Concerted effort to control malaria has had a substantial impact on the transmission of the disease in the past two decades. In areas where reduced malaria transmission is being sustained through insecticide-based vector control interventions, primarily long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), non-insecticidal complementary tools will likely be needed to push towards malaria elimination. Once interruption in local disease transmission is achieved, insecticide-based measures can be scaled down gradually and eventually phased out, saving on costs of sustaining control programs and mitigating any unintended negative health and environmental impacts posed by insecticides. These non-insecticidal methods could eventually replace insecticidal methods of vector control.House screening, a non-insecticidal method, has a long history in malaria control, but is still not widely adopted in sub-Saharan Africa. This study aims to add to the evidence base for this intervention in low transmission settings by assessing the efficacy, impact, and feasibility of house screening in areas where LLINs are conventionally used for malaria control.A two-armed, household randomized clinical trial will be conducted in Mozambique, Zambia, and Zimbabwe to evaluate whether combined the use of house screens and LLINs affords better protection against clinical malaria in children between 6 months and 13 years compared to the sole use of LLINs. Eight hundred households will be enrolled in each study area, where 400 households will be randomly assigned the intervention, house screening, and LLINs while the control households will be provided with LLINs only. Clinical malaria incidence will be estimated by actively following up one child from each household for 6 months over the malaria transmission season. Cross-sectional parasite prevalence will be estimated by testing all participating children for malaria parasites at the beginning and end of each transmission season using rapid diagnostic tests.CDC light traps and pyrethrum spray catches (PSC) will be used to sample adult mosquitoes and evaluate the impact of house screening on indoor mosquito density, species distribution, and sporozoite rates.This study will contribute epidemiological data on the impact of house screening on malaria transmission and assess the feasibility of its implementation on a programmatic scale.ClinicalTrials.gov PACTR202008524310568. Registered on August 11, 2020.The online version contains supplementary material available at 10.1186/s13063-021-05768-7. Note: the numbers in curly brackets in this protocol refer to SPIRIT checklist item numbers. The order of the items has been modified to group similar items  strategy to sustainably control, and ultimately, eliminate malaria  and espeMost malaria transmission in sub-Saharan Africa still occurs indoors despite increased vector control efforts with intra-domiciliary measures, LLINs, and IRS , with reHistorically, house screening studies have demonstrated a reduction in mosquito numbers indoors and subsequently reduced the risk of malaria transmission , 22. HowDespite its potential, house screening is yet to be embedded into the malaria control policy as a vector control tool. This is because with the advent of DDT in the 1950s, malaria control shifted focus to insecticide-based interventions and as a result, limited evidence on the epidemiological impact of environmental management, including house screening, was generated .icipe) are conducting a multi-country study in southern Africa on the additional impact of house screening on malaria transmission and clinical disease outcomes to support the formulation of policy guidelines around house improvement for malaria control. This study is being implemented by the Ministries of Health in Mozambique, Zambia, and Zimbabwe, through their respective National Malaria Control Programmes (NMCP) with technical assistance from WHO-AFRO and icipe.To date, only two randomized controlled trials have been conducted to evaluate the impact of house screening against malaria transmission , 26. AnoHouse screening presents a pragmatic tool for malaria control as housing conditions are improving in sub-Saharan Africa, and malaria control programs can tap into this opportunity to develop housing that protects against malaria transmission . In addiThe overall aim of the study is to assess the efficacy, impact, and feasibility of house screening as an additional anti-malaria intervention in areas where LLINs are conventionally used for malaria control in Zambia, Zimbabwe, and Mozambique.To evaluate whether house screening of all mosquito entry points (in semi-modern houses) in addition to LLIN use provides greater protection against clinical malaria in children between 6 months and 13 years than the sole use of LLINs.To evaluate the impact of house screening and LLIN use on malaria parasite prevalence in children (6 months–13 years) in the intervention compared to the control cohort of the study.To evaluate the impact of house screening and LLINs on mosquito host-seeking and indoor resting densities.To evaluate the impact of house screening and LLINs on the entomological inoculation rate.To assess the incremental costs of house screening as a supplementary mosquito abatement tool.To explore the acceptability of house screening as a malaria control tool in the community.This study is a multicounty, two-armed household randomized controlled trial using a generalized randomized block design, with the village as the block. Prior to the selection of the study households in each of the three study locations in the three countries, a baseline household census will be conducted in each village in the target areas. A database of study households that meet the eligibility criteria will then be generated.During the household census, household access to LLINs will be assessed to ensure that there is an LLIN for every sleeping place in the enumerated household, assuming two people use a single net. House type and potential mosquito entry points for each enumerated household will also be recorded. If the household does not have an LLIN for every sleeping space, this will be issued during the baseline household socio-demographic survey that will be conducted after the household census. The socio-demographic survey will be conducted in 800 randomly selected study households. After the baseline socio-demographic survey, one child between 6 months and 13 years will be randomly selected from the 800 study households to be included in the study cohort. This study cohort will then be screened for malaria parasites  at the beginning and end of the transmission season. Sentinel households, 120, will be selected from the enrolled study households for an entomological sampling of adult mosquitoes. Exposure to indoor mosquito bites will be estimated by routine surveillance of the sentinel households using CDC light traps and while indoor mosquito resting densities will be estimated using Pyrethrum Spray Catch (PSC) technique. Three months prior to the start of the transmission season (January-May), 400 enrolled households will be randomized to the intervention arm and all mosquito entry points  screened.The remaining 400 households will be assigned to the control (no-screening) arm. Households lost to follow-up will be replaced with non-randomized households enumerated during the household census.As malaria risk may vary between villages, the number of intervention and control households per village will be balanced. A maximum of 10% of all households per village will be enrolled for house screening to avoid the potential diversion of mosquitoes from screened to non-screened households. A similar number of households will be assigned to the control arm in the same village so that the number of intervention and control households will be evenly distributed in each village across the study sites.The main study outcome will be clinical malaria incidence monitored in the study children cohort by actively following each child enrolled in the study (active case detection-ACD), measuring their body temperature every fortnight for a period of 3–6 months during the high malaria transmission season (January to May) for 2 subsequent transmission seasons of the study. Children presenting with a fever above 37°C or reporting febrile illness in the past week will be tested for malaria using an RDT. The secondary outcome will be parasite prevalence assessed by screening study children at the beginning and end of each transmission season for the 2 consecutive transmission seasons of the study. Vector biting and resting densities indoors will be monitored throughout the study period using CDC light traps and PSC techniques in intervention and control sentinel houses. Acceptability of the proposed intervention, cost of house screening vis a vis LLINs, and the feasibility of scale-up to the community will be assessed using focus group discussions (FGDs) and key informant interviews (KII) with project stakeholders Fig. .Fig. 1SThe study will be conducted in three southern African countries: Zambia, Zimbabwe, and Mozambique. Study areas were selected in malaria transmission hotspots where the use of LLINs is promoted by the Ministry of Health, but IRS is not routinely implemented. The project countries had similar profiles in terms of malaria incidence/prevalence, primary malaria vectors, parasites transmitting malaria, vector resistance to insecticides, tools employed in malaria control, and transmission season Table .Table 1Plasmodium falciparum in Nyimba is vectored by An. funestus which mediates an entomological inoculation rate (EIR) of 70 infectious bites per unprotected user of LLIN [The house screening trial will be conducted in the Nyimba District located 350 km East of Lusaka in Eastern Province, Zambia. Nyimba has a cool dry winter from May to August and a warm wet season from December to April, which is the main malaria transmission season . Plasmod of LLIN . The ave of LLIN  Fig. 2)PlasmodiuAn. funestus. An. quadriannulatus was found to be the most predominant of the An. gambiae. s.l. complex. Other anophelines present in the area were An. pretoriensis, An. squamous, An. rufipes, An. coustani, and An. pharoensis. The major control measures employed in the area are IRS, LLINs, larval source management (LSM), and prompt case management [The evaluation of house-screening will be explored in Chiredzi District located in Masvingo province in South-Eastern Zimbabwe situated at 21° 02′ 20′′ S 31° 40′ 40′′ E. Chiredzi district receives on average 78 mm of annual rainfall and has an annual average temperature of 26°C . The mainagement  P. falciparum vectors are An. funestus and An. gambiae s.l. An. funestus are recorded to be highly resistant to pyrethroids in Chokwe [The house-screening trial will be conducted in Chokwe District, Gaza Province, specifically within the study area of the Chokwe Health Demographic Surveillance System (CHDDS), at 24° 33′ 37′′ S, 33° 1′ 20′′ E in southern Mozambique. Chokwe district receives on average 72 mm of annual rainfall and has an annual average temperature of 26°C. The major n Chokwe  and as an Chokwe  Houses that have either a tin or tiled roof; mud, stone, or wooden walls and earthen or cemented floors (*semi-modern) with few open spaces for potential mosquito entry and screening; the houses should not be in such a debilitated state that it is impossible to cover up the mosquito entry points.Houses are at least 50 m apart to prevent the diversion of mosquitoes from the treatment to control houses.Houses have at least 1 resident child between 6 months and 13 years of age.The resident child recruited into the study cohort