© 2024. The Author(s).)

Background: Malaria remains a significant cause of morbidity and mortality in Ethiopia with an estimated 3.8 million cases in 2021 and 61% of the population living in areas at risk of malaria transmission. Throughout the country Plasmodium vivax and Plasmodium falciparum are co-endemic, and Duffy expression is highly heterogeneous. The public health significance of Duffy negativity in relation to P. vivax malaria in Ethiopia, however, remains unclear. This study seeks to explore the prevalence and rates of P. vivax malaria infection across Duffy phenotypes in clinical and community settings., Methods: A total of 9580 and 4667 subjects from community and health facilities from a malaria endemic site and an epidemic-prone site in western Ethiopia were enrolled and examined for P. vivax infection and Duffy expression from February 2018 to April 2021. Association between Duffy expression, P. vivax and P. falciparum infections were examined for samples collected from asymptomatic community volunteers and symptomatic subjects from health centres., Results: Infection rate of P. vivax among Duffy positives was 2-22 fold higher than Duffy negatives in asymptomatic volunteers from the community. Parasite positivity rate was 10-50 fold higher in Duffy positives than Duffy negatives among samples collected from febrile patients attending health centres and mixed P. vivax and P. falciparum infections were significantly more common than P. vivax mono infections among Duffy negative individuals. Plasmodium vivax parasitaemia measured by 18sRNA parasite gene copy number was similar between Duffy positives and Duffy negatives., Conclusions: Duffy negativity does not offer complete protection against infection by P. vivax, and cases of P. vivax in Duffy negatives are widespread in Ethiopia, being found in asymptomatic volunteers from communities and in febrile patients from health centres. These findings offer evidence for consideration when developing control and intervention strategies in areas of endemic P. vivax and Duffy heterogeneity., (© 2024. The Author(s).)

Background: Understanding of malaria ecology is a prerequisite for designing locally adapted control strategies in resource-limited settings. The aim of this study was to utilize the spatial heterogeneity in malaria transmission for the designing of adaptive interventions., Methods: Field collections of clinical malaria incidence, asymptomatic Plasmodium infection, and malaria vector data were conducted from 108 randomly selected clusters which covered different landscape settings including irrigated farming, seasonal flooding area, lowland dryland farming, and highlands in western Kenya. Spatial heterogeneity of malaria was analyzed and classified into different eco-epidemiological zones., Results: There was strong heterogeneity and detected hot/cold spots in clinical malaria incidence, Plasmodium prevalence, and vector abundance. The study area was classified into four zones based on clinical malaria incidence, parasite prevalence, vector density, and altitude. The two irrigated zones have either the highest malaria incidence, parasite prevalence, or the highest malaria vector density; the highlands have the lowest vector density and parasite prevalence; and the dryland and flooding area have the average clinical malaria incidence, parasite prevalence and vector density. Different zones have different vector species, species compositions and predominant species. Both indoor and outdoor transmission may have contributed to the malaria transmission in the area. Anopheles gambiae sensu stricto (s.s.), Anopheles arabiensis, Anopheles funestus s.s., and Anopheles leesoni had similar human blood index and malaria parasite sporozoite rate., Conclusion: The multi-transmission-indicator-based eco-epidemiological zone classifications will be helpful for making decisions on locally adapted malaria interventions., (© 2024. The Author(s).)

Background: Timely molecular surveillance of Plasmodium falciparum kelch 13 (k13) gene mutations is essential for monitoring the emergence and stemming the spread of artemisinin resistance. Widespread artemisinin resistance, as observed in Southeast Asia, would reverse significant gains that have been made against the malaria burden in Africa. The purpose of this study was to assess the prevalence of k13 polymorphisms in western Kenya and Ethiopia at sites representing varying transmission intensities between 2018 and 2022., Methods: Dried blood spot samples collected through ongoing passive surveillance and malaria epidemiological studies, respectively, were investigated. The k13 gene was genotyped in P. falciparum isolates with high parasitaemia: 775 isolates from four sites in western Kenya (Homa Bay, Kakamega, Kisii, and Kombewa) and 319 isolates from five sites across Ethiopia (Arjo, Awash, Gambella, Dire Dawa, and Semera). DNA sequence variation and neutrality were analysed within each study site where mutant alleles were detected., Results: Sixteen Kelch13 haplotypes were detected in this study. Prevalence of nonsynonymous k13 mutations was low in both western Kenya (25/783, 3.19%) and Ethiopia (5/319, 1.57%) across the study period. Two WHO-validated mutations were detected: A675V in three isolates from Kenya and R622I in four isolates from Ethiopia. Seventeen samples from Kenya carried synonymous mutations (2.17%). No synonymous mutations were detected in Ethiopia. Genetic variation analyses and tests of neutrality further suggest an excess of low frequency polymorphisms in each study site. Fu and Li's F test statistic in Semera was 0.48 (P > 0.05), suggesting potential population selection of R622I, which appeared at a relatively high frequency (3/22, 13.04%)., Conclusions: This study presents an updated report on the low frequency of k13 mutations in western Kenya and Ethiopia. The WHO-validated R622I mutation, which has previously only been reported along the north-west border of Ethiopia, appeared in four isolates collected from eastern Ethiopia. The rapid expansion of R622I across Ethiopia signals the need for enhanced monitoring of the spread of drug-resistant P. falciparum parasites in East Africa. Although ACT remains currently efficacious in the study areas, continued surveillance is necessary to detect early indicators of artemisinin partial resistance., (© 2024. The Author(s).)

Background: Anopheles stephensi is an emerging exotic invasive urban malaria vector in East Africa. The World Health Organization recently announced an initiative to take concerted actions to limit this vector's expansion by strengthening surveillance and control in invaded and potentially receptive territories in Africa. This study sought to determine the invasion of An. stephensi in southern Ethiopia., Methods: A targeted entomological survey, both larvae and adult, was conducted in Hawassa City, southern Ethiopia between November 2022 and February 2023. Anopheles larvae were reared to adults for species identification. CDC light traps and BG Pro traps were used indoors and outdoors overnight at selected houses to collect adult mosquitoes in the study area. Prokopack aspirator was employed to sample indoor resting mosquitoes in the morning. Adults of An. stephensi was identified using morphological keys and then confirmed by PCR., Results: Larvae of An. stephensi were found in 28 (16.6%) of the 169 potential mosquito breeding sites surveyed. Out of 548 adult female Anopheles mosquitoes reared from larvae, 234 (42.7%) were identified as An. stephensi morphologically. A total of 449 female anophelines were caught, of which 53 (12.0%) were An. stephensi. Other anopheline species collected in the study area included Anopheles gambiae sensu lato (s.l.), Anopheles pharoensis, Anopheles coustani, and Anopheles demeilloni., Conclusion: This study confirmed the presence of An. stephensi in southern Ethiopia. The presence of both larval and adult stages of this mosquito attests that this species established sympatric colonization with native vector species such as An. gambiae (s.l.) in southern Ethiopia. The findings warrant further investigation on the ecology, behaviour, population genetics, and role of An. stephensi in malaria transmission in Ethiopia., (© 2023. The Author(s).)

Background: Water resource development projects are essential for increasing agricultural productivity and ensuring food security. However, these activities require the modification of pre-existing environmental settings, which may alter mosquito larval habitat availability and seasonality. The intensive utilization of current adult vector control tools results in insecticide resistance among the main vectors. When coupled with behavioural resistances, a shift in malaria vector feeding and resting behaviours could compromise the effectiveness of the current adult vector control strategies. Thus, it is important to look for new or alternative vector control interventions for immatures to complement adult control by focusing on different larval habitats and their seasonal availability. Thus, this study investigated larval habitat seasonality and seasonal larval abundance and distribution in irrigated sugar cane plantation settings in Ethiopia., Methods: Anopheles mosquito larval habitats were surveyed and visited twice a month for a period of 14 months. Anopheline larvae and pupae were collected, reared, and fed finely ground fish food. Adults were provided with sucrose solution and kept under standard conditions. Female Anopheles mosquitoes were identified morphologically and using a species-specific PCR assay. Environmental parameters, which include habitats' physico-chemical characteristics, were assessed. Larval habitat diversity and larval abundance and distribution were determined across different seasons., Results: The study revealed that Anopheles gambiae sensu lato (s.l.) was the most predominant 4197(57%) vector species, followed by Anopheles coustani complex 2388 (32.8%). Molecular analysis of sub-samples of An. gambiae s.l. resulted in Anopheles arabiensis (77.9%) and Anopheles amharicus (21.5%), and the remaining 1.1% (n = 7) sub-samples were not amplified. Physico-chemical parameters such as temperature (t = 2.22, p = 0.028), conductivity (t = 3.21, p = 0.002), dissolved oxygen (t = 7.96, p = 0.001), nitrate ion (t = 2.51, p = 0.013), and ammonium ion (t = 2.26, p = 0.025) showed a significant and direct association with mosquito larval abundance. Furthermore, mosquito larval abundance was correlated with distance to the nearest houses (r = - 0.42, p = 0.001), exposure to sunlight (r = 0.34, p = 0.001), during long and short rainy season animal hoof prints, truck tires/road puddles and rain pools were negatively correlated (r = - 0.22, p = 0.01) and types of habitat (r = - 0.20, p = 0.01). Significant habitat type productivity were observed in man-made pools (t = 3.881, P = 0.01163), rain pools, animal hoof prints, (t = - 4.332, P = 0.00749 in both short and long rainy season, whereas, during dry seasons habitat type productivity almost similar and have no significance difference., Conclusion: The study found that different larval habitats had variable productivity in different seasons, and that physical and physicochemical features like ammonium and nitrate, as well as the distance between larval habitats and households, are related to larval production. As a result, vector control should take into account the seasonality of Anopheles larval habitat as well as the impact of pesticide application on larval source management., (© 2023. The Author(s).)

