Infants under 6 months of age are often excluded from malaria surveillance and observational studies. The impact of malaria during early infancy on health later in childhood remains unknown.Plasmodium falciparum infections and clinical malaria. Poisson regression and linear mixed effects models measured the effect of exposure to malaria in infancy on subsequent malaria incidence, weight-for-age z-scores (WAZ), and haemoglobin concentrations after 6 months.Infants from two birth cohorts in Malawi were monitored at quarterly intervals and whenever they were ill from birth through 24 months for P. falciparum infection during their first 6 months had increased incidence ratio (IRR) of P. falciparum infection  and clinical malaria  compared to infants without infection. Infants with clinical malaria had increased risk of P. falciparum infection incidence between 6 and 24 months  and clinical malaria . Exposure to malaria was associated with lower WAZ over time (p = 0.02) and lower haemoglobin levels than unexposed infants at every time interval (p = 0.02).Infants with at least one Infants experiencing malaria infection or clinical malaria are at increased risk of subsequent infection and disease, have poorer growth, and lower haemoglobin concentrations. Plasmodium falciparum infection and clinical malaria disease remains highest among under-five children in many regions of sub-Saharan Africa. In these settings, children aged 6 months to 2 years may have up to five episodes of clinical malaria per year [P. falciparum infection and clinical malaria is more common than expected in the under 6-months population in sub-Saharan Africa [The burden of per year , 2. Infaper year , 4. Howen Africa –7. AssocP. falciparum infections in growth and development remains poorly defined and the literature is inconclusive [Plasmodium falciparum infection causes both red blood cell destruction and reduced production), resulting in low haemoglobin levels. The association is strong enough that anaemia prevalence has been proposed as an indicator of acute malaria prevalence in malaria endemic regions, though less is understood about the impact on subsequent anaemia in early childhood [P. falciparum infections are not well documented in infants, although there is evidence of an association between Plasmodium vivax and anaemia in infants [Malaria exposure in infancy may impact the health of young children, beyond the morbidity of acute infection. Infections of all varieties are known to impair nutritional status resulting in growth faltering, however the role of early nclusive –10. Malanclusive , 12. Plahildhood –15. Mala infants .P. falciparum infection and clinical malaria during early childhood. The study further evaluated the hypothesis that infants with P. falciparum infection or clinical malaria in the first 6 months may have lower weight-for-age z-scores and lower haemoglobin concentrations over time compared to unexposed infants.The study was designed to use longitudinal data collected from two birth cohorts in Malawi to measure malaria infection and disease in early infancy and the association this exposure has with subsequent malaria incidence in early childhood. Because malaria infection during early infancy may enhance or interfere with immunity and subsequent clinical response to malaria infection, the study tested the hypothesis that clinical malaria in the first 6 months of life would be associated with a statistically significant increase in incidence of Study participants from two prospective cohorts conducted in southern Malawi were included in this study because they were conducted during the same timeframe and with similar follow-up procedures. The Liwonde cohort enrolled infants born to mothers who participated in a clinical trial that randomly allocated 602 pregnant women to receive monthly administration of either sulfadoxine-pyrimethamine or dihydroartemisinin-piperaquine as intermittent preventive treatment during pregnancy (NCT03009526). Women aged 16 years or older, with a viable singleton pregnancy at less than or equal to 28 gestational weeks were included in the clinical trial if they were HIV-negative and were not experiencing a high-risk pregnancy or experiencing severe anaemia. During pregnancy, women were offered the opportunity to enroll their babies in the infant cohort after completing the clinical trial. Inclusion criteria for mothers enrolling their babies in the infant cohort included intention to stay in the study area for at least one year following delivery and provision of informed consent. Participant enrollment in the infant cohort began in May 2017 and follow up was completed in May 2019. Details of this cohort were described previously [The Mfera cohort enrolled 100 infants at delivery. Infant’s mothers were recruited for the study at the antenatal clinic or the well-child clinic. Enrollment criteria included: provision of informed consent by the parent or guardian, infant born to a mother with documented HIV-negative status, intention to reside in the study region for two years, and willingness to comply with study procedures and attend the health center for regular medical care. Enrollment for the Mfera cohort ended in January 2016 and the final participant completed follow-up in November 2018.Both studies were reviewed and approved by the University of Maryland Institutional Review Board and the Kamuzu University of Health Sciences College of Medicine Research Ethics Committee. Parents/guardians provided written informed consent for participation of the infant.All women in the Liwonde clinical trial delivered in the maternity wards at Machinga District Hospital in Liwonde, while infants in the Mfera cohort were born at the Mfera Health Centre in Chikwawa, both in southern Malawi. Data collected at enrollment included demographic characteristics and pregnancy history, including infant sex, maternal age, gravidity, and parity.Follow-up procedures were the same in both cohorts. Infants visited their respective study clinics for scheduled visits at 3-month intervals for up to 24 months. Growth measurements including weight, length, and head circumference were taken at each scheduled visit. Haemoglobin concentration was only measured in infants enrolled in the Mfera cohort. Mothers were encouraged to bring infants to the clinic in the event of illness. If an infant presented with signs and symptoms consistent with malaria at any visit , a Paracheck-Pf  rapid diagnostic test (RDT) for malaria was performed. All cases of malaria detected by RDT were treated with artemether-lumefantrine according to the national treatment policy in Malawi. Dried blood spots on 3M Whatman filter paper were collected at every scheduled and unscheduled visit from a finger prick.P. falciparum [Asymptomatic and submicroscopic infections were detected from the dried blood spots collected at every visit. Dried blood spots underwent DNA extraction and real time quantitative polymerase chain reaction (qPCR) at the University of Maryland malaria laboratory for detection of the 18S ribosomal RNA gene of lciparum . The qPCA)P. falciparum infection includes infants with a positive qPCR but excludes infants with signs and symptoms of malaria accompanied by a positive RDTAsymptomatic B)Clinical malaria was defined as signs or symptoms of malaria recorded at the clinic visit and a positive RDT, andC)P. falciparum infections include all infants with clinical malaria or a positive qPCR.All Exposure to malaria in the first 6 months of life was assessed and defined three ways:P. falciparum infection and clinical malaria cases, and incidence of clinical malaria between 6 months of age and the end of follow-up (up to 2 years of age) were measured by the total number of malaria cases divided by the number of person-years at risk. Mean weight-for-age Z-score (WAZ) was assessed at three-month intervals during follow-up (6–24 months) classified according to WHO child growth standards. Mean haemoglobin concentration was assessed at three-month intervals during follow-up (6–24 months) in the Mfera cohort only.Three outcomes were measured. Incidence of any malaria, including both asymptomatic Season of birth was classified as rainy (December–March) or dry (April–November) using historical definitions of rainy and dry seasons in the region. Length of follow-up times varied for participants, so an additional measure of seasonal exposure was included that measured the percent of study time each infant spent in the rainy season. A covariate measured malaria exposure during the follow-up period in the longitudinal analyses as a cumulative count of all malaria infections per individual.Infants with follow up that ended prior to 9 months of age were excluded from these analyses due to inadequate follow up time. Covariates assessed include infant sex, study site, season of birth, percent of follow-up time in the rainy season, low birth weight (< 2.5 kg), maternal age at delivery, and maternal gravidity. Although data from the two studies were combined, study site was included because of varying environmental factors between the two sites. Maternal age at delivery and gravidity were assessed continuously. Bivariate