Background: African children with severe malaria are susceptible to Gram-negative bacterial co-infection, largely non-typhoidal Salmonellae, leading to a substantially higher rates of in-hospital and post-discharge mortality than those without bacteraemia. Current evidence for treating co-infection is lacking, and there is no consensus on the dosage or length of treatment required. We therefore aimed to establish the appropriate dose of oral dispersible azithromycin as an antimicrobial treatment for children with severe malaria and to investigate whether antibiotics can be targeted to those at greatest risk of bacterial co-infection using clinical criteria alone or in combination with rapid diagnostic biomarker tests.Methods: A Phase I/II open-label trial comparing three doses of azithromycin: 10, 15 and 20 mg/kg spanning the lowest to highest mg/kg doses previously demonstrated to be equally effective as parenteral treatment for other salmonellae infection. Children with the highest risk of bacterial infection will receive five days of azithromycin and followed for 90 days. We will generate relevant pharmacokinetic data by sparse sampling during dosing intervals. We will use population pharmacokinetic modelling to determine the optimal azithromycin dose in severe malaria and investigate azithromycin exposure to change in C-reactive protein, a putative marker of sepsis at 72 hours, and microbiological cure (seven-day), alone and as a composite with seven-day survival. We will also evaluate whether a combination of clinical, point-of-care diagnostic tests, and/or biomarkers can accurately identify the sub-group of severe malaria with culture-proven bacteraemia by comparison with a control cohort of children hospitalized with severe malaria at low risk of bacterial co-infection.Discussion: We plan to study azithromycin because of its favourable microbiological spectrum, its inherent antimalarial and immunomodulatory properties and dosing and safety profile. This study will generate new data to inform the design and sample size for definitive Phase III trial evaluation.Registration: ISRCTN49726849 (27th October 2017). E. coli:Escherichia coli; ETAT: emergency triage assessment and treatment; FEAST: Fluid Expansion As a Supportive Therapy; GCP: Good Clinical Practice Hb: haemoglobin; IBI: invasive bacterial infection; ICREC: Imperial College Research Ethics Committee; KWTRP: KEMRI Wellcome Trust Research Programme; MIC: minimum inhibitory concentration MRRH: Mbale Regional Referral Hospital; MRRH-REC: Mbale Regional Referral Hospital Research Ethics Committee; MRC: Medical Research Council; NTS: non-typhoidal salmonella; PCT: procalcitonin; PD: pharmacodynamic;P. falciparum HRP2:Plasmodium falciparum histidine-rich protein 2; PK: pharmacokinetic; PKPD: pharmacokinetic-pharmacodynamic; RDT: rapid diagnostic test; TRACT: TRansfusion and Treatment strategies of severe Anaemia in African children Trial; WHO: World Health Organization.AQUAMAT: Artesunate versus Quinine in the treatment of severe falciparum Malaria in African children AUC24h: area under the curve at 24 hours; AVPU: alert, voice, pain, unresponsive: system of recording patient’s level of consciousness; CRF: case report form; CRP: C-reactive protein;Plasmodium faliciparum malaria remains to be a common cause of hospital admission in much of sub-Saharan Africa, and plays a substantial role in under five-year mortality. Strategies to control or prevent malaria (including vaccines) have so far offered limited short-term protection. Over the past decade some African countries have either documented no decline, an increase in hospitalisations with severeP. falciparum malaria or a resurgence of severe malaria following a period of sustained control. Thus, prevention strategies alone in many parts of Africa will not adequately address the burden that malaria poses on health services, particularly in countries where transmission is high. Even with the best (evidence-based) antimalarial treatment artesunate, children in the Artesunate versus Quinine in the treatment of severe falciparum malaria in African children (AQUAMAT) trial had an overall mortality of 6–8.5%. The trial population were selected pragmatically  for generalisability of the results. However, mortality in children with severe malaria would almost certainly be substantially higher outside a Good Clinical Practice (GCP)-run trial and outside selected centres with a strong track record of research; and with more stringent clinical criteria defining severe malaria. Thus, there is substantial potential for clinical trials addressing the safety and efficacy of adjuvant supportive therapies to not only close existing gaps in the severe malaria treatment algorithm, but also substantially improve outcomes.. An estimated one third of all severe malaria deaths in African children are attributable to bacterial co-infection. Current guidance and evidence for treating co-infection in children is lacking, and there is no consensus on the dosage or length of treatment required. Identifying which children genuinely have bacterial co-infection is practically impossible. The indiscriminate use of antibiotics is both financially costly and may perpetuate the rise of antibiotic resistance. Establishing which children with malaria are at greatest risk of bacteraemia is critical to pragmatically inform a policy for targeted antibiotic therapy that could substantially reduce malaria-associated mortality while minimising the risks of excess antibiotic prescribing.Children with severe malaria and bacterial co-infection, largely due to enteric Gram-negative organisms with a predominance of non-typhoidal salmonella (NTS) species, have substantially higher rates of in-hospital and post-discharge mortality. We identified a total of 25 studies across 11 African countries which fulfilled the inclusion criteria. These comprised of 20 cohort analyses, two randomised controlled trials and three prospective epidemiological studies. We initially compared the prevalence of IBI in 20,889 children who were hospitalised with all-severity malaria to 27,641 children hospitalised with a non-malarial febrile illness. The mean prevalence of IBI was 5.58 (95% CI 5.5 to 5.66%) in children with malaria and 7.77% (95% CI 7.72 to 7.83%) in non-malaria illness. In the meta-analysis involving 10 studies  of children with severe malaria the mean prevalence of IBI was 6.4%  5.81 to 6.98%). Ten of the 25 studies reported mortality stratified by bacterial infection status. Case fatality was higher at 81 of 336, 24.1% (95% CI 18.9 to 29.4) in children with malaria/IBI co-infection compared to 585 of 5,760, 10.2% (95% CI 9.3 to 10.98) with malaria alone. In seven of 14 hospital studies, NTS was the commonest isolate and one other study listed enteric Gram-negative organisms as the most common isolates in malaria-infected children. Thus, enteric Gram-negative organisms, and NTS in particular, cause the vast majority of bacteraemia co-infections. Moreover, the poor sensitivity of blood cultures (due to low blood volumes in children and previous antibiotic use) means that the true prevalence of bacteraemia co-infection is likely to be significantly higher and ‘nosocomial bacteraemia’ will be acquired in a further 2–3.5% of children receiving a transfusion  due to poor quality.We conducted a systematic review in 2013 examining studies among children with malaria admitted to hospitals or outpatient clinics in sub-Saharan Africa reporting invasive bacterial infection (IBI). However, many hospitals in Africa lack culture facilities or access to such data; thus, best practice is not obvious. In one study almost 50% of bacterial isolates were resistant to the antibiotics most commonly recommended . In the specific case of NTS, the efficacy of gentamicin is doubtful and susceptibility testing unreliable due to this infection’s intracellular nature. For newer and broad-spectrum antimicrobials, apart from financial considerations, there are concerns that over-use could lead to resistance. A systematic review of studies and trials of supportive therapy in severe malaria was undertaken. Whilst definitive evidence for parenteral antimalarial treatment has now been provided, progress on supportive therapeutics has been limited. To date, of the 34 clinical trials involving adjunctive therapies in severe malaria, including 20 (59%) in African children, none of these trials have targeted bacterial co-infection. Searching ClinicalTrials.gov and ISRCTN we found no trials addressing anti-microbial treatment of bacterial co-infection in severe malaria.Current recommendations indicate that antibiotics should be given to ‘all children with suspected severe malaria in areas of moderate and high transmission until a bacterial infection is excluded… and should be based on culture and sensitivity results or, if not available, local antibiotic sensitivity patterns’, against which currently recommended antimicrobials are ineffective . It is also active against a range of other gram-negative and gram-positive organisms, although it is less active againstEscherichia coli and Klebsiella spp. The alternative (effective) antimicrobial therapies we considered, given that they would likely be active against NTS, were, first, a full course (~seven days) of parenteral third generation cephalosporins. This has feasibility concerns as it would prolong hospital stay (median four-five days), and would be difficult  to blind. In contrast to macrolides, which are not widely used in other childhood diseases, adding a major new indication for third-generation cephalosporins  also risks widening their use even further, and thus increasing the threat of antimicrobial resistance to a second-line antibiotic recommended for other common childhood diseases. Quinolones (e.g. ciprofloxacin) are another alternative strategy, given their superior antimicrobial activity against Salmonella species,E. coli and Klebsiella spp. However, resistance is generated through single mutations in the housekeeping genes gyrA or gyrB; and can be detected shortly after administration in healthy volunteer studies, suggesting resistant variants are commonly present at low levels or easily acquired. A recent study found pathogenic ciprofloxacin-resistantE. coli were highly prevalent in healthy children and their mothers, raising the possibility that widespread use in children with severe malaria without evidence of bacterial infection could have serious unintended consequences. Moreover, in most countries the use of fluoroquinolones in children is relatively contraindicated  except for use in multi-drug resistant infections where there are no suitable alternatives. Finally, although cephalosporin and quinolone resistance rates are currently relatively low in relevant organisms in some African countries, they are much higher in Asia, limiting generalisability of the trial’s findings worldwide.There are several reasons why we consider that azithromycin represents an attractive option in such an approach. Given the worldwide threat of antimicrobial resistance, the underlying principle is to test the narrowest spectrum antibiotic that would be practical, generalisable and plausibly have reasonable efficacy. There are several reasons for selecting azithromycin as the targeted antimicrobial invention. Primarily, it is active against NTS, the commonest cause of bacterial co-infection in severe malariaOther favourable properties of azithromycin include:1.   