Background: Water resource development projects, such as dams and irrigation schemes, have a positive impact on food security and poverty reduction. However, such projects could increase prevalence of vector borne disease, such as malaria. This study investigate the impact of different agroecosystems and prevalence of malaria infection in Southwest Ethiopia., Methods: Two cross-sectional surveys were conducted in the dry and wet seasons in irrigated and non-irrigated clusters of Arjo sugarcane and Gambella rice development areas of Ethiopia in 2019. A total of 4464 and 2176 study participants from 1449 households in Arjo and 546 households in Gambella enrolled in the study and blood samples were collected, respectively. All blood samples were microscopically examined and a subset of microscopy negative blood samples (n = 2244) were analysed by qPCR. Mixed effect logistic regression and generalized estimating equation were used to determine microscopic and submicroscopic malaria infection and the associated risk factors, respectively., Results: Prevalence by microscopy was 2.0% (88/4464) in Arjo and 6.1% (133/2176) in Gambella. In Gambella, prevalence was significantly higher in irrigated clusters (10.4% vs 3.6%) than in non-irrigated clusters (p < 0.001), but no difference was found in Arjo (2.0% vs 2.0%; p = 0.993). On the other hand, of the 1713 and 531 samples analysed by qPCR from Arjo and Gambella the presence of submicroscopic infection was 1.2% and 12.8%, respectively. Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale were identified by qPCR in both sites. Irrigation was a risk factor for submicroscopic infection in both Arjo and Gambella. Irrigation, being a migrant worker, outdoor job, < 6 months length of stay in the area were risk factors for microscopic infection in Gambella. Moreover, school-age children and length of stay in the area for 1-3 years were significant predictors for submicroscopic malaria in Gambella. However, no ITN utilization was a predictor for both submicroscopic and microscopic infection in Arjo. Season was also a risk factor for microscopic infection in Arjo., Conclusion: The study highlighted the potential importance of different irrigation practices impacting on submicroscopic malaria transmission. Moreover, microscopic and submicroscopic infections coupled with population movement may contribute to residual malaria transmission and could hinder malaria control and elimination programmes in the country. Therefore, strengthening malaria surveillance and control by using highly sensitive diagnostic tools to detect low-density parasites, screening migrant workers upon arrival and departure, ensuring adequate coverage and proper utilization of vector control tools, and health education for at-risk groups residing or working in such development corridors is needed., (© 2023. The Author(s).)

Background: The rise of insecticide resistance against malaria vectors in sub-Saharan Africa has resulted in the need to consider other methods of vector control. The potential use of biological methods, including larvivorous fish, Bacillus thuringiensis israelensis (Bti) and plant shading, is sustainable and environmentally friendly options. This study examined the survivorship of Anopheles arabiensis and Anopheles funestus larvae and habitat productivity in four permanent habitat types in Homa Bay county, western Kenya., Methods: Predator densities were studied in a laboratory setup while habitat productivity and larval survivorship was studied in field setup., Results: Fish were observed as the most efficient predator (75.8% larval reduction rate) followed by water boatman (69%), and dragonfly nymph (69.5%) in predation rates. Lower predation rates were observed in backswimmers (31%), water beetles (14.9%), water spiders (12.2%), mayflies (7.3%), and tadpoles (6.9%). Increase in predator density in the field setup resulted in decreased Culex larval density. Larval and pupa age-specific distribution was determined and their survivorship curves constructed. Combined larvae (Stage I-IV) to pupa mortality was over 97% for An. arabiensis and 100% for An. funestus. The highest larval stage survival rate was from larval stages I to II and the lowest from larval stage IV to pupa. Stage-specific life tables indicated high mortality rates at every developmental stage, especially at the larval stage II and III., Conclusion: Determination of the efficiency of various larval predators and habitat productivity will help with the correct identification of productive habitats and selection of complementary vector control methods through environmental management and/or predator introduction (for instance fish) in the habitats., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: In western Kenya, not all malaria cases are reported as stipulated in the community case management of malaria (CCMm) strategy. This underreporting affects the equity distribution of malaria commodities and the evaluation of interventions. The current study aimed to evaluate the effectiveness of community health volunteers' active case detection and management of malaria in western Kenya., Methods: Cross-sectional active case detection (ACD) of malaria survey was carried out between May and August 2021 in three eco-epidemiologically distinct zones in Kisumu, western Kenya: Kano Plains, Lowland lakeshore and Highland Plateau. The CHVs conducted biweekly ACD of malaria household visits to interview and examine residents for febrile illness. The Community Health Volunteers (CHVs) performance during the ACD of malaria was observed and interviews done using structured questionnaires., Results: Of the total 28,800 surveyed, 2597 (9%) had fever and associated malaria symptoms. Eco-epidemiological zones, gender, age group, axillary body temperature, bed net use, travel history, and survey month all had a significant association with malaria febrile illness (p < 0.05). The qualification of the CHV had a significant influence on the quality of their service. The number of health trainings received by the CHVs was significantly related to the correctness of using job aid (χ 2  = 6.261, df = 1, p = 0.012) and safety procedures during the ACD activity (χ 2  = 4.114, df = 1, p = 0.043). Male CHVs were more likely than female CHVs to correctly refer RDT-negative febrile residents to a health facility for further treatment (OR = 3.94, 95% CI = 1.85-5.44, p < 0.0001). Most of RDT-negative febrile residents who were correctly referred to the health facility came from the clusters with a CHV having 10 years of experience or more (OR = 1.29, 95% CI = 1.05-1.57, p = 0.016). Febrile residents in clusters managed by CHVs with more than 10 years of experience (OR = 1.82, 95% CI = 1.43-2.31, p < 0.0001), who had a secondary education (OR = 1.53, 95% CI = 1.27-1.85, p < 0.0001), and were over the age of 50 (OR = 1.44, 95% CI = 1.18-1.76, p < 0.0001), were more likely to seek malaria treatment in public hospitals. All RDT positive febrile residents were given anti-malarial by the CHVs, and RDT negatives were referred to the nearest health facility for further treatment., Conclusions: The CHV's years of experience, education level, and age had a significant influence on their service quality. Understanding the qualifications of CHVs can assist healthcare systems and policymakers in designing effective interventions that assist CHVs in providing high-quality services to their communities., (© 2023. The Author(s).)

Background: Despite notable progress in the control and prevention of malaria in the Horn of Africa, the disease continues to cause significant morbidity and mortality in various regions of Ethiopia, and elsewhere in the region. The transmission of malaria is affected by genetic, sociocultural, and ecological factors. Lare is an Ethiopian district adjacent to the Ethio-South Sudan border, in Gambella region. The region currently has the highest prevalence of malaria in Ethiopia. This study assesses the burden and spatiotemporal patterns of disease transmission, including the effect of climatic factors on the occurrence of malaria, across an international border crossing. This understanding can assist in crafting informed programmatic and policy decisions for interventions., Methods: This study was conducted in Lare district, Southwest Ethiopia, a temperate zone. A retrospective descriptive analysis was conducted using clinical service data collected between 2011 and 2021 from the 9 health facilities of the district. Both clinically diagnosed patients and those identified using microscopy and rapid diagnostic testing (RDT) were included in the study. Additionally, climate data was incorporated into analyses. Examples of analyses include malaria burden, positivity rate, incidence, species frequency, and an ANOVA to assess inter-annual case number and meteorological factor variation., Results: Between 2011 and 2021, a total of 96,616 suspected malaria cases were tested by microscopy or RDT, and 39,428 (40.8%) of these cases were reported as positive. There were 1276 patients admitted with 22 deaths recorded. There were further more significant fluctuations in positivity rates across years, the highest being 74.5% in 2021. Incidence varied from 18.0% in 2011 to 151.6% in 2016. The malaria parasite species most detected was Plasmodium falciparum, followed by a smaller proportion of Plasmodium vivax. The greatest proportions of P. falciparum cases were observed in 2018 and 2019, at 97.4% and 97.0% prevalence, respectively. There was significant seasonal variation in case number, the highest observed in July through September of each year. Climatic conditions of annual rainfall, temperature and humidity favored the increment of malaria cases from June until October., Conclusion: The study shows that the burden, i.e. morbidity and mortality (with fluctuating patterns) of malaria are still significant public health problems and can pose serious consequences in the district. This has implication for cross-border malaria transmission risk due to considerable border crossings. The predominant cause of the disease is P. falciparum, which causes severe complications in patients. The district has to prepare to deal with such complications for better patient care and outcomes., (© 2023. The Author(s).)

Background: Artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment by the World Health Organization to treat uncomplicated Plasmodium falciparum malaria. However, the emergence and spread of P. falciparum resistant to artemisinins and their partner drugs is a significant risk for the global effort to reduce disease burden facing the world. Currently, dihydroartemisinin-piperaquine (DHA-PPQ) is the most common drug used to treat P. falciparum, but little evidence about the resistance status targeting DHA (ACT drug) and its partner drug (PPQ) has been reported in Shandong Province, China., Methods: A retrospective study was conducted to explore the prevalence and spatial distribution of Pfk13 and Pfcrt polymorphisms (sites of 72-76, and 93-356) among imported P. falciparum isolates between years 2015-2019 in Shandong Province in eastern China. Individual epidemiological information was collected from a web-based reporting system were reviewed and analysed., Results: A total of 425 P. falciparum blood samples in 2015-2019 were included and 7.3% (31/425) carried Pfk13 mutations. Out of the isolates that carried Pfk13 mutations, 54.8% (17/31) were nonsynonymous polymorphisms. The mutant alleles A578S, Q613H, C469C, and S549S in Pfk13 were the more frequently detected allele, the mutation rate was the same as 9.7% (3/31). Another allele Pfk13 C580Y, closely associated with artemisinin (ART) resistance, was found as 3.2% (2/31), which was found in Cambodia. A total of 14 mutant isolates were identified in Western Africa countries (45.2%, 14/31). For the Pfcrt gene, the mutation rate was 18.1% (77/425). T 76 T 356 and T 76 were more frequent in all 13 different haplotypes with 26.0% (20/77) and 23.4% (18/77). The CVIET and CVIKT mutant at loci 72-76 have exhibited a prevalence of 19.5% (15/77) and 3.9% (3/77), respectively. The CVIET was mainly observed in samples from Congo (26.7%, 4/15) and Mozambique (26.7%, 4/15). No mutations were found at loci 97, 101 and 145. For polymorphisms at locus 356, a total of 24 isolates were identified and mainly from Congo (29.2%, 7/24)., Conclusion: These findings indicate a low prevalence of Pfk13 in the African isolates. However, the emergence and increase in the new alleles Pfcrt I356T, reveals a potential risk of drug pressure in PPQ among migrant workers returned from Africa. Therefore, continuous molecular surveillance of Pfcrt mutations and in vitro susceptibility tests related to PPQ are necessary., (© 2022. The Author(s).)