analysis using Chi-square tests and Student’s t-tests were conducted to describe participant characteristics.Poisson regression was employed to estimate the incidence rate of exposure to malaria in the first 6 months of life and the incidence rate ratio (IRR) for malaria up to 24 months of age comparing those with and without malaria exposure in the first 6 months of life. To adjust for the effect of covariates on the association between the predictors and outcomes, multivariable Poisson regression models were developed with offset of follow up time to control for varying infection and disease rates.Unadjusted mean WAZ and haemoglobin were plotted over time and adjusted linear mixed effects models, with participant as the random effect, were built and graphed. An interaction term of exposure and infant age was included in every model, regardless of statistical significance, to visualize the difference in slope between the exposed and unexposed infants.All analyses were conducted using SAS software, Version 9.4 of the SAS System for Microsoft Windows.P. falciparum infection was not different. While infants in the Mfera cohort comprised 21% of the total study population, they made up 47% of the infants with clinical malaria.A total of 437 infants with complete data and sufficient follow-up time were included in the final analyses Table . Infant P. falciparum infections in the first 6 months of life, the incidence of any P. falciparum infections after 6 months of life was 2 cases per person-year and incidence of clinical malaria after 6 months of life was 0.9 cases per person-year. Among the 49 (11%) infants with clinical malaria in the first 6 months of life, incidence of asymptomatic P. falciparum infections was 3.8 cases per person-year and incidence of clinical malaria was 2.3 cases per person-year after the first 6 months of life. Results are summarized in Table Among 127 (27%) infants in the first 6 months of life, there were 241 episodes of any malaria, with an incidence rate of 1.1 episodes per person-year and 69 episodes of clinical malaria with an incidence rate of 0.3 cases per person-year, all in the first 6 months of life. Among 99 (26%) infants with asymptomatic P. falciparum infection during their first 6 months was 27% higher than unexposed infants . Across all 3 analyses, mean WAZ decreased over time in both the exposed and unexposed infants, but never fell to more than two standard deviations below the mean  and the difference steadily increased over time so that while the mean haemoglobin levels of unexposed infants reached the normal range (> 11 g/dL) around 12 months, exposed infants only reached that threshold around 21 months. The difference in mean haemoglobin levels between infants exposed to clinical malaria during their first 6 months and unexposed infants was -0.57 g/dL (p = 0.03).Unadjusted mean haemoglobin concentrations for exposed and unexposed infants were below 11 g/dL from 6 months of age until at least 12 months of age, indicating mild to moderate anaemia in the first year of life Fig. . Mean haP. falciparum 6 months of life, whether symptomatic or asymptomatic, was associated with higher incidence of both malaria infection and clinical malaria in early childhood. Additionally, any malaria infection during the first 6 months of life is associated with lower haemoglobin levels over time and lower weight-for-age z-scores compared to children uninfected during the first 6 months.This study found that infection with P. falciparum infection before reaching 6 months, confirming that infants are susceptible to malaria during early infancy. Nearly twice as many infants had asymptomatic P. falciparum infections before 6 months as infants who experienced clinical malaria during that period, which is consistent with other studies demonstrating relatively high rates of parasitaemia and lower rates of symptomatic malaria illness before 6 months [Infants under 6 months of age have historically been excluded from malaria studies and thus the results about the burden of infection and disease in this population is an important contribution to the literature. One-third of infants in our study had at least one episode of clinical malaria or 6 months .P. falciparum, however despite their early infection or even multiple infections, the exposed infants in this study did not experience reduced incidence during follow-up suggesting that acquired immunity does not develop this early in life, mirroring the poor immunogenicity of live viral and certain antibacterial vaccines which do not induce immunity in young children. Alternatively, these higher rates may be a marker of environmental exposure to malaria, as demonstrated by the difference in malaria incidence observed in the study sites, with higher infection and clinical malaria rates in observed in the Mfera cohort, or a result of differential exposure to P. falciparum in utero.Several possible causes can explain our finding of higher rates of malaria infection and diseases among infants infected with malaria in the first 6 months of life. Acquired immunity to malaria develops after repeated infection with P. falciparum infection at 24 months compared to without malaria in their first 6 months. This may have been observed because infants with asymptomatic P. falciparum infection were classified as unexposed in the clinical malaria model, thereby raising the risk of infants classified as unexposed when assessing the risk posed by clinical malaria in the first 6 months of life and confirmed this with a sensitivity analysis that excluded infants with asymptomatic P. falciparum infection in the first 6 months of life.Mean WAZ scores in infants infected with clinical malaria in their first 6 months are higher than unexposed infants at 24 months, while these measures are lower in infants with asymptomatic Infants with clinical malaria in their first 6 months were diagnosed and treated so that their infections were cleared, whereas infants with asymptomatic infections could have prolonged infections, resulting in chronic infection causing slower growth and lower haemoglobin. This is consistent with recent research showing that asymptomatic malaria infections are prevalent in anaemic children .P. falciparum endemic areas [The analysis identified a trend of decreasing mean weight-for-age z-scores over time in all infants regardless of malaria exposure in their first 6 months. A systematic review of this topic surmised that most studies found no association between malaria exposure and malnutrition (WAZ ≤ − 2.0) among populations in ic areas . HoweverP. falciparum infections during their first 6 months of life already have low haemoglobin levels at 6 months of age and continue to have lower haemoglobin levels compared to their unexposed peers through 2 years of age. The data analysis approach tried to control for the possibility that this trend may be due to repeated infections during follow-up by adjusting the models for cumulative malaria infections between 6 and 24 months. Malaria infections cause haemoglobin concentrations to drop rapidly after initial infection and it can take 4–6 weeks to completely normalize [Malaria is a known cause of low haemoglobin; however, this study suggests that infants exposed to ormalize . Infant The primary limitation of this study is the inability to control for the confounding of environmental malaria exposure that is associated with both the exposure of interest and infection outcomes. If infants live in an environment with increased likelihood of acquiring malaria infection, they will have high rates of malaria throughout their childhood. This limits the ability to assess a causal relationship between early malaria infection and subsequent burden of infection and may confound our finding of decreased WAZ and haemoglobin after early malaria infection. An additional limitation in this analysis was missing data on socioeconomic status, nutrition, or breastfeeding. Socioeconomic status, in particular, may be a predictor of both malaria risk and food insecurity though the sample population belonged to the same socioeconomic group. Mothers in the Mfera cohort all reported breastfeeding until the end of follow-up, so dietary variation were unlikely to be different between exposed and unexposed infants in the haemoglobin analysis.This study demonstrated that infants with malaria infection in the first 6 months of their lives have chronically lower haemoglobin levels than uninfected infants in the same cohort and higher incidence of malaria overall during early childhood. These early malaria episodes do not appear to elicit protective immunity. Rather, malaria infection before 6 months of age may be an indicator of increased environmental exposure risk suggesting a need to implement additional preventative measures targeting the household or neighbourhood, for example, focal indoor residual spraying or provision of chemoprophylaxis to these children. These results demonstrate the long-term impact of early malaria infection exposure on early childhood health and suggest a need for additional public health guidance for monitoring haemoglobin levels in young children with a history of malaria infection.


