Plasmodium falciparum and/or Plasmodium vivax among all age groups compared to LLINs or IRS alone.Conflicting results exist on the added benefit of combining long-lasting insecticidal nets (LLINs) with indoor residual spraying (IRS) to control malaria infection. The main study objective was to evaluate whether the combined use of LLINs and IRS with propoxur provides additional protection against This cluster-randomized, controlled trial was conducted in the Rift Valley area of Ethiopia from September 2014 to January 2017 (121 weeks); 44 villages were allocated to each of four study arms: LLIN + IRS, IRS, LLIN, and control. Each week, 6071 households with 34,548 persons were surveyed by active and passive case detection for clinical malaria. Primary endpoints were the incidence of clinical malaria and anaemia prevalence.P. falciparum and 25.3% were P. vivax, and 19.6% were mixed infections of P. falciparum and P. vivax. The overall malaria incidence was 16.5 per 1000 person-years of observation time (PYO), and similar in the four arms with 17.2 per 1000 PYO in the LLIN + IRS arm, 16.1 in LLIN, 17.0 in IRS, and 15.6 in the control arm. There was no significant difference in risk of anaemia among the trial arms.During the study, 1183 malaria episodes were identified, of which 55.1% were The clinical malaria incidence and anaemia prevalence were similar in the four study groups. In areas with low malaria incidence, using LLINs and IRS in combination or alone may not eliminate malaria. Complementary interventions that reduce residual malaria transmission should be explored in addition to LLINs and IRS to further reduce malaria transmission in such settings.Trial registration PACTR201411000882128 (08 September 2014)The online version of this article (10.1186/s12936-019-2775-1) contains supplementary material, which is available to authorized users. Despite remarkable achievements in the fight against malaria over the last decade, the World Health Organization (WHO) recommends universal coverage of populations at risk with long-lasting insecticidal nets (LLINs) and targeted indoor residual spraying (IRS) with an insecticide for the control of malaria , 2. BothReviews by Pluess et al. in 2009 and WHO in early 2014 documented that historical and programme documentation had clearly established the impact of IRS , 8. HoweDespite an increasing interest in the simultaneous use of both interventions, no clear guidelines existed at the start of this study on how these interventions should be combined . At the Recent reviews indicated that only a few of the published randomized controlled trials showed additional protection against fighting malaria when the use of LLINs was combined with IRS, compared to either method alone , 11. A mP. falciparum and/or P. vivax) among all age groups in the study area compared to LLINs or IRS alone; and, (2) determine whether LLINs + IRS improves haemoglobin (Hb) concentration and reduces anaemia among children under 5 years of age compared with children in LLINs or IRS arms alone.The specific objectives of this intervention study were: (1) to determine whether the combined use of LLINs and IRS with propoxur provides additional protection against malaria  in the East Shewa Zone of the Oromia Regional State in Ethiopia. The woreda is a local administrative unit in the country, which consists of several kebeles . Administratively, the district comprises 48 kebeles, each with a population size of approximately 1000 to 5000 people.This study was carried out in the Adami Tullu part of the Adami Tullu-Jiddo-Kombolcha The projected population size of the district for 2014 was approximately 173,000 people . The maikebele has at least one health post staffed by two health extension workers (HEWs) reporting to the health centre.In 2014, there was one public and one non-governmental organization hospital, 9 public health centres, and 43 health posts in the district. Each Plasmodium falciparum and P. vivax are the main causes of malaria infection in the area. Anopheles arabiensis is the major malaria vector in the district and An. pharoensis is considered to have an auxiliary role  two times a day for 3 days based on body weight, according to national guidelines [P. vivax infections were treated with chloroquine, 25 mg/kg for 3 days . Treatment of other conditions was performed in accordance with national guidelines, or referred to higher-level health facilities. Patients with severe illness at the time of visit  were referred to the nearest health facility.Individuals who were found to be positive for idelines . AL is aThe Hb concentration was measured in children 6–59 months old in the study households at three time points during the study  to assess the prevalence of anaemia. Through house-to-house visits, a single finger-prick sample was taken from each child, and height and weight were measured. Children with Hb values less than 11 g/dl were defined as anaemic.To validate the weekly incidence data, a community-based malaria prevalence survey was done on a randomly selected sample of households taking part from each arm of the trial during the main transmission season in November 2015 among all age groups. All household members were eligible and volunteered to be included in the study. The heads of the households were interviewed using a pre-tested structured questionnaire, and tested individuals for malaria parasites using RDT. In this survey, 4450 persons were included, and 0.46% (21 persons) of those who volunteered to contribute had malaria as assessed by RDT. There was no significant difference among the intervention arms, and the prevalence at week 57 was similar to the number of cases reported during the same week by the malaria incidence assessment.The data collection was done using standardized paper-based forms and questionnaires according to standardized operating procedures. Data were entered by trained data entry clerks and verified by range and consistency checks, and data cleaning was performed weekly. Any discrepancies were corrected by cross-checking against the corresponding original forms and subsequently amended in the final dataset.To minimize any loss to follow-up, all residents were followed and recorded if they moved out of the trial area or moved from one cluster to another cluster with a different intervention. For residents or respondents who were present at the time of the visit by project staff, basic information about dates and reasons for absence were recorded from other community members, such as friends or neighbours.P. falciparum or P. vivax using RDTs. All analyses were done on an intention-to-treat basis, regardless of whether the individual household members used LLINs, IRS or neither. All analyses were conducted using Stata version 13 . Outcomes were compared between study arms.The primary health outcome measure was malaria incidence determined by the detection of To control for potential confounding factors, the clustering effect of villages and the effect of repeated measurement in the same individual and individual-level covariates  were taken into consideration during the analysis. Building materials (roof type) is another potential confounding factor, and was also adjusted for in the regression analysis, which was estimated by a proportional hazards model. For ease of analysis, corrugated iron was merged with cement/concrete roof type. Though the main analysis plan was intention-to-treat, considering known protective effect of LLINs, there was a need to see if the rate of malaria infection for an average LLIN user was different from non-user. Thus, to determine if LLINs provided individual-level protection against malaria, a generalized estimating equation with Poisson log linear model was used to adjust for within-cluster correlation of measurements. Principal component analysis was used to construct a wealth index, as has been described before .http://www.satscan.org/) software was used for spatial and space–time statistical analysis, to identify statistically significant retrospective space–time malaria clusters.SatScan v9.1.1  of the College of Health Sciences at Addis Ababa University, the Ministry of Science and Technology, Ethiopia (ref: 3.10/446/06) and the Regional Committee for Medical and Health Research Ethics, Western Norway (ref: 2013/986/REK Vest).This study contains a control group, which did not receive any additional interventions except for the routine malaria work carried out by the district health office. The three ethical review boards accepted that such a group was included provided that the malaria incidence was followed closely, and if malaria incidence was not higher than expected. The study regularly monitored the malaria incidence in all four groups throughout the study, and the research did not observe higher incidences in the control group, nor any epidemics.kebele selection and randomization, implementation procedures, and expected outcomes of the trial to the communities, with representatives from the Oromia Regional Health Bureau, the East Shewa Zone Health Department, the Adami Tullu District Health Office and the District Administration. Study communities were sensitized prior to randomization through meetings and discussions with community leaders, kebeles, village leaders, and community elders.Prior to the implementation of interventions, a consultative workshop and several meetings were held to explain the objectives, Afan Oromo language. Verbal consent was used because many of the participants could not read and write. This consenting procedure was approved by the three ethical committees. Information sheets were provided about the purpose of the study, and the participants were informed that involvement in the study was voluntary and that they had the right to withdraw at any time regardless of reason. At each data collection, verbal consent was obtained from all study participants, and verbal assent was obtained from parents or guardians for children using the local language. Assurance was given that a refusal to participate in this study would not affect their access to services at the health posts in the study villages in the community. After completion of the study, households in the IRS and control groups received LLINs according to the national guidelines for bed net distribution [Verbal informed consent to participate in the study was obtained from the study participants and from parents or guardians for children under 18 years old using the local ribution .Ethical approval and pilot study were conducted in 2013, followed by trial registration, actual intervention and outcome measurement  had corrugated iron roofs.Baseline data collections done before the start of the study in 2014 showed that the study groups were comparable, except for house design  with an average of 196 people per cluster  were P. vivax infections, and 232 (19.6%) were mixed P. falciparum and P. vivax infections  were P falciparum infection was 9.1 per 1000 PYO (95% CI 8.4–9.8), for P. vivax 4.2 (3.7–4.6), and for mixed P. falciparum and P. vivax infection 3.3 per 1000 PYO (95% CI 2.8–3.6). There was no difference in malaria incidence among the four arms adjusting for roof type. The hazard rate of malaria infection for those living in thatched roofs was 18% higher than in households with corrugated iron roofs . Incidence rates were similar in the four arms with 17.2 per 1000 PYO in the LLIN + IRS arm, 16.1 in the LLIN arm, 17.0 in the IRS arm, and 15.6 in the control arm Table . The inc95% CI 8.–9.8, forOf the 1059 households with first episodes of malaria, 484 episodes occurred in areas with malaria clustering. The incidence of malaria in the clustered areas was 38.2 per 1000 PYO, and higher than 9.2 per 1000 PYO in the non-clustered areas; the incidence risk ratio (IRR) was 3.93 (95% CI 3.48–4.38). However, the IRR between the intervention groups in the clustered areas were similar.The prevalence of anaemia was 28.2% (95% CI 26.6–29.8) in 2014 and increased to 36.8% (95% CI 35.1–38.5) in 2015, and fell to 29.8% (95% CI 28.2–31.4) at the end of the study. There was no significant difference in risk of anaemia among the trial arms  and stable throughout the study [An. arabiensis was high [An. arabiensis to piperonyl butoxide (PBO), implicating the role of mixed function oxidases in the resistance of this insecticide [An. arabiensis. In another study, the PBO long-lasting insecticidal net and non-pyrethroid indoor residual spraying interventions showed improved control of malaria transmission compared with standard long-lasting insecticidal nets where pyrethroid resistance is prevalent [The bio-efficacy of LLINs was acceptable for at least 24 months . Neverth periods . The frehe study . The biowas high . Susceptecticide . Recent ecticide , and intrevalent . As a reThe study showed a low LLIN ownership after 2 years, and a low LLIN use, despite 100% net coverage at baseline . The useA strength of this study is that it was based on a random selection of villages: typical rural communities in Ethiopia. Moreover, the study included a large sample with high power and an adequate follow-up period. However, as the incidence rate of malaria was lower than expected at the start of the study, this could affect the statistical power of the study. The research was based on a hypothesised effect size of 30–50% reduction in malaria incidence. Using an effect size of 30% between the LLIN + IRS and the Control arm, a sensitivity analysis showed that the statistical power is 82%. In the study, except for housing type and the interventions, the baseline characteristics of the study arms were balanced Table . In addiAn. arabiensis, and the effectiveness of the two interventions combined was better than LLINs alone in reducing densities and human biting rates of the vector [An. arabiensis compared to either intervention alone remains unknown and warrants further research and action.Another limitation of the study could be the potential spill-over effect between clusters, and such an effect could have diluted any difference in the outcome measure. However, the IRS + LLINs were as effective as IRS alone in reducing densities and human biting rates of e vector . Added iThe study shows that malaria infection is a risk factor for anaemia , but theThe clinical malaria incidence and anaemia prevalence were similar in the four study groups. In areas with low malaria incidence, using LLINs and IRS in combination or alone may not eliminate malaria. Complementary interventions that reduce residual malaria transmission should be explored in addition to LLINs and IRS to further reduce malaria transmission in such settings.Additional file 1: Figure S1. Schedule of enrolment, interventions and assessments.