Background: Human mobility behaviour modelling plays an essential role in the understanding and control of the spread of contagious diseases by limiting the contact among individuals, predicting the spatio-temporal evolution of an epidemic and inferring migration patterns. It informs programmatic and policy decisions for effective and efficient intervention. The objective of this research is to assess the human mobility pattern and analyse its implication for malaria disease epidemiology., Methods: In this study, human mobility patterns in Benishangul-Gumuz and Gambella regions in Western Ethiopia were explored based on a cellular network mobility parameter (e.g., handover rate) via real world data. Anonymized data were retrieved for mobile active users with mobility related information. The data came from anonymous traffic records collected from all the study areas. For each cell, the necessary mobility parameter data per hour, week and month were collected. A scale factor was computed to change the mobility parameter value to the human mobility pattern. Finally, the relative human mobility probability for each scenario was estimated. MapInfo and Matlab softwares were used for visualization and analysis purposes. Hourly travel patterns in the study settings were compared with hourly malaria mosquito vector feeding behaviour., Results: Heterogeneous human movement patterns were observed in the two regions with some areas showing typically high human mobility. Furthermore, the number of people entering into the two study regions was high during the highest malaria transmission season. Two peaks of hourly human movement, 8:00 to 9:00 and 16:00 to 18:00, emerged in Benishangul-Gumuz region while 8:00 to 10:00 and 16:00 to 18:00 were the peak hourly human mobility time periods in Gambella region. The high human movement in the night especially before midnight in the two regions may increase the risk of getting mosquito bite particularly by early biters depending on malaria linked human behaviour of the population., Conclusions: High human mobility was observed both within and outside the two regions. The population influx and efflux in these two regions is considerably high. This may specifically challenge the transition from malaria control to elimination. The daily mobility pattern is worth considering in the context of malaria transmission. In line with this malaria related behavioural patterns of humans need to be properly addressed., (© 2022. The Author(s).)

Background: Long-lasting insecticidal nets (LLINs) have been the primary vector control strategy until indoor residual spraying (IRS) was added in Homa Bay and Migori Counties in western Kenya. The objective of this study was to evaluate the impact of LLINs integrated with IRS on the prevalence of asymptomatic and submicroscopic Plasmodium infections in Homa Bay County., Methods: A two-stage cluster sampling procedure was employed to enroll study participants aged ≥ 6 months old. Four consecutive community cross-sectional surveys for Plasmodium infection were conducted in residents of Homa Bay county, Kenya. Prior to the start of the study, all study households received LLINs, which were distributed between June 2017 and March 2018. The first (February 2018) and second (June 2018) surveys were conducted before and after the first round of IRS (Feb-Mar 2018), while the third (February 2019) and fourth (June 2019) surveys were conducted before and after the second application of IRS (February-March 2019). Finger-prick blood samples were obtained to prepare thick and thin smears for microscopic determination and qPCR diagnosis of Plasmodium genus., Results: Plasmodium spp. infection prevalence by microscopy was 18.5% (113/610) before IRS, 14.2% (105/737) and 3.3% (24/720) after the first round of IRS and 1.3% (11/849) after the second round of IRS (p < 0.0001). Submicroscopic (blood smear negative, qPCR positive) parasitaemia reduced from 18.9% (115/610) before IRS to 5.4% (46/849) after IRS (p < 0.0001). However, the proportion of PCR positive infections that were submicroscopic increased from 50.4% (115/228) to 80.7% (46/57) over the study period (p < 0.0001). Similarly, while the absolute number and proportions of microscopy positives which were asymptomatic decreased from 12% (73/610) to 1.2% (9/849) (p < 0.0001), the relative proportion increased. Geometric mean density of P. falciparum parasitaemia decreased over the 2-year study period (p < 0.0001)., Conclusions: These data suggest that two annual rounds of IRS integrated with LLINs significantly reduced the prevalence of Plasmodium parasitaemia, while the proportion of asymptomatic and submicroscopic infections increased. To reduce cryptic P. falciparum transmission and improve malaria control, strategies aimed at reducing the number of asymptomatic and submicroscopic infections should be considered., (© 2022. The Author(s).)

Background: Evolutionary pressures lead to the selection of efficient malaria vectors either resistant or susceptible to Plasmodium parasites. These forces may favour the introduction of species genotypes that adapt to new breeding habitats, potentially having an impact on malaria transmission. Thioester-containing protein 1 (TEP1) of Anopheles gambiae complex plays an important role in innate immune defenses against parasites. This study aims to characterize the distribution pattern of TEP1 polymorphisms among populations of An. gambiae sensu lato (s.l.) in western Kenya., Methods: Anopheles gambiae adult and larvae were collected using pyrethrum spray catches (PSC) and plastic dippers respectively from Homa Bay, Kakamega, Bungoma, and Kisumu counties between 2017 and 2020. Collected adults and larvae reared to the adult stage were morphologically identified and then identified to sibling species by PCR. TEP1 alleles were determined in 627 anopheles mosquitoes using restriction fragment length polymorphisms-polymerase chain reaction (RFLP-PCR) and to validate the TEP1 genotyping results, a representative sample of the alleles was sequenced., Results: Two TEP1 alleles (TEP1*S1 and TEP1*R2) and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2) were identified. TEP1*S1 and TEP1*R2 with their corresponding genotypes, homozygous *S1/S1 and heterozygous *R2/S1 were widely distributed across all sites with allele frequencies of approximately 80% and 20%, respectively both in Anopheles gambiae and Anopheles arabiensis. There was no significant difference detected among the populations and between the two mosquito species in TEP1 allele frequency and genotype frequency. The overall low levels in population structure (F ST  = 0.019) across all sites corresponded to an effective migration index (Nm = 12.571) and low Nei's genetic distance values (< 0.500) among the subpopulation. The comparative fixation index values revealed minimal genetic differentiation between species and high levels of gene flow among populations., Conclusion: Genotyping TEP1 has identified two common TEP1 alleles (TEP1*S1 and TEP1*R2) and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2) in An. gambiae s.l. The TEP1 allele genetic diversity and population structure are low in western Kenya., (© 2022. The Author(s).)

Background: Accurate malaria diagnosis and appropriate treatment at local health facilities are critical to reducing morbidity and human reservoir of infectious gametocytes. The current study assessed the accuracy of malaria diagnosis and treatment practices in three health care facilities in rural western Kenya., Methods: The accuracy of malaria detection and treatment recommended compliance was monitored in two public and one private hospital from November 2019 through March 2020. Blood smears from febrile patients were examined by hospital laboratory technicians and re-examined by an expert microscopists thereafter subjected to real-time polymerase chain reaction (RT-PCR) for quality assurance. In addition, blood smears from patients diagnosed with malaria rapid diagnostic tests (RDT) and presumptively treated with anti-malarial were re-examined by an expert microscopist., Results: A total of 1131 febrile outpatients were assessed for slide positivity (936), RDT (126) and presumptive diagnosis (69). The overall positivity rate for Plasmodium falciparum was 28% (257/936). The odds of slide positivity was higher in public hospitals, 30% (186/624, OR:1.44, 95% CI = 1.05-1.98, p < 0.05) than the private hospital 23% (71/312, OR:0.69, 95% CI = 0.51-0.95, p < 0.05). Anti-malarial treatment was dispensed more at public hospitals (95.2%, 177/186) than the private hospital (78.9%, 56/71, p < 0.0001). Inappropriate anti-malarial treatment, i.e. artemether-lumefantrine given to blood smear negative patients was higher at public hospitals (14.6%, 64/438) than the private hospital (7.1%, 17/241) (p = 0.004). RDT was the most sensitive (73.8%, 95% CI = 39.5-57.4) and specific (89.2%, 95% CI = 78.5-95.2) followed by hospital microscopy (sensitivity 47.6%, 95% CI = 38.2-57.1) and specificity (86.7%, 95% CI = 80.8-91.0). Presumptive diagnosis had the lowest sensitivity (25.7%, 95% CI = 13.1-43.6) and specificity (75.0%, 95% CI = 50.6-90.4). RDT had the highest non-treatment of negatives [98.3% (57/58)] while hospital microscopy had the lowest [77.3% (116/150)]. Health facilities misdiagnosis was at 27.9% (77/276). PCR confirmed 5.2% (4/23) of the 77 misdiagnosed cases as false positive and 68.5% (37/54) as false negative., Conclusions: The disparity in malaria diagnosis at health facilities with many slide positives reported as negatives and high presumptive treatment of slide negative cases, necessitates augmenting microscopic with RDTs and calls for Ministry of Health strengthening supportive infrastructure to be in compliance with treatment guidelines of Test, Treat, and Track to improve malaria case management., (© 2022. The Author(s).)

Background: Malaria causes significant mortality and morbidity in sub-Saharan Africa, especially among children under five years of age and places a huge economic burden on individuals and health systems. While this burden has been assessed previously, few studies have explored how malaria comorbidities affect inpatient costs. This study in a malaria endemic area in Western Kenya, assessed the total treatment costs per malaria episode including comorbidities in children and adults., Methods: Total economic costs of malaria hospitalizations were calculated from a health system and societal perspective. Patient-level data were collected from patients admitted with a malaria diagnosis to a county-level hospital between June 2016 and May 2017. All treatment documented in medical records were included as health system costs. Patient and household costs included direct medical and non-medical expenses, and indirect costs due to productivity losses., Results: Of the 746 patients admitted with a malaria diagnosis, 64% were female and 36% were male. The mean age was 14 years (median 7 years). The mean length of stay was three days. The mean health system cost per patient was Kenyan Shilling (KSh) 4288 (USD 42.0) (95% confidence interval (CI) 95% CI KSh 4046-4531). The total household cost per patient was KSh 1676 (USD 16.4) (95% CI KSh 1488-1864) and consisted of: KSh 161 (USD1.6) medical costs; KSh 728 (USD 7.1) non-medical costs; and KSh 787 (USD 7.7) indirect costs. The total societal cost (health system and household costs) per patient was KSh 5964 (USD 58.4) (95% CI KSh 5534-6394). Almost a quarter of patients (24%) had a reported comorbidity. The most common malaria comorbidities were chest infections, diarrhoea, and anaemia. The inclusion of comorbidities compared to patients with-out comorbidities led to a 46% increase in societal costs (health system costs increased by 43% and patient and household costs increased by 54%)., Conclusions: The economic burden of malaria is increased by comorbidities which are associated with longer hospital stays and higher medical costs to patients and the health system. Understanding the full economic burden of malaria is critical if future malaria control interventions are to protect access to care, especially by the poor., (© 2021. The Author(s).)