Approximately 40% of children aged 6–59 months worldwide are anaemic. Iron-containing multiple micronutrient powders (MNPs) and iron supplements (syrup/drops) are used to combat anaemia in children in different parts of the world. However, evidence for functional benefits of iron supplementation in children is scarce, and potential risks remain poorly defined, particularly concerning diarrhoea and malaria. This trial aims to determine if: (1) the efficacy of iron supplements or MNPs (containing iron) given with malaria chemoprevention is superior to malaria chemoprevention alone, or (2) if the efficacy of malaria chemoprevention alone is superior to placebo on child cognitive development.IRMA is a four-arm, parallel-group, double-blinded, placebo-controlled, triple-dummy, randomised trial in Southern Malawi. The study recruits 2168 infants aged 6 months, with an intervention period of 6 months and a post-intervention period of a further 6 months. Children are randomised into four arms: (1) No intervention (placebo); (2) malaria chemoprevention only; (3) MNPs and malaria chemoprevention; and (4) iron syrup and malaria chemoprevention. The primary outcome, cognitive development (Cognitive Composite Score (CogCS)), is measured at the end of the 6 months intervention. Secondary outcomes include CogCS at a further 6 months post-intervention, motor, language and behavioural development, physical growth and prevalence of anaemia and iron deficiency. Safety outcomes include incidence of malaria and other infections, and prevalence of malaria parasitaemia during and post-intervention period.The trial is approved by the National Health Sciences Research Committee (#19/01/2213)  and the Human Research Ethics Committee (WEHI: 19/012) . Written informed consent in the local language is obtained from each participant before conducting any study-related procedure. Results will be shared with the local community and internationally with academic and policy stakeholders.ACTRN12620000386932. The trial setting represents malaria-endemic sub-Saharan African countries where anaemia and malaria coexist, which is an ideal place to study the benefits versus risks of iron supplementation  to establish a generalisable clinical rationale.The participant screening and inclusion criteria involve all infants within the recruitment age range across a large geographical catchment area which enables the generalisability of the study results to most national programmes in malaria-endemic countries.The trial compares two major delivery modes for iron in young children: multiple micronutrient powders and iron syrup which will help identify the ideal and most effective iron source for children.The trial uses a functional clinical health outcome—child cognitive development—as its primary outcome, ensuring the trial provides evidence of the functional relevance of universal iron interventions and malaria prevention.The trial is powered to detect a small but clinically relevant effect for the primary outcome of child cognitive development; the use of a post-intervention follow-up period enables the assessment of both the immediate and medium-term impact of the intervention.Anaemia remains a critical global health problem affecting up to 39.8% of children aged 6–59 months worldwide and 60.2% in Africa.1WHO anaemia prevention guidelines recommend two universal iron supplementation strategies for children, namely; (1) iron supplement drops or syrups —given daily to children 6–24 months in areas where the prevalence of anaemia is 40% or higher; and (2) iron-containing multiple micronutrient powders (MNPs): 10–12.5 mg of iron—given daily over 6 months in areas where the prevalence of anaemia is 20% or higher.et al assessed cognitive development, and no significant beneficial effect was seen (mean difference 1.65 (95% CI −0.63 to 3.94)), but data were sparse.7Universal iron supplementation programmes have shown benefits in haematological outcomes. A meta-analysisWhile the evidence for functional benefits of iron supplementation is scarce, there has been emerging evidence of potential harm, particularly regarding increased malaria risk in endemic areas. The seminal Pemba randomised controlled trial (RCT) in Tanzania was stopped early due to increased death, hospitalisation and malaria in iron replete children receiving iron compared with placebo and called for revisions of universal iron supplementation recommendations.7Plasmodium falciparum parasites are less effective at invading iron deficient red cells and prefer invading reticulocytes (which are increased in the recovery phase of iron deficiency anaemia when iron supplementation is given).P. falciparum using red cells from anaemic children.21 22There is evidence to suggest that iron deficiency is protective against malaria. A study of preschool children in Malawi found that iron-deficient children were at lower risk of clinical malaria and parasitaemia than iron-replete children.Malaria is a significant cause of severe anaemia, hospitalisation and death in endemic areas.28Despite few studies, the majority with relatively small sample sizes and varying methods of assessing cognitive function, evidence suggests that severe and uncomplicated malaria may be independent risk factors for cognitive impairment.30 31Intermittent preventive treatment in children (IPTc) involves the empiric treatment of malaria at regular intervals and is a potential strategy to decrease malaria rates and sequelae. A Cochrane review that included seven RCTs evaluated the effect of chemoprophylaxis and intermittent treatment for malaria in preschool children in areas with seasonal malaria and showed a reduction in clinical malaria and moderate-to-severe anaemia.11P. falciparum malaria is with artemisinin-based combination therapy (ACT). Dihydroartemisinin-piperaquine (DP) is an ACT and is among WHO’s five recommended first-line treatments.32Recommended treatment for Despite important gains made worldwide in malaria control since 2010, worldwide cases increased in 2016. In Malawi, there were a total of 5.2 million presumed and confirmed malaria cases in 2016, 231 970 more cases than the year prior, resulting from a gradual increase in cases since 2014.36Both malaria and iron deficiency anaemia remain important causes of morbidity and mortality in Malawi. In Malawian children under 5 years of age, malaria is the leading cause of death,Several fundamental questions concerning the benefits and safety of iron-intervention programmes in malaria-endemic regions remain unanswered. The IRMA trial will assess functional health outcomes from iron supplementation and identify the ideal mode of iron delivery (MNPs vs iron syrup/drops). It will also allow evaluation of the effect of malaria chemoprevention alone on anaemia and child development.The primary objective is to determine (1) if the efficacy of iron supplements or MNPs (containing iron) given with malaria chemoprevention is superior to malaria chemoprevention alone, or (2) if the efficacy of malaria chemoprevention alone is superior to placebo, on child cognitive development  Cognitive Composite ScoresThe specific objectives are:Determine if the efficacy of (a) iron (as supplements or MNPs) given with malaria chemoprevention is superior to malaria chemoprevention alone or (b) if the efficacy of malaria chemoprevention alone is superior to placebo on.Child cognitive development, child motor, language, behavioural development, temperament and physical growth at the end of the 6 months intervention and a further 6 months post-intervention.Anaemia (and haemoglobin concentration), iron deficiency (and ferritin) and iron deficiency anaemia after 6 months of intervention and 6 months post-intervention.Compare the effects of iron given as supplements to MNPs on the above outcomes after 6 months of intervention and a further 6 months post-intervention.Determine the safety of iron (as supplements or MNPs) given with malaria chemoprevention on the risk of malaria and other morbidities versus malaria chemoprevention alone or placebo during and post-intervention.Determine the efficacy of malaria chemoprevention alone compared with placebo on clinical malaria, parasitaemia and malaria-specific clinical visits and all-cause sick visits during and after intervention.Determine the safety of iron (as supplements or MNPs) given with malaria chemoprevention compared with placebo on infection risk in young children, during and post-intervention.IRMA is a four-arm parallel-group, double-blinded, placebo-controlled, triple-dummy, individually randomised trial comparing: (1) No intervention, (2) malaria chemoprevention only, (3) MNPs+malaria chemoprevention and (4) iron syrup+malaria chemoprevention at the end of the 6 months intervention and at a further 6 months post-intervention. The trial design is summarised in P. falciparum parasitaemia.The trial is set in Chikwawa district, in Southern Malawi, a rural area where >70% of children 6–12 months are anaemic,The criteria to select participants considers participants’ safety, the generalisability to national programmes and the feasibility of the trial. Children are randomly assigned to a study intervention group only if they meet all the inclusion and none of the exclusion criteria.Infant aged 6 months (±14 days) at randomisation.Availability of a legally acceptable guardian/representative and providing written consent on the participant’s behalf.Severe anaemia (haemoglobin (Hb)<7 g/dL).Clinical signs of current infective illness ; however, children will be screened after recovery and recruited if they meet the age eligibility criteria at the second screening.Severe malnutrition (weight-for-length z-score <–3) or mid-upper arm circumference (MUAC)<11.5 cm.Established haemoglobinopathy diagnosis  thalassaemia).Known congenital anomaly, developmental disorder or severe developmental delay.Children with physical or behavioural problems that will make it impossible to undertake trial assessments.Children of multiple births, for example, twins.Children currently consuming iron supplements or MNPs, for example, from the National MNPs programme.Enrolment in another trial.Participants are randomly assigned to receive one of the following interventions:No intervention (placebo DPplusplacebo MNPsplusplacebo iron syrup): Placebo DP : three consecutive days every 4 weeks for 6 months plus placebo MNPs  daily for 6 months plus placebo iron syrup  daily for 6 months. Malaria chemoprevention is not given routinely to children under 2 years of age in this setting. Thus, the placebo arm is the current standard of care in the country and no treatment is being withheld from the participants.Malaria chemoprevention only (active DPplusplacebo MNPsplusplacebo iron syrup): Active DP  dosed by body weight (2/16 mg/kg) for three consecutive days every 4 weeks for 6 months plus placebo MNPs daily for 6 months, plus daily placebo iron syrup for 6 months.chemoprevention : Active DP as above, plus daily active MNPs containing 10.0 mg iron and 14 other micronutrients : Active DP as above, plus placebo MNPs, plus active iron syrup containing 10.0 mg ferrous sulphate  daily for 6 months.10.1136/bmjopen-2022-069011.supp1Supplementary dataParticipants are randomly allocated to one of the four study arms with 1:1:1:1 allocation via a computer-generated randomisation schedule of randomly permuted blocks stratified by sex and health facility/study sites  to achieve a balance between the intervention arms. An independent statistician from the University of Melbourne  generated the randomisation list.An independent trial pharmacist pre-packed and pre-labelled participant packs containing all three investigational products based on the randomisation list. Each participating child who meets the inclusion criteria is sequentially allocated to the next available study number indicated on the