It is licenced for use and has a good safety profile in children..2.   Azithromycin’s pharmacokinetic (PK) properties include extensive tissue distribution, prolonged phagocyte concentrations, and a longer elimination half-life (~50hours) than other macrolides, thus enabling once-daily dosing (versus more frequent dosing required for clindamycin).3.   Its long half-life is also likely to protect children at increased risk of NTS bacteraemia following a malaria episode. It is thus plausible that azithromycin may also have a beneficial effect on immune activation and gut inflammation.4.   An accumulating body of evidence indicates some macrolides have beneficial properties independent of their antimicrobial effects, and has proved effective in treatingP. falciparum malaria in early phase trials when used in combination with faster-acting antimalarials.5.   A further consideration is that azithromycin is a weak antimalarial6.   Dispersible azithromycin is currently widely available at an affordable price, costing 0.40 euros per 100mg dose in an oral formulation. i.e. two-six Euros per five-day paediatric treatment course.. However, WHO definitions of severe malaria are very broad, incorporating high parasitaemia as a single criterion, and are thus applicable to a large proportion of paediatric admissions in such regions, with a relatively overall low case fatality (1–2%), who are unlikely to benefit from antimicrobial therapy as a group. For this study  we aim to target antibiotics to children with the highest risk of bacterial co-infection, meeting ‘Teule’ criteria: that is, with malaria ; temperature > 38°C or < 36°C; and ≥1 of prostration, respiratory distress, haemoglobin (Hb) <5g/dl or HIV. These criteria identified 85% of bacterial co-infections, with a three-fold higher mortality than children admitted with malaria without these criteria.Current recommendations indicate that antibiotics should be given to ‘all children with suspected severe malaria in areas of moderate and high transmission until a bacterial infection is excluded’et al., the Teule criteria identifies 85% of malaria cases with culture-proven bacteraemia: a pathogen was isolated from 20% children meeting these criteria. Simple and objective criteria identifying those at risk would be more generalizable outside research centres and would be validated in this study. (ii) Potential biomarkers: even when quality-controlled blood-culture facilities are available, low sensitivity  and long times to culture-positivity  mean that alternative approaches for identifying concurrent gram-negative bacteraemia would be highly valuable.A major legitimate concern is whether future management guidelines can be implemented which try to reduce the overuse of antibiotics where microbiology services are poor or non-existent, thus an approach to targeting only those with IBI is not feasible in much of Africa. Within the context of this study and to inform a future trial, we aim to investigate approaches  to targeting antibiotics. We propose to use (i) simple clinical criteria: proposed by NadjmISRCTN49726849 (registered on 27th October 2017).Trial Registrationth October 2018.Protocol Version 1.2: Date 9This trial, designed to inform a key research gap, proposes targeted and appropriate antimicrobial treatment of children with severe malaria at greatest risk of bacterial co-infection. Incorporated in the design are experimental data to demonstrate that the mechanisms that azithromycin is targeting are biologically reasonable, that the dosing is providing the right exposure, and that there is a pharmacokinetic-pharmodynamic (PKPD) link between azithromycin exposure and the potential mechanisms for reducing both short-term and microbiological cure (seven-day) and longer-term mortality (28-day). Comparison with a control group will also enable us to identify whether antimicrobial treatment can be further targeted..At present pharmacological data are lacking on the correct dosage of oral azithromycin for treatment of bacterial co-infection in severe malaria in African children. We will therefore conduct a Phase I/II trial comparing three doses of azithromycin: 10, 15 and 20 mg/kg (prescribed for feasibility by weight-bands) spanning the lowest to highest mg/kg doses demonstrated to be equally effective as parenteral treatment for other salmonellae infection1. Azithromycin given to children once-daily for five days in addition to standard treatment of severe malaria  can provide adequate dosing in children admitted to hospital with severe malaria.2. Children with severe malaria and culture-proven bacteraemia can be accurately identified using clinical criteria alone or in combination with a rapid diagnostic biomarker tests, in comparison with a control cohort of children hospitalized with severe malaria but not meeting Teule criteria, at low risk of bacterial co-infection.Our principal objectives are to:(i)   establish the appropriate dose of oral dispersible azithromycin as an antimicrobial treatment for children with severe malaria;(ii)   investigate whether antibiotics can be targeted to those at greatest risk of bacterial co-infection using clinical criteria alone or in combination with a rapid diagnostic biomarker test.(i)   To conduct a Phase I/II trial comparing three doses of azithromycin: 10, 15 and 20 mg/kg (prescribed for feasibility by weight-bands) spanning the lowest to highest mg/kg doses demonstrated to be equally effective as parenteral treatment for other salmonellae infection.(ii)   To determine, via PKPD modelling, the optimal azithromycin dose in severe malaria, and investigate associations between azithromycin exposure and pathogen susceptibility  with treatment outcome. Outcome measures will consist of changes in C-reactive protein (CRP), a putative marker of sepsis at 72 hours (continuous) and microbiological cure (seven-day) (binary), alone and as a composite with seven-day survival, while providing preliminary data on longer-term survival (to day 90).(iii)   To evaluate whether a combination of clinical, point-of-care diagnostic tests, and/or biomarkers can accurately identify the sub-group of severe malaria with culture-proven bacteraemia by comparison with a control cohort of children hospitalized with severe malaria but not meeting Teule criteria, at low risk of bacterial co-infection., in an area with hyperendemic malaria and paediatric admissions of >20,000/year, approximately 50% with malaria parasitaemia. MRRH is now an established research hub of the KEMRI-Oxford-Wellcome Trust Major Overseas Programme, assisted by trial, data and laboratory management from Kilifi, Kenya.The trial will be conducted in Mbale Regional Referral Hospital (MRRH), Eastern Uganda, where we already have an on-going collaborationsA Phase I/II open-label trial comparing three doses of azithromycin: 10, 15 and 20 mg/kg (prescribed for feasibility by weight-bands) spanning the lowest to highest mg/kg doses demonstrated to be equally effective as parenteral treatment for other salmonellae infection.Children will be considered eligible for enrolment in this trial if they fulfil all the inclusion criteria and none of the exclusion criteria. Cases:P. falciparum malaria (on either blood film or ParacheckTM RDT) and all of the following:105 children aged six months to 12 years at admission to hospital withi) Axillary temperature >38°C or <36°C.: any one of the following: prostration; respiratory distress; severe anaemia (haemoglobin<5g/dL) or HIV infection.ii) Teule severity criteriaiii) Parents willing/able to provide consent.The control cohort of children (n=50) are those hospitalised with severe malaria but not meeting Teule criteria  whose parents are willing/able to provide consent.Major contraindications to azithromycin, e.g. strong existing clinical diagnosis of QT-prolongation. Concomitant use of interacting drugs: drugs that may cause QT-prolongation or drugs that may cause a pharmacokinetic interaction with azithromycin, like strong CYP3A/P-GP inducers and concomitantly administered antacids. A formal sample size was not calculated. Phase I/II trials are an important step in the assessment of new or existing interventions providing the first data on the feasibility of using a product in a new application. They assess the effect of products (how well they work), and the side effects they may have. They are not usually designed to compare superiority of different products or interventions.The aim is to generate pilot efficacy data on the optimal azithromycin dose for children with severe malaria, which will inform the design of a later larger Phase III trial. The numbers required to address the trial objectives are therefore balanced against the exposure of children in these settings to a therapeutic intervention (dose) for which there are limited data to date.The overall sample size for the trial will be 105 children randomised 1:1:1 to receive 10, 15 or 20mg/kg azithromycin (based on weight-bands). This is sufficient for the PKPD sampling and modelling to determine an optimal dose in children with severe malaria using change in CRP at 72 hours, and microbiological cure (seven-day) alone and with seven-day survival. This is under the assumption that 20% of enrolled children (meeting Teule severity criteria) will have bacteraemia and that 80% of these infections will be caused by NTS or other enteric gram-negative organisms based on previous research. , although there are no data on absorption in severe malaria, which is characterised by impaired gut function, hence the need for this pilot. Along with the 105 children with Teule criteria we will enrol 50 children meeting other inclusion criteria but without Teule criteria (and without any exclusion criteria) into the control group.Based on Monte Carlo simulations, we expect that all dose groups should reach the population reference target (adult) AUC24h of at least 3.4 h*mg/LP. falciparum histidine-rich protein 2, HRP2) to determine malaria status and eligibility.Eligible children will be identified by the nurse and clinician on duty and registered in the eligibility screening log. A member of the trial team will then perform a rapid structured assessment of heart rate, oxygen saturation (pulse oximetry), respiratory rate, axillary temperature, blood pressure, markers of shock  and severity (conscious level and respiratory distress). Children who are potentially eligible with suspected severe malaria will have a rapid bedside malaria test  Clinical Trials Unit at University College London, London. The randomisation envelopes will be prepared before the trial, using the lists at the Clinical Trials