will stay in the study area for the whole study period and will not be allowed to travel out of the study area during the project period.Houses in which the household head/guardian has signed the study informed consent.The households to be enrolled in the study will be assigned the following inclusion criteria:Once all houses (800) to be enrolled in the study have been identified, consent will be sought from the household heads/guardians for a resident child between 6 months and 13 years, to join the study cohort for the malaria incidence  and prevalence (parasite density) surveys.Written and verbal information will be provided to household heads/guardians on the objectives, benefits, and potential risks of the study using a local language with which they are familiar. The household heads/guardians will be advised that participation is voluntary. Only children whose parents have given written consent for their child to be included in the study will be enrolled.The participant child will be selected randomly among those aged 6 months to 13 years from the 800 houses enrolled. During the blood specimen collection for clinical malaria infection and parasite prevalence, assent will be sought from children above 8 years, and if the assent is denied, this child will be excluded from data collection. No distinctions will be made regarding the gender or ethnic group of the household.Consent will also be sought from household heads for their houses to be used as sentinel stations for the sampling of adult mosquitoes using CDC light traps and PSC techniques.Any child testing positive for malaria parasites will be treated as per respective national malaria diagnosis and treatment guidelines .On the actual day of blood specimen collection, the household heads/guardians will be presented with another consent form before a blood specimen is collected from the study children. If consent is obtained, then the field workers will collect the data. However, if consent is denied, blood specimens will not be collected from the child.Long-lasting insecticidal nets (LLINs) will be the comparator in this study. This is because LLINs are the currently recommended intervention by the study countries’ NMCPs and by WHO. Universal coverage of LLINs for personal protection is also the gold standard malaria intervention and the only ethical comparator that can be used when testing new malaria control tools as has been demonstrated to be both effective and readily available. Therefore, we will ensure universal access to LLINs and then evaluate the additional benefits of house screening against malaria transmission.In the target areas of all three countries, 400 houses will be screened with non-insecticide PVC-coated fiberglass netting by trained artisans recruited from each study village. The installations are expected to take about 2–3 months to complete. House residents will be trained on the care needed to keep the screens intact and avoid activities that could damage the screens. Screened houses will be examined by field assistants during entomological sampling of adult mosquitoes in sentinel households when conducting active case detection of malaria incidence every fortnight during the transmission season and during the cross-sectional parasite density surveys . The integrity of the screens will be recorded during these observations. Other routine procedures to reduce mosquito entry into houses, such as shutting the door whenever an occupant enters or leaves the house between 18.00h and 06.00h, will be emphasized and adherence to these practices monitored passively by the study field team during the above activities. These observations will be recorded in structured forms in order to quantify the frequency of their occurrence over time.Withdrawals may also occur if a study subject has any significant adverse events such increase in upper respiratory tract infections (URTis) in household members in screened households compared to non-screened households.Household heads and residents will be trained on the care needed to keep the screens effective, such as avoiding activities that may poke holes onto the nets or cause damages that will create spaces that would allow mosquito entry into the houses. Other routine procedures that reduce mosquito entry into houses such as closing windows and doors early will be emphasized and adherence to this practice monitored passively by the study team during entomological, clinical, and parasitological data collection as described above. The community members in the study areas will also be continually engaged on the benefits of participating in the study and the importance of following the recommended procedures above through community meetings to encourage adherence to malaria and parasitological surveys.As this study aims to assess the additional impact of house screenings to LLINs in preventing malaria transmission, the use of IRS in the study target area will be prohibited.At the end of the study, households in the control arm will be given the option of screening their households at project cost. Also, households in the intervention arm will be given the option of having the screens removed from their houses if they so wish.The primary endpoint will be the incidence of clinical malaria in children between 6 months and 13 years. Clinical malaria will be assessed by active case detection over the main transmission season (January-April). The axillary temperature will be taken every fortnight by CHWs from all enrolled study children and if ≥ 37.5°C or history of fever in the past 48 h is reported, the child will be tested for malaria using a rapid diagnostic test kit (RDT). Blood specimens will also be collected using thick and thin blood slide smears for parasite identification and a dry blood spot (DBS) using a filter paper for later malaria analysis using PCR techniques.The secondary endpoint will be the prevalence of malaria parasitemia in the study children, and the average parasite density in each study arm will be determined. Cross-sectional surveys will be done before (November) and after the main transmission season (May). A finger prick sample will be taken for an RDT and a thick and thin blood smear and DBS prepared for microscopy and molecular analysis in the laboratory.Artemether-lumefantrine (AL) for treatment by a trained health worker following in-country national guidelines [RDT positive children will be provided with a full dose of idelines .The primary entomological endpoint will be the relative indoor mosquito density, expressed as the average number of mosquitoes caught per trap night in each study arm. The secondary endpoint will be the entomological inoculation rate (EIR) incorporating the blood meal source (human biting rate) and the mosquito sporozoite positivity per study arm.N=60) per country will be randomly selected for adult mosquito collections. The household selection will be stratified by village and geographical area so that an equal number of sentinel houses are selected in each village and equally spread over the study area. This stratified randomization by the village will reduce the likelihood of imbalances between study arms. Indoor host-seeking mosquitoes will be collected for 5 days each month using CDC UV light traps from 15 sentinel households. The CDC light trap will be placed next to the sleeping space of the study enrolled child and will be raised 1.5 m from the ground. The light trap will be set up at 7:00 p.m. and mosquitoes collected at 7:00 a.m. the next morning.A total of 30 sentinel houses per study arm (Indoor resting mosquitoes will be collected using pyrethrum spray catches (PSC) in the remaining 15 sentinel households in each arm of the study. The collections will be done inside the room where the child recruited into the study cohort sleeps. Mosquito sampling using PSC will be conducted early in the morning between 06:00 and 08:00 h. All the exit points to the sleeping area will be shut and a white sheet spread on the floor. An insecticide will then be sprayed in the room to knock down mosquitoes which will then be collected after 10 min. This sampling will be conducted 1 day for each month in the fixed sentinel households throughout the study period.Anopheles mosquitoes. A material transfer agreement (MTA) will be signed between the Ministries of Health of the project countries and icipe to allow for a sample of the collected mosquitoes to be transported to icipe, in Nairobi Kenya for quality assurance. In case there is no in-country capacity to process the collected samples, then all collected mosquitoes will be transported to icipe for morphological and molecular analysis.All the mosquito collections will be preserved in silica gel and transported to the respective national health laboratories where they will be stored in a freezer for identification using morphological and molecular methods. Morphological identification will use morphological keys for afro tropical vectors . MoleculTo determine the cost-effectiveness of house screening, the costs associated with house screening will be collected from the implementing organizations (WHO and The NMCPs of the project countries). The per capita costs of house screening will be estimated by dividing the average cost of house screening per house by the household size, and this will be compared to the per capita costs of LLINs. Daily Adjusted Life Years (DALY) will be estimated for each household using standard methods on the basis of the number of malaria cases averted . The cosThe acceptability of house screening as a mosquito abatement tool will be evaluated using qualitative surveys that include key informant interviews and focus group discussions as the secondary endpoint.Hayes and Bennet et al. for incidence rates [The sample size used in this study is based on simulation models described in ce rates . Routinece rates , we estice rates . A totalData collected from all health facilities in Chokwe district, Mozambique, estimated an incidence rate of 0.325 cases per person from January to June. Using the same formula, we estimated that 324 children were required per treatment arm to observe a 35% treatment effect with 80% power at the 5% significance level . A totalHayes and Bennet model for sample size calculation when using means to determine the number of households required to demonstrate a 50% reduction of indoor-entering mosquitoes associated with house screening, with 80% power at the 5% level of significance [An. funestus were caught using CDC light traps per night [House screening is projected to reduce indoor malaria vector collections by at least 50% . We useder night . Using EMosquito data from Chokwe in Mozambique was not available, and there we used the same sample size as that of Zambia of 30 households per treatment arm.Study participants will be recruited by the national project team in consultation with local health workers. The project staff will obtain verbal and written informed consent using ethical review committee (ERC)-approved language that outlines the objectives, benefits, and potential risks of the study and the data and biological samples that will be collected. Household heads will be briefed about the study, provided with an information sheet, and asked for their written consent to have their children involved in the study. Assent will be sought from all children above the age of 10 years. Household heads/guardians who do not want their children to participate in the study will be free to refuse participation. On the survey day, children will be informed about the survey procedures, making it clear that their participation is voluntary and that they might opt-out at any time if they chose to. Children unwilling to