randomisation list. The participant is then given a pre-labelled pre-packed Ziplock bag containing the study medication corresponding to the study number assigned to them. Hence, participants and their families and all study team members involved in collecting and analysing outcome data, including field workers, developmental assessors, laboratory scientists, statisticians and investigators, are blinded to the treatment allocation until the database is locked and before unblinding.All levels of the local leadership , Health Surveillance Assistants (HSAs) and District Health Assembly) are continuously engaged by the trial’s Community Engagement Team (CET). CHAGs and HSAs were engaged to ensure that we reach the whole community via it’s resident health ‘gate-keepers’. Additionally, the CET conducted community sensitisation meetings to explain the research objectives, what it means to participate, benefits for the individual or the community and overall contribution to science. Finally, community members are regularly invited to contribute ideas that could help improve the success of the research thereby increasing the probability of success.Study recruitment opened on 17 April 2020. Study activities are as detailed below  of all potential participants (children under 6 months of age) living in the target area and generated a study population database. New births are identified and reported by HSAs or registered at the health facilities’ under-5 clinics throughout the trial recruitment period and added to the database. Research assistants visit all children approaching 6 months of age in the community. After taking written informed consent, the team reviews the child’s medical history to determine potential eligibility. Eligible children are invited to visit a selected health facility for further screening.On a predetermined day, the child, accompanied by their parent(s) or legal guardian, visit the selected health centre. HemoCue Hb301+  is used to assess capillary Hb level to exclude severely anaemic children. Additionally, anthropometric measurements: weight and length  documentation of episodes of infection , (2) recording any adverse events/effects, (3) documentation of details of unplanned hospital and healthcare facility attendance, for example, to a doctor, traditional healer, clinic or hospital, (4) provision of educational information to participants on the correct use of the study interventions and encouraging adherence to study interventions, (5) adherence monitoring  and (6) advice on future visits.The research assistants issue the investigational products monthly during a home visit. The following activities (including data collection) are undertaken: issuance of the study interventions according to study protocol and adherence monitoring . In addition, unused investigational products are collected.The participating child and their parent/legal guardian attend the health facility at the end of the 6 months intervention for developmental assessments using locally adapted Bayley Scales of Infant and Toddler Development, Wolke’s Behavioural Rating (WBR) and Child Temperament using the Bates’ Infant Characteristics Questionnaire. Anthropometric measurements are conducted on both the child and the mother. Venous blood (3 mL) for Hb testing, determination of iron indices, malaria microscopy  and malaria filter paper for future PCR analyses is collected from the child.Research assistants visit the homes of participants once per month after completion of the midline visit for assessment of episodes of infection  in the previous 1 month and documentation of unplanned hospital and/or healthcare facility attendance  and record of healthcare usage.The procedures are similar to the midline visit described above and in Mothers or legal guardians are asked to attend the research site/clinic when their child is symptomatically unwell. They are managed according to national standard treatment guidelines by a trained healthcare provider. Blood samples for malaria rapid diagnostic test (mRDT) and blood culture are taken if clinically indicated. The clinical diagnosis of the unscheduled visit is recorded in the participant’s record and captured on the study’s interim visit case report form (CRF) and adverse event form. If the child requires inpatient care, they are referred to the Chikwawa district hospital.Plasmodium infection by RDT (mRDTs);  at baseline. In addition, up to 3 mL venous blood sample is collected from all enrolled participants at baseline, midline, endline and unscheduled visits (if necessary) for Hb testing, determination of iron indices , thick and thin malaria slides for malaria microscopy testing and filter paper for Plasmodium PCR. Families are encouraged to permit the collection of venous blood for venous Hb and iron and inflammation biomarkers at all time points; however, if a family refuses venous blood collection or if collection was unsuccessful, data for the primary outcome and other non-laboratory secondary outcomes will still be collected.A capillary blood sample is collected by finger prick from all age-eligible participants to measure the Hb concentration by HemoCue  and An independent Data Safety and Monitoring Committee (DSMC) comprised of international experts in clinical trials, child health, infectious diseases, epidemiology and statistics was set-up to review regularly, safety data of the ongoing trial. The DSMC may recommend to the sponsor and investigators whether to continue, modify or terminate the trial on ethical grounds.Cognitive development: assessed by the differences in Bayley-III Cognitive Composite Score (CogCS) between intervention arms measured after 6 months intervention (midline) and 12 months post-intervention (endline). The Bayley-III is a validated index of child development that can be administered in ~0.5–1 hour and comprises cognitive, motor and language development scales. Assessors are specially trained to administer the tests, and inter-rater reliability is established before starting data collection. In addition, we perform 10% interobserver testing to monitor inter-rater reliability of the measure over the study period.Secondary outcomes include: motor and language development assessed by Bayley-III Motor (MotCS) and Language (LangCS) Composite Scores , behaviour assessed by WBR, temperament, growth assessed by weight-for-age z-score, length-for-age z-score, weight-for-length z-score, head circumference z-score, anaemia (Hb<11 g/dL), Hb concentration, iron deficiency (ferritin <12 ng/µL adjusted for inflammation assessed by CRP or AGP), ferritin concentration and iron deficiency anaemia.Safety will be assessed by prevalence of malaria parasitaemia and CRP and inflammation (CRP>5 mg/L) at the end of the intervention and a further 6 months post-intervention; episodes of caregiver reported symptoms  determined by the biweekly and monthly morbidity questionnaire; episodes of unplanned clinical visits and hospitalisations and deaths.Non-serious adverse events and serious adverse events (SAEs) are collected from the time consent is given until the participant completes the study . These are identified through spontaneous reports by the participant, unplanned visits to the research site or any participating health centres, observation by the study staff and standard questioning at each visit (biweekly during the intervention period and monthly during the post-intervention period). All SAEs are reported to the sponsor and the appropriate ethics committee, whether or not they are considered causally related to the investigational products.The primary objective will be met if either (1) MNPs+DP is superior to DP alone, or (2) iron+DP alone is superior to DP alone or (3) DP alone is superior to placebo on the Bayley-III CogCS immediately after completion of the intervention (midline). A previous study in 2853 Bangladeshi children showed that a 0.2 standardised mean difference (SMD) in Bayley-II Mental Development Index at 7 months translates to a 0.7 SMD at 18 months and a 1.2 SMD at 64 months age.A stand-alone statistical analysis plan will be detailed before breaking the blind. This plan will detail all principles and precise methods of analysis, including primary and secondary treatment effects of interest; alternative analyses if model assumptions do not hold; and additional analyses. Participants will be analysed according to their randomised treatment group for efficacy objectives and actual treatment group for safety objectives. The primary outcome of Bayley-III CogCS will be analysed using a mixed-model repeated measures analysis including in the model treatment group, time point and group by time point interaction. The stratification variables sex and site will be included in all models as main effects. In addition, the CREDI variable (collected at baseline) will be included as a main effect in the models for childhood development as it is considered prognostic for childhood development outcomes. Secondary repeated continuous outcomes  will be analysed similarly to Bayley-III CogCS. The outcome may be appropriately transformed before fitting the analysis model if considered skewed . Secondary repeated binary outcomes  will be analysed using a log-binomial model and account for the repeated measures with a random intercept for participants. Safety  will be summarised during the intervention and post-intervention periods separately and compared between the groups. Analysis of binary repeated outcome safety data  and continuous repeated outcome safety data  will be analysed similarly to the secondary outcomes detailed above. Episode safety data  will be analysed using negative binomial regression models. Subgroup analyses  will be performed to explore the heterogeneity of the results independent of the study findings.Data collected from the subjects are recorded in digital CRFs using Research Electronic Data Capture (REDCap) software  on tablets. Relevant hard copy patient hospital files are scanned for reference and stored digitally, and securely. The trial is independently monitored by an external monitoring service. No interim analysis is planned.The National Health Science Research Committee, Malawi, and the Walter and Eliza Hall Institute of Medical Research Human Research Ethics Committee, Australia, approved this study. The Malawian Pharmacy and Medicines Regulatory Authority also approved the trial. Additionally, the protocol is registered with the Australian and New Zealand Clinical Trials Registry. Protocol modifications are reported to the ethics committees, regulatory authorities in Malawi, investigators, trial participants and trial registries. Written informed consent in the local language see  is obtai10.1136/bmjopen-2022-069011.supp3Supplementary dataThe IRMA trial will provide valuable evidence on the immediate and medium-term impacts of iron interventions (iron supplements and MNPs) and malaria prevention with DP on critical functional outcomes, most importantly, child cognitive development. These findings will complement high-quality data recently reported in South Asia to provide generalisable evidence for or against universal iron interventions across sub-Saharan Africa and other malaria-endemic settings.10More than two-thirds of African children aged 6–59 months are anaemic. Preventing anaemia is considered important within the first 1000 days and aligns with WHO policy for children 6–23 months of age,49E) provides much greater protection from malaria attacks in children compared to IPTi or RTS, S/AS01E given alone.Recent clinical trials have indicated that the combination of intermittent preventive treatment of malaria in infants (IPTi) and malaria vaccine  ensured that all clinical malaria cases were picked up by ensuring that all children with fever and other symptoms could access hospital care and get a rapid malaria test done.