Facilty, KEMRI Wellcome Trust Research Programme (KWTRP), Kilifi, Kenya. These will be opaque and sealed and will contain a card with allocation. Children will be randomised (1:1:1) to receive 10, 15 or 20 mg/kg azithromycin (based on weight-bands). The cards will be numbered consecutively and opened in numerical order. Clinicians will be aware of the treatment-group dose assignments, but the laboratory tests are to be performed in a blinded manner. and for which we have received ethical approval. It proposes to use a ‘two-stage’ consent process in this circumstance. Verbal assent will be sought from parents or guardians by the admitting medical team if it is considered that the full consent process would significantly delay treatment allocation, and consequently could be detrimental to the child’s health. Full consent will be sought once the child’s clinical condition has been stabilized. Caregivers will be provided with a brief verbal description of the trial and will be given the opportunity to “opt out”. As in the FEAST trial, if following an assent process a child died prior to full written consent, full consent would not be sought. This process of emergency consent was approved for FEAST and has been subsequently approved for use in a TRACT transfusion trial in Uganda and Blantyre.Once eligibility has been confirmed, authorised trial staff will approach parents/guardians to invite their child to take part in the trial. An information sheet will be provided to the parent/guardian in their usual language containing details of the TABS trial. The sheet will be read aloud to those who are unable to read. The doctor/nurse will check that the information has been fully understood and parents/guardians will be encouraged to ask questions they may have about their child’s participation in TABS trial. The information sheet and consent includes details of the clinical trial, follow up and additional biological samples taken for the trial and permissions for sample storage. Where possible, prospective written informed consent will be sought from parents/guardians who will then be asked to sign the consent form. Consent will include permission for the collection of admission and follow-up blood samples for later aetiological investigations. If parents/guardians are unable to sign, a thumbprint will be taken in lieu of a signature. A copy of the consent form will be given to the parent/guardian, the original placed in the patient’s medical notes, and a copy kept in the investigator site file. A number of children will present as emergencies where delay in study enrolment, and thus treatment, will not be practical or indeed humane. We will use a modified form of deferred consent; used in the FEAST trialsee Inclusion criteria above) eligible for the trial will be randomised to one of three doses of adjunctive once daily oral azithromycin doses, which will be given for five days in addition to a three-day course of parenteral artesunate.The comparator cohort with non-severe malaria will receive usual standard of care . See Study Flow , the trial will use weight bands see all usin and will be validated by the nested PKPD sub-study . Children generally tolerate a wide range of azithromycin doses (5-20mg/kg); however, when given in combination with artesunate, doses of 20mg/kg more commonly resulted in vomiting (versus artemether-lumefantrine). Based on Monte Carlo simulations, we expect that all dose groups should reach the population reference target (adult) AUC24h of at least 3.4 h*mg/L, although there are no data on absorption in severe malaria, which is characterised by impaired gut function, hence the need for this pilot.This weight-band dosing has been developed based on existing dataChildren will be intensively monitored during admission by the clinical team at least eight hourly in the first 72 hours following recruitment, and daily thereafter. Locator maps and contact numbers will be obtained to facilitate follow-up. All participants will then be seen at seven-days, 28-days and 90-days post-admission at outpatient clinics attached to each centre for evaluation of morbidity and toxicity. Any patient not returning for a study visit will be traced for vital status ascertainment .P. falciparum HRP2), genetic studies (including sickle cell and glucose-6-phosphatase deficiency status) and immune activation studies (transcriptome and cellular assays). For the latter, red and white cell pellets will be prepared and stored. Further blood samples will be taken at 72 hours, seven days and 28 days (for biomarker assays and immune activation studies); and at day seven for repeat blood culture , blood culture and other clinically indicated investigations (not required by the study protocol). Urine will be taken as soon as possible after trial enrolment for multi-stick analysis for assessment of cola or red colour urine and additional storage for later analysis of gut barrier function. In accordance with national guidelines, HIV testing will be performed during admission procedures since this is one of the eligibility criteria. Pre- and post-test counselling will be done in accordance with routine practice. At admission, a venous blood sample will be collected into Heparin and EDTA tubes and stored for subsequent cytokine and biomarker assays, parasitological  clinicians will be permitted to prescribe broad-spectrum antimicrobials guided by the admission blood culture results (species and/or susceptibility). The Uganda severe malaria guidelines give no specific guidance on the use of antibiotics in severe malaria. Children or their carers will be provided with an appropriate supply of azithromycin drugs if discharged before day five.All trial patients will receive standard of care including  anti-malarial drugs following national guidelines, based on WHO syndromic patient managementThe clinical coordinator is responsible for ensuring the discharge check-list is complete and for chasing up inpatient notes at discharge. Any relevant information, especially with regard to date of discharge, serious adverse events (SAEs), treatments, blood transfusions, use of intravenous fluids, oxygen or non-routine treatments or investigations, will be recorded. Children will be assessed on day seven, day 28 and day 90 following recruitment. The parent/guardian will receive a follow up invitation on a card. A symptom checklist and targeted physical examination will be performed at each clinic visit post-discharge. Medical history since last visit including hospital re-admissions, transfusions and grade three or four adverse events related to the antibiotic interventions including severity and likely relationship of any adverse events will be documented by a doctor. Any child lost to follow-up before 90 days will be traced for vital status. In order to minimise losses to follow-up, locator data (maps and identifiable landmark) and mobile phone numbers will be taken on discharge and verified at every review. During the 90 day study period attempts will be made to contact the patient via phone (if available) and to follow-up with home visits, if clinic visits are not attended. In the statistical analysis, a patient will be regarded as ‘lost to follow-up’ if they were not seen in clinic at the day 90 visit and were not known to have died.The product to be tested in the trial is azithromycin 100 mg dispersible tablets, which are a commercial product and were obtained for the trial as a donation from Cipla Pharmaceuticals, Mumbai, India. Azithromycin is approved for use in children in Uganda where the trial will take place. Cipla Ltd is fully GMP compliant and has provided full certificates for the manufacture of these products. Azithromycin will be stored at room temperature and not exceeding 30°C (86°F). It will be stored in these conditions for three years as packaged. The trial coordinator at the site will maintain accountability logs for the antibiotic intervention . These will be kept securely until verified by the external monitor’s visit.For PK analysis, model-based sampling strategies will enable the description of both intra- and inter-patient variability. A maximum of 3x 0.2ml samples will be taken from each subject on two occasions between day one to five around meaningful time points to describe the PK throughout a dosing interval plus one sample at follow up on day seven to determine the elimination of azithromycin after end of treatment . In the first year we will develop highly sensitive bioanalytical assays using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS) for determination of azithromycin in stored microvolumes of plasma .  and the treatment outcomes . Based on Monte Carlo simulations, we expect that all dose groups should reach the population reference target (adult) AUC24h of at least 3.4 h*mg/L, although there are no data on absorption in severe malaria, which is characterised by impaired gut function, hence the need for this pilot. The different doses in 105 children provides variability in exposure so that we identify whether all doses are in the flat part of the exposure-response curve, in which case the lowest dose will be chosen. Other secondary endpoints include mortality at 48 hours, length of hospital stay (days), re-hospitalisation and other adverse events. Quality-control of stored bacterial isolates, non-culture (molecular) assessment of bacterial infection including the determination of MIC to inform PKPD studies will be batch-processed at KWTRP, Kenya.Initially a population PK model will be developed for azithromycin and will investigate possible covariates for its PK. This will be followed by an investigation of the relationship between plasma PK and PD using the developed PK model, known MIC-values since the area under the curve (AUC):MIC ratio is the pharmacodynamic driver for azithromycin and endotoxin, to identify whether any of the above markers, alone or in combination, can predict which at-risk children with severe malaria have bacterial co-infections to improve the sensitivity of clinical/laboratory criteria and enable antimicrobial therapy to be further targeted. We will investigate associations between pathological concentrations of these markers and clinical, parasitological and microbiological data in cases (meeting Teule criteria) and controls (not meeting Teule criteria) to test their accuracy for stratification in the main trial. The samples will also allow us to analyse markers of gut barrier dysfunction – a requisite for bacteraemia  and the ileal-bile acid binding protein (I-BABP) and establish whether these can accurately predict children with blood-culture proven bacteraemia alone or in combination with clinical criteria/RDTs. In addition, we will analyse the peripheral blood mononuclear cell transcriptome in a subset of children with and without bacterial co-infection to verify the significant changes in transcriptome we observed in a small pilot study of children with malaria and bacterial co-infection compared to children with malaria alone and to confirm observed changes on the cellular level in a second set of children. We will investigate whether changes in gene expression profile can be translated into rapid and affordable biological tests predicting children with bacterial co-infection with high specificity and sensitivity. This later analysis is subject to additional funding.In 105 trial participants and 50 children with controls  we plan to investigate from admission samples the utility of a range of potential biomarkers including CRP, PCT•  Change in CRP, a putative marker of sepsis at 72 hours (continuous) and microbiological cure (seven-day) (binary), alone and as a composite with seven-day survival.