participate or whose parents did not give consent will be excluded from the selection procedure, with assent obtained from selected children before samples are collected. Participants’ names will be removed from the final database to ensure anonymity. Children with a positive malaria RDT will be treated for malaria according to national guidelines.Participants will be free to withdraw from the study at any time without giving a reason. In the unlikely event that homeowners require their house to be returned to the pre-intervention state, items added will be removed. No replacements will be made during the surveillance period in either year, but in year 2, children who have withdrawn or are no longer in the study for any reason will be replaced by a child within the age range of 6 months—13 years in the same household. If another child within this age range is not present in that house, then another household with a child within this age range will be sampled in the same village. If a household withdraws consent, no further follow-up will be made in that household. All data collected up until the point of withdrawal will be kept for the per-protocol analysis at the end of the study. If the house was participating in the entomology collections, it will be replaced by a neighboring house of the same type and intervention status.Households were used as the unit of randomization. For allocation of the households, a computer-generated list of random numbers was used. Households were randomly assigned following simple randomization procedures (computerized random numbers) to either the intervention or control group. The allocation was stratified by the village. The randomization sequence was created using Excel 2007  by the Co-PI’s in the project countries.All eligible households will be uploaded into an Ms Excel spreadsheet. The simple randomization formula will then be used to assign the households to the intervention and control groups. The Co-PI’s therefore did not know which household would be assigned to the intervention or control group before the allocation of the interventions.The simple randomization was done by a computer-generated random number list that was prepared by the Co-PI’s in each of the study countries. The randomization was stratified by the village so that there was an equal number of intervention and control households in each village.This study cannot be blinded since house screening will be visible to all including data collectors and the researchers involved. However, staff involved in reading the blood slides will be blinded to the intervention. Statisticians analyzing the primary endpoints of the study will also be blinded to the intervention.Datasets will be unblinded after all primary and secondary analysis have been conducted.Study endpoints will include clinical, entomological, and cost-effectiveness data. Data will be collected electronically using Open Data Kit (ODK) software which was programmed and uploaded on mobile phones. Once households have been enrolled in the study, a household survey adapted from a WHO standardized Malaria Indicator Survey (MIS) questionnaire  and in subsequent malaria transmission seasons at similar time points after the intervention implementation Fig. .Once a household and child are enrolled or randomized, the study team will make every reasonable effort to follow the child for the entire study period.Demo project. A unique ID number will be assigned to each participant. The NCMP/NMEC team will be responsible for overseeing data management. Data will be collected electronically and synced daily through 3G enabled Wi-Fi hotspot with the database at the NMCP/NMEC offices by the study supervisors. Collected data will remain on the tablets until the end of data collection to ensure that it is backed up. The NCMP/NMEC team will also ensure that the questionnaires uploaded to the database are backed up regularly to prevent any loss of data. At the end of the survey, all the data will be in an aggregated master database. The NCMP/NMEC team, WHO, and icipe staff will check the data for inconsistencies and will make necessary corrections.Data will be stored by the NCMP/NMEC at their national offices in a secure database as they are the custodians of the After all, the primary and secondary analysis of the project data has been conducted, the database will be archived for the minimum NMCP/NMEC retention period and disposed of as per ERC guidelines. The ERC will be notified in writing of the intention to dispose of the project data by filling the ERC destruction form outlining the data disposal method and invited to witness the destruction of the hard disc that contains the project database.Data confidentiality will be strictly kept with the data stored on an access-restricted/password-locked database accessible only to authorized personnel, and the data used for analysis will be de-identified so that they cannot be traced back to any individual. To maintain confidentiality, a system of coding will be used to link the name and data.Plasmodium parasites. Blood films will be stained in the field by project staff, transported to the in-country laboratory, and read independently by two microscopists blinded to the identity of the child. Any discrepancy will be resolved by a third senior microscopist. A sample of the blood films will be transported to icipe for quality assurance after signing the material transfer agreement (MTA) between icipe and the Ministries of Health of the Project countries. Children with malaria will be referred for treatment if they have not received any treatment already.During the cross-sectional surveys, clinical surveys will be done on all study children. A finger prick sample will be taken and an RDT used to test for the presence of malaria parasites. Thick and thin blood smears will be taken for later detection and quantification of icipe for quality assurance. Blood slides and mosquito DNA will be stored in a −20°C non-frost-free freezer until DNA extraction. This data will be made available for future studies after all the guidelines, and conditions for secondary data use have been met at both icipe and the project countries.All the mosquito collections will be preserved in Eppendorf tubes with silica gel and transported to the laboratories where they will be identified to species level and examined for sporozoite infection and blood meal analysis. A sample of the mosquito specimen will also be transported to A mixed-effects model will be used to compare clinical malaria incidence rates between the intervention and control arms, to allow for repeated measures within households, villages, and the effect of year. Possible confounders such as the age of child, gender, ethnicity, and season will be included in the models.Differences in the abundance of mosquitoes caught indoors and outdoors will be made between the study aims to evaluate the impact of house screening on mosquito density. Mixed-effects models will be used to estimate differences in the numbers of mosquitoes, adjusting for repeated measures , and other possible covariates such as the child’s age, gender, ethnicity, and rainfall. A Poisson distribution model with a log link function will be used, and a random factor for each data point will be included in the model to adjust for excess variation between data points (over-dispersion). All mean counts per treatment and their 95% confidence intervals (CIs) will be modeled as the exponential of the parameter estimates for models with no intercept included. No additional analysis will be conducted.Interim statistical data analyses will be implemented annually to monitor the progress of the project and a comprehensive data analysis will be implemented at the end of the project.A per-protocol and an intention-to-treat analysis will be conducted.The study team members will have access to records. The authorized representatives of the sponsor, the ethics committee(s), or regulatory bodies may inspect all documents and records required to be maintained by the PI, including but not limited to medical records  for the participants in this study. The clinical study site will permit access to such records. The results of the study will be made publicly available.A regional Project Steering Committee (PSC) of 6 members was established to provide oversight of this multi-country study. The PSC’s main mandate is the monitoring of the participating countries’ progress towards the implementation of the planned project activities as outlined in the protocol. The PSC will also ensure WHO and national patient safety guidelines are followed and adhered to, review reports on adverse events reported in the study, and patient’s rights are upheld.https://www.mrc.ac.uk/documents/pdf/good-clinical-practice-in-clinical-trials/). In particular, the DSMC will concentrate on the progress of the trial, adherence to the protocol, patient safety, and the consideration of new information, and the SSC will formally report to the sponsor (UNEP). The DSMC will determine if additional interim analyses of trial data should be undertaken and assess any additional safety issues that may arise during the study. They will ensure the safety, rights, and well-being of the study participants and will report to the SCC at regular intervals.A DMSC will provide overall supervision of the study and ensure that it is conducted to the standards set out in Good Clinical Practice (Adverse events and serious adverse events will be recorded during both transmission seasons. Sick children will receive referral notes to the nearest health facility, and treatment will be documented.The trial implementation and progress will be reviewed yearly during the project steering committee meetings and way forward agreed upon.In case of protocol amendments, this will be communicated to participants through community meetings and field assistants. This information will also be communicated to the DSMC through the established organogram.The results of the study will be made publicly available through peer-reviewed publications and presentations at international conferences.Progress made against malaria control has stalled in the past 5 years . To get In addition, recent evidence suggests that even with optimal coverage of core interventions, it will not be possible to eliminate malaria in high burden countries . This isHouse improvements have been shown to reduce malaria morbidity by half , and thiAfrica’s economy is experiencing rapid growth and with it is the exponential population growth and rapid urbanization . With thThis study will therefore assess the cost-effectiveness of the intervention while also exploring avenues through which the capital costs of house screening can be subsidized to promote its uptake. Further, a roadmap through which this intervention can be implemented will be explored and advocated by the community and project stakeholders.This study, therefore, aims to provide the much-needed high-quality entomological and epidemiological evidence-based impact of house screening on malaria transmission from 3 well-designed randomized controlled trials from 3 eco-epidemiological settings in southern Africa. This study will provide high-quality data for evidence-based policy adoption of house screening a mosquito abatement tool.This is protocol version 7. The participant recruitment started in February in Zambia 2019 and March 2019 in Mozambique. The recruitment process was completed in April and May 2019 for Zambia and Mozambique, respectively. This participant recruitment started in December 2020 in Zimbabwe and is currently ongoing. The delays in recruitment were due to the emergence of the COVID-19 pandemic.Additional file 1.