Background: Despite increasing documentation of insecticide resistance in malaria vectors against public health insecticides in sub-Saharan Africa, there is a paucity of information on the potential fitness costs of pyrethroid resistance in malaria vectors, which is important in improving the current resistant management strategies. This study aimed to assess the fitness cost effects of insecticide resistance on the development and survival of immature Anopheles gambiae from western Kenya., Methods: Two-hour old, first instar larvae (L1) were introduced and raised in basins containing soil and rainwater in a semi-field set-up. Each day the number of surviving individuals per larval stage was counted and their stage of development were recorded until they emerged as adults. The larval life-history trait parameters measured include mean larval development time, daily survival and pupal emergence. Pyrethroid-resistant colony of An. gambiae sensu stricto and susceptible colony originating from the same site and with the same genetic background were used. Kisumu laboratory susceptible colony was used as a reference., Results: The resistant colony had a significantly longer larval development time through the developmental stages than the susceptible colony. The resistant colony took an average of 2 days longer to develop from first instar (L1) to fourth instar (L4) (8.8 ± 0.2 days) compared to the susceptible colony (6.6 ± 0.2 days). The development time from first instar to pupa formation was significantly longer by 3 days in the resistant colony (10.28 ± 0.3 days) than in susceptible colony (7.5 ± 0.2 days). The time from egg hatching to adult emergence was significantly longer for the resistant colony (12.1 ± 0.3 days) than the susceptible colony (9.6 ± 0.2 days). The pupation rate (80%; 95% (CI: 77.5-83.6) vs 83.5%; 95% (CI: 80.6-86.3)) and adult emergence rate (86.3% vs 92.8%) did not differ between the resistant and susceptible colonies, respectively. The sex ratio of the females to males for the resistant (1:1.2) and susceptible colonies (1:1.07) was significantly different., Conclusion: The study showed that pyrethroid resistance in An. gambiae had a fitness cost on their pre-imaginal development time and survival. Insecticide resistance delayed the development and reduced the survivorship of An. gambiae larvae. The study findings are important in understanding the fitness cost of insecticide resistance vectors that could contribute to shaping resistant management strategies.

Background: Despite extensive irrigation development in Ethiopia, limited studies assessed the impact of irrigation on malaria vector mosquito composition, abundance and seasonality. This study aimed to evaluate the impact of sugarcane irrigation on species composition, abundance and seasonality of malaria vectors., Methods: Adult Anopheles mosquitoes were collected using CDC light traps from three irrigated and three non-irrigated clusters in and around Arjo-Didessa sugarcane irrigation scheme in southwestern Ethiopia. Mosquitoes were surveyed in four seasons: two wet and two dry, in 2018 and 2019. Mosquito species composition, abundance and seasonality were compared between irrigated and non-irrigated clusters. Anopheles mosquitoes were sorted out to species using morphological keys and molecular techniques. Chi square was used to test the relationships between Anopheles species occurrence, and environmental and seasonal parameters., Results: Overall, 2108 female Anopheles mosquitoes comprising of six species were collected. Of these, 92.7% (n = 1954) were from irrigated clusters and 7.3% (n = 154) from the non-irrigated. The Anopheles gambiae complex was the most abundant (67.3%) followed by Anopheles coustani complex (25.3%) and Anopheles pharoensis (5.7%). PCR-based identification revealed that 74.7% (n = 168) of the An. gambiae complex were Anopheles arabiensis and 22.7% (n = 51) Anopheles amharicus. The density of An. gambiae complex (both indoor and outdoor) was higher in irrigated than non-irrigated clusters. The overall anopheline mosquito abundance during the wet seasons (87.2%; n = 1837) was higher than the dry seasons (12.8%; n = 271)., Conclusion: The ongoing sugarcane irrigation activities in Arjo-Didessa created conditions suitable for malaria transmitting Anopheles species diversity and abundance. This could drive malaria transmission in Arjo-Didessa and its environs in both dry and wet seasons. Currently practiced malaria vector interventions need to be strengthened by including larval source management to reduce vector abundance in the irrigated areas.

Background: Surveillance of outdoor host-seeking malaria vectors is crucial to monitor changes in vector biting behaviour and evaluate the impact of vector control interventions. Human landing catch (HLC) has been considered the most reliable and gold standard surveillance method to estimate human-biting rates. However, it is labour-intensive, and its use is facing an increasing ethical concern due to potential risk of exposure to infectious mosquito bites. Thus, alternative methods are required. This study was conducted to evaluate the performance of human-odour-baited CDC light trap (HBLT) and human-baited double net trap (HDNT) for outdoor host-seeking malaria vector surveillance in Kenya and Ethiopia., Methods: The sampling efficiency of HBLT and HDNT was compared with CDC light trap and HLC using Latin Square Design in Ahero and Iguhu sites, western Kenya and Bulbul site, southwestern Ethiopia between November 2015 and December 2018. The differences in Anopheles mosquito density among the trapping methods were compared using generalized linear model., Results: Overall, 16,963 female Anopheles mosquitoes comprising Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l., Anopheles pharoensis, Anopheles coustani and Anopheles squamosus were collected. PCR results (n = 552) showed that Anopheles arabiensis was the only member of An. gambiae s.l. in Ahero and Bulbul, while 15.7% An. arabiensis and 84.3% An. gambiae sensu stricto (s.s.) constituted An. gambiae s.l. in Iguhu. In Ahero, HBLT captured 2.23 times as many An. arabiensis and 2.11 times as many An. funestus as CDC light trap. In the same site, HDNT yielded 3.43 times more An. arabiensis and 3.24 times more An. funestus than HBLT. In Iguhu, the density of Anopheles mosquitoes did not vary between the traps (p > 0.05). In Bulbul, HBLT caught 2.19 times as many An. arabiensis as CDC light trap, while HDNT caught 6.53 times as many An. arabiensis as CDC light trap. The mean density of An. arabiensis did not vary between HDNT and HLC (p = 0.098), whereas the HLC yielded significantly higher density of An. arabiensis compared to HBLT and CDC light trap. There was a significant density-independent positive correlation between HDNT and HLC (r = 0.69)., Conclusion: This study revealed that both HBLT and HDNT caught higher density of malaria vectors than conventional CDC light trap. Moreover, HDNT yielded a similar vector density as HLC, suggesting that it could be an alternative tool to HLC for outdoor host-seeking malaria vector surveillance.

Background: G6PD enzyme deficiency is a common enzymatic X-linked disorder. Deficiency of the G6PD enzyme can cause free radical-mediated oxidative damage to red blood cells, leading to premature haemolysis. Treatment of Plasmodium vivax malaria with primaquine poses a potential risk of mild to severe acute haemolytic anaemia in G6PD deficient people. In this study, the prevalence and distribution of G6PD mutations were investigated across broad areas of Ethiopia, and tested the association between G6PD genotype and phenotype with the goal to provide additional information relevant to the use of primaquine in malaria treatment., Methods: This study examined G6PD mutations in exons 3-11 for 344 febrile patient samples collected from seven sites across Ethiopia. In addition, the G6PD enzyme level of 400 febrile patient samples from Southwestern Ethiopia was determined by the CareStart™ biosensor. The association between G6PD phenotype and genotype was examined by Fisher exact test on a subset of 184 samples., Results: Mutations were observed at three positions of the G6PD gene. The most common G6PD mutation across all sites was A376G, which was detected in 21 of 344 (6.1%) febrile patients. Thirteen of them were homozygous and eight were heterozygous for this mutation. The G267+119C/T mutation was found in 4 (1.2%) individuals in South Ethiopia, but absent in other sites. The G1116A mutation was also found in 4 (1.2%) individuals from East and South Ethiopia. For the 400 samples in the south, 17 (4.25%) were shown to be G6PD-deficient. G6PD enzyme level was not significantly different by age or gender. Among a subset of 202 febrile patients who were diagnosed with malaria, 11 (5.45%) were G6PD-deficient. These 11 infected samples were diagnosed with Plasmodium vivax by microscopy. Parasitaemia was not significantly different between the G6PD-deficient and G6PD-normal infections., Conclusions: The prevalence of G6PD deficiency is modest among febrile patients in Ethiopia. G6PD deficiency testing is thus recommended before administrating primaquine for radical cure of P. vivax infected patients. The present study did not indicate a significant association between G6PD gene mutations and enzyme levels.

Background: The impact of large dams on malaria has received widespread attention. However, understanding how dam topography and transmission endemicity influence malaria incidences is limited., Methods: Data from the European Commission's Joint Research Center and Shuttle Radar Topography Mission were used to determine reservoir perimeters and shoreline slope of African dams. Georeferenced data from the Malaria Atlas Project (MAP) were used to estimate malaria incidence rates in communities near reservoir shorelines. Population data from the WorldPop database were used to estimate the population at risk of malaria around dams in stable and unstable areas., Results: The data showed that people living near (< 5 km) large dams in sub-Saharan Africa grew from 14.4 million in 2000 to 18.7 million in 2015. Overall, across sub-Saharan Africa between 0.7 and 1.6 million malaria cases per year are attributable to large dams. Whilst annual malaria incidence declined markedly in both stable and unstable areas between 2000 and 2015, the malaria impact of dams appeared to increase in unstable areas, but decreased in stable areas. Shoreline slope was found to be the most important malaria risk factor in dam-affected geographies, explaining 41-82% (P < 0.001) of the variation in malaria incidence around reservoirs., Conclusion: Gentler, more gradual shoreline slopes were associated with much greater malaria risk. Dam-related environmental variables such as dam topography and shoreline slopes are an important factor that should be considered in efforts to predict and control malaria around dams.