•  The changes in CRP between day 0 and day 3 as the main endpoint for the PKPD studies.The secondary outcome measures are:•  Mortality at 48-hours and longer-term survival (to day 28 and to day 90).•  Length of hospital stay (days)•  Re-admission to hospital by 90 days•  Adverse events•  The population PK of azithromycin and their relation (combined with pathogen susceptibility) with treatment outcome (PD).SAEs will be reviewed immediately by a designated physician (SAE reviewer) and reported to the appropriate ethics and regulatory committees within one week. The Chief Investigator will inform the Trial Steering Committee (TSC) and Data Monitoring Committee (DMC) for review on a regular basis (as deemed necessary).An independent DMC (see composition at the end of the protocol) will review data on enrolment, safety, adherence to the trial protocol and efficacy at regular intervals and in strict confidence. The terms are covered in the DMC charter, signed by the chair and trial statistician. There are no fixed ‘stopping rules’. The DMC will receive and review information on the progress and accruing data of the trial and provide advice on the conduct of the trial to the TSC. The DMC would inform the Chair of the TSC if, in their view the results are likely to convince a broad range of clinicians, including those supporting the trial and the general clinical community, that, on balance, one trial arm is clearly indicated or contraindicated for all participants or a particular category of participants. The DMC is comprised of a chair and two other independent members. None declared conflicts of interest.The Mbale site will use a web enabled trial database and will be responsible for data entry and local trial management. The site will retain the original case report forms (CRFs). Data stored on the database will be checked for missing or unusual values (range checks) and checked for consistency within participants over time. If any such problems are identified, the site will be contacted and asked to verify or correct the entry. Changes will be made on the original CRF and entered into the database at the site. Kilifi Clinical Trial Facility will also send reminders for any overdue and/or missing data with the regular inconsistency reports of errors.This trial will be monitored according to a monitoring plan which will set out the frequency of visits, the degree of source document verification against the CRFs and the requirements for triggered on-site monitoring visits. This plan will also detail the procedures for review and sign-off. The monitoring will adhere to the principles of ICH GCP.The site initiation visits will include training in the trial procedures, as well as practical training in administration of trial interventions, reporting guidelines for adverse events of study interventions as well as other trial procedures. All staff at sites involved in the trial will receive formal training in GCP through a dedicated training programme during site initiation visit and will also be required to complete an on-line course. The Clinical Trial Facility in Kilifi oversees standards and quality of all trials conducted through the KWTP and through its monitoring systems and standard operating procedures are organised to ensure that all sites can be monitored with equal independence and rigor. All monitors will be appropriately qualified and trained.At each monitoring visit the monitors will:▪   verify completeness of Trial Master File▪   confirm adherence to protocol▪   review eligibility verification and consent procedures▪   look for missed clinical event reporting▪   verify completeness, consistency and accuracy of data being entered on CRFs▪   evaluate drug accountability▪   provide additional training as neededThe monitors will require access to all patient medical records including, but not limited to, laboratory test results and prescriptions. The investigator (or delegated deputy) should work with the monitor to ensure that any problems detected are resolved.OpenClinica. All data will be regularly backed up and backup copies stored both on and off site. Paper records will be archived in locked cabinets. These cabinets will have limited access with prior authorisation . All data will be partially anonymised prior to presentation or publication of any results. Study participants will be identified by a unique subject identification number but patient identifiable information will not be recorded on the study database in compliance with GCP requirements. The data will be examined for inconsistencies during the trial by the statistician and fed back to study sites for corrections following GCP procedures.All clinical and laboratory data will be recorded in the CRF and stored with a unique serial number identifier. Data will be entered (double data entry) ontoAll clinical data will be held confidentially, and personal identifiers will be removed before analysis of the data and presentation of the results..After completion of the study, requests for data access from researchers outside the study team will be considered by the trial management team and clinical trials unit (Data Governance Committee), and where indicated, requestors will be asked to develop scientific protocols for approval of secondary analyses. The potential to share data will be included in the participant Information and Consent Form (see extended data section)The primary analysis for the trial will describe baseline parameters (stratified by study arm) and compare primary and secondary end points by trial arm. The primary analysis will be by intention to treat and secondary analysis will include those treated per protocol .The co-primary outcomes are change in CRP  and microbiological cure (seven day) alone and as a composite with seven-day survival . Analysis of adverse events and re-admission will use time-to-event methods through day 90 counting in-hospital death as a competing risk. Changes in CRP and other inflammatory markers will be analysed using normal linear regression , using generalised estimating equations to jointly model changes at 72 hours and day-seven. Adverse events will also be summarised by body system.th October 2017 and updated on 12th August 2020 .Ethical approval has been obtained Mbale Regional Hospital Research Ethics committee (MRRH_REC 095/2017) and from Imperial College Research Ethics Committee (17IC3965), the sponsor of the study. The trial was registered on ISRCTN (ISRCTN49726849) on 27The randomised trial will be conducted in children who are most likely to benefit from the treatment. We will minimise the risks of cannula insertion and phlebotomy by pretrial training in phlebotomy technique and regular cannula site inspection which is included in a SOP. We have calculated that no more than 1ml/kg of blood will be drawn for research purposes at any one time.Children with severe malaria often develop complications such as convulsions, severe anaemia and hypoglycaemia which through close clinical monitoring will be identified at the earliest opportunity and appropriate therapy initiated. Mbale Regional Referral Hospital trial has considerable experience with managing severe malaria and this will serve to minimise the risks to the patients in the trial. Pretrial training of the dedicated study team will include specific training on general management of severe malaria and its complications. A manual of operations will provide clear management guidelines as well as the details of trial conduct and procedures. Children enrolled in the trial will therefore receive a higher quality of care than those managed routinely.Results from this trial will be disseminated locally through community meetings and national meetings with the wider healthcare professional community. These systems have been developed for dissemination of MRC FEAST and TRACT trial results and will be adapted to dissemination for the TABS PKPD trial.The site principal investigator has discussed the study with Ministry of Health staff. When the results are available, we will provide a summary briefing highlighting the trial results and what then next steps will be. Whilst the current study will go some way towards addressing whether azithromycin could be used as an adjunctive treatment in severe malaria a future trial should include a pragmatic design to ensure results are applicable to health services in Africa. The approach we propose may also have utility for syndromic management of other conditions, especially those whose clinical features overlap with severe malaria and in areas where microbiological diagnostic facilities are non-existent. ) is commonly practiced in high-income countries. However, poor microbiology services and overloaded health systems mean that this approach is not feasible in much of Africa and a trial designed around it would not be generalisable. Thus, in many parts of Africa antibiotic prescribing in children with severe malaria is highly variable across hospitals, with most physicians essentially using antibiotics “blind”, and thus are unable to follow WHO guidelines recommending stopping broad-spectrum antibiotics when bacterial infection has been ruled out. A more pragmatic approach would be to use a standard short-course of a moderately broad-spectrum antibiotic that is not already recommended for empiric use in a large number of other conditions with the potential for improving both early and post-discharge mortality. Relevant to a future trial and to current guidelines, is that many hospitals in Africa lack microbiological culture facilities, meaning best practice is not obvious given the clear threat posed by antimicrobial resistance. To address this within the context of this study and to inform a future trial, we aim to investigate potential approaches  to targeting antibiotics where microbiological faciliaties are not present or reliable.Severe malaria remains a common cause of paediatric admission in many countries in Africa. Children who have bacterial co-infection are at high risk of poor outcome. Establishing which children with malaria are at greatest risk of bacteraemia is critical to pragmatically inform a policy for targeted antibiotic therapy that could substantially reduce malaria-associated mortality while minimising the risks of excess antibiotic prescribing. Short-course empiric therapy with early termination guided by culture results  are the most widely used antimicrobials but