The gametocyte prevalence was also significantly higher in school-age children (11.9%) compared to the under-fives (3.7%). Adjusted for covariates, school-age children were 2.9 times  more likely to carry the asexual parasite, compared to the under-fives. Malaria was moderate and stable endemic in this area and school-age children play a key role in the spread of the disease. The WHO conditional recommendation for intermittent preventive treatment of malaria in school-aged children living in malaria-endemic settings with moderate to high perennial or seasonal transmission should be implemented.Continuous monitoring of malaria epidemiology is needed in malaria-endemic settings to inform malaria control and elimination strategies. This study aimed to compare the malariometric indices between the under-fives and school-age children. We surveyed children aged 1.5 to 12 years for plasmodia carriage with the aim of including them in a longitudinal follow-up cohort. The survey took place from 7–11 September 2020 in a southwest area of Burkina Faso. Clinical and demographic data including malaria control measures were collected. A finger prick blood sample was taken for haemoglobin testing, and blood smears and dried blood spot preparation. The malariometric indices were calculated and compared between school-age children and those under the age of five. Multiple logistic regression was fitted to assess the association between malaria parasite carriage and age categories. Based on the PCR results, the parasite prevalence was 21.4% in the under-fives versus 44.2% in school-age children ( Joint public health efforts have led to a decrease in the malaria burden in recent decades. Indeed, globally, malaria cases decreased from 241 million in 2000 to 227 million in 2019 while malaria deaths reduced from 896,000 in 2000 to 558,000 in 2019 ). This prevalence using microscopy results was 24.53% (130/530) . The proportion of sub-microscopic parasitemia (defined as parasitemia not detected by microscopy but detected by PCR) was 27.33% (47/172), IC = . Considering the PCR which is the most accurate test, parasite prevalence was 21.43% (57/266) in the SMC target population against 44.23% (115/260) in school-age children (After the end of SMC (after the age of 5 years), parasite prevalence appears to be an increasing trend as shown in p-value = 0.0042). The GMPD was 4122 trophozoites/µL in children experiencing a clinical malaria episode (6 cases) against 753 trophozoites/µL in asymptomatic children (166 cases) (p < 0.0001).Overall the GMPD was 815 trophozoites/µL . The GMPD was 2008 trophozoites/µL  in the SMC target population (32 positive participants) while it was 605 trophozoites/µL  in the school-age children (98 positive participants) , which corresponds to six clinical malaria episodes that were diagnosed during the survey within the study population (530 children). Most of the parasite carriers were asymptomatic (166/172 = 96.51%).P. falciparum was the main parasite species. It was found in about 96% of the infection . This specific infection proportion was comparable between age groups. P malariae and P. ovale were found in, respectively, 6.92% (9 cases) and 0.77% (1 case) of the study population. The unique case of P. ovale was a mono-infection while the six cases of P. malariae were found in co-infection with P. falciparum.p < 0.0001).In total, the gametocyte (regardless of species) carriers as diagnosed by microscopy represented 7.74%  of the study population. This prevalence was significantly higher in school-age children (11.88%) compared to the SMC target population (3.72%) (p = 0.098). Overall, 45.18% (239/529) of the children  were anaemic of whom 41.4% (99/239) were mild, 57.3% (137/239) moderate, and 1.23% (3/239) severe. The interquartile range (IQR) of the haemoglobin level was  with a median of 11.4 g/dl .In this study, 48.7% (131/269) of SMC target children were anaemic, while in school-age children this proportion was 41.5% (108/260)  more likely to be infected with the malaria parasite , compared to the SMC target population. A logistic regression was also fitted to assess the factors associated with gametocyte carriage. The results indicate that the school-age children were 3.55 times  more at risk of carrying gametocytes compared to the SMC target population, adjusting for sex, anaemia, usage of malaria prevention methods, and parent’s educational status. Malariometric indices are key indicators of malaria endemicity in an area. They should be continuously monitored as they inform on the parasite reservoir and its transmission and could give insights on the bottlenecks towards disease elimination. In Burkina Faso, the latest malaria indicator survey was conducted during the years 2017–2018 . This stP. falciparum (91–98.4%). This result indicated that malaria was a moderate and stable endemic  in the area [The parasite prevalence in this study was 32.7% and was mostly due to the area . After rthe area . In the um 91–98.%. This rthe area ,15. Howeum 91–98.%. This rThe study found that one-quarter of the parasitemia was sub-microscopic (27.33%). This reflects the magnitude of parasite carriers not detectable by routine diagnostic tools yet contributing to the spread of the disease. Targeting sub-microscopic parasitemia with innovative interventions is a requirement for the achievement of the malaria elimination goal.P. falciparum infection compared to the under-fives [The study has shown that asexual parasite prevalence as well as gametocyte prevalence was significantly higher in school-age children compared to the SMC target population. Furthermore, adjusting for covariates  school-age children were about three times more at risk of carrying asexual parasites than SMC target children. Likewise, the odds of carrying the sexual stage of the malaria parasite were 3.55 times higher in school-age children compared to those under the age of five years. This study’s results corroborate with those of several studies assessing malaria burden in school-aged children ,8,16,17.er-fives . In 2013er-fives  might haWith regard to the fact that school-age children contribute widely to the malaria parasite reservoir and that this group may suffer from more uncomplicated and severe malaria cases, many authors claimed more malaria intervention towards them ,8,16,17.Some limits should be considered while interpreting the results of this study. The convenience sample could hamper the results’ generalisability. It has been presumed that the SMC target population has received two rounds of SMC before the survey but this was not documented and could bias the interpretation of the results. Moreover, some household characteristics like the level of sanitation and the level of wealth were not included in our analyses and this could affect the relationship between malaria indices and age groups. Nevertheless, we used PCR results for assessing the relationship between malaria infection and age group, and this helped in mitigating the misclassification bias (with regard to the parasite carriage status).Despite the implementation of SMC, malaria transmission remains moderate and stable in the Health District of Banfora. School-age children are three to four times more likely to carry both the asexual and sexual stages of the parasite compared to the under-fives targeted by SMC. The positive impact of SMC in reducing the burden of malaria in children under five can therefore be inferred. The study indicated that school-age children represent a large reservoir of parasites and could significantly contribute to the spread of the disease. Further epidemiological studies are needed to determine whether this parasite carriage in school-age children leads to higher morbidity or mortality than in children under five years of age, and thus to conclude that there is a shift in malaria burden. To meet the goal of malaria elimination, effective control interventions should be implemented in school-age children.