Background: Understanding the complex heterogeneity of risk factors that can contribute to an increased risk of malaria at the individual and household level will enable more effective use of control measures. The objective of this study was to understand individual and household factors that influence clinical malaria infection among individuals in the highlands of Western Kenya., Methods: This was a matched case-control study undertaken in the Western Kenya highlands. Clinical malaria cases were recruited from health facilities and matched to asymptomatic individuals from the community who served as controls. Each participant was screened for malaria using microscopy. Follow-up surveys were conducted with individual households to collect socio-economic data. The houses were also checked using pyrethrum spray catches to collect mosquitoes., Results: A total of 302 malaria cases were matched to 604 controls during the surveillance period. Mosquito densities were similar in the houses of both groups. A greater percentage of people in the control group (64.6%) used insecticide-treated bed nets (ITNs) compared to the families of malaria cases (48.3%). Use of ITNs was associated with lower level of clinical malaria episodes (odds ratio 0.51; 95% CI 0.39-0.68; P < 0.0001). Low income was the most important factor associated with higher malaria infections (adj. OR 4.70). Use of malaria prophylaxis was the most important factor associated with less malaria infections (adj OR 0.36). Mother's (not fathers) employment status (adj OR 0.48) and education level (adj OR 0.54) was important malaria risk factor. Houses with open eaves was an important malaria risk factor (adj OR 1.72)., Conclusion: The identification of risk factors for clinical malaria infection provides information on the local malaria epidemiology and has the potential to lead to a more effective and targeted use of malaria control measures. These risk factors could be used to assess why some individuals acquire clinical malaria whilst others do not and to inform how intervention could be scaled at the local level.

Following publication of the original article [1], it came to the authors' attention that unfortunately the last name of one of the authors is spelled incorrectly in the published article.

Background: The trend analysis of malaria data from health facilities is useful for understanding dynamics of malaria epidemiology and inform for future malaria control planning. Changes in clinical malaria characteristics, like gender and age distribution are good indicators of declining malaria transmission. This study was conducted to determine the malaria trend at Arjo-Didessa sugar development site and its vicinity, southwest Ethiopia, from 2008 to 2017., Methods: Monthly malaria confirmed case data from 2008 to 2017 was extracted from 11 health facilities based on clinical registers at Arjo sugar development site and its vicinity, southwest Ethiopia. Both positivity rate and malaria incidence rate were calculated. Changes in malaria parasite species and seasonality were analysed; age structure and gender distribution were compared between different study periods. Trend in malaria incidence and climatic impact were analysed and past LLIN and IRS campaigns were used as dynamics modifier., Results: Over a period of 10 years, 54,020 blood film were collected for malaria diagnosis in the health facilities at the area, of which 18,049 (33.4%) were confirmed malaria cases by both microscopically and RDT. Plasmodium falciparum, Plasmodium vivax, and mixed infection (P. falciparum and P. vivax) accounted for 8660 (48%), 7649 (42.4%), and 1740 (9.6%) of the malaria cases, respectively. The study also revealed that P. vivax was the predominant over P. falciparum for 4 years (2010, 2014, 2015 and 2016). There was a remarkable reduction of overall malaria infection during the 10 years. Malaria has been reported in all age groups, but age distribution showed that vast majority of cases were adults age 15 years and above 13,305 (73.7%). In all age groups, males were more significantly affected than females (χ 2  = 133.0, df = 2, P < 0.0001). Moreover, malaria positivity rate showed a strong seasonality (χ 2  = 777.55, df = 11, P < 0.0001). However, malaria cases were reported in all seasons across 10 years in the study area., Conclusion: In general, malaria positivity showed a declining trend over 10 years period in the area. However, current prevalence shows it is public health burden and needs attention for further intensification of interventions. In the study area, both P. falciparum and P. vivax co-exist and P. vivax is more prevalent than P. falciparum in almost half of the years. Therefore, malaria interventions should be strengthened in the study area.

Background: Physiological characteristics (age and blood feeding status) of malaria vectors can influence their susceptibility to the current vector control tools that target their feeding and resting behaviour. To ensure the sustainability of the current and future vector control tools an understanding of how physiological characteristics may contribute to insecticide tolerance in the field is fundamental for shaping resistance management strategies and vector control tools. The aim of this study was to determine whether blood meal and mosquito age affect pyrethroid tolerance in field-collected Anopheles gambiae from western Kenya., Methods: Wild mosquito larvae were reared to adulthood alongside the pyrethroid-susceptible Kisumu strain. Adult females from the two populations were monitored for deltamethrin resistance when they were young at 2-5 days old and older 14-16 days old and whether fed or unfed for each age group. Metabolic assays were also performed to determine the level of detoxification enzymes. Mosquito specimens were further identified to species level using the polymerase chain reaction (PCR) method., Results: Anopheles gambiae sensu stricto was the predominant species comprising 96% of specimens and 2.75% Anopheles arabiensis. Bioassay results showed reduced pyrethroid induced mortality with younger mosquitoes compared to older ones (mortality rates 83% vs. 98%), independently of their feeding status. Reduced mortality was recorded with younger females of which were fed compared to their unfed counterparts of the same age with a mortality rate of 35.5% vs. 83%. Older blood-fed females showed reduced susceptibility after exposure when compared to unfed females of the same age (mortality rates 86% vs. 98%). For the Kisumu susceptible population, mortality was straight 100% regardless of age and blood feeding status. Blood feeding status and mosquito age had an effect on enzyme levels in both populations, with blood fed individuals showing higher enzyme elevations compared to their unfed counterparts (P < 0.0001). The interaction between mosquito age and blood fed status had significant effect on mosquito mortality., Conclusion: The results showed that mosquito age and blood feeding status confers increased tolerance to insecticides as blood feeding may be playing an important role in the toxicity of deltamethrin, allowing mosquitoes to rest on insecticide-treated materials despite treatment. These may have implications for the sustained efficacy of indoor residual spraying and insecticide-treated nets based control programmes that target indoor resting female mosquitoes of various gonotrophic status.

Background: Sub-microscopic and asymptomatic infections could be bottlenecks to malaria elimination efforts in Ethiopia. This study determined the prevalence of malaria, and individual and household-level factors associated with Plasmodium infections obtained following detection of index cases in health facilities in Jimma Zone., Methods: Index malaria cases were passively detected and tracked in health facilities from June to November 2016. Moreover, family members of the index houses and neighbours located within approximately 200 m from the index houses were also screened for malaria., Results: A total of 39 index cases initiated the reactive case detection of 726 individuals in 116 households. Overall, the prevalence of malaria using microscopy and PCR was 4.0% and 8.96%, respectively. Seventeen (43.6%) of the index cases were from Doyo Yaya kebele, where parasite prevalence was higher. The majority of the malaria cases (90.74%) were asymptomatic. Fever (AOR = 12.68, 95% CI 3.34-48.18) and history of malaria in the preceding 1 year (AOR = 3.62, 95% CI 1.77-7.38) were significant individual-level factors associated with detection of Plasmodium infection. Moreover, living in index house (AOR = 2.22, 95% CI 1.16-4.27), house with eave (AOR = 2.28, 95% CI 1.14-4.55), area of residence (AOR = 6.81, 95% CI 2.49-18.63) and family size (AOR = 3.35, 95% CI 1.53-7.33) were main household-level predictors for residual malaria transmission., Conclusion: The number of index cases per kebele may enhance RACD efforts to detect additional malaria cases in low transmission settings. Asymptomatic and sub-microscopic infections were high in the study area, which need new or improved surveillance tools for malaria elimination efforts.

Background: Parasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity. Traditional methods often underestimate the frequency and diversity of multiclonal infections due to technical sensitivity and specificity. Next-generation sequencing techniques provide a novel opportunity to study complexity of parasite populations and molecular epidemiology., Methods: Symptomatic and asymptomatic Plasmodium vivax samples were collected from health centres/hospitals and schools, respectively, from 2011 to 2015 in Ethiopia. Similarly, both symptomatic and asymptomatic Plasmodium falciparum samples were collected, respectively, from hospitals and schools in 2005 and 2015 in Kenya. Finger-pricked blood samples were collected and dried on filter paper. Long amplicon (> 400 bp) deep sequencing of merozoite surface protein 1 (msp1) gene was conducted to determine multiplicity and molecular epidemiology of P. vivax and P. falciparum infections. The results were compared with those based on short amplicon (117 bp) deep sequencing., Results: A total of 139 P. vivax and 222 P. falciparum samples were pyro-sequenced for pvmsp1 and pfmsp1, yielding a total of 21 P. vivax and 99 P. falciparum predominant haplotypes. The average MOI for P. vivax and P. falciparum were 2.16 and 2.68, respectively, which were significantly higher than that of microsatellite markers and short amplicon (117 bp) deep sequencing. Multiclonal infections were detected in 62.2% of the samples for P. vivax and 74.8% of the samples for P. falciparum. Four out of the five subjects with recurrent P. vivax malaria were found to be a relapse 44-65 days after clearance of parasites. No difference was observed in MOI among P. vivax patients of different symptoms, ages and genders. Similar patterns were also observed in P. falciparum except for one study site in Kenyan lowland areas with significantly higher MOI., Conclusions: The study used a novel method to evaluate Plasmodium MOI and molecular epidemiological patterns by long amplicon ultra-deep sequencing. The complexity of infections were similar among age groups, symptoms, genders, transmission settings (spatial heterogeneity), as well as over years (pre- vs. post-scale-up interventions). This study demonstrated that long amplicon deep sequencing is a useful tool to investigate multiplicity and molecular epidemiology of Plasmodium parasite infections.

Multiplicity of infection (MOI), also termed complexity of infection (COI), is defined as the number of genetically distinct parasite strains co-infecting a single host, which is an important indicator of malaria epidemiology. PCR-based genotyping often underestimates MOI. Next generation sequencing technologies provide much more accurate and genome-wide characterization of polyclonal infections. However, complete haplotype characterization of multiclonal infections remains a challenge due to PCR artifacts and sequencing errors, and requires efficient computational tools. In this review, the advantages and limitations of current molecular approaches to determine multiplicity of malaria parasite infection are discussed.