resistance is becoming widespread. Moerover, few children will receive an ‘adequate’ therapeutic dose of cephalosporins, since NTS, the commonest cause of bacterial co-infection, requires ~seven days parenteral therapy thus requiring prolonged hospital stay (median four-five days) with economic implications. Further, adding a major new indication for third generation cephalosporins  risks substantially expanding their use, with downstream threats of AMR to a second-line antibiotic currently recommended for other common childhood diseases. Newer antimicrobials, apart from financial considerations, have similar concerns that over-use could lead to resistance. We favoured investigating azithromycin since one study found that almost 50% of bacterial isolates were resistant to the antibiotics most commonly recommended for empirical useWe therefore plan to study azithromycin because of its favourable microbiological spectrum, its inherent antimalarial and immunomodulatory properties and dosing and safety profile. Its long half-life is also likely to protect children at increased risk of NTS bacteraemia during the month following a malaria episode. At present pharmacological data are lacking on the correct dosage of oral azithromycin for treatment of bacterial co-infection in severe malaria in African children. The TABS trial will therefore conduct a Phase I/II trial comparing three doses of azithromycin: 10, 15 and 20 mg/kg (prescribed for feasibility by weight-bands) spanning the lowest to highest mg/kg doses demonstrated to be equally effective as parenteral treatment for other salmonellae infection. Treatment will be given over five days in children with severe malaria at highest risk of bacterial co-infection (meeting Teule criteria) in order to generate relevant PK data by sparse sampling during dosing intervals. The goal is to determine, via population PK modelling, the optimal azithromycin dose in severe malaria, and investigate associations between azithromycin exposure and potential mechanisms PKPD using change in CRP, a putative marker of sepsis at 72 hours (continuous) and microbiological cure (seven-day) (binary), alone and as a composite with seven-day survival, while providing preliminary data on longer-term survival (to day 90). We will also evaluate whether a combination of clinical, point-of-care diagnostic tests, and/or biomarkers can accurately identify the sub-group of severe malaria with culture-proven bacteraemia by comparison with a control cohort of children hospitalized with severe malaria but not meeting Teule criteria, at low risk of bacterial co-infection., but would also be informed by this pilot trial. The primary outcome would be mortality at either day seven, day 28 or day 90 mortality which is also be informed by this pilot trial (other timepoints would be secondary outcomes). Other secondary outcomes include mortality at 48 hours, length of hospital stay (days); subsequent hospital readmission; adverse events, faecal changes in antimicrobial resistance and cost-effectiveness.A future Phase III trial may therefore consider two strata: first comparing a pharmacologically-informed dosage of oral azithromycin to standard-of-care  in children at highest risk of bacterial co-infection and second comparing standard of care versus no antibiotics in children with severe malaria but minimal risk of bacterial co-infection to establish whether a policy for targeted antibiotic therapy could substantially reduce malaria-associated mortality while minimising the risks of excess antibiotic prescribing. Risk of co-infection could be determined by Teule criteriath October 2021 and follow up on 4th. January 2022. The clinical trial data and PKPD are currently being analysed.Trial enrolment started in January 2021. The delayed start of recruitment was due to the requirement for CIPLA to donate the trial drug which was mandated by the National Drug Authority (NDA), Uganda. Although we received support from CIPLA (2019) and the relevant approvals for importing the drug for the trial was then secured from NDA there was significant delays to importing the dispersible azithromycin for the trial and starting the trial due to the COVID epidemic. The trial completed recruitment on 4th November 2017 following MRRH approval (27th October 2017) of the revised version 1.0 Protocol : College of Medicine, Blantyre Malawi; Dr Jane Crawley: University of Oxford; Dr Irene Lubega: Department of Paediatrics Makerere University College of Health Sciences, Uganda. Prof. Timothy Peto (Chair): University of Oxford; Dr Jennifer Thompson : London School of Hygiene and Tropical Medicine; and Professor Philippa Musoka:Makerere University College of Health Sciences, Uganda.Imperial College London is the main research Sponsor for this study. For further information regarding the sponsorship conditions, please contact the Head of Regulatory Compliance at:Joint Research Office, Room 221b, Medical School Building, St Mary’s Campus, Norfolk Place, London, W2 1PG. Telephone: +44 (0) 020 7594 1872. The abstract does not clearly state the primary objective of the study, which is to determine the appropriate dose of oral azithromycin for treating African children with severe malaria and bacterial co-infection. It also does not clearly state the secondary objective, which is to investigate the use of clinical criteria and biomarker tests to identify children at the highest risk of bacterial co-infection.The article does not provide detailed information on the study design, such as the number of participants, the inclusion and exclusion criteria, and the randomization process. This information is important for readers to understand the validity and generalizability of the study findings.The article does not provide information on the statistical methods that will be used to analyze the data. This information is important for readers to understand how the study findings will be interpreted and to assess the validity of the conclusions.The article briefly mentions that current evidence for treating co-infection is lacking and that there is no consensus on the dosage or length of treatment required. However, it does not discuss other potential limitations of the study, such as potential biases or confounding factors, which could affect the validity of the study findings.This study aims to determine the appropriate dose of oral dispersible azithromycin for treating children with severe malaria and investigate whether antibiotics can be targeted to those at high risk of bacterial co-infection using clinical criteria and rapid diagnostic biomarker tests. The trial will compare three doses of azithromycin and collect pharmacokinetic data to determine the optimal dose. The study will also evaluate the use of clinical criteria, diagnostic tests, and biomarkers to identify severe malaria patients with bacterial co-infection. The results will inform the design of a Phase III trial. However, there are a few areas that could be adjusted; However, while the study protocol is of interest and addresses an important clinical question, the article could be improved by providing more clarity and detail on the study design, statistical analysis, and potential limitations.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?PartlyAre the datasets clearly presented in a useable and accessible format?PartlyReviewer Expertise:Malaria chemotherapy, ImmunopathologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I am satisfied with the authors’ responses to my review.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?PartlyAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Malaria in pregnancy and childhood. Pathogenesis and immunity. Collaborations with clinical pharmacologists but o specific expertise.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dr Olupot-Olupot and colleagues present a protocol for a trial that recently commenced recruitment in Uganda, comparing different doses of oral azithromycin as adjunctive treatment for children with severe malaria. Although all doses would be expected to be effective if used parenterally for the commonest pathogen, non-typhoidal salmonellae, impaired gut function might affect drug absorption, justifying the range of doses under study. Given this appears primarily to be a dose-finding study for a proposed definitive study, some of the hypotheses, aims and the primary outcomes do not seem to be particularly aligned with this, focusing instead on biomarker and bacterial sepsis endpoints, which the sample size is unlikely to be powered to address effectively. With 35 children per group, and a possible bacterial sepsis rate of 20% only a handful of children in each group are likely to have sepsis. By contrast, the justification for the pharmacokinetic aspects of the study is strong. The trial was originally registered in 2017, but logistical delays including COVID-related disruptions led to delayed initiation to January 2021. The descriptions of the trial procedures  are mostly clear, but there are some points for clarification.Major comments:The co-primary endpoint of “change in CRP at 72 hours” needs better explanation. Malaria itself elevates CRP, so what is the evidence to support using this as a discriminating factor between malaria and malaria+ bacteraemia? There is no discussion to justify this.Control group: 50 children are to be recruited and not treated with azithromycin. At different points these are described as “those hospitalised with severe malaria but not meeting Teule criteria” ; and elsewhere as “non-severe malaria”, Figure 1 and “treatment allocation”, Page 7. Given most severe malaria in Uganda is due to severe anaemia, cerebral malaria and/or respiratory distress and few children who do not meet the Teule criteria but do have severe malaria may be expected, please clarify which children will be in the “control” group.  et al.,  J Antimicrob Chemother 1996).On page 4 the authors claim that the azithromycin half life is such that it is likely to offer protection from bacteraemia for a month. My reading of the literature is that drug concentrations in target tissues are expected to be above MICs for ~10 days ? Or “a total of 7 samples per child” ?Under “statistical analysis” there is no discussion of analysis and interpretation of the PK data which to my mind is the main justification for the study.In the discussion, the authors state “Establishing which children with malaria are at greatest risk of bacteraemia is critical to pragmatically inform a policy for targeted antibiotic therapy that could substantially reduce malaria-associated mortality while minimising the risks of excess antibiotic prescribing.” Whilst true, the present study does not address this question, rather it gives all children in established high risk groups treatment.Minor comments:The methods section of the abstract is hard to follow, particularly the sentence which reads “and investigate azithromycin exposure to change in C-reactive protein, a putative marker of sepsis at 72 hours, and microbiological cure (seven-day) alone and as a composite with seven-day survival.”The list of abbreviations is quite incomplete.In the Introduction, there is a very long sentence that needs revision, starting with “Even when with the best (evidence-based)…”There is no mention of molecular diagnosis of malaria. Will this be performed?Is the LC/MS set up now in Nijmegen? There are published methods for this, using small volumes of plasma.  