Plasmodium spp. More than a century after the discovery of malaria parasites, this disease continues to pose a global public health problem and the pathogenesis of the severe forms of malaria remains incompletely understood. Extracellular vesicles (EVs), including exosomes and microvesicles, have been increasingly researched in the field of malaria in a bid to fill these knowledge gaps. EVs released from Plasmodium-infected red blood cells and other host cells during malaria infection are now believed to play key roles in disease pathogenesis and are suggested as vital components of the biology of Plasmodium spp. Malaria-derived EVs have been identified as potential disease biomarkers and therapeutic tools. In this review, key findings of malaria EV studies over the last 20 years are summarized and critically analysed. Outstanding areas of research into EV biology are identified. Unexplored EV research foci for the future that will contribute to consolidating the potential for EVs as agents in malaria prevention and control are proposed.Malaria is a complex parasitic disease, caused by  Plasmodium falciparum and Plasmodium vivax species, as well as by zoonotic Plasmodium knowlesi infections [P. vivax [Plasmodium falciparum is the most medically important Plasmodium spp. and is known to be highly associated with CM [P. falciparum accounted for over 90% of the globally estimated 400,000 malaria deaths [Malaria is one of the oldest diseases known to man  yet it rfections . Major sfections . CM remafections , 6, but P. vivax –11. Plas with CM , 12. CM P. falciparum and host immune responses that converge on a compromised brain microvasculature [P. vivax is a fairly new concept and remarkably less is known about the pathogenesis of the severe forms of vivax malaria [The pathogenic basis of CM is poorly understood but involves profound adhesive properties of culature , 13–15.  malaria . Towards malaria –19.Since the earliest observations of EVs in human plasma were made , 21, theOver the last two decades, EVs in malaria have been investigated in human field studies as well as in in vivo, in vitro and ex vivo experimental studies. In this review, these studies and how they have advanced the understanding of the importance of host and parasite derived EVs in malaria pathogenesis and biology are examined. an analysis of the evidence supporting malaria EVs as potential biomarkers and therapeutic tools is provided. Until recently, the majority of these studies designated EVs with respect to their biogenesis as microparticles, microvesicles or exosomes without clearly demonstrating their cellular origin. Taking this into consideration, these vesicles will be referred to generically as EVs throughout this review. For studies that adopted the International Society for Extracellular Vesicles (ISEV) nomenclature guidelines with reference to size i.e., small EVs and medium EVs  these wiExosomes and microvesicles are released from viable cells. While both are membrane bound structures, exosomes originate from multivesicular bodies (MVB) and are released as vesicles of 50-100 nm in diameter from the cell following fusion of the MVB and plasma membrane; microvesicles on the other hand, are formed directly from the plasma membrane by outward budding and have a diameter of 100-350 nm though may be larger .In parasitic infections, EVs may be released directly by the parasite, by parasite-infected host cells, and/or by effector cells in response to infection , 31. InvElevated circulating EEV levels were the first to be observed in a clinical study of Malawian children, where there was a sixfold increase in EEVs in patients with CM . This waArguably, there have been discrepancies in reports of the host cell sources of EVs and, therefore, discrepancies in the distribution of plasma EV populations in the different malarial infections across human populations in malaria endemic regions, as well as in murine malaria models. This observed variance, particularly in human field studies of plasma EVs in malaria –36, 42 mA salient observation made by the human studies was that the increased EV titres detected during acute malaria significantly decreased at the time of patient recovery, returning to baseline levels in most cases. Thus, an association between EVs and malaria was established with the consensus that EVs are potential biomarkers for disease, specifically falciparum and vivax malaria. In the case of falciparum malaria, certain published data suggested that plasma EVs may be a biomarker for CM—to be used for disease management and monitoring cure rates—since abnormally high levels of EVs correlated specifically with neurological symptoms of CM, but not other forms of severe malaria or uncomplicated falciparum malaria , 33. In So far, the exception for an association between severe malaria and plasma EV levels has been placental malaria, which occurs in pregnancy. Nevertheless, EVs have been flagged as potential biomarkers for placental malaria due to overexpression of specific EV bound molecules in active placental malaria cases .EVs as biomarkers for severe malaria is an enticing prospect achievable only with intensified clinical and experimental studies to substantiate their potential role. Since EVs are released under physiological and many pathological conditions, and the nature of EVs varies within and among individuals, the cargo of EVs in malaria are likely quite heterogenous and needs to be investigated thoroughly. This is imperative in determining whether or not malaria derived EVs may serve as diagnostic, monitoring, pharmacodynamic, predictive or prognostic markers. An in-depth knowledge of the diversity of EV cargo, as well as an understanding of the differences between biomarkers and their applications, will guide malaria EV research in the right direction towards validating malaria EVs as biomarkers, particularly of cerebral malaria.Nevertheless, the assertion that plasma EVs in malaria may fulfil applications as multifunctional biomarkers has spawned several scientific questions, setting in motion a succession of functional studies centred on the pathogenic role EVs may play in severe malaria. These are presented below.Membrane vesiculation is a cellular process that is largely dependent on the ATP-binding cassette transporter A1 (ABCA1) , 48. LocIn vitro, these EVs had a potent procoagulant activity, the strength of which increased with increasing amounts of EVs [Plasmodium berghei infected mice with CM, localized in the brain microvasculature of recipient mice with CM but not in healthy controls or mice with non-CM [With high plasma EV levels, there is an abundance of circulating tissue factor and phosphatidylserine (PS), both of which are procoagulant molecules that initiate coagulation and are enriched on the EV membrane . In expes of EVs . Coagulos of EVs . EVs in h non-CM . This sth non-CM .P. berghei-induced structures protein 1 and merozoite surface protein-1), is strongly suggestive of selective packaging into ECM-EVs [The cargo EVs carry is equally important as high EV titres in implicating EVs in malaria pathogenesis. Malaria EV cargo has been investigated in experimental malaria models –58 and h ECM-EVs  thereby P. falciparum similarly identified a myriad of host proteins compared with uninfected controls [P. falciparum proteins associated with parasite biology and virulence were identified, of which heat shock 70 kDa protein, Pf-enolase and Pf-actin had the highest frequencies of detection [Proteomic analysis of plasma EVs from out-patients infected with controls . Many ofcontrols . RAB procontrols . 29 P. fetection .Plasmodium infection evidently alters miRNA expression, as distinct plasma EV-miRNA profiles in murine malaria models [P. berghei ECM, the expression of miR-146a and miR-193b was found to be significantly dysregulated compared to EVs in Plasmodium yoelii non-CM and non-infected mice; while miR-146a was markedly upregulated in EVs, miR-193b was downregulated [miRNAs are important regulators of gene expression. Protectively packaged into EVs, miRNAs are now known to play vital roles in protozoan and other infectious diseases , 64. Plaa models  and humaa models  have beeegulated . In humaegulated . These aegulated , adherenegulated . Furtheregulated .Plasmodium (discussed below), suggest a regulatory role for miRNAs in this disease. Moreover, miRNAs have been found to be differentially expressed in the brains of mice with CM and non-CM [Although the role of miRNAs in CM pathogenesis is unknown, the notable degree of altered expression in EVs from mice with CM and human malarial infection, as well as preliminary data from in vitro EV experiments on d non-CM , 66. In d non-CM . In a fid non-CM .P. falciparum malaria either in natural human infections or through extrapolations from murine CM studies, the data from gene knockouts, omics studies and gene association have cumulatively inferred a potential role for plasma derived EVs in malaria. Importantly, a study using P. vivax has rendered the first evidence of a direct pathophysiological mechanism by which plasma EVs in natural malaria infection may contribute to the pathogenesis of this disease [Focusing on  disease .P. vivax infection are comprised mainly of a CD71+ EV (<200 nm) sub-population that carries human and parasite proteins, cautiously suggesting infected reticulocytes might be the main source of EVs in P. vivax infection [P. vivax infection derived EVs are actively taken up by human spleen fibroblasts, followed by translocation of the NF-kB transcription factor from the cytoplasm to the nucleus. This induces a TNF-independent upregulated surface expression of ICAM-1 on spleen fibroblasts, which results in the adherence of mature schizont stage P. vivax-infected reticulocytes to these cells [P. vivax EVs by spleen cells had been previously demonstrated [P. falciparum and P. berghei can infect reticulocytes, the discovery of this EV-directed pathway in CM would be quite compelling and stimulate new research foci.It was noted that plasma EVs from acute human nfection . Based ose cells . The actnstrated . This EVP. vivax may have in common with P. falciparum [P. vivax derived EVs may have a crucial role to play and set forward a paradigm shift in the understanding of P. vivax biology, its interaction with the human host and pathogenesis. Indeed, previously unknown aspects of P. falciparum biology and its communication with host cells have come to light in in vitro EV studies and will be discussed in later sections.Only recently has the potential severity and lethality of vivax malaria been acknowledged , the patlciparum , 69. As lciparum , P. vivaThe biomolecular constitution of EVs and their parent cells are as similar as they are different. This paradox is testament to a host of EV features, including (1) selective EV cargo packaging, (2) differential biodistribution and target cell-specificity, (3) distinct downstream cellular signalling pathways activated by EVs, and (4) specialized functions different from their cells of origin , 70–72. While the scientific evidence for a potential role of EVs in malaria gathered from clinical and experimental cerebral malaria studies is compelling, they are generalizations of plasma EVs from humans and mice. Over the last 2 decades, there have been a number of studies on cell specific EVs derived from the vascular endothelium, platelets, and RBCs. While