SAM) has been reported to be associated with increased malaria morbidity in Sub‐Saharan African children and may affect the pharmacology of antimalarial drugs. This population pharmacokinetic (PK)‐pharmacodynamic study included 131 SAM and 266 non‐SAM children administered artemether‐lumefantrine twice daily for 3 days. Lumefantrine capillary plasma concentrations were adequately described by two transit‐absorption compartments followed by two distribution compartments. Allometrically scaled body weight and an enzymatic maturation effect were included in the PK model. Mid‐upper arm circumference was associated with decreased absorption of lumefantrine (25.4% decreased absorption per 1 cm reduction). Risk of recurrent malaria episodes  were characterized by an interval‐censored time‐to‐event model with a sigmoid maximum‐effect model describing the effect of lumefantrine. SAM children were at risk of underexposure to lumefantrine and an increased risk of malaria reinfection compared with well‐nourished children. Research on optimized regimens should be considered for malaria treatment in malnourished children.Severe acute malnutrition ( WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?☑ Artemether‐lumefantrine  is the most prescribed antimalarial drug worldwide. However, exposure to lumefantrine is lower in children compared with adults with standard dosing recommendations. Moreover, altered physiological properties in children with severe acute malnutrition (SAM) might reduce drug absorption and further contribute to subtherapeutic exposures. Hitherto, the pharmacology of lumefantrine has been poorly defined in malnourished children, and its population pharmacokinetic (PK) properties have not been studied previously in SAM children. In addition, a lack of PK‐pharmacodynamic (PD) information on antimalarial drugs, when given to malnourished patients, makes it very difficult to dose these children adequately.WHAT QUESTION DID THIS STUDY ADDRESS?☑ What is the population PK and PD properties of lumefantrine in SAM children?WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?z‐score, and weight‐for‐age z‐score) influenced the pharmacological properties of lumefantrine in a similar way. The MUAC was the most significant covariate, resulting in substantially reduced absorption of lumefantrine with increasing malnourishment (25.4% decreased absorption per 1 cm reduction in MUAC). Lower exposure to lumefantrine resulted in an increased risk of acquiring a new Plasmodium falciparum infection during the follow‐up period.☑ This study is the first population PK‐PD study of lumefantrine in SAM children. Evidently, all malnutrition indicators , weight‐for‐height HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?☑ This work allows for an in‐depth understanding of the differences in PKs and PDs between SAM children and well‐nourished children. SAM children achieved a significantly lower exposure to lumefantrine than normal children. Lumefantrine dose optimization is needed urgently in this population.z‐score , mid‐upper arm circumferences , or presence of nutritional edema.Young children (<5 years of age) are especially vulnerable to malaria, and around 61% of all malaria deaths worldwide occur in this population.e WHZ; <−, mid‐uppMost clinical studies exclude severely malnourished children resulting in limited evidence on the PKs of drugs in this population, and the few studies available report contradictory results, as illustrated in a recent systematic review.in vitro antimalarial activity with geometric mean 50% inhibitory concentrations (IC50) of 65.2 nM : 42.3−101 nM) and 9.0 nM (95% CI: 5.7−14.4 nM), respectively.in vivo concentrations.Artemether‐lumefantrine  is one of the artemisinin‐based combination therapies recommended by the WHO for the treatment of malaria, and it is the most common antimalarial drug used worldwide.falciparum malaria, aged between 6 and 59 months. Only 160 of 266 non‐SAM children provided blood samples, and these children were included in the PK analysis. Full demographic characteristics of study participants are presented in TableThis clinical trial was an open‐labeled comparative intervention study of artemether‐lumefantrine in 131 SAM and 266 non‐SAM children with uncomplicated Lumefantrine capillary plasma concentrations were transformed into their natural logarithms and modeled using nonlinear mixed‐effects modeling. The PK properties of lumefantrine were described best by a two‐compartment disposition model  = −753, compared with a one‐compartment disposition model; FigureThe fraction of observed concentrations below the lower limit of quantification (LLOQ) were low  but evaluated using M1, M3, and M6 methods to avoid possible bias on account of censored data.z‐score (WAZ)) had a significant impact on the relative bioavailability, irrespective of the assumed parameter distribution of the relative bioavailability. Of these three malnutrition covariates, MUAC had the largest drop in ΔOFV (−64.4) and was retained in the final PK model. Addition of any other indicators associated with malnutrition together with MUAC did not result in any additional improvement in model fit. Impact of malnutrition‐associated indicators was further investigated using a full covariate approach. The median bioavailability was 38.6% (95% CI: 74.5−27.4%) lower in SAM children compared with non‐SAM children, and the median bioavailability was reduced by 21.0% (95% CI: 19.5−29.3%) per 1 cm reduction of MUAC or 15.5% (95% CI: 3.07−33.2%) per 1 unit WAZ reduction. Impact of the malnutrition‐associated indicators on other PK parameters was not statistically significant , but it was retained in the final model due to the strong biological prior evidence and previously published results.nt Figure2.Figuren = 2,000) resulted in 1.85% (95% CI: 1.39−3.79%) and 3.01% (95% CI: 1.62−3.48%) of lumefantrine observations being below and above the simulated 95% prediction interval, respectively. Bootstrapping  indicated a robust PK model with high precision in parameter estimates. Final primary and secondary PK parameter estimates of lumefantrine are summarized in TablesandThe final model showed a satisfactory goodness‐of‐fit Figure1. IntePlasmodium vivax infections, and 12 children lost to follow‐up or with unidentifiable malaria species were excluded from the PD analysis. Thus, the PD analysis was based on 380 children. The final PK model and individual parameter estimates were fixed and added into the interval‐censoring time‐to‐event model describing the risk of having a reinfection. A sigmoid maximum effect (Emax)‐function of the predicted lumefantrine concentrations improved the model fit significantly when compared with a model without antimalarial drug effect (ΔOFV = −62.3). No other covariates were found to have a significant impact in the PD model. A visual predictive plot of the interval‐censoring time‐to‐event model exhibited an appropriate predictive performance of time to parasite breakthrough during a malaria reinfection, time to malaria detection, and the recurrent parasite density . Thus, observed recurrent malaria was back‐extrapolated to the starting interval of the blood stage infection . Children in the PK‐PD arm and PD arm were combined and used for the development of the PD model. Weekly malaria screening identified 95 patients with a reinfection of malaria  who were included in the PD modeling. Four children with recrudescent malaria, three children with ty Figure3. Bootin vivo minimum inhibitory concentration (MIC) of lumefantrine was estimated based on the predicted lumefantrine concentration at the start of new blood stage infection, using the same back‐extrapolation methodology as described above. The 95th percentile of these predicted lumefantrine concentrations were assumed to be the highest possible concentrations, which still allowed parasite replication. In the patients with recurrent malaria, the predicted MIC values were between 164 and 182 ng/mL, based on the start and the end of the likely time period of new infection emerging from the liver , and day 7 concentration) of the increased dose regimen were approximately equivalent to standard dosing, because of a relatively lower bioavailability of the increased dose . However, both the intensified and extended dosing regimens were able to increase the exposure to lumefantrine in SAM children to similar levels as that seen in non‐SAM children receiving standard dosing , peak plasma concentration  days and 9.33 (95% CI: 6.60−39.3) days for SAM and non‐SAM children, respectively, after standard dosing. Time above MIC in SAM children could be expanded to 9.81 (95% CI: 6.67−50.8) days and 12.3 (95% CI: 8.10−52.0) days after the intensified and extended dosing regimens, respectively.PD protective efficacy of different dosing regimens was evaluated by simulating the total incidence of malaria reinfections during the 42 days of follow‐up Table S1Comorbidity of malaria and malnutrition is common, reaching high prevalence in the Sahel region where acute malnutrition episodes among children <5 years of age occur frequently during the malaria season. The review of the literature does not provide a clear picture of the effect of malnutrition on the malaria risk, but some studies show that malnutrition increases mortality due to malaria in children <5 years of age.The PK properties of lumefantrine were explained by a two‐distribution compartment model, which is similar to what has been reported previously.FigureEven though only 6.26% of samples were below the LLOQ, different methods were evaluated to incorporate these samples.FigureAllometrically scaled body weight on all clearance and volume parameters, and an enzyme maturation effect were included in the final model due to prior knowledge and a strong biological support.P. falciparum malaria were used as the PD end point, in order to describe the protective effect of lumefantrine. Only a few patients showed recrudescent malaria , and they were, therefore, excluded from the PD analysis. In addition, P. vivax infections have a different mechanism compared to P. falciparum infections, and these patients were excluded from the analysis. Both the PK‐PD and the PD arms were included into the PD data analysis and modeled simultaneously to maximize the amount of data. Commonly, time‐to‐event models are used to describe the time to malaria detection and to quantify the impact of drug concentration by linking