Under “Trial monitoring” the first sentence is unclear.. It reads: “The Mbale site on to a web enabled trial database will be responsible for its own data entry and local trial managementIs the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?PartlyAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Malaria in pregnancy and childhood. Pathogenesis and immunity. Collaborations with clinical pharmacologists but o specific expertise.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. First of all may I apologise for the late response to Dr Rogerson as we were waiting for a second review so any revisions requested would be included in a single revision. We have still not been able to get an additional review therefore we now provide this response and revised manuscript. Dr Olupot-Olupot and colleagues present a protocol for a trial that recently commenced recruitment in Uganda, comparing different doses of oral azithromycin as adjunctive treatment for children with severe malaria. Although all doses would be expected to be effective if used parenterally for the commonest pathogen, non-typhoidal salmonellae, impaired gut function might affect drug absorption, justifying the range of doses under study. Given this appears primarily to be a dose-finding study for a proposed definitive study, some of the hypotheses, aims and the primary outcomes do not seem to be particularly aligned with this, focusing instead on biomarker and bacterial sepsis endpoints, which the sample size is unlikely to be powered to address effectively. With 35 children per group, and a possible bacterial sepsis rate of 20% only a handful of children in each group are likely to have sepsis. By contrast, the justification for the pharmacokinetic aspects of the study is strong. The trial was originally registered in 2017, but logistical delays including COVID-related disruptions led to delayed initiation to January 2021. The descriptions of the trial procedures  are mostly clear, but there are some points for clarification.We thank Dr Rogerson for his very careful consideration of our trial protocol. To be clear the major objectives are to (i) establish the appropriate dose of oral dispersible azithromycin (by PKPD) as an antimicrobial treatment for children with severe malaria and (ii) investigate whether antibiotics can be targeted to those at greatest risk of bacterial co-infection using clinical criteria alone or in combination with a rapid diagnostic biomarker tests. This study will provide supporting data to inform a future trial and/or policy for hospitals without reliable microbiological facilities.Here are our specific responses to the comments.  Major comments:The co-primary endpoint of “change in CRP at 72 hours” needs better explanation. Malaria itself elevates CRP, so what is the evidence to support using this as a discriminating factor between malaria and malaria+ bacteraemia? There is no discussion to justify this. The major aim was to see if there were differences in CRP   between the intervention (Azithromycin) and control. We understand that CRP is elevated in severe malaria, however the data on why CRP is raised is not clear. The most recent review of CRP in malaria only found a handful of studies conducted in Africa, many focused onP. Vivax or malaria in pregnancy. (DOI: 10.1038/s41598-021-01556-0). Whilst risk of co-infection could be determined by Teule criteria, this pilot trial will also study a number of other potential biomarkers (which could be done as point of care test) which could identify a group with a higher risk of bacterial sepsis. A continuous numeric (delta change in CRP) endpoint rather than a categorical endpoint reduces the overall number needed. Control group: 50 children are to be recruited and not treated with azithromycin. At different points these are described as “those hospitalised with severe malaria but not meeting Teule criteria” ; and elsewhere as “non-severe malaria”, Figure 1 and “treatment allocation”, Page 7. Given most severe malaria in Uganda is due to severe anaemia, cerebral malaria and/or respiratory distress and few children who do not meet the Teule criteria but do have severe malaria may be expected, please clarify which children will be in the “control” group.   We apologise for the use of the term non-severe malaria, what was implied was cases admitted with severe malaria who do not have Teule criteria. Those children were identified as the group with the highest risk of bacterial infection. With regards to other cases of severe malaria we include the WHO classification of severe malaria in the PDF file linkedhere, which considers three groups for admission. Quite a number of other features are not covered by the Tuele critieria.et al.,  J Antimicrob Chemother 1996).1On page 4 the authors claim that the azithromycin half-life is such that it is likely to offer protection from bacteraemia for a month. My reading of the literature is that drug concentrations in target tissues are expected to be above MICs for ~10 days ? Or “a total of 7 samples per child” ?  There were a total of 7 samples per child. The Figure has “D0, D1-D5 (2 x 0.2ml), D7” where D1-D5 is inclusive of D2, D3 and D4. Under Figure 1 where we state D0, D1- (2x0.2ml*), D7 the asterix was missing a label, which now indicates D1-D5 inclusive.  Under “statistical analysis” there is no discussion of analysis and interpretation of the PK data which to my mind is the main justification for the study.Of methods used for the PK analysis is described under the PKPD substudy section of the paper. More details will be given in the trial report since the model will need to be built on the clinical data.In the discussion, the authors state “Establishing which children with malaria are at greatest risk of bacteraemia is critical to pragmatically inform a policy for targeted antibiotic therapy that could substantially reduce malaria-associated mortality while minimising the risks of excess antibiotic prescribing.” Whilst true, the present study does not address this question, rather it gives all children in established high risk groups treatment. We agree; this is a pilot study aimed at informing a future trial with those aims.  Minor comments:The methods section of the abstract is hard to follow, particularly the sentence which reads “and investigate azithromycin exposure to change in C-reactive protein, a putative marker of sepsis at 72 hours, and microbiological cure (seven-day) alone and as a composite with seven-day survival.”has been revised to ‘and investigate the impact of different levels of azithromycin exposure on change in C-reactive protein (a putative marker of sepsis at 72 hours), as well as on microbiological cure alone and as a composite with survival at 7 days’.The list of abbreviations is quite incomplete. We have updated these.In the Introduction, there is a very long sentence that needs revision, starting with “Even when with the best (evidence-based)…” This has been revised.  There is no mention of molecular diagnosis of malaria. Will this be performed? There is no need to perform molecular diagnosis of malaria – this is a clinical study where children will be enrolled pragmatically using clinical criteria, point of care malaria diagnostics and a malaria slide. We are also performing PfHRP2 on admission samples to characterise those with true severe malaria from those with incidental parasitaemia.  Is the LC/MS set up now in Nijmegen? There are published methods for this, using small volumes of plasma.    Under “Trial monitoring” the first sentence is unclear.. It reads: “The Mbale site on to a web enabled trial database will be responsible for its own data entry and local trial management   We have corrected this sentence to provide more clarity.  This is currently being set up in Nijmegen; the methods will be published separately.


Plasmodium vivax, one of the major species associated with human malaria, continues to be a major public health problem in many parts of the world. Numerous studies related to vivax malaria have described quantitative haematological findings , but diverse morphological changes of parasite forms within infected red blood cells (iRBCs) have been mentioned only in few studies. Here we report a case of a 13-year-old boy who presented with fever, significant low platelet counts and hypovolaemia that created a diagnostic dilemma. Detection of microgametocytes by microscopic examinations, further confirmed by multiplex nested PCR assays and response to anti-malarials, helped to make the diagnosis. We present an atypical case of vivax malaria with a review of morpho-variations of iRBCs and have summarized the characteristics that aid in creating increased awareness among laboratory health professionals and public health workers. No supporting data, software or code was generated or re-used.Plasmodium falciparum and Plasmodium vivax co-endemicity, vivax malaria is relatively more widely distributed, causing singnificant morbidity and mortality [P. vivax as intrinsically benign, but such a description may no longer be viewed as a paradigm of vivax malaria [P. vivax infects reticulocytes and young red blood cell (RBCs) [P. vivax malaria from eight different countries revealed an association of severe malaria, severe anaemia, acute respiratory distress syndrome (ARDS) and cerebral malaria [P. vivax malaria can be attributed to long duration of the hepatic stage that allows the parasite to remain in the host even after interruption of transmission and successful treatment of the primary infection [In geographical areas with ortality . In the  malaria . P. vival (RBCs)  and rarel (RBCs) . Classic malaria . Severe nfection . Furthernfection .P. vivax observed during microscopic examination of stained peripheral blood smears include (i) crisis forms, (ii) pseudo-parthenogenesis forms, (iii) equatorial trophozoites, (iv) phagocytosed parasites, (v) malaria pigment-containing phagocytes, (vi) microgametocytes with or without ex-flagellation, (vii) multiple invasions of iRBCs and (viii) macrogametocytes. Crisis forms appear as blackish-brown pigmented and are characterized by an extra-erythrocytic bluish amoeboid structure of the parasites. These are disintegrating forms of parasites and have been reported from India, often in co-infections with lymphatic filariasis [Various morphological changes in infected RBCs (iRBCs) due to lariasis . The undlariasis .P. vivax malaria were first described by Garnham et al. in 1966 where the simultaneous presence of both asexual and sexual forms were detected in the same iRBC [et al. in 1997 [‘Pseudo-parthenogenesis forms’ in ame iRBC . These fame iRBC . In equa in 1997 .et al. in 1986 [et al. in 1983 using dark-field microscopy [Intra-leucocytic malaria pigment was first observed by Meckel in viscera and blood of patients who died of pernicious fever . Such fi in 1986  using pocroscopy . MalariaPhagocytosed malaria parasites have been reported in patients with extremely acute pernicious malaria. Almost a century ago, Golgi depicted circulating leucocytes containing malarial parasites either well preserved or in various stages of disintegration .in vitro ex-flagellation [P. falciparum malaria [Plasmodia species have been reported [Plasmodia occur as thin filamentous structures with an oval-shaped dark blue nucleus [et al. [P. falciparum malaria, have also been reported in P. vivax. As many as five rings in a single iRBC in a neonate just 15 h after birth [More commonly reported variations are microgametocytes with or without ex-flagellation. Such events occur often in definitive hosts but are rarely seen in peripheral blood smears of intermediate hosts. A heat-stable molecule derived from mosquito head and gut, namely mosquito ex-flagellation factor (MEF), is believed to be vital in inducing ellation . During ellation . This fo malaria . At the reported . The occreported . With de nucleus . In cont [et al. . Multipler birth  and singer birth .