these studies have generated more questions than answers, they have offered new insights into EV characteristics, and the potential involvement of EVs in the pathology of malaria. Importantly, a critical evaluation of the findings from these studies will direct future research.in vitro, is the shedding of EVs from TNF-activated ECs in thrombotic disorders [Endothelial cell (EC) activation is a core component of the pathophysiological events that occur in falciparum malaria. This is consequent of the distinctive cytoadherence of mature-stage infected RBCs (iRBCs) to the microvascular endothelium of various tissues and organs, including the brain in CM , 77. ECsisorders . Coupledisorders , 80, thiisorders .ex vivo with significantly increased shedding of EVs in the former [in vitro generated EEVs were injected into healthy mice, they were rapidly cleared from circulation and induced lesions in the brain and lungs, similar to those seen in human CM (HCM) and ECM [Patient derived-ECs in CM and non-CM have been shown to respond differently to TNF stimulation e former . Considee former . When in and ECM . These fin vitro inhibition study, where ECs were treated with pantethine before TNF activation [P. berghei did not display any signs of CM, which was attributed to two crucial events: diminished platelet reactivity, and downregulated EV production [The correlation between EEVs and TNF, particularly in HCM , 35, 81,tivation . Pantethtivation . In vivooduction . Anotheroduction . CiticolTo adequately investigate the function of EEVs, specifically in plasma, they must be properly characterized in the midst of EVs from other cell sources. However, EEV characterization is problematic and poses a challenge for downstream EEV studies . In the P. berghei-infected mice [Platelets, like endothelial cells, play an important role in the pathogenesis of CM. Studies have shown platelets to be involved both functionally (such as by releasing potent inflammatory cytokines) and physically (by accumulating in the brain and directly promoting iRBC cytoadherence) reviewed in , 41. Abnted mice . Correspted mice . Piguet ted mice . It is uted mice .in vitro model of HCM by Faille and colleagues [P. falciparum erythrocyte membrane protein 1—PfEMP1, (3) PEVs adhere to the surface of iRBCs and human brain ECs but only translocate across the membrane of the latter, (4) PEVs can transfer PECAM-1 & GPIV to the surface of human brain ECs, and (5) iRBCs are more adherent to ECs in the presence of PEVs [A clear relevance for PEVs has been described in various pathologic conditions, including those caused by infectious diseases . In expelleagues . Among t of PEVs . The resin vitro by Faille and colleagues. Sequestration in vital organs other than the brain is an important element of the pathological picture of CM [These findings have not been directly corroborated in vivo. However, in a clinical study in Cameroon, elevated PEV levels in children correlated with the severity of coma . It may re of CM  and proare of CM  and undere of CM  is the ere of CM , 96. ThuPlasmodium spp. for the most part of their life cycle in the vertebrate host during which the parasite extensively modifies the structure and function of the erythrocyte. Plasmodium spp. synthesize and export numerous proteins that are presented on the RBC surface membrane [Plasmodium-infected RBCs have been found to induce systemic inflammation in ECM [RBCs accommodate membrane . Systemimembrane , 15, 49.membrane , 99. Sinn in ECM .P. berghei-infected RBC derived EVs (pb-iREVs) where they found that pb-iREVs carried distinct parasite cargo and activated macrophages to release TNF in vitro, specifically through a TLR-4/MyD88 dependent pathway [P. berghei-infected RBCs, and the most immunogenic pb-iREVs were detected at the onset of clinical signs, which, in turn, correlated with peak pb-iREV levels. The authors of this study concluded that an accumulation of pb-iREVs with a distinct pro-inflammatory phenotype contribute to the development of severe malaria [P. falciparum demonstrated the active uptake of P. falciparum-infected RBC derived EVs (pf-iREVs) by monocyte-derived macrophages. The pf-iREVs not only had a potent proinflammatory effect on macrophages, they also induced the expression of anti-inflammatory cytokines [Couper and colleagues performed studies on  pathway . These p malaria . In-vitrytokines . Mantel ytokines .P. falciparum DNA cargo from these EVs is released into the cytosol, where it induces the secretion of type 1 interferons (IFN) and other cytokines in the cell via STING (Stimulator of Interferon Genes) activation [P. falciparum DNA [P. berghei infected mice that are otherwise susceptible to CM [Demonstrating with monocytes, iREVs have been implicated as central players in a novel mechanism of immune cell activation by plasmodial molecules. Nucleic acid-containing pf-iREVs are actively taken up by monocytes , 102. Witivation , 104. Tharum DNA . IRF3 trarum DNA . How plaarum DNA , 107 is arum DNA . DNA delP. berghei-iRBCs that likely contain iREVs [There is evidence from a recent in vitro study suggesting that parasite derived EVs are more directly involved in the immunopathogenesis of CM. pf-iREVs activated human monocyte-derived microglia, resulting in an upregulation of TNF-α and simultaneous downregulation of IL-10 . Microglin iREVs . Furtherin iREVs .P. falciparum in vitro studies have comprehensively analysed the protein [P. yoelii infections [P. vivax infections of liver-chimeric humanized mice [Altogether, these findings implicate iREVs as facilitators of immunopathology and vascular dysfunction in malaria by virtue of the biomolecular cargo they carry. Several  protein , 114, nu protein , 116, an protein , 118  that mediate ‘direct’ cell-cell interactions between parasite-infected RBCs and other host cells . VSAs ariewed in , 120.P. falciparum strains to enhance survival and gametocytogenesis under drug pressure [Plasmodium spp. propagate their life cycle in natural infections. The implication of this pathway, should it occur in humans infected with malaria, is the potential of tackling drug resistance via novel strategies and developing drugs and vaccines that target iREVs to block the development of transmission stages of the parasite [Using co-cultures of diverse parasite strains, Regev-Rudzki and colleagues demonstrated that ring stage pf-iREVs transfer episomal drug resistance genes from resistant to susceptible pressure . This inpressure . Rather,pressure  and the parasite .P. falciparum responds to high density stress in vitro by releasing a subpopulation of EVs with differentially abundant parasite lactate dehydrogenase (LDH) that, in recipient parasites, induces apoptosis, thereby regulating parasite population [Contrary to increased sexual differentiation under drug pressure and a resultant low or controlled parasitaemia, pulation . Programpulation  and evidP. berghei rodent infection, compared to iREVs from HRF and Ef-1α knock-out mice, wild type sourced iREVs efficiently inhibited T cell proliferation in vitro and in vivo [Other iREV-bound parasite proteins—histidine rich factor (HRF), elongation factor 1α (Ef-1α)  and PfEM in vivo . Over 3  in vivo , 126. Th in vivo , 124 andThe presence of parasite biomolecules in iREVs is apparently purposeful and it has been proposed that malaria parasites direct cargo-specific loading of iREVs to exert effects in target cells for their benefit , 116. ThP. falciparum may signal peak EV release from infected RBCs [As the malaria parasite matures within the RBC from ring stages, through trophozoites to schizonts, iREV production increases steadily , 100. Whted RBCs . Moreoveted RBCs  exists tPlasmodium spp. Studies of iREVs have identified novel malaria pathways with potential drug targets and anti-malarial host-derived EV cargo, as well as EV-associated parasite molecules already under evaluation in vaccine trials. In fact, a few studies have demonstrated the potential of whole malaria derived EVs as vaccines.Vector control and targeted chemoprophylaxis are the main malaria prevention and control strategies . While wP. yoelii-infected reticulocytes mounted a parasite-specific humoral immune response [P. yoelii infections [Plasmodium yoelii-infected reticulocyte-derived EVs revealed over 70 Plasmodium proteins, including the immunogenic rhoptry proteins and merozoite surface protein 1 (MSP-1) [P. berghei infected mice survived an initial lethal infection and developed long-lasting immune memory that conferred protection against a second infection [In an experimental model of malaria, mice vaccinated with CpG-ODN adjuvanted EVs derived from non-lethal response  accompanresponse . Ultimatfections , 55. Pla (MSP-1) , 55. Simnfection . The autnfection . Althougnfection , 128.P. falciparum cultures and corresponding uninfected RBC derived EVs (uREVs), have been evaluated for their potential as natural antimalarial drug delivery systems [P. falciparum in vitro more efficiently than when the parasites were exposed to free unencapsulated drugs [A number of earlier malaria vaccine studies focused on developing synthetic nanoparticles and microparticles for the delivery of parasite genes or proteins and a majority of the formulations included the biocompatible polymer poly (lactic-co-glycolic acid)—PLGA –134. For systems . Two liped drugs .Amongst other advantages of natural EVs, iREVs were bound by iRBCs and uRBCs with significantly greater avidity than uREVs, making this novel targeted drug delivery system a promising approach to improving antimalarial therapy and tackling drug resistance . It alsoPlasmodium [Uptake of iREVs by natural killer (NK) cells is particularly appealing for anti-malarial therapy and deserves thorough investigation. When pf-iREVs, NK cells and iRBCs were co-cultured, iREVs (in a dose-dependent fashion) altered the phenotype of a ‘non-responsive’ population of NK cells to a ‘responsive’ phenotype with concomitant reduction in parasitaemia . NK cellasmodium  and dataasmodium , 139. Idasmodium  as an NKasmodium .P. falciparum virulence [P. falciparum isolates [On the other hand, functional human miR-451 and miR-140 complexes in EVs from uRBCs have been shown to negatively impact irulence . Malariairulence . The unfirulence  contrastirulence  mentioneisolates , 140 andisolates  to gain isolates  that canP. yoelii-infected mice [P. yoelii infection and these miRNAs directly inhibited expression of an essential regulatory gene for growth and migration of vascular endothelial cells [Malaria-derived EVs may hold potential beyond antimalarial therapeutics. In a murine model of cancer, intriguing pathological and immunohistochemical data showed the tumor-suppressive and anti-angiogenic properties of plasma derived EVs from ted mice . The autal cells .The EV network in malaria is complex, with extensive crosstalk at a molecular and cellular level among numerous host cells, within parasite populations, and between host cells and parasites. The correlation between circulating EVs and disease severity in natural infections, as well as protection against ECM by gene knockouts and pharmacological inhibition, supports the potential for EVs as biomarkers and suggests a pathogenic role in disease. A protective function of some EV populations has also been proposed. The many attributes of EVs are bestowed by their biomolecular cargo. Although the understanding of malaria EV biology is gradually advancing, there remains significant gaps in the knowledge of the distinct nature of EVs released by the different life stages of malaria parasites as well as how this may vary from one individual to another. More so, the mechanisms of specialised cargo loading, EV release and EV uptake are yet to be elucidated. This is essential for deeper understanding of the pathogenic and protective pathways of EVs, which in turn is necessary for the successful application of EVs as therapeutics and vaccines to intercept or promote these pathways. Furthermore, the notion that EVs may be used in the development of malaria vaccines requires substantially more scientific evidence. Technologies need to be developed that allow the manufacturing of homogenous EV populations and the purification of EVs from malaria cultures in large enough scale for vaccine production.P. falciparum and P. vivax, novel details have emerged from numerous studies over the last 2 decades. Much remains to be learned, and the field of malaria will likely see more research focusing on the characterization of distinct malaria EV populations, their biogenesis and fate, and critically investigating their role in disease.Nevertheless, as for understanding the involvement of cell specific EVs in malaria pathogenesis, host-parasite interactions, and the complex biology of