the PK model with a PD Emax‐model. However, the estimated IC50 in such models will underpredict the true IC50 value because the model assumes that the recurrent malaria appears exactly on the day of the follow‐up visit. In reality, the blood stage infection started several days before the time of microscopy detection and the parasites have managed to grow through whatever drug concentrations that were present at that time. Bergstrand et al. and Chotsiri et al.50 closer to the true value. Therefore, the present study took this into account by determining the likely time of the emergence of the blood stage malaria infection and used this time interval as the event time. Observed parasite density at reinfection and the individual microscopic detection limit were used to determine the individual parasite growth rate. However, the average or median of estimated individual growth rates will not represent the true growth rate in the population because an estimated slow parasite growth rate might be a result of a recent infection or a lower parasite density than the individual detection limit at the previous malaria‐free visit. Therefore, it is expected that the true population value is skewed toward the higher estimated growth rates. Thus, the overall parasite growth rate in the population was defined by the 95th percentile of individually estimated parasite growth rates, resulting in an 11‐fold increase in parasite densities per asexual reproductive cycle (48 hours). This value is close to what would be expected from historical data.P. falciparum parasites were predicted, and, thus, provided insight into the clinical MIC values in this population. The predicted MIC values were between 164 and 182 ng/mL, which is similar to previously reported cutoff day 7 values associated with therapeutic efficacy.Reinfections with Figureb) supported the interval‐censoring time‐to‐event model and showed a good prediction of the time to malaria detection. The final PD model and parameter estimates, including IC50, from this study were similar with a previous report studying Thai women infected with P. falciparum.The visual predictive check of the PK‐PD model Figure4b supin silico dose optimization. Due to the dose limited absorption of lumefantrine, an increased dose regimen could not compensate fully for the lower exposure observed in SAM children.Figure,This study showed that malnourishment had a dramatic effect on the absorption of lumefantrine when given to children. This threatens our ability to treat malaria in this group, as it will result in inadequate exposure to lumefantrine in SAM children. Alternative dosing regimens were evaluated using In conclusion, the population PK properties of lumefantrine in this study were successfully explained by a two‐compartment disposition model with two transit‐compartments in the absorption phase. Body weight as an allometric function and age as an enzyme maturation effect were included into the PK model. Malnutrition had a significant impact on the absorption of lumefantrine, resulting in substantially lower drug exposure with increasing malnutrition. A parasitemia‐corrected time‐to‐event model was developed to explain the post‐treatment prophylactic effect of lumefantrine against malaria reinfections. Research on altered dosing regimens should be considered for optimal treatment of malaria in malnourished children.n = 133) and non‐SAM children (n = 266). A subset of the whole study was chosen to be part of the PK study (n = 131 for SAM children and n = 160 for non‐SAM children). Details on the study protocol as well as the clinical efficacy and safety have been published previously.An open comparative intervention study was conducted to determine the clinical efficacy and PK‐PD properties of lumefantrine in African SAM , severe anemia, known underlying or chronic diseases, and other complications requiring hospitalization were excluded from the study.Children aged between 6 and 59 months with uncomplicated Fixed‐dose combination tablets of nondispersible artemether 20 mg and lumefantrine 120 mg (Coartem) were given according to the weight‐based manufacturer recommended dose (1 tablet < 15 kg and 2 tablets 15–25 kg), twice daily for 3 days. Study drugs were administered with fat  in the non‐SAM group, and RUTF; Plumpy'Nut, Société de Transformation Alimentaire (STA), Niamey, Niger one bag of 92 g containing 32.9 g of fat) in the SAM group. If the children vomited within 30 minutes of dose administration, a repeat dose was administered. If the children vomited after the second dose, they were given rescue oral medication  and were excluded from the study.A population‐based sparse sampling approach was used to limit the number of PK samples required per child.in silico lumefantrine dose optimization can be found in the Lumefantrine capillary blood concentrations were transformed into their natural logarithms and analyzed using nonlinear mixed‐effects modeling in NONMEM version 7.3 . Pirana version 2.9.0,The Mahidol‐Oxford Tropical Medicine Research Unit is supported by the Wellcome Trust of Great Britain. The pharmacokinetic and pharmacodynamic analysis was supported by a grant from the Bill and Melinda Gates Foundation to Professor Joel Tarning. The funding bodies did not have any influence on the collection, analysis, interpretation of data, writing of the manuscript, or in the decision in submitting the manuscript for publication.The authors declared no competing interests for this work.P.C., R.M.H., and J.T. wrote the manuscript. L.D.N., E.B., O.G., O.K.D., K.I.B., A.D., and J.F.E. designed the research. L.D.N., E.B., O.G., H.D., O.A., M.S., P.J.G., and O.K.D. performed the research. P.C. and J.T. analyzed the data. L.W. and K.I.B. contributed new reagents/analytical tools.Supplementary methodsFigure S1Figure S2Figure S3Figure S4Figure S5Table S1Supplementary referencesClick here for additional data file.


Malaria diagnosis among HIV-positive patients is uncommon in Nigeria despite the high burden of both diseases.We evaluated the performance of a malaria rapid diagnostic test (MRDT) against blood smear microscopy (BSM) among HIV-positive patients in relation to anti-retroviral treatment (ART) status, CD4+ count, fever, cotrimoxazole prophylaxis and malaria density count.A cross-sectional study involving 1521 consenting randomly selected HIV-positive adults attending two ART clinics in Kano, Nigeria, between June 2015 and May 2016. Venous blood samples were collected for testing with MRDT, BSM, and CD4+ T cells count by cytometry. Biodata and other clinical details were extracted from patient folders into an Excel file, cleaned, validated, and exported for analysis into SPSS version 23.0. Sensitivity, specificity, predictive values of MRDT were compared with BSM with a 95% confidence interval.p < 0.05).Malaria parasites were detected in 25.4% of enrollees by BSM and 16.4% by MRDT. Overall sensitivity of MRDT was 58% and specificity was 97%. Cotrimoxazole prophylaxis and fever status did not affect MRDT sensitivity and specificity. Unexpectedly, the sensitivity was highest at parasite density count of less than 500 cells/µL. At CD4+ T cells count over 500 cells/µL the sensitivity was higher (62.4%) compared to 56% at less than 500 cells/µL. In the non-ART group sensitivity was higher (65%) compared to those on ART (56%) but the specificity was similar. All differences were significant for all variables (Although the MRDT specificity was good, the sensitivity was poor, requiring further evaluation for use in malaria diagnosis among HIV-malaria co-infected persons in these settings. Opportunistic infections and other febrile illnesses mimic malaria in HIV patients.5A key factor for effective management of malaria is early and accurate diagnosis. The global impact of malaria has prompted an increase in the development of diagnostic strategies. The updated World Health Organization (WHO) recommendations indicate that all suspected cases of malaria must be confirmed with a laboratory diagnostic test before treatment.13 In many malaria-endemic areas like Nigeria, it is difficult to maintain the required technical skills, equipment and infrastructure particularly in rural areas,15 where the disease is more prevalent. Hence, the use of rapid diagnostic tests (RDT) in such endemic areas is recommended.17 Furthermore, part of the national malaria control strategy plan for 2014 to 2020, with a vision of having a malaria-free Nigeria, is to test all care-seeking persons suspected of suffering from malaria using RDT or microscopy.1Although microscopy is the current gold standard for the diagnosis of malaria, it is time consuming and labour intensive.18 Thus far, only one study has evaluated the performance of a malaria RDT (MRDT) among 387 HIV-positive persons in Nigeria.19 Overall MRDT sensitivity, specificity, positive and negative predictive value have not yet been properly evaluated especially among HIV-positive individuals.21Data from reviewed literature showed that very few studies have evaluated the validity of RDT for malaria diagnosis in the general population in Nigeria.P. falciparum histidine-rich protein type-2 and Plasmodium lactate dehydrogenase), the Standard Diagnostics (SD) Bioline MRDT, compared to the gold standard, microscopy, in the diagnosis of malaria among HIV-positive participants with respect to malaria parasite density, CD4+ T cells count, cotrimoxazole prophylaxis use, anti-retroviral treatment (ART) status and occurrence of fever. The choice of SD Bioline was on the premise that this rapid test kit is the most commonly used MRDT for routine malaria diagnosis in Kano State.The objective of the study was to evaluate the performance of a commercially available MRDT . Participant consent was obtained from all respondents.3 The facilities are Infectious Diseases Hospital Kano (IDH) and Murtala Mohammed Specialist Hospital Kano.This was a cross-sectional study carried out from June 2015 to May 2016 at two health facilities in Kano City, Nigeria, a malaria endemic area.The population of interest in this study were HIV-positive adult participants who routinely visit the two health facilities to access ART services. They consisted of male and female (non-pregnant) patients (on ART and non-ART) aged 18 years and older enrolled in an ART programme who had not been on an antimalarial drug in the past 14 days and who willfully consented to participate in the study.