Background: Identifying asymptomatic reservoirs of malaria parasites using index cases as entry points into the community is potentially a cost-effective way towards achieving malaria elimination., Methods: Within 1 year, 1430 confirmed malaria cases were identified in Marani hospital, western Kenya. Fifty cases were followed up, and 108 index case household members and 612 neighbours within a 100 m radius were screened. As controls, samples were collected from 510 individuals matched with index cases and located at a distance of ≥ 500 m from them. Infections were diagnosed by microscopy and PCR while simultaneously collecting malaria vectors indoor using pyrethrum spray catches., Results: In the index case and neighbour households, the prevalence of infection was approximately twice as high as in control households (by PCR: index cases households: 28.9%, neighbours: 25.3%, matched controls: 12.9%). In index case households, the indoor vector density (Anopheles gambiae and Anopheles funestus) was higher (0.46 female/house/night) than in neighbouring (0.31 f/h/n) and control houses (0.29 f/h/n)., Conclusions: Screening index case households and neighbours approximately doubles the chance to detect asymptomatic infections compared to randomly selected households. However, even if all cases were followed up, only a small proportion (˂ 10%) of the asymptomatic reservoir in the population would have been identified. Control programmes need to weigh the increased chance to find cases around index cases vs. the logistical challenges to target this subgroup within the population.

Background: In sub-Saharan Africa, malaria interventions over the last decades have been successful in reducing both mortality and morbidity. In western Kenya however some areas experience contrasting outcomes of the ongoing interventions while the causes for this observation remains not yet clearly known., Methods: The WHO insecticide (deltamethrin) susceptibility test of the common malaria vectors was studied. Multiple surveys on household use and hospital prescriptions of antimalarial drugs from 2003 to 2015 were done. Along with this, cross sectional surveys on their availability in the local drug dispensing outlets were also done in 2015. Monthly precipitations and air temperature data was collected along with systematic review on abundance and composition of common malaria vectors in the study area before and during interventions. The above factors were used to explain the possible causes of contrasting outcome of malaria interventions between the three study sites., Results: Areas with malaria resurgence or sustained high transmission (Kombewa and Marani) showed higher composition of Anopheles funestus sensu lato (s.l.) than the previously abundant Anopheles gambiae sensu stricto (s.s.) and the later had higher composition to an area with a sustained infection decline (Iguhu). Anopheles gambiae s.l. from Kombewa showed highest resistance (50% mortality) upon exposure to WHO deltamethrin discriminating dosage of 0.75% while those from Marani and Iguhu had reduced resistance status (both had a mean mortality of 91%). Sampled An. funestus s.l. from Marani were also highly resistant to deltamethrin as 57% of the exposed vectors survived. An increasing of mean air temperature by 2 °C was noted for Marani and Iguhu from 2013 to 2015 and was accompanied by an increased rainfall at Marani. Community drug use and availability in selling outlets along with prescription in hospitals were not linked to the struggling control of the disease., Conclusions: The malaria vector species composition shift, insecticide resistance and climatic warming were the likely cause of the contrasting outcome of malaria intervention in western Kenya. Surveillance of malaria parasite and vector dynamics along with insecticide resistance and vector biting behaviour monitoring are highly recommended in these areas.

Background: Monitoring and evaluation of entomological, parasitological and clinical data is an important component of malaria control as it is a measure of the success of the interventions. In many studies, clinical data has been used to monitor trends in malaria morbidity and mortality. This study was conducted to demonstrate age dependent prevalence of malaria in the pre- and post-interventions period., Methods: A series of cross-sectional malaria parasitological surveys were conducted in Iguhu, western Kenya. Participants were randomly selected school-aged children between 6 and 13 years. The study was conducted between June 2002-December 2003 and January 2012-February 2015. Sexual and asexual parasite prevalence and densities were determined using microscopy. Age-dependence in parasite infections was compared between 2002-2003 and 2012-2015., Results: Plasmodium falciparum had the highest prevalence of 43.5 and 11.5% in the pre- and post-intervention periods. Plasmodium malariae had a prevalence of 2.3 and 0.2%, while Plasmodium ovale had a prevalence of 0.3 and 0.1% during the pre- and post-intervention period, respectively. There was a 73.7% reduction in prevalence of P. falciparum in the post-intervention compared to the pre-intervention period. Plasmodium falciparum parasite density increased by 71.2% between pre- and post-intervention period from (geometric mean of) 554.4-949.2 parasites/µl. Geometric mean gametocytaemia in Iguhu was higher in the post-intervention period (106.4 parasites/µl), when compared to the pre-intervention period (54.1 parasites/µl). Prevalence and density of P. falciparum showed a lower age-dependency during post-intervention period when compared to pre-intervention period., Conclusion: The study provides evidence for reduction of malaria prevalence following the introduction of LLINs and ACT in western Kenya. Fewer people become infected but the few infected may be more infectious as suggested by higher gametocyte densities. The high parasite densities, which were not dependent on age, observed in the post intervention period imply that a more comprehensive integrated malaria management may be required to sustain the current interventions and hence reduce malaria transmission.

Background: The widespread use of indoor-based malaria vector control interventions has been shown to alter the behaviour of vectors in Africa. There is an increasing concern that such changes could sustain residual transmission. This study was conducted to assess vector species composition, feeding behaviour and their contribution to indoor and outdoor malaria transmission in western Kenya., Methods: Anopheles mosquito collections were carried out from September 2015 to April 2016 in Ahero and Iguhu sites, western Kenya using CDC light traps (indoor and outdoor), pyrethrum spray catches (PSCs) (indoor) and pit shelters (outdoor). Species within Anopheles gambiae s.l. and Anopheles funestus s.l. were identified using polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was used to determine mosquito blood meal sources and sporozoite infections., Results: A total of 10,864 female Anopheles mosquitoes comprising An. gambiae s.l. (71.4%), An. funestus s.l. (12.3%), Anopheles coustani (9.2%) and Anopheles pharoensis (7.1%) were collected. The majority (61.8%) of the anopheline mosquitoes were collected outdoors. PCR result (n = 581) revealed that 98.9% An. arabiensis and 1.1% An. gambiae s.s. constituted An. gambiae s.l. in Ahero while this was 87% An. gambiae s.s. and 13% An. arabiensis in Iguhu. Of the 108 An. funestus s.l. analysed by PCR, 98.1% belonged to An. funestus s.s. and 1.9% to Anopheles leesoni. The human blood index (HBI) and bovine blood index (BBI) of An. arabiensis was 2.5 and 73.1%, respectively. Anopheles gambiae s.s. had HBI and BBI of 50 and 28%, respectively. The HBI and BBI of An. funestus was 60 and 22.3%, respectively. Forage ratio estimate revealed that An. arabiensis preferred to feed on cattle, An. gambiae s.s. showed preference for both human and cattle, while An. funestus preferred human over other hosts. In Ahero, the sporozoite rates for An. arabiensis and An. funestus were 0.16 and 1.8%, respectively, whereas in Iguhu, the sporozoite rates for An. gambiae s.s. and An. funestus were 2.3 and 2.4%, respectively. In Ahero, the estimated indoor and outdoor entomological inoculation rate (EIR) was 108.6 infective bites/person/year (79.0 from An. funestus and 29.6 from An. arabiensis) and 43.5 infective bites/person/year (27.9 from An. arabiensis and 15.6 from An. funestus), respectively. In Iguhu, the estimated indoor and outdoor EIR was 24.5 infective bites/person/year (18.8 from An. gambiae s.s. and 5.7 from An. funestus) and 5.5 infective bites/person/year (all from An. gambiae s.s.), respectively., Conclusion: Anopheles gambiae s.s. showed an increasing tendency to feed on cattle. Anopheles arabiensis was highly zoophagic, whereas An. funestus showed anthropophagic behaviour. While the majority of malaria transmission occurred indoor, the magnitude of outdoor transmission was considerably high. Additional control tools that complement the existing interventions are required to control residual transmission.

Background: Outdoor malaria transmission is becoming an increasingly important problem in malaria control in Africa. Larval control is a promising intervention as it can target both indoor and outdoor biting mosquitoes. However, the currently available biolarvicide formulations have a short effective duration, and consequently larval control incurs a high operational expense due to the requirement for frequent re-treatment of larval habitats. Formulations of biolarvicides with long-lasting effects is highly desired. A recently developed FourStar® slow-release briquet formulation of Bacillus thuringiensis israelensis and Bacillus sphaericus was evaluated to test its efficacy on malaria vectors., Methods: The study evaluated FourStar™ briquets 180-days formulation under semi-natural and natural conditions to test their efficacy in reducing the mosquito population in western Kenya. The semi-natural habitats used the formulation dissolved in rainwater with appropriate concentrations, and second-instar larvae of Anopheles gambiae were introduced and the number of surviving larvae and pupae produced was recorded daily as the outcome. The briquets formulation was then tested in natural habitats for efficacy on pupal productivity reduction in highland and lowland sites in western Kenya. The formulation was finally tested for efficacy in reducing adult mosquito populations in randomized clusters in western Kenya highland., Results: In semi-natural conditions, the FourStar™ briquets 180-days formulation completely inhibited mosquito pupal production in the first 3 months, and then reduced pupal productivity by 87-98% (P < 0.001) 4-6 months after application. In natural habitats, during the first 2 months no pupae were detected from any of the treated habitats in highland sites, and Anopheles spp. pupal density was reduced by 60-90% in the next 3-5 months (P < 0.001). In the lowland site, pupal productivity reduction was 100% in the first 3 months, and 75-90% in the next 4-5 months (P < 0.001). The randomized cluster trial found that the application of the briquets formulation reduced mean densities of indoor-biting mosquitoes by 76-82% (P < 0.001) and by 67-75% (P < 0.001) for outdoor-biting mosquitoes., Conclusion: This study demonstrated that long-lasting biological larviciding was effective in reducing pupal productivity of larval habitats, and reducing indoor and outdoor resting mosquitoes. The study suggests that long-lasting microbial larviciding may be a promising complementary malaria vector control tool and warrants further large-scale evaluation.