Aedes mosquitoes and these are prevalent in Ghana. Despite its global significance, the true burden of chikungunya virus infection in Ghana is largely unknown and the threat of outbreak remains high owing to international travel. This study sought to determine chikungunya virus infection among febrile patients suspected of having malaria infections at some selected health facilities in the Ashanti, Bono East, and Bono Regions of Ghana.Chikungunya is now of public health concern globally due to its re-emergence in endemic areas and introduction into new areas of the world. Worldwide, the vectors for transmission of the chikungunya virus are This cross-sectional study recruited six hundred (600) febrile patients suspected of having malaria who submitted their clinical samples to the laboratories of the selected health facilities for the diagnosis of their infections. Five to ten millilitres (5-10ml) of venous blood were collected from each study participant. Sera were separated and tested for anti-chikungunya (IgM and IgG) antibodies using InBios ELISA kit following the manufacturer’s instruction. Samples positive for chikungunya IgM and IgG were selected and tested for chikungunya virus RNA using Reverse Transcription-quantitative Polymerase Chain Reaction. Malaria Rapid Diagnostic Test kits were used to screen the participants for malaria. Structured questionnaires were administered to obtain demographic and clinical information of the study participants.Of the 600 samples tested, the overall seroprevalence of chikungunya was 6%. The seroprevalence of chikungunya IgM and IgG antibodies were 1.8% and 4.2% respectively. None of the chikungunya IgM and IgG positive samples tested positive for chikungunya RNA by RT-qPCR. Of the 600 samples, tested 32.3% (194/600) were positive for malaria parasites. Malaria and chikungunya co-infection was detected in 1.8% (11/600) of the participants.Findings from the current study indicate low-level exposure to the chikungunya virus suggesting the virus is circulating and potentially causing morbidity in Ghana. Aedes) responsible for the transmission is present in the country. Ghana’s neighbours (Ivory Coast and Burkina Faso) have also reported isolated cases of CHIKV infections. The authors sought to find evidence of the circulation of CHIKV among suspected malaria patients in Ghana and to shed light on the importance of the investigation of non-malaria causes of fever such as chikungunya fever. And the possible inclusion of chikungunya fever in the differential diagnoses of unexplained febrile cases in Ghana. We found out that 6% of the 600 people studied had antibodies to the CHIKV i.e. they were previously infected with CHIKV. Malaria prevalence was 32.3% among the study participants. The rate of malaria-chikungunya co-infections was 1.8% among the study participants. Our findings show a low-level exposure to the CHIKV suggesting the virus is circulating and potentially causing morbidity in Ghana that may be misdiagnosed as malaria.Chikungunya fever is a mosquito-borne disease of Chikungunya virus (CHIKV) origin that shares similar symptoms with malaria fever. The symptoms include fever, joint pains, muscle pains, skin rashes and headache. There is a paucity of information about CHIKV epidemiology in Ghana although the mosquito vector ( Aedes species predominantly Aedes aegypti and Aedes albopictus [Aedes aegypti mosquitoes are actively circulating in Ghana and could potentially promote outbreaks of viral haemorrhagic fevers [Chikungunya virus (CHIKV) which was first reported in Tanzania in 1952 is a spherical, enveloped virus with a diameter of about 60–70 nm . The virbopictus ,12. Two c fevers ,14.In Africa, sporadic and epidemic cases of CHIKV infections have been reported in several countries including Cameroon, Senegal, Central African Republic, South Africa, Angola, Nigeria, Burkina Faso, Ivory Coast, Benin, Guinea . The disEven though CHIKV infections have not been officially reported in Ghana, autochthonous cases or outbreaks have been reported in Ghana’s neighbouring countries such as Burkina Faso, Ivory Coast, Benin, and Nigeria in recent years ,19. ThisEthical approval was sought from the Committee on Human Research, Publications, and Ethics of School of Medicine and Dentistry (SMD), Kwame Nkrumah University of Science and Technology (KNUST), Kumasi-Ghana . Permission was also sought from the Institutional Review Board (IRB) of the selected health facilities. Formal written consent was obtained from individual participants after explaining the purpose of the study in a language each participant understood.This hospital-based cross-sectional study was conducted at the Holy Family Hospital, Techiman, Bono East Region; Sunyani Municipal Hospital, Sunyani, Bono Region; and the Manhyia District Hospital, Kumasi, Ashanti Region between September 2018 and November 2019.et al., 2017 [The sample size was estimated based on the seroprevalence of 41.8% of chikungunya virus infections in two states of Southwest Nigeria reported by Olajiga l., 2017 . The detWhere:n = sample size; Z = z-score or standard score value from the standard normal distribution reflecting the confidence level ; M = margin of error of 5.0%; P = estimated seroprevalence of 41.8%.The estimated sample size was 374, which implied the minimum number of participants to be recruited.The study recruited 661 febrile patients who submitted their clinical samples to the laboratories of the selected health facilities for laboratory diagnosis of malaria. However, 61 (9.22%) of the samples taken haemolysed and were not suitable for the serological test so were excluded from the study. Therefore, the total number of subjects recruited for the study was 600 and the breakdown was 213, 227 and 221 from Manhyia Hospital, Kumasi, Sunyani Municipal Hospital, Sunyani and Holy Family Hospital, Techiman, respectively.Inclusion criteria for enrolment included patients suspected of having malaria with the onset of symptoms not exceeding 15 days and having one or more of the following symptoms: arthralgia, myalgia, fever, and other malaria-like symptoms including headache, nausea, vomiting, chills, and shivering.Structured questionnaires were administered to obtain demographic information of the study participants. Five to ten millilitres (5–10 ml) of whole blood samples were collected from each participant by venipuncture into serum separation tubes. The samples were allowed to clot and then centrifuged at 1500 rpm for 5 minutes to separate the sera. The serum samples were aliquoted into two separate vials of 1 ml and temporarily stored at -20°C at the laboratories in the selected health facilities before being transported to the Virus Research Laboratory at the Department of Clinical Microbiology, SMD-KNUST where the samples were stored frozen at -20°C until testing.Plasmodium falciparum indicative of the presence of the parasites in the patient. A line on only the ‘C’ indicated a negative result. No line at all or on the ‘C’, indicated an invalid result, and the test was performed again.Malaria Rapid Diagnostic Test kits  were used to screen the participants for malaria. Each cassette was labelled with the patient’s serial number. Five microliters (5 μl) of the blood sample was placed into the sample (S) pad. Two drops of the sample diluent assay (buffer) were dispensed into the diluent pad and allowed to run. The results were interpreted within 20 minutes. Lines on both the control (C) and test (T) indicated a positive result, which signified the presence of histidine-rich protein 2 (HRP 2) of Serological detection of anti-chikungunya IgM and IgG were done using CHIKjj Detect IgM and CHIKjj Detect IgG ELISA kits  following instructions and recommendations from the manufacturer. The reagents and the samples were allowed to equilibrate at room temperature before the assay was performed. CHIKV IgM and IgG positive and negative controls as well as the serum samples were first diluted with the sample dilution buffer (1:100). Fifty microliters (50 μl) of the diluted controls and samples were added to the designated wells of the micro-titre plates and then incubated at 37°C for 30 minutes. After washing, 50 μl of CHIKV antigen was added to each well and incubated at 37°C for 30 minutes. Washing was repeated after which each well (except for the blank) was treated with 50 μl of enzyme conjugate for CHIKV and then incubated at 37°C for 30 minutes. After subsequent washing, substrate solution 3,3’,5,5’-tetramethylbenzidine (TMB) hydrogen peroxide was added to each well (including the blank well) and incubated in the dark at room temperature (20–25°C) for 10 minutes. A stop solution was added to each well and incubated at room temperature for 1 minute after which the optical density (OD) of each well was measured at 450 nm using iMark Microplate Reader . The ELISA results were interpreted following the instructions of the manufacturer of the test kits used. The sensitivity and specificity of the test kits were greater than 90% according to the manufacturer.Serum samples that tested positive for CHIKV IgM and IgG were selected and further tested using RT-qPCR. CHIKV RNA was extracted and purified from the serum samples using the QIAamp Viral Mini Extraction kit , following the kit manufacturer’s instructions. The quantity and the purity of the extracted RNA were verified by spectroscopy .Genesig multiplex kit  was used to test for the presence or otherwise of chikungunya, dengue, and zika viruses’ RNA in a single reaction . The kithttps://www.genesig.com/assets/files/dengue_chikv_zikv_multiplex_handbook.pdf)(2) test was used to determine significant differences between categorical variables, while Fisher exact test was used in cases when expected counts were less than 5. A p-value of less than 0.05 was considered statistically significant.The data generated were analysed with IBM Statistical Package for Social Sciences (SPSS) version 20 . Continuous data were expressed as mean ± standard deviation (SD). Categorical variables were