®) software with the support of trained data entry clerks. We ensured data confidentiality through participant coding and restricting access to the data to only research team members.A total of 1521 consenting participants were randomly selected and blood specimens were collected and analysed for the presence of malaria parasite using both RDT and microscopy techniques and density of malaria parasites was determined. Additionally, relevant socio-demographic and clinical data including clients’ code, age, gender, use of co-trimoxazole prophylaxis, ART status (receiving or not receiving treatment), and episodes of fever were retrieved from both patients’ folders and Lafiya Management Information System  anticoagulant tube. The blood samples were mixed properly to avoid clotting before laboratory investigations.We used the MRDT kit (Code 05FK30) as described by the manufacturers .23 To ensure the quality of testing, MRTD and microscopy were performed by independent laboratory personnel and MRTD results were blinded to microscopists. All MRTD kits were stored according to manufacturer recommendation and used before the expiry date. The two microscopists responsible for slide examinations in both facilities have attended WHO training on malaria microscopy. Additionally, 10% of the slides were randomly selected for re-testing. About 2% of the results were discordant between the first and second readers and were subjected to further evaluation by a third reader before a final decision was made.For microscopy slide method, both thin and thick smear slides were prepared and examined. The microscopy procedure adopted was described by WHO.Malaria parasite density for each positive smear was calculated using individual white blood cell count according to this formula:p < 0.05.Data were reviewed, cleaned and validated in a Microsoft 2013 Excel file  and analysed using Statistical Package for Social Scientists . The sensitivity, specificity, positive predictive value and negative predictive value of MRDT were estimated against blood smear microscopy (BSM), the gold standard, at 95% confidence interval in relation to (1) malaria parasite density count, (2) fever (presence or absence), (3) CD4+ T cells count, (4) co-trimoxazole prophylaxis use, and (5) ART status (currently on ART or not taking ART). In addition, differences in performance of MRDT against BSM were analysed  based on level of ‘equality of marginal positives’ in disease classification between the two using McNemar tests and significance level was set at This study included a total of 1521 HIV-positive participants consisting of 1074 (70.6%) women and 447 (29.4%) men. The mean age was 37.20 with a standard deviation (SD) of 10.41 years.n = 772, 50.8%) were on daily dose co-trimoxazole prophylaxis for prevention against opportunistic infection including malaria parasite infection while 749 (49.2%) were not on co-trimoxazole prophylaxis. About (7%) complained of fever. The mean current CD4+T cells count of the participants was 401.35 cells/µL ± 239.24 cells/µL, and ranged from 6 cells/µL to 1736 cells/µL. The majority  of the respondents had a CD4+T cells count lower than 500 cells/µL, while 31.8% (n = 483) had CD4+ T cell counts of 500 cells/µL or higher. The mean malaria parasite density was 265 cells/µL ± 31.8 (SD) cells/µL with a range of 20 cells/µL to 2500 cells/µL. A majority (87.8%) had malaria parasite density less than 500 cells/µL, while the rest (12.2%) had parasite density 500 cells/µL or higher.Data on clinical information showed that the majority (84.7%) were on ART. An almost equal proportion of the participants  participants  and only 16.4% with MRDT. It is worth nothing that participants were recruited at routine ART follow-up visits, with or without symptoms of malaria. Almost all (99.2%) malaria infections were icipants . The oveicipants . A 2.5% p < 0.001), with the MRDT demonstrating a ‘fair’ performance and discriminatory capacity of 0.775  of the studied population.Overall, there was significant difference in MRDT performance compared to BSM .MRDT performance was significantly different from BSM findings, regardless of malaria parasite density count of participants (p < 0.0)The MRDT showed slightly lower but similar sensitivity in cases with and without fever, but similar specificity for both groups of participants. However, positive predictive value was higher (95.6%) among clients that complained of fever compared to those without (88.1%) fever . In thosp < 0.001) in both groups.MRDT performed similarly in sensitivity and specificity in both groups, although higher positive predictive value was observed among participants who were not on co-trimoxazole . Regardlp < 0.001).MRDT revealed higher  diagnostic accuracy among participants with CD4+T cells count greater than 500 cells/µL. This was lower  among participants with CD4+T cells count under 500 cells/µL . The obsp < 0.001).The MRDT revealed a higher (65%) sensitivity among HIV participants who were not on ART compared (56%) to those on ART. A similar pattern of higher positive predictive value (98%) was observed among the non-ART group compared to 87% among the ART group . For botWe detected malaria parasites in 25.4% of participants by BSM and 16.4% by MRDT. Overall sensitivity of MRDT was 58% and specificity was 97%. The sensitivity and specificity were similar irrespective of co-trimoxazole and fever status. At malaria parasite density count of under 500 cells/µL, sensitivity was 73% and between 500 and 999 cells/µL, sensitivity was 34%. At CD4+ T cell count over 500 cells/µL the sensitivity was higher (62.4%) compared to 56% at under 500 cells/µL. In the non-ART group sensitivity was higher (65%) compared to those on ART (56%) but the specificity was similar.15 Since WHO recommended the use of MRDT, several test kits have flooded the market, hence the need for constant field validation to verify manufacturers’ claims. This is critical, particularly among HIV-positive patients in whom other febrile illnesses and infections mimic malaria.5Although blood smear microcopy still remains the gold standard for malaria diagnosis, several limitations ranging from infrastructure and equipment to competence still exist in endemic areas like Nigeria. These justify the need for an alternate method like MRDT.24. However, our findings varied from a similar study in Lagos, Nigeria, involving a smaller sample size of 387 HIV patients where a malaria prevalence of about 19% was reported for both BSM and MRDT of another brand19. Another study in Burkina Faso among 114 HIV-positive participants showed a malaria prevalence of about 45% for MRDT and 42% for BSM.21 It is worth noting that in this study, participants were recruited at routine follow-up visits, regardless of suspicion of malaria unlike the other studies comparing performance of BSM and MRDT in HIV-positive patients suspected of suffering from or having symptoms of malaria. For example, the Tanzania24 and Burkina Faso21 studies looked at HIV-positive children suspected of malaria, while those from Lagos,19 Uganda,20 and Malawi25 looked at adults with HIV attending outpatient clinics suspected of having malaria. Thus, the direct comparison of proportions positive for malaria in our studies with the others must be considered with caution.The prevalence of malaria in HIV-positive patients in this study was 25.4% based on microscopy compared to 16.4% based on MRDT method. This was similar to a Tanzanian study reporting a malaria prevalence of 23.8% using microscopy method among HIV-positive patients compared to 17.5% with MRTD19 Contrary to our results, a 100% sensitivity and similar specificity of about 95.4% was documented in a Burkina Faso study among HIV-positive participants.21 Other studies on MRDT performance among HIV-positive population in Malawi,25 and two different studies in Uganda20 have shown higher sensitivity of over 94%. In these studies, the specificities averaged over 97% like in ours except for the Malawi study that reported about 51% specificity.25MRDT overall sensitivity and specificity reported in this study was similar to a 55.4% sensitivity reported in Lagos, Nigeria, but varied in specificity (90.3%).26 Using MRDT, a false positivity rate of 5.9% had been reported in Burkina Faso among HIV-positive patients.The 2.5% false positive results observed with the use of MRDT may be an indicator of residual antigenemia. The average time that histidine rich protein-2 (pFHRP-2) remains positive after resolution of parasitaemia is about 2 weeks, although it has been shown that the protein can take over a month to clear.28 It has been reported to exhibit a high level of polymorphism.30 This is an important factor that may affect the performance of MRDT based on antigen detection.32 In addition, false negative results have been associated with blood samples taken beyond the period of malaria fever paroxysms.33P. falciparum isolates without the HRP2 gene have been shown to be important contributors to false negative HRP2-based MRDT testing.35 Moreover, only Plasmodium falciparum releases HRP2; therefore, the presence of non-falciparum infection as another malaria species co-infection in Plasmodium falciparum may also give a false negative result. Other factors that might possibly affect MRDT field performance are shelf life and heat stability.37 In this study, the test kits were stored based on manufacturers’ recommendations and used before the expiry date on the MRDT kits.The 27.55% false negative results observed might be partly due to deletion or mutation of HRP2 gene in the malaria parasite, which is the most common target for MRDT.19 Generally, at lower parasitaemia, variability in sensitivity is more common.39 Similar observation of false negative MRDT results at parasite counts even higher than 1000 cells/µL have been reported in other studies41 and may be attributed to prozone effect observed with immunochromatographic tests such as MRDT.42 All the same, many MRDTs today can achieve excellent sensitivity and specificity for P. falciparum at parasite density lower than 500 cells/µL.11Unexpectedly, with the MRDT, we observed a lower sensitivity of 34% among HIV-positive participants with a malaria density count between 500 cells/µL and 999 cells/µL compared to a 73% sensitivity when the malaria density count was less than 500 cells/µL. However, the specificity at between 500 cells/µL and 999 cells/µL was 97.5%. This was contrary to a previous study in