–3 blood; normal: 1.5–4.5 mm–3 blood), low haematological parameters of haemoglobin , haematocrit , mean corpuscular volume  and mean corpuscular haemoglobin . Blood urea  and random blood sugar  levels were significantly raised. Peripheral blood smear examination was positive for P. vivax. Other tests such as Dengue  NS1, scrub typhus card tests, Widal test and COVID 19 reverse transcriptase (RT)-PCR were negative. Despite thrombocytopaenia, the child had no bleeding manifestations and was administered a first dose of intravenous artesunate and intravenous fluids.A 13-year-old boy was referred to our hospital with complaints of high-grade fever, non-bilious, non-bloody projectile vomiting, light headedness, generalized weakness, reduced appetite and passing black-coloured urine. Prior to his admission, the child was febrile (40 °C) with blood pressure of 110/40 mmHg , along pO2) was well maintained. Liver was enlarged up to 2 cm below the right costal margin. Other systemic examinations were within normal limits. Upon laboratory investigations, his haemoglobin level was 11 g dl−1, with a low haematocrit (34.2 %), mean corpuscular volume (71 fl) and mean corpuscular haemoglobin (22.9 pg) values with platelet count of 36 000 mm–3 blood. Serum direct bilirubin and liver enzymes were elevated. Giemsa-stained peripheral blood smears revealed normal sized erythrocytes with microgametocytes containing malarial pigments  , with high pulse rate (110 bpm) and facial swelling. There was reduction in urine output of around 150 ml per day, but the oxygen saturation  . Buffer P. vivax infection can manifest clinically as that of P. falcipraum infection and has been previously reported in the literature. A case series [P. vivax malaria simulating that of P. falciparum infection. In each case, blood pressure was 80/50 mmHg, haemoglobin 9 g dl−1, platelets 70 000 mm–3 blood, and both chloroquine and artesunate was continued along with other supportive medications, which resulted in improvement of vital signs over the next 48 h in these patients. In another case, severe P. vivax malaria mimicked sepsis in a neonate, with low hemoglobion (4.4 g dl−1), hematocrit (13.1 %) and platelet count (95 000 mm–3 blood). The child improved with administration of injectable artesunate [P. vivax proteins such as Pv RBP1 and Rp RBP2 that are thought to restrict P. vivax to reticulocytes [Severe and complicated e series  describetesunate . Two othtesunate . Such evulocytes .P. vivax have been increasingly reported, suggesting a diversity of P. vivax isolates in different parts of the world [P. vivax cases in duffy antigen receptor for chemokines (DARC)-negative individulas further adds to the changing paradigm [P. vivax isolates supported the distinctiveness of transmissibility and spectre of P. vivax related manifestations [P. vivax infection can be identified with the help of latency, patency and relapse. The periodicity of P. vivax varies systematically in different regions, indicating adaptations in the variability of iRBCs. Presentations mimicking dengue in the present case with significant thrombocytopaenia and without haemorrhagic manifestations is another facet, although is not unusual. Malaria and dengue co-infections have been reported most frequently from the Indian subcontinent and often cause misdiagnosis and/or misinterpretation of mono-infections [P. vivax malaria indicate interactions among host, parasite and external factors, of which parasite virulence seems to play a major role [P. vivax polymorphisms altering cytokine production are other important factors [P. vivax shows its distinctiveness with underlying pathogenetic mechanisms. The recognition of such morphological changes of iRBCs is important to avoid diagnostic dilemmas.In recent years, severe forms of malaria due to he world . The groparadigm . In thisstations . P. vivafections . Variatijor role . Anti-ma factors . PeripheWhat makes our case interesting is that our patient had a very low thrombocytopaenia with no bleeding diathesis but with severe hypovolaemia without manifesting with any features of shock or sepsis. In addition, a collective investigative algorithm was able to conclusively achieve a diagnosis.


Plasmodium falciparum is common. It is critical to decipher the role of single or co-infected disease scenarios on immune system regulation in such individuals and how such co-infections can either ameliorate or complicate immune response and the consequent disease outcome. First, 10 ml of urine samples, collected between 10:00 am and 2:00 pm, was filtered for diagnosis of schistosomiasis, while egg count, indicative of disease severity, was determined by microscopy. Furthermore, genomic DNA samples extracted from dried blood spots collected on filter paper from one hundred and forty-four Schistosoma haematobium-infected school-children was tested for P. falciparum parasite positivity by an allele-specific nested-PCR analysis of merozoite surface protein (msp)-1 and -2 genes and a real-time PCR assay. In addition, among P. falciparum parasite-positive individuals, we carried out a Taqman SNP genotyping assay to extrapolate the effect of host CD14  genetic variants on schistosome egg count.Schistosomiasis remains a chronic disease of global importance, especially in many rural areas of the world where co-infection with P. falciparum parasite positivity with msp-1 gene were 34%, 43% and 55% for MAD20, RO33 and K1 alleles respectively. Of the co-infected individuals, CD14 genetic variants ranged from 18.8% vs 21.5%, 33.3% vs 44.4%, 9.7% vs 11.8% for single versus schistosome co-infection for the wild type (CC), heterozygous (CT) and mutant (TT) variants respectively. Though the mean egg count for co-infected individuals with CD14 wild type (33.7 eggs per 10 ml of urine) and heterozygote variants (37.5 eggs per 10 ml of urine) were lower than that of schistosome infection alone (52.48 and 48.08 eggs/10 ml of urine respectively), it lacked statistical significance , possibly reflecting the benefit of the CD14 activation in schistosome plus malaria co-infection and not schistosome infection alone. In addition, the lower mean egg count in co-infected individuals reveal the benefit of downstream Th1 immune response mitigated by CD14 innate activation that is absent in schistosome infection alone.Of the 144 individuals recruited,  Schistosoma, whose global burden as of 2017, was estimated at 1.4 million disability-adjusted life-years [S. haematobium and S. mansoni are the most predominant species in many endemic regions [S. haematobium is the most common in Nigeria, presenting with disease-associated complications such as hematuria, hydronephrosis and predisposition to the human immunodeficiency virus infection [Schistosomiasis is one of the most common water-borne disease caused by parasitic trematodes of the genus  regions . S. haemnfection , 6–8. Manfection . Local rnfection . Many deHost genetics and immune response have been shown to be mitigating factors driving infectivity and disease severity among individuals exposed to the same rates of infection . This geP. falciparum malaria parasitemia [P. falciparum infection. While Chakraborty et al. [The homozygous mutant allele of the CD14 promoter gene polymorphism rs2569190) has been shown by our group to be associated with susceptibility to disease and regulation of asitemia . Our res69190 hasy et al.  detectedP. falciparum positivity or severity, along with the high rates of schistosomiasis and malaria endemicity in sSA, it is necessary to delineate how schistosome infection alone or co-infection with malaria modulates immune response and disease outcome. Given the prevalence, severity and impact of S. haematobium infection in rural Nigeria, including the potential for co-infection with P. falciparum, we set out to demonstrate the role of CD14 genetic variants on disease severity and outcome variables among school-children infected with schistosomiasis alone or co-infected with malaria in Nigeria.Beyond This was a cross-sectional study conducted between January and October, 2015. Study participants were from Ore and Ilie communities in Osun state, Nigeria where schistosomiasis has been reported to be endemic. The lake, harboring the snail intermediate host, is freely accessible for all forms of water contact activities, including recreational use by school children. Inclusion criterion is residency in the study area for more than a year, and school-children must be below the age of 19 years, as previously described . All metS. haematobium positive, an egg count was conducted. To do this, 10 ml of well-mixed urine was aspirated and carefully forced through a filter membrane. The filter was removed, placed on a slide and examined under a 10X objective of a light microscope. The number of eggs on the filter was counted and recorded as the number of eggs per 10 ml urine. From all the collected slides, 10% were randomly selected and re-examined by an independent microscopist for quality control. In addition, demographic data and other correlates of disease  were collected using a short questionnaire, as previously described [Urine samples, collected between 10:00 AM and 2:00 PM from all consenting individuals or school-children whose parents and guardians had provided consent, were screened for schistosome infection. For those classified as Finger pricked blood was spotted onto filter papers  from each patient whose urine sample was collected. Genomic DNA was extracted from filter paper blood spot with the Qiagen Blood Mini Kit  following manufacturer’s instruction, eluted in 100 μl total vlume and stored at –20 °C until further analysis. All methods were carried out in accordance with relevant guidelines and regulations or declaration of