expressed in percentages and frequencies, and Chi-square (χData on 600 participants comprising 226 (37.7%) males and 374 (62.3%) females were analysed in this study. More females than males participated in the study; 63% (126/200) were from the Holy Family Hospital, 62.5% (125/200) from the Sunyani Municipal Hospital, and 61.5% (123/200) from Manhyia District Hospital. The mean age of the participants was 26.5 years (±20.1) and the ages ranged between 1 and 91 years. The majority of the participants were less than 11 years (29%), and most were from the Holy Family and Sunyani Municipal Hospitals with 41.5% and 40.0% respectively, with the least (5.5%) from the Manhyia District Hospital. The age group with the least participants was the above-60-years group representing 5.8% of the total study participants; Manhyia District Hospital had the highest proportion (7.0%), followed by Sunyani Municipal Hospital (6.5%), and the least was Holy Family Hospital (4.0%).p-value = 0.003), but not between IgG and the sampling sites . The prevalence of malaria detected in the current study was 32.3% (194/600). Malaria prevalence was higher (46%) at Manhyia District Hospital and the distribution of malaria positivity at the hospitals was significant, (p < 0.001)  detected was 6% (36/600). The seroprevalences for CHIKV IgM and IgG antibodies were 1.8% (11/600) and 4.2% (25/600) respectively; however, 8.3% (3/36) of the overall seroprevalence (representing 0.5% of 600) had both IgM and IgG antibodies. The IgM (4.5%) and IgG (12%) seropositivities were both highest at the Manhyia District Hospital. There was a significant association between CHIKV IgM antibodies and the sites of sampling (< 0.001) .p-values = 0.112, 0.149, and 0.153 respectively)  CHIKV IgG seropositives and 58.2% malaria positives. A Chi-square test did not reveal statistically reliable differences between gender and with the various CHIKV IgM and IgG antibodies and malaria positivity (ctively) .p = 0.005). A larger proportion (35%) of malaria positives were detected among participants within the age group of 11–20 years (Participants within the age group of 21–30 years were observed to have a higher proportion (36.4%) of CHIKV IgM seropositives. Those within the age group of 31–40 years had a higher proportion (24%) of CHIKV IgG positives and a Chi-square test did reveal a statistically significant difference between IgG and the age groups . Lower proportions of patients with the reported symptoms were positive for CHIKV antibodies and malaria parasites; 0.4% for joint pains, 1.8% for muscular pains, 2.0% for chills, 2.0% for headache, and 3.0% for nausea and vomiting. No significant differences were observed among patients positive for both CHIKV antibodies plus malaria parasites and reported symptoms (The most common symptoms reported among the patients were headache (57.9%), muscular pains (46.0%), chill, shivering & sweating (42.2%), joint pain (41.7%), nausea/vomiting (16.5%), and rashes (7.3%). Most of the patients’ positive for IgM (2.0%), IgG (5.5%), and malaria (34.2%) presented with headaches, however, no statistical differences were found in these distributions . Lower psymptoms .et al., (2019) in Accra Ghana [et al., (2013) [et al., (2017) [et al., (2019) [et al., (2017) in Nigeria [et al., (2018) [et al., (2008) [et al., (2013) [et al., (2017) [The current study has provided evidence of CHIKV exposure among the study participants in the Ashanti, Bono, and Bono East Regions of Ghana. The overall CHIKV specific antibodies (IgM and IgG) seropositivity was 6%. IgM and IgG seropositivities were 1.8% and 4.2% respectively. The overall CHIKV specific seropositivity reported in the current study was lower than the 27.69% reported by Manu ra Ghana , 50.17% , (2013) , 41.8% bt al., 20 in Accra, 2019)  in separ Nigeria . The low, (2018) . The det, (2013) , and Ola  in sepaet al., (2017) [et al., (2018) [Aedes mosquitoes which prefer daytime and dusk to feed [et al., (2018) [Conversely, IgG seropositivity was higher in females and was similar to that reported by Olajiga , (2017)  but diff, (2018) . Seropre to feed ,30. Notw, (2018) .et al., (2013) [et al., (2014) [et al., (2016) [et al., (2016) [et al., (2018) [et al., (2018) [Anopheles mosquitoes, vectors of the malaria parasite, is a night-biting mosquito while the Aedes mosquitoes, vectors of chikungunya virus, is daytime feeder [Aedes mosquitoes are actively circulating and could potentially promote outbreaks of viral haemorrhagic fevers [Aedes mosquitoes could potentially be the source of the low-level circulating chikungunya virus identified in this study.Chikungunya-malaria co-infection, however, was lower at 1.8% (11/600) in this study. This low co-infection rate observed was similar to studies done in different areas across sub-Sahara Africa reported as; 6.7% by Baba , 2013) , 0.54% b, 2014) , 15% by , (2016) , 0.02% b, (2016) , 7.14% b, (2018)  and 1.23, (2018) . However, (2018) . The Anoe feeder . Studies , 0.54%  , 15% byet al., [et al., (2011) pointed out that the detection of chikungunya viral RNA is from the day of infection or onset of symptoms to day 7, after which acute infections detection is limited [With no statistical associations, the most commonly reported clinical symptoms among the chikungunya and malaria infections aside from the febrile condition were headache, muscular pains, chills, shivering & sweating, joint pains, and nausea/vomiting. This result is in line with that of other studies ,26,29,31et al., . Dash et limited . The non limited . It is rThe details of primers and probes sequences were not provided by the manufacturer although the authors requested for the details. Moreover, the RDT test used for malaria detection among the study participants is of lower sensitivity compared to the PCR and so inference based on CHIKV-malaria co-infection should be made with caution.This study report evidence of chikungunya virus exposure among Ghanaians in the study areas. The detection of chikungunya antibodies or exposure points to an unnoticed transmission intensity of the virus among patients who patronised health services at the health facility. The true burden of the virus and its associated disease is still not known though the country is endemic with the vector implicated in the transmission of this virus. It is anticipated that these findings would provoke a wider discussion about the burden of chikungunya virus infections. This will help guide policymakers to develop effective and affordable early warning and outbreak response systems for Ghana.S1 Questionnaire(DOCX)Click here for additional data file.S1 FigChikungunya, Dengue and Zika viruses RNAs were amplified and detected with Genesig Real Time PCR detection kit using CFX 96 Touch Real-Time PCR detection system. The chikungunya virus RNA was detected using CY5 fluorescent channel while Dengue and Zika viral RNAs were detected using VIC and FAM fluorescent channels respectively. All the samples tested were below the RNA detection threshold. However, the positive controls for the three viruses tested came out positive. The pink curve represent positive control for chikungunya virus while the green and blue curves represent the positive controls for Zika and Dengue viruses respectively.(TIF)Click here for additional data file.


Despite the growing evidence that malaria and anaemia are two interlinked health problems of school-aged children (SAC) in developing countries, there is scarce information about malaria among SAC in Ethiopia. Moreover, anaemia-related studies were more concentrated in easily accessible areas. This study aimed to assess the prevalence of malaria and anaemia and corresponding risk factors among SAC in Dara Mallo and Uba Debretshay districts, in hard to reach areas, so as to inform appropriate integrated interventions for both diseases.This study was part of baseline data collected for a cluster-randomized trial registered in Pan African Clinical Trials Registry (PACTR202001837195738). Data were collected from 2167 SAC and their households through face-to-face interview; malaria was diagnosed by using rapid diagnostic test (RDT); haemoglobin concentration was determined using hemoCue hb 301 and adjusted for altitude to determine anaemic status; helminth infections were determined by using kato-katz, and anthropometric measurements were made to determine nutritional status of children. Generalized mixed effects logistic regression model was used to assess the association between predictor variables and malaria and anaemia using school as a random variable.Plasmodium falciparum, Plasmodium vivax and mixed infections of P. falciparum and P. vivax, respectively. Malaria was significantly lower among children from literate household head  and residence house located at an altitude range above 1100 masl . The prevalence of anaemia was 22.00% (95% CI 20.3–23.8%) (477/2167) and was significantly reduced by eating legumes, nuts or seed group of food in their 24-h dietary diversity recall .The overall prevalence of malaria was 1.62% (95% CI 1.15–2.27%) (35/2167). Of the 35 children positive for malaria, 20 (57.14%), 3 (8.57%) and 12 (34.29%) were due to The prevalence of malaria was low and unevenly distributed per school while the overall prevalence of anaemia was moderate. It is important to implement integrated interventions targeting both malaria and anaemia, with special emphasis given to children from illiterate households and living at an altitude below 1100 masl. The micronutrient content of locally grown legumes should be further investigated to recommend specific interventions to overcome anaemia. Plasmodium species of protozoan parasites. There are five known Plasmodium parasites causing malaria in humans. Plasmodium falciparum and Plasmodium vivax are the two dominant species causing malaria in Ethiopia [Malaria is an infectious disease caused by Ethiopia , 2. Due Ethiopia –3. AccorEthiopia .Plasmodium species [Plasmodium species [. [Plasmodium infection was 38% higher in SAC than children under 5 years old.Nowadays, there is an age shift in the susceptibility to malaria. Malaria prevention measures targeting children under 5 years old leads to decreased exposure of them to  species . Less fr species . As comp species . The othecies [.  