Background: The Greater Mekong Subregion is aiming to achieve regional malaria elimination by 2030. Though a shift in malaria parasite species predominance by Plasmodium vivax has been recently documented, the transmission of the two minor Plasmodium species, Plasmodium malariae and Plasmodium ovale spp., is poorly characterized in the region. This study aims to determine the prevalence of these minor species in the China-Myanmar border area and their genetic diversity., Methods: Epidemiology study was conducted during passive case detection in hospitals and clinics in Myanmar and four counties in China along the China-Myanmar border. Cross-sectional surveys were conducted in villages and camps for internally displaced persons to determine the prevalence of malaria infections. Malaria infections were diagnosed initially by microscopy and later in the laboratory using nested PCR for the SSU rRNA genes. Plasmodium malariae and P. ovale infections were confirmed by sequencing the PCR products. The P. ovale subtypes were determined by sequencing the Pocytb, Pocox1 and Pog3p genes. Parasite populations were evaluated by PCR amplification and sequencing of the MSP-1 genes. Antifolate sensitivity was assessed by sequencing the dhfr-ts and dhps genes from the P. malariae and P. ovale isolates., Results: Analysis of 2701 blood samples collected from the China-Myanmar border by nested PCR targeting the parasite SSU rRNA genes identified 561 malaria cases, including 161 Plasmodium falciparum, 327 P. vivax, 66 P. falciparum/P. vivax mixed infections, 4 P. malariae and 3 P. ovale spp. P. vivax and P. falciparum accounted for >60 and ~30% of all malaria cases, respectively. In comparison, the prevalence of P. malariae and P. ovale spp. was very low and only made up ~1% of all PCR-positive cases. Nevertheless, these two species were often misidentified as P. vivax infections or completely missed by microscopy even among symptomatic patients. Phylogenetic analysis of the SSU rRNA, Pocytb, Pocox1 and Pog3p genes confirmed that the three P. ovale spp. isolates belonged to the subtype P. ovale curtisi. Low-level genetic diversity was detected in the MSP-1, dhfr and dhps genes of these minor parasite species, potentially stemming from the low prevalence of these parasites preventing their mixing. Whereas most of the dhfr and dhps positions equivalent to those conferring antifolate resistance in P. falciparum and P. vivax were wild type, a new mutation S113C corresponding to the S108 position in pfdhfr was identified in two P. ovale curtisi isolates., Conclusions: The four human malaria parasite species all occurred sympatrically at the China-Myanmar border. While P. vivax has become the predominant species, the two minor parasite species also occurred at very low prevalence but were often misidentified or missed by conventional microscopy. These minor parasite species displayed low levels of polymorphisms in the msp-1, dhfr and dhps genes.

Background: Internally displaced persons (IDP) represent vulnerable populations whose public health conditions merit special attention. In the China-Myanmar border area, human movement and resettlements of IDP can influence malaria transmission. Comparison of disease incidence and vector densities between IDP camps and surrounding local villages allows for better understanding of current epidemiology and to evaluate the effectiveness of interventions in the region., Methods: Malaria and vector surveillance was conducted in three IDP camps and three local villages neighbouring the camps along the China-Myanmar border in Myanmar. Clinical malaria cases were collected from seven hospitals/clinics from April 2011 to December 2014. Malaria vector population dynamics were monitored using CDC light traps. The use of malaria preventive measures and information on aid agencies and their activities was obtained through questionnaire surveys., Results: Malaria was confirmed in 1832 patients. Of these cases, 85.4 % were Plasmodium vivax and 11.4 % were Plasmodium falciparum malaria. Annual malaria incidence rates were 38.8 and 127.0 cases/1000 person year in IDP camps and local villages, respectively. Older children of 5-14 years had the highest incidence rate in the camps regardless of gender, while male adults had significantly higher incidence rates than females in local villages and females child-bearing age had significantly lower risk to malaria in IDP camps compare to local villages. Seasonal malaria outbreaks were observed both in the IDP camps and in the local villages from May to August 2013. The proportion of P. vivax remained unchanged in local villages but increased by approximately tenfold in IDP camps from 2011 to 2014. Anopheles vector density was tenfold higher in local villages compared to IDP camps (2.0:0.2 females/trap/night). Over 99 % of households in both communities owned bed nets. While long-lasting insecticidal nets accounted for 61 % of nets used in IDPs, nearly all residents of local villages owned regular nets without insecticide-impregnation. There were more active aid agencies in the camps than in local villages., Conclusion: Malaria in IDP camps was significantly lower than the surrounding villages through effective control management. The observation of P. vivax outbreaks in the study area highlights the need for increased control efforts. Expansion of malaria intervention strategies in IDP camps to local surrounding villages is critical to malaria control in the border area.

Background: Despite largely successful control efforts, malaria remains a significant public health problem in Thailand. Based on microscopy, the northwestern province of Tak, once Thailand's highest burden area, is now considered a low-transmission region. However, microscopy is insensitive to detect low-level parasitaemia, causing gross underestimation of parasite prevalence in areas where most infections are subpatent. The objective of this study was to assess the current epidemiology of malaria prevalence using molecular and serological detection methods, and to profile the antibody responses against Plasmodium as it relates to age, seasonal changes and clinical manifestations during infection. Three comprehensive cross-sectional surveys were performed in a sentinel village and from febrile hospital patients, and whole blood samples were collected from infants to elderly adults. Genomic DNA isolated from cellular fraction was screened by quantitative-PCR for the presence of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasma samples were probed on protein microarray to obtain antibody response profiles from the same individuals., Results: Within the studied community, 90.2 % of Plasmodium infections were submicroscopic and asymptomatic, including a large number of mixed-species infections. Amongst febrile patients, mixed-species infections comprised 68 % of positive cases, all of which went misdiagnosed and undertreated. All samples tested showed serological reactivity to Plasmodium antigens. There were significant differences in the rates of antibody acquisition against P. falciparum and P. vivax, and age-related differences in species-specific immunodominance of response. Antibodies against Plasmodium increased along the ten-month study period. Febrile patients had stronger antibody responses than asymptomatic carriers., Conclusions: Despite a great decline in malaria prevalence, transmission is still ongoing at levels undetectable by traditional methods. As current surveillance methods focus on case management, malaria transmission in Thailand will not be interrupted if asymptomatic submicroscopic infections are not detected and treated.

Background: Malaria intervention in Ethiopia has been strengthened significantly in the past decade. The Ethiopian government recently stratified the country based upon annual parasite incidence into malaria free, low, moderate and high transmission strata. Districts with low transmission were targeted for indigenous transmission elimination. Surveillance on malaria disease incidence is needed for planning control and elimination efforts., Methods: Clinical malaria was monitored prospectively in health facilities in Jimma town, Oromia Region, southwestern Ethiopia from July 2014 to June 2015. Seasonal cross-sectional parasite prevalence surveys in local communities were conducted in 2014 and 2015 in eight kebeles. Case report forms were administered to obtain sociodemographic and epidemiological information from patients., Results: A total of 1434 suspected malaria cases were examined from the health facilities and 428 confirmed malaria cases were found. Among them, 327 (76.4 %) cases were Plasmodium vivax, 97 (22.7 %) were Plasmodium falciparum, and 4 (0.9 %) were mixed infection of P. vivax and P. falciparum. The annual malaria incidence rate was 1.7 cases per 1000 people at risk. Parasite prevalence in the community was less than 3 %. Household ownership of insecticide-treated nets (ITNs) was 47.3 % (1173/2479) and ITN usage was 37.9 %. All ITNs were long-lasting insecticidal nets, and repellent use was not found in the study area. Being male and traveling were the significant risk factors for P. falciparum malaria. For P. vivax malaria, risk factors included occupation and history of malaria illness during the preceding 30 days., Conclusion: Epidemiological evidence suggested low clinical malaria incidence and prevalence in Jimma town. More aggressive measures may be needed to further suppress vivax transmission. Strategies should be planned targeting sustained control and elimination.

Background: The growing threat of insecticide resistance in mosquitoes and drug resistance in the Plasmodium parasites increases the importance of ensuring appropriate malaria case management and enabling positive health-seeking behaviour. Treatment-seeking behaviours are poorly characterized in malaria-endemic regions that have been the focus of intensive control and elimination campaigns. This study uses a comprehensive approach to shed light on the determinants of malaria treatment-seeking behaviours from different perspectives., Methods: The authors conducted cross-sectional surveys from 832 households, fifteen health centers, and 135 retailers across three sites in the Emuhaya and Kakamega districts of the western Kenyan highlands. Participants were recruited via random sampling and data were collected with the use of a structured questionnaire about malaria treatment-seeking behaviour. All households, healthcare facilities, and retailers were mapped using a handheld GPS and a GIS algorithm was used to calculate "walk distance" based on the Tobler rule; an estimate of this distance was used to calculate the travel time used in the analyses., Results: Across the three sites, 47.5-78.9% of the residents sought diagnosis and treatment at hospitals, clinics, or dispensaries; 6.3-26.1% of the residents sought malaria care only at pharmaceutical retailers. Overall, 40.3-59.4% of residents reported delaying seeking care for more than 24 h after fever onset. After adjustment, residents who chose to visit a pharmaceutical retail facility rather than a hospital were 121 and 307% more likely to delay seeking medical care after fever onset than those who reported choosing a healthcare facility for treatment. No significant association was found between travel time and delay in seeking care. The surveys of the healthcare facilities indicated an average total cost per patient per visit was 112 KES ($1.40 US) for public facilities and 165 KES ($2.06 US) for private facilities., Conclusion: Understanding the local health behaviours that perpetuate transmission of malaria will help develop targeted preventive measures and educational interventions that can empower the residents with the knowledge needed to combat malaria in a safe and effective manner. Ensuring patient access to health care facilities in countries with high disease burdens has broader implications on measures of equity and on public health prevention methodologies.

Background: Malaria is a great public health burden and Africa suffers the largest share of malaria-attributed deaths. Despite control efforts targeting indoor malaria transmission, such as insecticide-treated bed nets (ITNs) and deployment of indoor residual spraying, transmission of the parasite in western Kenya is still maintained. This study was carried out to determine the impact of ITNs on indoor vector densities and biting behaviour in western Kenya., Methods: Indoor collection of adult mosquitoes was done monthly in six study sites in western Kenya using pyrethrum spray collections from 2012 to 2014. The rotator trap collections were done in July-August in 2013 and May-June in 2014. Mosquitoes were collected every 2 h between 18.00 and 08.00 h. Human behaviour study was conducted via questionnaire surveys. Species within Anopheles gambiae complex was differentiated by PCR and sporozoite infectivity was determined by ELISA. Species distribution was determined and bed net coverage in the study sites was recorded., Results: During the study a total of 5,469 mosquito vectors were collected from both PSC and Rotator traps comprising 3,181 (58.2%) Anopheles gambiae and 2,288 (41.8%) Anopheles funestus. Compared to all the study sites, Rae had the highest density of An. gambiae with a mean of 1.2 (P<0.001) while Kombewa had the highest density of An. funestus with a mean of 1.08 (P<0.001). Marani had the lowest density of vectors with 0.06 An. gambiae and 0.17 An. funestus (P<0.001). Among the 700 PCR confirmed An. gambiae s.l. individuals, An. gambiae s.s. accounted for 49% and An. arabiensis 51%. Over 50% of the study population stayed outdoors between 18.00 and 20.00 and 06.00 and 08.00 which was the time when highest densities of blood fed vectors were collected. Anopheles gambie s.s. was the main malaria parasite vector in the highland sites and An. arabiensis in the lowland sites. Bed net ownership in 2012 averaged 87% across the study sites., Conclusions: This study suggests that mass distribution of ITNs has had a significant impact on vector densities, species distribution and sporozoite rate. However, shift of biting time poses significant threats to the current malaria vector control strategies which heavily rely on indoor controls.