Salmonella spp. isolates to commonly used antimicrobial agents in Ghana.Clinicians in areas where malaria and typhoid fever are co-endemic often treat infected patients irrationally, which may lead to the emergence of drug resistance and extra cost to patients. This study determined the proportion of febrile conditions attributable to either malaria and/or typhoid fever and the susceptibility patterns of Salmonella isolates to antimicrobial agents was performed using the Kirby-Bauer disk diffusion method with antibiotic discs on Müller Hinton agar plates. For each sample, conventional Widal test for the detection of Salmonella spp was done as well as blood film preparation for detection of Plasmodium spp. Data on the socio-demographic and clinical characteristics of the study participants were collected using an android technology software kobo-collect by interview.One hundred and fifty-seven (157) febrile patients attending the Ga West Municipal Hospital, Ghana, from February to May 2017 were sampled. Blood samples were collected for cultivation of pathogenic bacteria and the susceptibility of the Salmonella typhi O and H antigens were detected in 23/157 (14.6%) of the samples. The detection rate of Salmonella spp in febrile patients was 10/157 (6.4%). Malaria and typhoid fever coinfection using Widal test and blood culture was 9 (5.7%) and 3 (1.9%), respectively. The isolates were highly susceptible to cefotaxime, ceftriaxone, ciprofloxacin, and amikacin but resistant to ampicillin, tetracycline, co-trimoxazole, gentamicin, cefuroxime, chloramphenicol, and meropenem.Of the total number of patients aged 2–37 years , 82 (52.2%) were females. The proportion of febrile patients with falciparum malaria was 57/157 (36.3%), while Plasmodium falciparum and Salmonella spp coinfections were only up to 1.9%, while malaria and typhoid fever, individually, were responsible for 36.3% and 6.4%, respectively. Treatment of febrile conditions must be based on laboratory findings in order not to expose patients to unnecessary side effects of antibiotics and reduce the emergence and spread of drug resistance against antibiotics. Salmonella typhi and common in malaria-endemic settings [Salmonella in these settings with mostly weak peripheral health systems and resource constraints, cases of acute febrile illnesses are usually treated presumptively as malaria by clinicians [Malaria and typhoid fever are two febrile illnesses that cause varying degrees of illness and death in the tropics and sub-tropics, where malaria and typhoid fever are co-endemic. According to the World Health Organization (WHO) global data on malaria published in 2021, about 241 million cases of malaria were diagnosed, with 627, 000 attributable deaths [settings  resultedinicians . HoweverThe predisposition to malaria and typhoid fever coinfection is usually influenced by similar socio-epidemiological factors and dynamics such as dense populations with poor hygiene and sanitation practices ,9. In GhSalmonella isolates to commonly used antimicrobial agents in patients attending the municipal health facility.Malaria POC-RDT lacks sensitivity at low parasitemia levels below 50 parasite/μl of blood . Only inThe Ghana Health Service Ethics Review Committee approved this study (GHS-ERC 52/12/16). Consent was obtained from each patient or guardian of patients aged less than 18 years. Privacy and confidentiality of the study participants were ensured by conducting the interviews in a private and conducive environment.The study was conducted at the Ga West Municipal Hospital (GWM) in Amasaman, Accra. The GWM is one of the 29 districts in the Greater Accra Region. The district is 60% rural and 40% peri-urban and urban. The municipality was carved out of the erstwhile Ga District, which was created in 1988 in pursuance of the government’s decentralization and local government reform policy . In 2004Salmonella typhi and other Salmonella species [The study sample comprised febrile patients aged 2 years and above. Samples were collected from February to May, 2017 at the outpatients’ department (OPD) in Ga West Municipal Hospital, Amasaman in the Greater Accra region of Ghana. Participation in the study was voluntary and those who refused to take part in the study were still given appropriate attention by the health personnel without any bias. Patients with fever > 37.5°C (temperature taken by an infrared thermometer) presenting to the health facility at the OPD and having consent were included in the study. Patients who met the above criteria but were found to be on antibiotics or antimalarial therapy and those in critical conditions such as convulsion were excluded. Ten (10) milliliters of venous blood were collected aseptically from patients over the age of ten. Out of this volume of blood, 7 mL was inoculated immediately into 45 mL of brain heart infusion (BHI) broth and the remaining 3 mL was transferred into a sterile EDTA tube for malaria diagnosis and typhoid fever serodiagnosis. Similarly, 3–4 mL of blood was collected from children under 10 years old, and 1.5–2 mL of blood was inoculated into 9 mL of BHI broth to isolate  species .Plasmodium falciparum histidine rich protein II (PfhrpII) and Plasmodium lactate dehydrogenase (pLDH) antigens. Also, standard size thick and thin blood films were prepared, thoroughly air-dried and the thin films fixed with absolute methanol for malaria species identification. The blood films were stained with 10% (v/v) Giemsa solution for 20 minutes for the detection of Plasmodium parasites and speciation. Malaria microscopy and RDT were done by medical laboratory scientists certified by Ghana Health Service through the On-site Training and Supportive Supervision (OTSS) training program.Malaria POC-RDT was done with the SD Bioline rapid diagnostic test kit . This kit determines both Salmonella antigens; somatic (O) and flagella (H) antigens . Briefly, 50 μL of test serum was placed in two circles on a glass slide and equal volumes each of positive control and normal saline in each of the last two circles respectively. A drop each of O and H antigens was added to the test serum in each circle and then to the negative and positive controls. The content of each circle was mixed using the disposal mixing sticks provided and spread to the entire circle, after which it was rocked gently for 1 minute and observed for agglutination. Antibody titration was performed for the slide reactive samples using the tube technique. According to the manufacturer’s instructions, an antibody titer of > 1: 80 was considered to be significant and usually suggestive of infection, according to the manufacturer.The Widal agglutination test  was performed on all blood samples using the rapid slide titration method to detect 2) for 24–48 hours. This protocol was adapted from a previous study [Blood in BHI broth was incubated aerobically at 35–37 °C for 7 days with intermittent cultivation on solid media plates, namely, MacConkey agar (MAC), blood agar (BA) and chocolate agar (CA) on day 2, day 4, and day 6. In the case where BHI broth showed no growth up to day 6, sub-cultures were repeated from the broth on day 7 before it was discarded. Also, on each day of sub-cultivation, broth tubes were examined for turbidity of blood-broth mixture, growth of micro-colonies, haemolysis, colour changes and gas production. For 24–48 hours, the BA and MAC agars were incubated aerobically at 35–37 °C and anaerobically at CA in micro-aerophilic conditions , citrate utilization and urease production were used to identify pathogens in positive cultures. A sterilized vertical wire loop was used to pick about two colonies and inoculated into the various biochemical tests . Gram-negative rods that were oxidase negative with the characteristic red slope/yellow butt reaction on triple sugar iron agar either with or without production of Hublished .Salmonella isolates to antimicrobial agents was performed using the Kirby-Bauer disk diffusion method with antibiotic discs on Müller Hinton agar plates . The antibiotics that were used include: Ampicillin (10μg), Co-trimoxazole (25 μg), Tetracycline (10μg), Chloramphenicol (10μg), Ciprofloxacin (5μg), Amikacin (30ug), Gentamicin (10μg), Meropenem (10μg), Cefuroxime (30μg), Ceftriaxone (30μg) and Cefotaxime. About 2–3 colonies of the same morphological type were picked and emulsified in a test tube containing 2.5 mL of sterile peptone water to form a