noted thPlasmodium species [The prevalence of malaria and its determinants varies widely in the different contexts. Up to 73.9% of SAC in SSA were infected by  species . It was  species , 10.8% i species , 1.7–18. species –15, 6.4– species –18, 40%  species , and 73. species . Malaria species , 19–23. Plasmodium infections cause anaemia through direct destruction of the red blood cells, clearance of infected and uninfected red blood cells by the spleen and impaired production of the red blood cells by the bone marrow [The other consequences of malaria are life-threatening anaemia and metabolic acidosis . Anaemiae marrow , 27.Anaemia was used as an important health indicator in the population. It is more common in economically deprived populations where malaria and other infectious diseases were highly prevalent. For each gross domestic product (GDP) increase in China, there was a 40% reduction in the prevalence of anaemia among SAC . The majDespite the dramatic reduction in malaria in the last two decades, malaria remains a major public health and economic problem in Ethiopia . AccordiThis study was conducted in Dara Mallo and Uba Debretshay districts in Gamo and Gofa Zones, respectively Fig. . The popThis study was part of baseline data collected for a cluster-randomized trial evaluating the effect of malaria prevention education on malaria, anaemia and cognitive development of SAC. The trial was registered in Pan African Clinical Trials Registry (PACTR202001837195738). Study design, sample size estimation and sampling technique for the trial were described elsewhere .The data collection process was carried out between October and December 2019. A pre-tested, structured questionnaire was uploaded to the Open Data Kit (ODK) data collection tool for observation and face-to-face interview with the mother of the child (caretaker). Socio-demographic factors, household assets, housing conditions, 24-h dietary diversity of SAC, sanitation and hygiene facilities, bed-net ownership and utilization-related factors were noted. The 24-h dietary diversity questionnaire was adapted from guidelines for measuring household and individual dietary diversity, developed by the Food and Agricultural Organization . In addiExcept the face-to-face interview with mothers (caretakers) of the children, other components of the data were collected at the school compound by trained data collectors from Arba Minch University and health professionals from Dara Mallo and Uba Debretsehay districts, under close supervision of the principal investigators.Plasmodium infection status by using a rapid diagnostic test (RDT) (CareStart™ PF/PV(HRP2/pLDH)Ag Combo), which is 98 and 96% sensitive for P. falciparum and P. vivax, with equal specificity of 97.5% for both species. The auxiliary temperature of all children was taken by using a digital thermometer. Auxiliary body temperature above 37.5 °C and positive in RDT was considered as a clinical malaria case, otherwise the child was considered as an asymptomatic infection. Plasmodium-infected individual were treated according to the national malaria treatment guideline [The finger prick blood specimen was collected to determine haemoglobin concentration by using HemoCue hb 301  and Plasuideline .Anthropometric measurements were taken according to WHO guidelines . ChildreWhen measuring the height of a child, he/she stood with his/her back against the board, his/her heels, buttocks, shoulders, and head touching a flat upright sliding head piece. The child’s legs were placed together with the knees and ankles brought together. Children were asked to take a deep breath, and the height measurement was taken at maximum inspiration. The head piece was brought down onto the uppermost point on the head and the height was recorded to the nearest 0.1 cm at the examiner’s eye level. Heights were measured by two data collectors and difference of 0.5 cm was tolerated. In cases when the difference between the two measurements was greater than 0.5 cm, a third measurement was taken and the nearest two measurements were recorded.After instructing children on how to collect stool specimen without contamination with soil or urine and any dirty material, a polyethylene screw-capped stool container was given to all children enrolled in the study in order to bring their stool specimen. Stool samples were collected at the school compound. The collected stool specimens were transported to Wacha and Beto health centres for laboratory diagnosis within the same day of collection. Stool smears were prepared following manufacturer’s instruction of Kato-Katz thick smear preparation. Prepared stools were left for 30 min after preparation and examined under bright field microscope within an hour. The template used for preparation of the smear was 41.7 mg . The numData collected by using the ODK data collection tool were converted into a comma-separated value (CSV) files by using ODK briefcase. The average weight and height of a child was taken to represent their weight and height. The WHO AntrhroPlus software was used to calculate anthropometric indices such as body mass index (BMI)-for-age-z-score (BAZ), height-for-age-z-score (HAZ) and weight-for-age-z-score (WAZ). Children whose indices were less than − 2 were considered as underweight, stunted or wasted , respectively for the previous indices. A child whose z-score was less than − 2 in any of the above indices was considered undernourished .The haemoglobin concentration was adjusted for altitude based on WHO recommendations. Anaemia was defined based on WHO classification of haemoglobin (Hb) concentration: Hb  <  115 g/L for children aged 5–11 years; Hb  <  120 g/L for children aged 12–14 years . The dieMultiple factor analysis by using household assets, housing conditions, source of drinking water, agricultural land area, and the number of domestic animals was used to generate the wealth index of a household. The first dimension was classified into tertiles to classify household economic status into poor, medium and high.Descriptive statistical analysis was performed; univariable and multivariable mixed effects logistic regression models were used to assess the association between malaria infection status and anaemia among SAC taking into account the cluster effect. The analysis was made using glmer function in lme4 R package. Odds ratio (OR) and corresponding 95% confidence interval (CI) were used to assess the strength of association between the outcome variables and predictor variables. Variables with P-value less than 0.25 in univariable analysis were potential candidates for multivariable mixed effect logistic regression. The fit of the model in predicting the outcome variables was checked by Akaike Information Criterion (AIC). Selection of variables for multivariable mixed effect logistic regression was made through backward stepwise variable selection method in which variable with the largest P value is removed from the model and checked for AIC. If removal of a variable from the model improves the AIC value, it is removed from the model, otherwise it is maintained in the model. For multivariable mixed effects models, P-value less than 0.05 were considered statistically significant.A total of 2167 SAC participated in the study, which gave a response rate of 94.1% . Of these, 64.0% were from Uba Debretshay district, 85.0% were from rural areas, 50.1% were boys and the mean (SD) age of children was 8.75 (1.53) years. About 39.2, 28.3 and 32.5% of children were attending grades 1, 2 and 3 education, respectively. Household residence lies at an altitude between 754 and 1988 masl with mean (SD) altitude of 1192 m (145 m) masl  of SAC were RDT-positive with 95% CI of 1.15–2.27%. Of the total, 35 SAC-positive for malaria 20, 3 and 12 were due to Plasmodium infection among SAC residing at an altitude above 1500 masl. Household ownership of bed nets was not associated with malaria in univariable analysis , but none of the children who slept under bed net the night before the survey was positive for malaria. The prevalence of malaria was 1.79% in rural areas while it was 0.62% in other areas although the difference was not statistically significant . The prevalence of malaria was 1.75% in boys and 1.48% in girls with no statistically significant difference . Literate educational status of household head , malnourished children , wasted children , and being resident in a household located at an altitude above 1,109.89 masl  significantly influenced malaria among SAC in univariable mixed effect logistic regression. However, after fitting the multivariable mixed effect logistic regression and taking care of collinearity between malnourishment and wasted children, only literate educational status of household head  and being resident in a household at an altitude above 1109.89 masl  remained statistically significant  g/dL. The prevalence of anaemia was 22.0% (95% CI 20.3–23.8) (477/2167). Of the 477 anaemic SAC, 216, 256 and 5 were mild, moderate and severely anaemic, respectively. The proportion of mild, moderate and severe anaemia among anaemic children is depicted in Fig. Schistosoma mansoni infection, infection by Plasmodium species and co-infection of Plasmodium species with STH or S. mansoni were statistically significantly associated with anaemia  infections or infection by particular species of STHs, ia Table .Table 3UThe mean DDS of children from 24-h dietary recall was 3.57 (SD: 1.74) from the nine food groups included in the analysis  for malaria, 2016–2030. There was a high heterogeneity in the burden of malaria in schools participating in the study, which calls for well-tailored malaria prevention interventions where prevalence of malaria is high. High uneven spatial distribution of malaria was also demonstrated in the previous epidemiology study in Ethiopia .The prevalence of malaria in the present study area was in agreement with the last national malaria indicator survey of Ethiopia, the recently stratified estimate made in Arba Minch Zuria district  and one Plasmodium-infected children and subsequent loss of power to detect the difference of anaemia among Plasmodium-infected and uninfected children. The prevalence of anaemia was moderate among SAC in Dara mallo and Uba Debretsehay districts. It was lower than prevalence of anaemia in similar population groups in Jima town [Unlike the previous hypothesis that malaria was significantly associated with anaemia, this was not demonstrated in the present study. Failure to demonstrate significant association between malaria and anaemia in this study might be due to the low number of ima town , Arba Miima town  and the ima town , while iima town . This fiima town  while itima town , Uganda ima town  and Kenyima town . The difima town , and lowima town  and nortima town . This diApart from parasitic infections , 52, 53,Plasmodium parasites per μl of blood or the percentage of Plasmodium-infected red blood cells was not calculated. The fourth limitation is due to recruiting only school-enrolled children into the study as the situation may be different for children not enrolled to a school. The strength of this study is the area of the study being conducted in hard-to-reach areas, not previously explored.There were certain limitations associated with this study. The first was due to the design in which both outcome and predictor variables were assessed at the same time. Such studies were not strong in generating causal evidence. Secondly, the low power of the study to detect significant difference of malaria in its predictor variables and malaria as predictor for anaemia. The third limitation was related to the biomass of malaria-positive individuals. The number of The overall prevalence of malaria among SAC in Dara Mallo and Uba Debretsehay districts was low, but there was uneven distribution of malaria in the schools. Malaria was more common among children from household heads who were illiterate and where the residence was located at an altitude range below 1100 masl. This calls for targeted intervention in schools where the prevalence of malaria is high with due emphasis given to schools at an altitude below 1100 masl and households with illiterate household heads. Anaemia was moderate in the area, being eligible for interventions to improve the haemoglobin concentration of SAC. Anaemia was negatively affected by 24-h dietary recall of eating legumes, nuts and seeds. Studies evaluating the micronutrient content of these foods and their daily intake for nutritional adequacy shall be studied.