Lagos, Nigeria, that observed an increase in sensitivity from 90.9% at parasite densities greater than 200 cells/µL to 97.6% at densities greater than 500 cells/µL.10 MRDT performance in our study showed similar sensitivity and specificity as reported by Mills et al.,10 in febrile or non-febrile cases in rural Uganda. Apparently, MRDT reliability is independent of febrile status.Besides this study, and to the best of our knowledge, only one other study has assessed performance of MRDT among HIV-positive patients based on febrile status in Africa.43MRDT performance was similar in terms of sensitivity, specificity and positive predictive value regardless of whether a participant was on co-trimoxazole prophylaxis or not. Information on similar studies is not available; however, with microscopy a relatively higher prevalence has been reported on those without co-trimoxazole prophylaxis compared with those on it.19MRDT performance revealed a higher sensitivity among participants with a CD4+ T cell count greater than 500 cells/µL compared to a lower sensitivity among participants with a CD4+ T cell count under 500 cells/µL. Although information on similar studies is not available, it has been reported in a similar setting that HIV-positive participants with CD4+ T cell counts 250 cells/µL or higher are significantly less likely to have patent parasitaemia.44 as well as reports of antimalarial properties of some antiretroviral drugs.46The MRDT revealed a higher sensitivity among HIV-positive participants who were non-ART compared to those on ART. Lower incidence of malaria among HIV-positive adults on ART have been reported in different settings47 For some of these reasons, such recommendation must be taken with caution in HIV-malaria co-infected persons, even though no differential effect of HIV infection on MRDT performance  has been reported in literature.25Based on other reports in the general population, the use of MRDT method as an alternative to BSM in malaria endemic areas is recommended for epidemiological studies. However, the performance varied depending on species of the malaria parasite, level of parasitaemia, and immunity.This study was sufficiently powered having a large sample size of 1521 HIV-positive participants across the two health facilities. Apparently, this may be the first study to assess the performance of MRDT in an African setting of significantly high HIV and malaria co-morbidity in association with variables like immune status, parasitaemia and some clinical  factors.The cross-sectional design of this study was a limitation as it was difficult to determine how the potential antimalarial effect of co-trimoxazole prophylaxis and ART could have affected MRDT performance over time. Note that we observed better diagnostic accuracy of MRDT among participants who were neither on co-trimoxazole prophylaxis nor on ART. Secondly, we did not measure individual body temperature to correlate it with participants’ reports of fever.P. falciparum histidine-rich protein type-2 and Plasmodium lactate dehydrogenase, is lower than WHO recommendation for MRDT.48 Thus, further evaluation is required to determine its suitability in malaria diagnosis among HIV-malaria co-infected patients in these settings.As observed in this study, the sensitivity of SD Bioline, which is based on It is worth speculating that a similar observation with the MRDT could have been observed in non–HIV-positive persons.The data reported in this current article reflect findings of work done on evaluation of standard diagnostic Bioline rapid test kits for malaria diagnosis among HIV patients in Kano, Nigeria, by our research team members who all participated in the study design, execution, collection, analysis of data and report writing.The research outcomes presented in this report were from the diagnostic evaluation of standard diagnostic Bioline rapid test kits for malaria diagnosis among HIV-positive participants in Kano, Nigeria, at Infectious Diseases Hospital and Murtala Mohammed Specialist Hospital Kano. Although previous studies have reported on the evaluation of rapid malaria diagnosis against gold standard microscopy methods with a focus on the general population, it is worth noting that data among HIV-positive persons under different conditions are not readily available. Hence, this study sought to address paucity of data on performance evaluation of standard diagnostic Bioline among HIV-positive patients in relation to various factors such as fever status, CD4+ cell count, ART status, co-trimoxazole prophylaxis and malaria density.The outcomes of this study and proposed recommendations could be useful in public health programme planning in such settings where HIV and malaria burden is high. Apart from disease prevalence data reported, malaria density count and performance of rapid test kits was also evaluated in addition to other clinical and useful variables. This may serve as a baseline upon which comparison may be made in future.


Intermittent preventive treatment (IPT) of malaria is recommended as policy for certain high-risk populations, but not currently for schoolchildren. A cluster-randomized trial was conducted to evaluate the effect of IPT with dihydroartemisinin–piperaquine (DP) on primary schoolchildren in Jinja, Uganda. Results of the impact of IPT of schoolchildren on community-level transmission have been reported previously. Here, secondary outcomes from a school-based survey are presented.Eighty-four clusters (one primary school plus 100 households) were randomized to intervention and control (1:1 ratio). Participants from intervention schools received monthly IPT with DP for up to 6 rounds (June–December 2014). At endline (November–December 2014), randomly selected children from all 84 schools were surveyed (13 per school) and thick blood smears were done. Those with fever or history of fever were tested with rapid diagnostic tests (RDTs) for malaria. Haemoglobin was measured in every fifth participant. Outcome measures included prevalence of asexual parasites and gametocytes (by microscopy), and prevalence of anaemia. Prevalence outcomes were analysed using generalized linear Poisson models with log link function, incorporating a cluster-level random intercept and quantified using prevalence risk ratios.Among 23,280 students listed on the 42 intervention school registers, 10,079 (43.3%) aged 5–20 years were enrolled into the IPT intervention and received at least one dose of DP; of these, 9286 (92.1%) received at least one full (3-day) course. In total, 1092 children were enrolled into the final school survey (546 per arm) and had a thick blood smear done; of these, 255 had haemoglobin measured . Children in the intervention arm were less likely to have asexual parasites , gametocytes , fever , or symptomatic malaria . Prevalence of anaemia and mean haemoglobin were similar in both study arms.School-aged children are a major reservoir of malaria parasites. Delivering IPT to schoolchildren would benefit individual children and may reduce transmission. School-based IPT could help to intensify malaria control toward elimination, and should be considered for policies and programmes.Trial registration Clinicaltrials.gov (NCT02009215), Registered 11 December 2013. https://clinicaltrials.gov/ct2/show/NCT02009215 Malaria remains a major global health problem. Despite malaria control achievements over the last 20 years , recent Children under five are at high risk of malaria in endemic areas and have typically been targeted for malaria control interventions, along with pregnant women. However, this approach overlooks school-aged children whose burden of disease is also important ; they arIntermittent preventive treatment (IPT) is a well-established malaria control intervention, which is recommended for pregnant women and infants. IPT of malaria in children under-five has also been operationalized as seasonal malaria chemoprevention (SMC) in West Africa. Chemoprevention is not currently policy for school-aged children, but extending SMC programmes to include older children has been investigated within the Sahel region of Africa , 19. EviTo further evaluate IPT of malaria in schoolchildren, a cluster randomized trial was conducted in Jinja, Uganda in 2014–2015. Children enrolled from intervention schools received monthly IPT with dihydroartemisinin-piperaquine (DP) for up to 6 rounds of treatment. The primary objective of the trial was to evaluate the impact of IPT of schoolchildren on community-level indicators of malaria transmission, as measured in a post-intervention cross-sectional community survey and continuous entomological surveillance; these results have been reported previously . Here, tThe study was carried out in Jinja district in eastern Uganda, where malaria transmission is perennial Fig. . At baseDigitally enumerated maps were used to define clusters, including one primary school plus the 100 closest households surrounding the school . One schPrior to the study, team members met with key stakeholders in health and education at the national and district levels to sensitize them to the trial using an information sheet. Verbal consent to participate was obtained from headteachers of all schools, and copies of the school registers were obtained from intervention schools. Recruitment for the IPT intervention was carried out continuously from March to December 2014.Study personnel met with parents/guardians to review initial eligibility criteria, including: (1) able to locate parent/guardian, (2) enrolled in intervention school, (3) age ≥ 5 years (4) no known allergy to DP, (5) no menarche , (6) no history of cardiac problems or fainting, (7) no family history of long QT syndrome, (8) not currently using medications known to prolong the QT interval, and (9) willingness of parent/guardian to provide written informed consent. If these criteria were met, children were reviewed individually at school by study personnel for the final criteria, including: (1) able to locate student, (2) no menarche , (3) weight ≥ 11 kg, and (4) provision of written assent by student (aged 8 years or above). Children who passed screening underwent a brief physical examination, including measurement of weight and were fingerprinted to facilitate identification for future IPT treatment.Participants in the IPT intervention received DP  monthly for up to 6 rounds of treatment (June to December 2014). DP was administered by study