bacterial suspension . A sterile swab stick was dipped into the suspension and uniformly spread on the surface of the Muller-Hinton agar by streaking the entire agar surface [The susceptibility of the After taking the body’s temperature, those whose temperature reading was ≥37.5°C and had consented were randomly selected. A questionnaire designed and deployed using an android technology, ordinary data kit (ODK) was administered to all participants in the study in English or the two main local languages (Twi or Ga) for those who could not speak English. Each subject was assigned an identification number. The questionnaire was made up of a section on the date of interview and individual serial number; a demographic section including age, sex, marital status, educational level, occupation, and location; and a clinical section covering fever, vomiting, diarrhoea, abdominal pain, joint pain, fatigue, headache, history of transfusion, and antibiotic use; and an environmental section on water source, toilet facility availability, bed net use, hand washing habits, and habit of eating outside home. Randomly selected participants were interviewed, with parents or guardians answering for children aged under 10 years. The questionnaire, which took an average of 15 minutes to administer, was applied by two trained nurses who can communicate in all the three languages.Data were double-entered and cleaned in MS Excel before exporting to Stata 13.0 for analysis. Descriptive statistics were presented as percentages. Continuous variables were presented as mean with standard deviation (SD) and median with Interquartile range (IQR), while the Chi square test was used to determine the association of infection status and socio-demographic and clinical variables. Multivariate logistic regression analysis was performed to determine the magnitude and direction of these factors, setting p-value at 0.05 for statistically significant associations.Most of the participants positive for febrile malaria were males (54.4%), and aged 18 years and below (89.5%). Most participants had only primary education (75.4%) and majority (82.5%) were employed. Most lived in an urban setting (66.7%), used bed net (66.7%) and did not have a history of travel (87.7%). Similar trends were observed for subjects with typhoid fever. The common source of household water was borehole (80.0%), most infrequently ate outside (90%) and slightly more than two-thirds (70.0%) had a toilet facility as seen in PfhrpII antigens. Twenty-three (14.6%) of patients had antibody titers (both O and H antigens) for Salmonella spp while 10 (6.4%) were positive for blood culture. Diagnosing typhoid fever with Widal test and blood culture, coinfection with malaria was 9 (5.7%) and 3 (1.9%), respectively.The commonest presenting signs and symptoms were fever (100%), headache (68.8%), abdominal pain (45.9%), vomiting (36.9%), anorexia (29.3%), joint pain (20.4%), fatigue (15.9%), diarrhea (15.9%), and with having previously used antibiotic during disease onset (100.0%). Of the 157 febrile patients, the prevalence of malaria as determined by RDT and confirmed by microscopy was 36.3% (57/157). In all the parasites identified, mRDT detected All isolates exhibited resistance against ampicillin, tetracycline, co-trimoxazole, gentamicin, cefuroxime, chloramphenicol and meropenem while the isolates showed susceptibility to cefotaxime, ceftriaxone, ciprofloxacin, and amikacin .Malaria and typhoid fever continue to be major diseases of public health concern, especially in tropical countries, and are known to present clinically similar symptoms, with fever being the major presentation . Our invSalmonella antibodies in the patients [Salmonella H and O antigens with other pathogens [Even though the two diseases are endemic in Ghana, findings from this study show that malaria was implicated as the cause of most febrile illnesses. Studies conducted elsewhere in Ghana reported a marginally higher prevalence of malaria (18.6%) than typhoid fever (17%) . Howeverpatients  or crossathogens .Plasmodium antibodies [Typhoid fever prevalence in this study, using the Widal test, is comparable to findings in Northwest Ethiopia (19.0%)  and Ibadtibodies . Due to Mycoplasma pneumoniae infection [A previous study in Ghana , Cameroonfection , streptonfection  and meninfection  are alsonfection . These fIt was found that insecticide-treated bed net usage was not associated with malaria even though about 65% of the patients did not use insecticide-treated bed nets. Insecticide-treated nets have been shown to be highly efficient at reducing malaria incidences . Also, tSalmonella enterica serovar and Escherichia coli [Klebsiella pneumonia, Rickettsia felis, Acinetobacter spp., Klebsiella spp., Haemophilus influenza, Pseudomonas spp., Enterobacter cloacae, Serratia marcescens, Aeromonas spp and Campylobacter spp [Salmonella pathogens isolated in this study were found to be resistant to seven out of the eleven (63.6%) antibiotics tested. The antibiotics found not to be effective against the pathogens were the penicillins (ampicillin), the sulphonamide (co-trimoxazole), tetracycline, the aminoglycoside (gentamicin), cephalosporin (cefuroxime), chloramphenicol (inhibitor of protein synthesis) and the carbapenem (meropenem). However, another penicillin class of antibiotics, amikacin, two cephalosporins that are β-lactam agents (cefotaxime and ceftriaxone) and the only fluoroquinolone (ciprofloxacin) were highly sensitive against the isolated pathogens. As reported in this study, several other studies in Ghana and elsewhere have reported high prevalence of ampicillin, co-trimoxazole, tetracycline, gentamicin, cefuroxime, chloramphenicol, and meropenem resistant blood pathogens [Salmonella species has increasingly been reported in some studies [Salmonella species [Malaria and other bacterial coinfection have been previously reported. Pathogens such as hia coli , Klebsiecter spp  Surprisiathogens –49. The athogens –52. The athogens ,54. Mult studies ,56. Howe species –59. Cipr species .Salmonellae species was not done.Some limitations of this study are the small sample size which might have reduced the statistical power to detect statistical differences. However, given the greater stability and less variability of the assessed parameters the sample size remains adequate and unlikely to influence the conclusion of the study. Secondly, only the standard manual method (blood culture) was used in isolating the organisms and this could have been done alongside molecular techniques involving PCR which are more sensitive and would have been more accurate. Thirdly, serotyping for antigenic structure and grouping of Malaria and typhoid comorbidity were less than 2% of the total samples analyzed. Individually, more than five times malaria fever were presented compared to typhoid fever. Due to the low prevalence of malaria and typhoid fever coinfection, clinicians should not treat concurrently but rather stick to differential diagnosis to ensure judicious use of resources.The implications of these findings are enormous. Based on these findings and reports from other studies, malaria was the cause of fever among febrile patients rather than typhoid fever. Meanwhile, the prevalence of malaria and typhoid fever coinfection was also found to be low. Therefore, treating bacterial infections without previous determination of antibiogram does not help the control of bacterial infections and also leads to the creation of drug-resistant strains. It is recommended that all bacterial and parasitic infections be treated by clinicians based on a laboratory diagnosis and antibiogram profile. This is because there is a chance that most commonly available antibiotics or antimalarials may not be sensitive to the pathogen causing the infection. Again, to reduce the risk of antimicrobial resistance development and the spread of resistant bacteria, healthcare organizations like Ghana Health Service must enact or re-enforce procedures to ensure the optimal use of antibiotics. This can be achieved by implementing antimicrobial stewardship programs. Organizations can provide appropriate antibiotics to patients requiring antibiotic treatment. Additionally, these programs ensure the correct antibiotic is prescribed at the right time, dose, and duration. Collectively, these measures improve patient health outcomes and preserve the effectiveness of antimicrobial treatments.