Background: Endemic malaria in Thailand continues to only exist along international borders. This pattern is frequently attributed to importation of malaria from surrounding nations. A microgeographical approach was used to investigate malaria cases in a study village along the Thailand-Myanmar border., Methods: Three mass blood surveys were conducted during the study period (July and December 2011, and May 2012) and were matched to a cohort-based demographic surveillance system. Blood slides and filter papers were taken from each participant. Slides were cross-verified by an expert microscopist and filter papers were analysed using nested PCR. Cases were then mapped to households and analysed using spatial statistics. A risk factor analysis was done using mixed effects logistic regression., Results: In total, 55 Plasmodium vivax and 20 Plasmodium falciparum cases (out of 547 participants) were detected through PCR, compared to six and two (respectively) cases detected by field microscopy. The single largest risk factor for infection was citizenship. Many study participants were ethnic Karen people with no citizenship in either Thailand or Myanmar. This subpopulation had over eight times the odds of malaria infection when compared to Thai citizens. Cases also appeared to cluster near a major drainage system and year-round water source within the study village., Conclusion: This research indicates that many cases of malaria remain undiagnosed in the region. The spatial and demographic clustering of cases in a sub-group of the population indicates either transmission within the Thai village or shared exposure to malaria vectors outside of the village. While it is possible that malaria is imported to Thailand from Myanmar, the existence of undetected infections, coupled with an ecological setting that is conducive to malaria transmission, means that indigenous transmission could also occur on the Thai side of the border. Improved, timely, and active case detection is warranted.

Background: Malaria is a public health problem in parts of Thailand, where Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels., Methods: Three-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals., Results: Of 219 samples from the village, qPCR detected 25 (11.4%) Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others., Conclusion: These findings suggest that parasite prevalence is higher than currently estimated by local authorities based on the standard light microscopy. As transmission levels drop in Thailand, it may be necessary to employ higher throughput and sensitivity methods for parasite detection in the phase of malaria elimination.

Background: Malaria is the most prevalent communicable disease in Ethiopia, with 75% of the country's landmass classified as endemic for malaria. Accurate information on the distribution and clinical prevalence of Plasmodium vivax and Plasmodium falciparum malaria in endemic areas, as well as in Duffy-negative populations, is essential to develop integrated control strategies., Methods: A total of 390 and 416 community and clinical samples, respectively, representing different localities and age groups across Ethiopia were examined. Malaria prevalence was estimated using nested PCR of the 18S rRNA region. Parasite gene copy number was measured by quantitative real-time PCR and compared between symptomatic and asymptomatic samples, as well as between children/adolescents and adults from the local community. An approximately 500-bp segment of the human DARC gene was amplified and sequenced to identify Duffy genotype at the -33rd nucleotide position for all the clinical and community samples., Results: Plasmodium vivax prevalence was higher in the south while P. falciparum was higher in the north. The prevalence of P. vivax and P. falciparum malaria is the highest in children compared to adolescents and adults. Four P. vivax infections were detected among the Duffy-negative samples. Samples from asymptomatic individuals show a significantly lower parasite gene copy number than those from symptomatic infections for P. vivax and P. falciparum., Conclusions: Geographical and age differences influence the distribution of P. vivax and P. falciparum malaria in Ethiopia. These findings offer evidence-based guidelines in targeting malaria control efforts in the country.

Background: Epidemiological characteristics of clinical malaria may differ from asymptomatic infections, thus both cross-sectional parasite screening and longitudinal clinical case surveillance are necessary for malaria transmission monitoring and control., Methods: In order to monitor malaria transmission, surveillance of clinical malaria from two years of active case surveillance in three cohorts of 6,750 individuals, asymptomatic parasitaemia cases of 5,300 individuals and clinical cases in three study areas were carried out in the western Kenyan highlands in 2009 and 2010. Age distribution, seasonality and spatial clustering were analysed., Results: The results revealed a significant difference in the age distribution of clinical cases between passive and active case surveillance, and between clinical case rate and asymptomatic parasite rate. The number of reported cases from health facilities significantly underestimated clinical malaria incidence. The increase in asymptomatic parasite prevalence from low to high transmission seasons was significantly higher for infants (

Background: In African highland areas where endemicity of malaria varies greatly according to altitude and topography, parasitaemia accompanied by fever may not be sufficient to define an episode of clinical malaria in endemic areas. To evaluate the effectiveness of malaria interventions, age-specific case definitions of clinical malaria needs to be determined. Cases of clinical malaria through active case surveillance were quantified in a highland area in Kenya and defined clinical malaria for different age groups., Methods: A cohort of over 1,800 participants from all age groups was selected randomly from over 350 houses in 10 villages stratified by topography and followed for two-and-a-half years. Participants were visited every two weeks and screened for clinical malaria, defined as an individual with malaria-related symptoms (fever [axillary temperature≥37.5°C], chills, severe malaise, headache or vomiting) at the time of examination or 1-2 days prior to the examination in the presence of a Plasmodium falciparum positive blood smear. Individuals in the same cohort were screened for asymptomatic malaria infection during the low and high malaria transmission seasons. Parasite densities and temperature were used to define clinical malaria by age in the population. The proportion of fevers attributable to malaria was calculated using logistic regression models., Results: Incidence of clinical malaria was highest in valley bottom population (5.0% cases per 1,000 population per year) compared to mid-hill (2.2% cases per 1,000 population per year) and up-hill (1.1% cases per 1,000 population per year) populations. The optimum cut-off parasite densities through the determination of the sensitivity and specificity showed that in children less than five years of age, 500 parasites per μl of blood could be used to define the malaria attributable fever cases for this age group. In children between the ages of 5-14, a parasite density of 1,000 parasites per μl of blood could be used to define the malaria attributable fever cases. For individuals older than 14 years, the cut-off parasite density was 3,000 parasites per μl of blood., Conclusion: Clinical malaria case definitions are affected by age and endemicity, which needs to be taken into consideration during evaluation of interventions.

Background: Mass distribution of insecticide-treated nets (ITNs) is a cost-effective way to achieve universal coverage, but maintaining this coverage is more difficult. In addition to commonly used indicators, evaluation of universal coverage should include coverage of effective nets and changes in coverage over time., Methods: Longitudinal and cross-sectional household ITN surveys were carried out from 2010 to 2013 in six locations representing a variety of settings across western Kenya. Five indicators were used to evaluate the current status of universal coverage: 1) ITN ownership--proportion of households that own at least one ITN, 2) access index--ratio of the number of family members over the number of ITNs owned by that household, 3) operational coverage--proportion of the at-risk population potentially covered by ITNs, assuming one ITN for every two people, 4) effective coverage--population coverage of effective ITNs, and 5) usage--proportion of the population that used ITNs the previous night., Results: ITN ownership and operational coverage increased substantially from 2010 to 2013, but this increase was mostly due to the 2011 mass distribution campaign. In 2013, household ITN ownership was on average 84.4% (95% CI [78.4, 90.5]) across the six study areas, and operational coverage was 83.2% (95% CI [72.5, 93.8]). The ITN access rate was 59.1% (95% CI [56.6, 61.7]), and 40.8% (95% CI [38.3, 43.4]) of the people at risk needed more nets to achieve universal coverage. About 88.5% (95% CI [86.1, 90.9]) of the ITNs were below three years old and 16.5% (95% CI [12.1, 20.9]) of the ITNs had hole(s). The estimated effective long-lasting insecticide-treated net (LLIN) coverage was 70.5% (95 CI [58.7, 82.3]). Approximately 18.4% (95% CI [15.5, 21.4]) of the ITNs were shared by more than three persons, and the population ITN usage rate was about 75-87%. The reason for not using ITNs was almost exclusively "net not available"., Conclusion: Current methods of delivering ITNs, i.e., one mass campaign every five years and regular distribution of ITNs from health center can barely maintain the current effective coverage. Inaccessibility and loss of physical integrity of ITNs are major hindrances to achieving and maintaining universal coverage.

Background: Nested PCR is considered a sensitive and specific method for detecting malaria parasites and is especially useful in epidemiological surveys. However, the preparation of DNA templates for PCR is often time-consuming and costly., Methods: A simplified PCR method was developed to directly use a small blood filter paper square (2 × 2 mm) as the DNA template after treatment with saponin. This filter paper-based nested PCR method (FP-PCR) was compared to microscopy and standard nested PCR with DNA extracted by using a Qiagen DNA mini kit from filter paper blood spots of 204 febrile cases. The FP-PCR technique was further applied to evaluate malaria infections in 1,708 participants from cross-sectional epidemiological surveys conducted in Myanmar and Thailand., Results: The FP-PCR method had a detection limit of ~0.2 parasites/μL blood, estimated using cultured Plasmodium falciparum parasites. With 204 field samples, the sensitivity of the FP-PCR method was comparable to that of the standard nested PCR method, which was significantly higher than that of microscopy. Application of the FP-PCR method in large cross-sectional studies conducted in Myanmar and Thailand detected 1.9% (12/638) and 6.2% (66/1,070) asymptomatic Plasmodium infections, respectively, as compared to the detection rates of 1.3% (8/638) and 0.04% (4/1,070) by microscopy., Conclusion: This FP-PCR method was much more sensitive than microscopy in detecting Plasmodium infections. It drastically increased the detection sensitivity of asymptomatic infections in cross-sectional surveys conducted in Thailand and Myanmar, suggesting that this FP-PCR method has a potential for future applications in malaria epidemiology studies.