personnel orally once daily for 3 days, using full-strength DP tablets (40/320 mg), according to weight-based guidelines . All treatments were directly observed. The trial was open-label, and no placebo treatment was provided in the control schools.A cross-sectional survey of children from each participating school was conducted toward the end of the intervention (November–December 2014). A random sample of children was selected from each school register. Recruitment and screening were stratified by class, to ensure equitable distribution of participants from all classes (P1–P7), until the target sample size of children per cluster was reached. A similar survey was conducted at baseline prior to the intervention delivery and was used to inform sample size calculations.Study personnel invited parents/guardians of selected children for a meeting at the school to screen for eligibility criteria, including: (1) enrolled in a participating primary school, and (2) agreement of parents/guardians to provide informed consent. If these criteria were met, study personnel interviewed each child individually at school for the final criteria, including: (1) able to locate student and (2) provision of written assent by student (aged 8 years or above). Children were excluded if they could not be located on more than 3 occasions.CareStart™ Malaria HRP-2 P. falciparum; ACCESSBIO). Children with a positive RDT were treated with artemether–lumefantrine unless they exhibited signs of severe malaria, in which case they were referred to appropriate health facilities. Detailed records of school attendance were collected from both intervention and control schools.If selection criteria were met, a brief questionnaire was administered to collect information on age, gender, bed net use, location of residence, and a brief food history. Participants had their temperature measured, and a finger-prick blood sample was obtained for a thick blood smear and haemoglobin measurement (in every fifth participant) using a portable spectrophotometer . RDTs were performed on participants with fever (tympanic membrane temperature of ≥ 38.0 °C) or history of fever in the past 48 h , assuming a leukocyte count of 8000/μl. A thick blood smear was considered negative when the examination of 100 high power fields did not reveal asexual parasites or gametocytes. For quality control, all slides were read by a second microscopist and a third reviewer settled discrepant readings.The primary outcome among school children  was prevalence of asexual parasitaemia, as measured by microscopy of individual blood smears. Secondary outcomes were prevalence of gametocytaemia, prevalence of anaemia , mean haSample size was determined for the trial’s secondary outcome, smear positive microscopy among school children, and was refined in October 2014 after results from the baseline survey were available. Thirteen randomly selected children in each of the 84 clusters , had over 80% power at significance level 5% to detect a relative reduction in parasite prevalence of 35%, corresponding to an absolute difference in parasite prevalence of 11.5% (33% vs 21.5%), assuming a coefficient of variation between clusters of 0.50.A secondary objective of the trial was to evaluate the impact of IPT for malaria in schoolchildren on clinical malaria indicators, aiming to test the hypothesis that the prevalence of asexual parasitaemia would be lower in the children enrolled in intervention schools than in those enrolled in control schools. The plans for the analysis presented here were outlined a priori in the statistical analysis plan for the trial. Data were analysed at the individual-level due to the large number of clusters per trial arm  and statThe trial was approved by the Ugandan National Council for Science and Technology (UNCST Ref HS 1530), Makerere University School of Biomedical Sciences Research & Ethics Committee (SBS REC 145), London School of Hygiene and Tropical Medicine Ethics Committee (LSHTM Ref 6509), School of Biological and Biomedical Sciences Ethics Committee, Durham University (DU SBBS/EC/STARTv5.0/June15) and University of California, San Francisco Committee on Human Research (UCSF CHR Ref 074826). Sponsorship and insurance were provided by the LSHTM’s Clinical Trials Sub-Committee (Ref QA380). The trial was overseen by an independent Data and Safety Monitoring Board and a Trial Steering Committee.The funders played no role in the design of the study; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.Of the 84 primary schools participating in the trial, most were public (85.7%). There were 23,280 children listed on the registers for the 42 intervention schools  vs 21,299 listed for the control schools . As previously reported , a totalThe final school survey was conducted from 13 November to 5 December 2014   is a well-established malaria control intervention that is recommended for certain high-risk populations, but is not current policy for schoolchildren. In this cluster-randomized trial, IPT with DP delivered monthly to primary schoolchildren was associated with a substantial reduction in parasitaemia in all ages, particularly in rural areas. Children in the intervention arm were significantly less likely to have fever and symptomatic malaria, and also less likely to have gametocytaemia. These results contribute to the growing body of evidence that targeting chemoprevention to school-aged children provides important health benefits to individual children. Moreover, by reducing parasitaemia and particularly gametocytaemia, which might otherwise go untreated, IPT of schoolchildren stands to reduce the infectious reservoir of parasites available for onward transmission of malaria. IPT could be integrated into existing school infrastructure and programmes thus expanding the tools available to control malaria in school-aged children and the community as a whole.In this study, a substantial number of children in the control arm were febrile and were subsequently diagnosed with symptomatic malaria by a positive RDT on the day of the survey; IPT markedly reduced the risk of fever and symptomatic malaria. There is growing awareness of the burden of malaria in school-aged children , and thePlasmodium falciparum in Burkina Faso and Kenya, found that gametocyte carriage was common in asymptomatic individuals and that children were more likely to infect mosquitoes than adults [P. falciparum and Plasmodium vivax [School-aged children are important contributors to the human infectious reservoir of malaria . In malan adults . After aum vivax . These fum vivax . In the um vivax . The resIn this study, IPT with DP was associated with a substantial reduction in parasitaemia in rural areas, but these differences were more modest in the urban area of Jinja town. Parasite prevalence in the control arm varied widely between the rural (51.2%) and urban (8.8%) areas. Reduced transmission and burden of malaria in urban settings is well-described , likely Schools provide a potential platform for delivery of health programmes to older children. Schools are already targeted for malaria control interventions in Uganda, acting as sites for targeted delivery of LLINs to schoolchildren. Selected schools located in hard-to-reach areas host facility outreach distribution programmes aiming to maintain universal coverage of LLINs achieved through mass LLIN distribution campaigns led by the Ministry of Health. Schools also serve as a focal point for societal and behaviour change communication. Ugandan schoolchildren have been engaged to deliver malaria intervention messages to their families and friends, thus acting as ‘change agents’ within their communities . AlthougP. falciparum malaria is a well-recognized risk factor for anaemia in malaria-endemic settings [In this study, IPT with DP did not have an impact on haemoglobin levels or prevalence of anaemia. The aetiology of childhood anaemia in low- and -middle income countries is multifactorial and complex . Howeversettings , 48, alosettings . The reasettings , 49.This study had several limitations. First, intervention coverage in the study population was suboptimal. Only 65.2% of children enrolled in the survey from the intervention arm received at least one full dose of DP, and only 2.8% received the full 3-day course of DP in all 6 rounds of treatment. However, despite this low coverage, a marked reduction in parasitaemia was achieved in the intervention arm, even in those children who did not receive DP. Of note, IPT coverage in the school surveys, although low, was still higher than in the main trial, which does not rule out potential selection bias. Secondly, in this single cross-sectional survey of schoolchildren, outcomes were only measured at a single timepoint. Prospective evaluation of children in a longitudinally followed cohort might have been preferable, as would longer term follow-up of children to assess sustainability of impact, but resources precluded this type of evaluation. Finally, parasitaemia and gametocytaemia were only measured using microscopy, which may have underestimated prevalence measures. Indeed, there is an increasing appreciation of sub-microscopic infection and more sensitive molecular tests could have been applied, including loop mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) for asexual parasites , 51, andIn this cluster-randomized trial, IPT with DP delivered to primary schoolchildren reduced parasitaemia, gametocytaemia, fever, and symptomatic malaria. These results contribute to the growing body of evidence that targeting chemoprevention to school-aged children would benefit individual children. Moreover, IPT of schoolchildren stands to reduce malaria transmission by reducing the infectious reservoir of malaria, which could be an important new tool for countries seeking to intensify malaria control on the pathway to elimination. School-based IPT could be integrated with school infrastructure and interventions, thus leveraging existing resources while expanding the toolbox for malaria control. Future research on IPT of schoolchildren should explore strategies to achieve high coverage, approaches to integrate IPT with other school-based interventions, and cost-effectiveness, particularly in urban settings. The potential impact of IPT of schoolchildren on community-level malaria transmission should also be investigated further, ideally at higher coverage levels and in different epidemiological settings.