ANC P. falciparum rates detected by quantitative polymerase chain reaction mirrored rates in children, regardless of gravidity and HIV status , with a 2–3 months lag. Only at rapid diagnostic test detection limits at moderate-to-high transmission, did multigravidae show lower rates than children . Seroprevalence against the pregnancy-specific antigen VAR2CSA reflected declining malaria trends . 60% (9/15) of hotspots detected from health facility data (n = 6662) using a novel hotspot detector, EpiFRIenDs, were also identified with ANC data (n = 3616). Taken together, we show that ANC-based malaria surveillance offers contemporary information on temporal trends and geographic distribution of malaria burden in the community.Pregnant women attending first antenatal care (ANC) visits represent a promising malaria surveillance target in Sub-Saharan Africa. We assessed the spatio-temporal relationship between malaria trends at ANC ( Pregnant people visiting antenatal clinics may represent a useful sentinel surveillance population for monitoring infections such as malaria. Here, the authors investigate the potential of this approach by comparing malaria prevalence in pregnant people and children living in the same area of southern Mozambique. Estimating malaria trends from clinical cases at health facilities remains challenging due to differences in care-seeking behaviour, unknown denominator populations, and asymptomatic infections2. These biases are minimised in nationally-representative cross-sectional surveys, but due to their high costs and complex logistics, they are typically only conducted every 2–3 years3.Surveillance is key to inform optimal and equitable resource allocation for malaria control and elimination5. In sub-Saharan Africa, 79% of pregnant women attend at least one ANC visit6, offering a good representation of the population. Since visits are unrelated to illness, malaria testing is not biased by care-seeking behaviour or testing decisions, and captures asymptomatic infections7.Pregnant women attending a first antenatal care (ANC) visit have been proposed as a potential convenience group for surveillance of malaria and other infectious diseases8. Less heterogeneity was found for low-prevalence settings , and less difference between women and children was found when restricting the analysis to primigravidae. However, only one study recruited women from an antenatal clinic, and small-scale trends could not be assessed due to pooling data obtained from different administrative levels. Studies using routine ANC data in Tanzania did not provide information on gravidity and were unable to reproduce a similar linear effect as the meta-analysis when comparing young women to children11. Another study analysed data from health centres in conflict settings in the Democratic Republic of Congo and found a strong but non-linear relationship between ANC prevalence and incidence in children12. However, all the studies used low-sensitivity microscopy or rapid diagnostic tests (RDT), missing the significant proportion of P. falciparum infections with parasite densities below the detection threshold of conventional field diagnostic tools13. Moreover, the effect of gravidity and other factors such as HIV was not consistently assessed. Also, spatial patterns in malaria burden have not been compared between both groups due to lack of geospatial data. Finally, none of the studies quantified correlation in low-transmission settings, where ANC-based surveillance might be particularly attractive due to local clustering of malaria cases which are difficult to monitor with traditional strategies2. Therefore, a better understanding of the validity of ANC prevalence data for monitoring transmission in the community and the factors that affect this relationship remains to be developed.A meta-analysis of pooled prevalence data from Sub-Saharan Africa found a strong correlation between malaria burden in pregnant women and children, with lower rates in the later but with large heterogeneity between studies16, can potentially increase sensitivity to detect recent exposure in low-transmission settings where detecting a sufficient number of active infections to estimate trends in burden is difficult17. Combined with novel clustering approaches, ANC data can increase the resolution to detect spatial patterns3, providing a cost-effective approach to target interventions to the most affected areas.New surveillance tools, such as antibodies against the pregnancy-specific antigen VAR2CSA that mediates parasite sequestration in the placentaIn this study, we estimate and compare malaria burden at first ANC visits with data from cross-sectional surveys and clinical cases in three settings in southern Mozambique with different transmission levels. We correlate temporal and spatial trends at both RDT- and quantitative polymerase chain reaction (qPCR)-detection levels, and characterise the effect of HIV and gravidity on the correlation. Finally, we assess the added value of antibody data obtained from a bead-based multiplex immunoassay against VAR2CSA and general malaria antigens, and a newly developed hotspot detection algorithm, as innovative tools to improve surveillance in malaria-endemic areas.P. falciparum parasite rates using qPCR (PfPRqPCR) in pregnant women and in children 2–10 years old were highly correlated (Pearson correlation coefficient [PCC] = 0.94 [95% CI 0.70–0.99]) and showed a consistent and linear relationship . Similar high correlations (PCC > 0.85), linear relationships (slope~1 and origin~0) and consistencies (χ²<1.1) were found regardless of gravidity and HIV status  in pregnant women and children showed lower correlations and weaker linear relationships , regardless of HIV status . Clinical cases showed an overall decline of 47% . Statistically significant declines in parasite rates among children 2–10 years old from cross-sectional surveys were only observed in Ilha Josina for PfPRqPCR . The PfPRqPCR temporal patterns in pregnant women and clinical cases were consistent (χ² < 1) and correlated (PCC = 0.87 [95%CI 0.69–0.91]) with a delay of ~90 days, regardless of gravidity  statistics (χ²<1.50) in all three years, regardless of gravidity and HIV status at both qPCR and RDT-detection levels Fig. . 2PCFs fThe performance of Epidemiological Foci Relating Infections by Distance (EpiFRIenDs), a novel malaria hotspot detector, and SaTScan to detect clusters on simulated data was compared for three different scenarios . In the n = 3616; Fig. n = 6662 visits from a total of 37,131, see p ≤ 0.054, and two with p ≤ 0.91; Supplementary Table n = 5049 first ANC visits at the three health facilities), four RDT hotspots  and 11 qPCR hotspots were found, regardless of whether HIV-positive or multigravid women were included , and the highest seroprevalence was found for MSP1 in Ilha Josina during the first year  (Supplementary Table PfPRqPCR in children (PCC > 0.7), with antigen-dependent linear regression parameters  and VAR2CSAP1 (PCC = 0.74 [95%CI 0.27–0.76]), remaining high when stratifying by gravidity  were succesfully analysed for the presence of antibodies against 14 χ² ≤ 1.6; Fig. DBL3-4, and combining all VAR2CSA peptides , more seroclusters of HSP40, PfTramp, RH5, VAR2CSADBL3-4 and combined VAR2CSA peptides were found than hotspots. In addition, eight seroclusters were found in year 1 in the south-west of Magude, which did not reflect hotspots of qPCR-positive cases. Finally, in the north of Manhiça we detected one hotspot from qPCR data in year 1. In this same area, two seroclusters  were detected in year 1, and 6 others  were detected in year 3. Seroclusters were more stable than qPCR hotspots, with nine persisting more than 20 days and one persisting for 197 days  were obtained from the District Health Information System 2 (DHIS2). In Manhiça district, 37,131 RDT and microscopy results from children <5 years attending health facilities were available from OPMSS. Data from 3,933 children aged 2–10 years was collected in age-stratified cross-sectional surveys conducted every May from 2015 to 2019 in the catchment areas corresponding to the three ANC health facilities of this study. Gender was not considered to define this population, providing an even representation with 50.3% of female and 49.2% of men (with 0.5% of unavailable information). Geo-localisation of the residence of pregnant women and children was obtained from a local health and demographic surveillance system using their permanent or family identification number32, from their household identification number or by registering the geo-localisation of the households  were analysed by qPCR . P. falciparum infection was detected and quantified in duplicate from DBS with a qPCR targeting the 18 S rRNA gene (using primers “18s-QF”: 5’ GTA ATT GGA ATG ATA GGA ATT TAC AAG GT 3’ and “18s-QR”: 5’ TCA ACT ACG AAC GTT TTA ACT GCA AC 3’) on an ABI PRISM 7500 HT Real-Time System (Applied Biosystems)33. Immunoglobulin Gs (IgG) were detected and quantified in DBS (n = 6038) using a multiplexed bead array, the xMAP© technology and the Luminex 100/200 System 31. In brief, magnetic beads were coupled to our panel , thrombospondin-related apical merozoite protein [PfTRAMP], and biomarkers of recent P. falciparum exposure . Information about antigens, procedures for reconstitution of DBS and quality control, bead-based immunoassay, and data normalisation are described in Finger prick blood drops were collected onto Whatman 903 filter paper (DBS) from pregnant women and from children in the cross-sectional surveys. Children were also tested by RDT .Study years 1 to 3 were defined from November to October in 2016–2017, 2017–2018 and 2018–2019, respectively. Infections with densities above 100 parasites/µL were defined as RDT-detectablePfPRRDT or PfPRqPCR detection limits in pregnant women and children was analysed using linear regressions, PCC, and χ2 statistics  is the 2PCF at distance r, P1P2(r) is the number of pairs of positive cases between populations 1 and 2 separated a distance r between them, and B1B2(r) is the number of background pairs (positive or negative) between both populations at distance r. P1P2(r) and B1B2(r) were normalised by nP1nP2 and nB1nB2 respectively, where nP1,2 and nB1,2 are the number of positive samples and the total number of samples respectively for population 1,2. The 2PCF measurements were done using 10 bins in distance in a range from 0 to 60 km. The choosing of these bins was based on the spatial range of pairwise distances and finding the balance between spatial granularity and the statistical power (sample size) of the measurements. The agreement between different 2PCFs was quantified with χ2 statistics.The relationship between atistics . Similarsing PCC . 2PCFs wP. falciparum infections (hotspots) and seropositivity against P. falciparum antigens (seroclusters) than statistically expected in a stable period of time2. EpiFRIenDs was designed to detect structures of arbitrary shapes and sizes that account for the background population distribution, a difference with the most commonly used scan statistics based on the SaTScan software28 that detects structures of pre-defined shapes. EpiFRIenDs detects hotspots and seroclusters by linking positive cases when they are closer than a given pre-defined distance and indirectly linking them to all the positive cases that are close to their connections. The negative cases are then included in the hotspots from their close positive cases. The EpiFRIenDs software is publicly available in Python35 and R36 and it is described in detail in the section EpiFRIenDs of A novel malaria hotspot detector, EpiFRIenDs, was developed to detect areas with higher levels of PfPRqPCR in our study population (Supplementary Table p < 0.05. χ2 values were interpreted as good consistency from thresholds corresponding to p < 0.05 in a null-hypothesis statistical test (All analyses were stratified by gravidity and HIV status, the main factors shown to affect cal test . Error bFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting Summary