Plasmodium falciparum-specific MHC class I-restricted CD8+ T cells are important for sterile protection against Plasmodium falciparum infection. Here, we present an interactive epitope map of all P. falciparum-specific CD8+ T cell epitopes published to date, based on a comprehensive data base (IEDB), and literature search. The majority of the described P. falciparum-specific CD8+ T cells were directed against the antigens CSP, TRAP, AMA1, and LSA1. Notably, most of the epitopes were discovered in vaccine trials conducted with malaria-naïve volunteers. Only few immunological studies of P. falciparum-specific CD8+ T cell epitopes detected in patients suffering from acute malaria or in people living in malaria endemic areas have been published. Further detailed immunological mappings of P. falciparum-specific epitopes of a broader range of P. falciparum proteins in different settings and with different disease status are needed to gain a more comprehensive understanding of the role of CD8+ T cell responses for protection, and to better guide vaccine design and to study their efficacy.Control of malaria is an important global health issue and there is still an urgent need for the development of an effective prophylactic vaccine. Multiple studies have provided strong evidence that  Plasmodium species, Plasmodium falciparum , accounts for the vast majority of the 445,000 deaths which occurred in 2016 due to malaria. Rising rates of drug (parasite-related) and insecticide (vector-related) resistance underscore the urgent need for an effective vaccine  and therefore prevents clinical disease but not the infection of hepatocytes , 7, 25, P. falciparum-specific antibodies, parasite-specific CD8+, and CD4+ T cells, as well as certain cytokines have been implicated as important effectors  total ] subject, P. falciparum-specific CD8+ T cell epitopes that were published at least in three different publications and/or showed a response frequency of 40% or greater or other important features will be listed according to each of these proteins. In the following section MHC class I-restrictedPlasmodium life cycle  is an important protein of the pre-erythrocytic stage of the parasite. It is the most abundant surface protein on sporozoites and has important roles in the fe cycle . The funatocytes –151. Viaatocytes . The funnstrated .Plasmodium species, suggesting conserved essential roles for each domain. The protein consists of the N-terminus, the central tandem repeat region and the C-terminus (The CSP protein is 397 amino acids in length and shows a similar structure among different terminus  153)..PlasmodiKLKQP) conserved in almost all Plasmodium species , is a very well conserved part of the CSP . It cont species . The N-t species , 155. Cl species , 157. An in vivo .MMRKLAILSV shows a high response frequency (RF) of 40% (6/15) in a study with volunteers from a malaria endemic area  in immunized but malaria-naïve volunteers (ILSVSSFLFV showed response frequencies of 50% (1/2) to 71.4% (10/14) in studies with malaria-naïve volunteers who were immunized with CSP, but the epitope could also be detected in inhabitants from a malaria endemic-area [RF: 5.9% (1/17) to 8.3% (3/36)] ] , 144.Within the N-terminus of CSP Sedegah et al. detected responses directed against 15 CD8+ T cell epitopes in 2013 . They peLRKPKHKKL was detected in both, vaccinated malaria-naïve volunteers and volunteers from an endemic area and was restricted to HLA-B8 (LRKPKHKKL showed a RF of 18.2% (2/11) in a malaria-endemic study population  domain , 167 Fi. Within Within the C-terminus of the protein, the following epitopes have been described :MPNDPNRNV was tested in malaria-naïve volunteers as well as in subjects from a malaria-endemic area. RF varied from 1/10 (10%) to 1/3 (33%) in malaria-naïve volunteers immunized with CSP  to 100% (8/8) (Y/KLN/KIQ/KNSL/I is the best characterized T cell epitope and was described in 8 publications (YLNKIQNSL was recognized in 23.5% (4/17) volunteers from a malaria-endemic area. In malaria-naïve volunteers who were immunized with a subunit vaccine RF was 13.3 (2/15) to 100% (5/5). YLKKIKNSL, YLKKIQNSL, and KLKKIKNSI were all detected in a malaria-endemic study population. RF for YLKKIKNSL was 60% (9/15) to 100% (1/1), for YLKKIQNSL 33.3% (5/15) and to KLKKIKNSI responded 41.2% (7/17) of the study population. The epitope VTCGNGIQVR is located within the region II-plus. This epitope was tested in subunit vaccine trials [RF = 10% (1/10) to 57.1% (4/7)], with irradiated sporozoites [RF = 100% (4/4)] and naturally exposed volunteers [RF = 17.8% (8/45) to 41.7% (5/12)]  , 144. Thications , 175. Itications . YLNKIQN (5/12)] , 144. ThHLA-A*11 .KPKDELDY, restricted to HLA-B*35:01 was detected in malaria-naïve [RF = 100% (1/1)] and naturally exposed subjects (RF = 12.5% (1/8) to 20% (3/15)] (KSKDELDY had a RF of 10% (1/10) to 12.5% (1/8) in a naturally exposed population. (3/15)] , 176. KSGLIMVLSFL was tested in subunit vaccine trials [RF = 6.7% (1/15) to 78.6% (11/14)], with irradiated sporozoites [RF = 100% (2/2)] and naturally exposed volunteers [RF = 9.1% (1/11) to 11.1% (4/36)] ( (4/36)] , 145.The CSP is the most frequently investigated antigen in terms of the number of different studies and number of described T cell epitopes. This is probably due to the fact that CSP was the first of the malarial proteins to be cloned, and as a result, significant efforts have been undertaken to investigate this protein . Ever siTRAP is a 559 AA long protein that induces antibody and T cell responses . This trThe N-terminus of TRAP contains the A domain and thrombospondin type-I repeat (TSR) which are two adhesive areas that can bind to heparan sulfate proteoglycans (HSPG) , 182 Fi. Upon coC-terminus of TRAP remains intracellularly located and is attached to the sporozoite motor system  to 100% (1/1). 11.1% (4/36) of volunteers from a malaria-endemic area and 50% (1/2) of volunteers immunized by irradiated sporozoites responded to the peptide  of volunteers naturally exposed to malaria responded to this epitope ] and in study participants who had been experimentally immunized with irradiated sporozoites [75% (3/4)] ].RF scores show that 28.6% (2/7) to 100% (1/1) of subjects from a malaria-endemic area responded to IAGGLALL , 63, 101% (3/4)] , 145. LLPlasmodia, attach to the host cell. Secondly, reorientation of the merozoites brings the apical end of the parasite toward the host cell. Finally, the erythrocytes are invaded (AMA1 is both blood stage as well as a sporozoite stage antigen . It is i invaded –194.In order to enter red blood cells (RBC), merozoites release specialized proteins out of secretory organelles called micronemes and rhoptries. AMA1 is one of the proteins, that are released to the surface of merozoites prior to invasion , 195. A AMA1 is a 622 AA long protein. At its N-terminus, AMA1 consists of a propeptide that is removed before translocation of the protein onto the merozoite's surface . This clApicomplexa, suggesting a conserved function for these domains, whereas domain III is divergent . In this study, the P. falciparum-specific CD8+ T cells were detected by ELISPOT. Five subjects were tested, and the response frequencies varied from 20% (1/5) to 40% (2/5). TLDEMRHFY and NEVVVKEEY are the only CD8+ T cell epitopes that were detected with tetramer-technology after immunization with the NMRC-M3V-Ad-PfCA vaccine (TLDEMRHFY had a response frequency of 66.7% (2/3) and NEVVVKEEY of 100% (1/1). Other epitopes were detected after immunization with DNA plasmids and adeno-viral vectors of CSP and AMA1   in in: 3D7) , is onlyin: 3D7) . The pot malaria   and APFISAVAA showed a RF of 28.6% (2/7). Both epitopes were detected in a naturally exposed study population.Two epitopes restricted by the protective HLA-B53 alleles have also been found in LSA3 : EPKDEIVPKDEIVEV  and APFIPFISAVAA . ResponsP. falciparum malaria life cycle is far from clear. EXP1 is transported out of the parasite into the parasitophorous vacuole membrane  separated from the cytosol by the PV membrane (PVM) . The PVMmembrane , 205 aftmembrane . It is tmembrane . EXP1 hamembrane  and its membrane . EXP1 semembrane , 210.EXP-1 is a 162 AA long protein consisting out of a signal sequence (AA 1–23), an N-terminus (AA 23–79), a transmembrane domain (AA 79–101), and a C-terminus (AA 101–162) . Six epiAll epitopes were detected in subjects from an endemic area. Interestingly, no epitope within the N- or C-terminus has been described so far.in vivo  are thought to be the primary ligands responsible for the low-affinity interactions between the merozoites and the erythrocytes [reviewed in Cowman and Crabb ]. The moin vivo , 214. Wiin vivo , only onin vivo : KLKEFIPnic mice .MPLETQLAI had a response frequency of 100% (4/4) in subjects immunized with irradiated sporozoites. It was also tested in volunteers from a malaria endemic area [RF: 6.7% (3/45)−30% (3/10)] ] , 74.P. falciparum sporozoite invasion of hepatocytes (MINAYLDKL is restricted by HLA-A*02:01 and *02:02. All studies that were conducted included subjects from endemic areas. Response frequency varied from 11.8% (2/17) to 75% (6/8) (Sporozoite threonine and asparagine-rich protein (STARP) is expressed on the surface of sporozoites that invade hepatic cells, which suggests that it plays a role during parasite entry into the hepatic cell and infection . The STAatocytes . So far,atocytes . MINAYLD5% (6/8) , 141.P. falciparum-specific CD8+ T cell epitopes published so far, and show their role in the Plasmodium life cycle, potential importance for vaccine development, point out areas where there are still knowledge gaps and outline future directions.Here we present a comprehensive overview of all human P. falciparum-specific CD8+ T cell epitopes that have been described in the literature, the respective study population and response frequency, methodology used for detection, and corresponding restricting HLA-molecule.We summarize the data of 132 different P. falciparum-specific CD8+ T cells, for immunomonitoring in vaccine trials, and finally to assist researcher interested in the development of a subunit vaccine. The different and detailed epitope maps give an overview of all epitopes within the antigens  study populations were tested (an average of 10.3 subjects). Notably, the response frequencies varied between the different study populations. The group that was immunized with irradiated sporozoites had the highest response frequency (72.6%). The group that was immunized with parts of the plasmodial antigens showed a response frequency of 26.8% and the volunteers from an endemic area had a RF of 16.0% only. The very high RF after immunization with irradiated sporozoites might explain why this is the most effective form of vaccination so far  the discovery of new specific malaria epitopes combined with the overall low frequency of  T cells , 226. Stchniques .P. falciparum-specific T cell answer was published before 2000  during an acute disease are lacking because specific CD8+ T cells have rarely been investigated in malaria patients.Most studies were performed either in the setting of a protective vaccine or in healthy exposed volunteers. Future studies should look at the kinetics of the priming of the CD8+ T cell response during a natural and acute malaria infection with longitudinal long-term follow up to understand the longevity of the Even though a T cell response is mounted against a certain epitope does not mean that this response will correlate with protection. E.g., RAS-vaccines are likely to induce CD8+ T cell responses to many sporozoite proteins, but only a subset of these will be presented by liver stage infected hepatocytes which are the target of protective immunity . In diffP. falciparum-specific T cells in humans are missing. Fine needle aspiration could be introduced as a useful tool to investigate liver-resident T cells because understanding of the CD8+ T cell response from peripheral blood samples has proven to be difficult  but it could also reflect the focus of scientific investigations .The biggest effort in vaccine development was put into the assessment of the humoral response . Neverthigations . It is aP. falciparum-specific CD8+ T cell epitopes is hampering optimization for a malaria vaccine.Further and more comprehensive studies using different cohorts, novel and more sensitive immunological techniques and assays and comprehensive, overlapping peptide sets for a number of different malaria proteins are warranted. In other words: the lacking knowledge of potential P. falciparum-specific immune response on an epitope level is of utmost importance for the detailed understanding of the role of CD8+ T cells for control of disease and will give us the tools to optimize vaccine design, immune monitoring of future malaria vaccine trials and to better understand naturally acquired immunity.In summary, the discovery of the full breadth and exact specificities of the All datasets generated for this study are included in the manuscript and/or the supplementary files.JH, TJ, and JSzW: conception. JH and JSzW: first draft. All authors: important contributions and proofread.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It is estimated that over a third of the world population is infected by malaria and helminthiases mainly among communities with high poverty indices. The distribution of these parasitic infections overlaps in many epidemiological settings and have varying outcomes in the host. In this paper we report the prevalence of malaria and intestinal helminthiases coinfections among malaria suspected patients and the association of helminthiases with the occurrence of malaria and its outcomes in Wondo Genet, southern Ethiopia.+). Difference in proportions and means were tested by Student’s t test and ANOVA while logistic regression analysis was used to determine the association between variables.In a cross-sectional study conducted from December 2009 to July 2010 in Kella, Aruma and Busa Health Centers in Wondo Genet, a total of 427 consenting febrile patients were screened for malaria and intestinal helminths infections. Malaria parasite detection and quantification were done using Giemsa stained thick and thin blood films. Helminth infections were screened and quantified by Kato-Katz thick smear method. Haemoglobin level was assessed using haemocue machine , Trichuris trichiura (21.55%), Schistosoma mansoni (13.35%), and hookworms (6.79%). The overall malaria-helminthiases coinfection was 33.96%. The prevalence of anaemia was 43.12%. Helminthiases coinfection showed a positive correlation with the occurrence of malaria . Schistosoma mansoni coinfection was associated with the increased risk of developing malaria associated anaemia .Of the total examined, 196 (45.90%) were positive for at least one helminth infection while 276 (64.64%) were positive for malaria. The prevalence of Malaria and helminth coinfections are important causes of morbidities among the population in Wondo Genet necessitating integrated control measures. Nevertheless, further detailed studies on the consequences and pathogenesis of these coinfections are needed to institute sound control and intervention measures. Malaria and intestinal helminths are co-existing important public health problems in many parts of sub-Saharan African , 2. AccoA. lumbricoides), 795 million (T. trichiura), and 740 million (hookworms) infections /d2, where: n = sample size, P = expected proportion in the study area, Z 1-α/2 = CI of 95%, d = marginal error to be tolerated. Accordingly, a total of 427 study participants were included in the study.Sample size was determined using single proportion formula at 95% confidence interval (CI) level (Z = 1.96) with the expected prevalence of 50% for malaria and 5% marginal error. The sample size was then calculated as Plasmodium species and determination of parasitaemia, respectively. Parasitaemia was calculated per 200 white blood cells assuming 8000 white blood cells per microliter of blood and classified as low, medium, high and hyperparasitaemia according to Garcia [Two slides were prepared for each febrile patient. The slides were free of dust, grease, soap, fingerprints and other debris so that the blood adheres to the slide for proper staining. On each slide, thick and methanol fixed thin blood films were prepared from capillary blood obtained by finger prick using disposable sterile lancet. Each blood smear was stained using 3% Giemsa stain for 30 min and examined according to WHO . The stao Garcia .Stool specimens were collected from the study participants using clean plastic vial and were processed for microscopic examination using Kato-Katz thick smear method (two slides for each specimen). Kato-Katz thick smears were prepared from stool specimen using 41.7 mg templates and examined quantitatively under 40x microscope objectives for ova of hookworms within 15–30 min. All the slides were carefully examined twice for the ova of other intestinal helminths after 30 min on the same day. The number of eggs per gram was calculated by multiplying the average number of eggs per slide by 24. The intensity of infections was classified as light, moderate and heavy .+ Ängelholm, Sweden) and anaemia was defined as per WHO criteria [Haemoglobin assays were carried out on finger-prick blood samples collected with sterile disposable lancet and the reading was carried out using a portable haemoglobinometer .SPSS version 17  was used for the data entry and statistical analysis. Student’s Overall, 427 malaria suspected febrile patients from three health centers of the study area were screened for malaria parasites and intestinal helminthiases between 2009 and 2010. Fifty five percent of the participants were males and 45% were females. The study participant’s age ranged from 6 to 77 years (mean ± SD = 20.8 ± 11.7) , P. vivax 71 (16.62%) and mixed infection by both Plasmodium species 3 (0.70%). There were no severe malaria cases presented during the study period.Out of a total 427 study participants screened for malaria, 276 (64.64%) were positive for malaria parasites. The P. falciparum and 39.44% of P. vivax infected study participants were anaemic with the mean haemoglobin level of 12.15 ± 1.98 g/dL and 12.48 ± 1.43 g/dL, respectively (range: 7.0 to 18.5 g/dL). There was no severe anaemia (haemoglobin < 5 g/dL) among the study participants. Among the P. falciparum infected study participants aged between five to < 12 years, 53.57% were anaemic (mean haemoglobin = 11.46 ± 1.98 g/dL). Likewise, 78.26% of children aged between 12 and 15, 29.63% of non-pregnant women age > 15 year old and 50.00% of males age greater than 15 years were found to be anaemic. There was statistically significant difference between the haemoglobin concentration of anaemic and non-anaemic P. falciparum infected study participants  , Trichuris trichiura (21.55%), Schistosoma mansoni (13.35%), hookworms (6.79%) and Hymenolepis nana (1.41%). According to the WHO [S. mansoni had 248.8 EPG whereas the mean intensity of T. trichiura, A. lumbricoides, hookworms and H. nana infections were 214.4, 2,003.6, 145.7 and 584 EPG, respectively.Out of the 427 study participants, 196 (45.90%) were positive for at least one helminth infection. Intestinal helminths observed were  the WHO  classes P < 0.001).The univariate and multivariate analysis for the association of gender, age or presence/absence of helminthiases coinfection on the course of malaria infection was presented in Table Schistosoma mansoni had increased the odds of developing anaemia  . The Multivariable logistic regression analysis with single helminth coinfection with malaria showed that Schistosoma mansoni  and Ascaris lumbricoides  had positive associations with the occurrence of malaria infection.In the present study, it was observed that helminth coinfection had no significant association with the level of haemoglobin concentration among malaria positive study participants. However, from a single helminth coinfection analysis among malaria infected participants, Malaria and helminths coinfection is a serious public health problem in developing countries where their distribution overlaps , 15, 25.A. lumbricoides, T. trichiura and S. mansoni in different parts of Ethiopia [A. lumbricoides (33.96%) and T. trichiura (21.55%) were lower than those of the national prevalence 37 and 30%, respectively [Similar to previous reports on Ethiopia , 11, 26,Ethiopia . Similarectively . Intestiectively  and 43.5ectively . Similarectively –34.P. falciparum among children aged five to < 12 years (53.57%) and those aged 12–15 years (78.26%), reported in the present study, falls within this range. Nutritional deficiency and other etiologies might have added to this burden of anaemia.Although the causes of anaemia are known to be complex and multi-factorial, it is a major complication of malaria . An inadS. mansoni and A. lumbricoides, was found to be positively associated with malaria infection. This finding is similar with a report from Dore Bafeno, southern Ethiopia [P. falciparum infection. Other reports from the country showed the opposite finding in that helminth coinfections resulted in higher mean level of parasitaemia [Helminth parasite coinfection, especially Ethiopia  and fromEthiopia . Howeversitaemia  and incrsitaemia .S. mansoni was found to increase the odds of developing anaemia. Schistosome coinfection might be exacerbating anaemia arising from malaria [S. mansoni coinfection might reduce the effectiveness of malaria treatment [Based on the information that malaria and intestinal helminth coinfections reduce haemoglobin levels, it is presumable that their combined presence might interact to enhance the risk of anaemia. Several reports from Kenya, Nigeria, Thailand and some other countries of Africa have also suggested the existence of an additive impact of helminth-malaria parasite coinfection on anaemia , 39–41.  malaria . This is malaria . Other rreatment  indicatiThe present study showed the occurrence of helminth parasite coinfections with malaria and its association with the occurrence of malaria infection and the development of anaemia. The finding has relevant implications for control interventions of malaria and helminth parasite infections. However, it was found difficult to reach at a definitive conclusion about the interaction of helminthiases and malaria based on the cross-sectional studies conducted. This is the limitation of the study and a longitudinal/cohort study design on a mixed infection model would be required for a better understanding of the effects of helminth-malaria parasite coinfections.Malaria and helminthiases are important public health problems in Wondo Genet. Lower prevalence of malaria compared with that of previous report  is attri