Helsinki.Primers for the polymorphic regions of interest (msp-1 block 2 and msp-2) was used for a two-step PCR amplification of the extracted genomic DNA from the blood samples, as previously described . For priP. falciparum parasite diversity, we also utilized the real-time PCR (RT-PCR) assay targeting the P. falciparum multi-copy var acidic terminal sequences (varATS) gene, for confirmation purposes [P. falciparum have been previously described [In addition to using the msp-1 and 2 allele specific typing for determining purposes . Primersescribed . A cycleTo determine the frequency of CD14 genetic variants, we carried out a single nucleotide polymorphism (SNP) assay. In brief, SNP genotyping was performed using Thermo Scientific Taqman SNP genotyping master mix, primers and protocols, as previously described . A totalS. haematobium egg count, age of participants and the packed cell volume (PCV) of individuals with schistosomiasis and malaria infection or schistosomiasis infection alone. Furthermore, we carried out a binary logistic regression model using schistosomiasis-malaria co-infection as outcome variable, and CD14 genetic variants as independent variable, while controlling for age, sex and PCV. In addition, the mean multiplicity of infection was calculated by dividing the total number of alleles detected for msp-1 and msp-2 genes by the total number of samples, as described [The genotypic and allelic frequencies of each variant  were estimated as fraction of the total CD14 variants. Independent t-test was carried out to evaluate the association between CD14 genetic variants and escribed . We usedescribed . For allThis study recruited a total of 144 participants, with a mean age of 10.97 years. There were more male (n = 77) than female (n = 67) participants, while the mean egg count of 38.36 eggs per 10 ml of urine was recorded  of the CD14 wild type allele (CC) in schistosomiasis infected individuals with or without malaria revealed an interesting result. Although, urine samples from individuals with schistosomiasis infection alone had a higher mean egg count (52.48 eggs per 10 ml of urine) than schistosomiasis and malaria co-infected individuals (33.70 per 10 ml of urine)  and heterozygous mutant (CT) alleles were combined and compared between both groups of either schistosome infection alone or in combination with malaria, similar pattern as in the wild type allele was observed. These variants were combined because they provide same level of protection against malaria and schistosome. Additionally, since the number of heterozygous individuals was limited, combining them provide an opportunity to assess their synergistic effect on reducing egg burden and other disease variables.Although, mean egg count (48.1 egg per 10 ml of urine) and PCV 37.6%) in individuals with schistosomiasis infection alone was higher than in those co-infected with malaria , there was no statistical difference  between the two groups. Nevertheless, we observed a significant difference in the mean age between schistosomiasis infected versus malaria co-infected group   and packed cell volume (37.6%) in individuals with schistosomiasis infection alone was higher than in those co-infected with malaria   and 3D7 17.4%) were the most prevalent alleles in both the msp-1 and msp-2 families respectively. This is followed by RO33 and MAD20 of msp-1 family, while FC27 of msp-2 was the least prevalent. Not surprisingly though, more malaria cases were observed with the real-time PCR assay (86.1%) among the schistosomiasis infected individuals  in schistosome-malaria co-infection, especially in younger individuals. This protection is completely abrogated in the schistosome-infected alone.Although, both infections co-circulate in endemic regions and among individuals, their interactions are driven specifically by host genetic and immune response and less by pathogen competition. In summary, the immune environment modulated by host CD14 gene in Our study is limited by its small patient number which may have contributed to the currently observed results. As a single center study, there is the potential that these findings may not be generalizable or representative enough to be applicable other areas with malaria and schistosomiasis co-infection in Africa and elsewhere. Therefore, we recommend that future studies should include a larger sample size covering parts of the continent with different transmission dynamics, and be conducted longitudinally to understand immune dynamics, especially as it relates with schistosomiasis and malaria co-infection.


Plasmodium falciparum transmission, malaria in pregnancy remains a significant cause of low birth weight, stillbirth, and severe anaemia. Previously, fetal sex has been identified to modify the risks of maternal asthma, pre-eclampsia, and gestational diabetes. One study demonstrated increased risk of placental malaria in women carrying a female fetus. We investigated the association between fetal sex and malaria in pregnancy in 11 pregnancy studies conducted in sub-Saharan African countries and Papua New Guinea through meta-analysis using log binomial regression fitted to a random-effects model. Malaria infection during pregnancy and delivery was assessed using light microscopy, polymerase chain reaction, and histology. Five studies were observational studies and six were randomised controlled trials. Studies varied in terms of gravidity, gestational age at antenatal enrolment and bed net use. Presence of a female fetus was associated with malaria infection at enrolment by light microscopy . Fetal sex did not associate with malaria infection when other time points or diagnostic methods were used. There is limited evidence that fetal sex influences the risk of malaria infection in pregnancy.In areas of moderate to intense  Plasmodium and transmitted by infected female Anopheles mosquitoes1. In 2020, about 241 million people had malaria and 627,000 died. The same year, 11.6 million pregnant women were exposed to malaria in sub-Saharan Africa, and malaria infection resulted in 819,000 neonates with low birth weight 2. Most malarial infections worldwide are attributed to Plasmodium falciparum and Plasmodium vivax, with P. falciparum accounting for most of the morbidity and mortality, especially in sub-Saharan Africa. In Africa, it is estimated that a quarter of cases of severe anaemia in pregnant women and a fifth of LBW and stillbirth cases can be linked to malaria5. Of note, P. falciparum-infected erythrocytes can sequester in the placental intervillous space, and placental malaria is associated with LBW and stillbirth3.Malaria is an infectious disease caused by parasites of the genus 3. Furthermore, parity-specific immunity acquired through pregnancies seems to contribute to a lower risk of placental malaria and the poor birth outcomes associated with it 3. Young maternal age may be an independent risk factor, as studies have shown that younger women are more susceptible to placental malaria and its poor outcomes, compared to older women with the same gravidity6.The risk of malaria in pregnancy is modulated by different factors. Transmission intensity drives the development of acquired immunity, and this leads to differences in disease risk and severity between areas of high and low transmission8. Male fetal sex has been associated with pregnancy complications such as term pre-eclampsia and gestational diabetes in meta-analysis of studies including over 12.5 million women8. Mothers of male fetuses are at increased risk of pre-eclampsia, but male fetuses may be more likely to maintain their growth trajectory compared to female fetuses. The mechanism could involve redirecting maternal blood flow to the placenta through vasoconstriction of maternal microvasculature, which is enhanced in pre-eclamptic compared normotensive women with a male fetus10. In contrast, there was no difference in the microvasculature of normotensive and pre-eclamptic women carrying female fetuses9. A study of asthma in pregnancy also demonstrated reduced growth in female compared to male fetuses. In the same study, the use of glucocorticoids as treatment for mild asthma was shown to improve growth in female fetuses, suggesting that the sex-specific growth impairment is associated with inflammation pathways11. It was demonstrated that placentas of asthmatic pregnant women with female  fetuses had an increased expression of pro-inflammatory cytokines, namely tumor necrosis factor α (TNFα) and interleukin 6 (IL-6), compared to healthy women12. Similarly, lipopolysaccharide-stimulated peripheral blood mononuclear cells obtained from women pregnant with females released more IL-6, TNFα and IL-1β than cells from women pregnant with males13. These findings suggest that the fetus and/or its placenta could influence maternal physiology in a sex-specific manner.There have been several studies exploring if and how fetoplacental sex might impact maternal health14. In women infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), maternal SARS-CoV-2-specific antibody titers were lower when the mother carried a male fetus15. So far, only one study has focused on maternal malaria risk. Adam et al. studied the relationship between placental malaria and fetal sex in women in eastern Sudan. Placental malaria was detected on placental histology and 88% of infections were past infections. The odds of placental malaria were 2.55 times higher when the fetus was female16. To complement the findings of this study, we conducted a meta-analysis of data collected from cohort studies and clinical trials that followed pregnant women in P. falciparum-endemic areas 17, to determine the association between fetal sex and malaria infection during pregnancy.Little is known about the effect of fetal sex on the risk of infection in the mother (the ‘host’), with most studies focusing on whether associations between maternal infection  and infant health outcomes  differ by sex19. Of the remaining 11 studies included, five were observational and six were clinical trials measuring the effect of chemoprevention or insecticide-treated nets31. Nine studies were conducted in sub-Saharan African countries and two in PNG. Among 12,830 women enrolled in these studies, 12,821 singleton pregnancies with data on fetal sex were included in this analysis.Of the 13 studies in the original pooled dataset, two studies were excluded. One was unavailable for this meta-analysis, and one did not record maternal malarial infection by LM, PCR or histologyApproximately two-thirds of the women included were aged 24 years or younger. Gravidity, trimester at antenatal enrolment, and bed net ownership varied across studies. Most of the participants lived in rural areas. In the studies where chemoprevention was used, the most commonly used strategy was intermittent preventive treatment with sulfadoxine-pyrimethamine. Details of each study participants’ characteristics are outlined in Table Ten studies n = 11,729) assessed peripheral malaria status at antenatal enrolment using LM  assessed peripheral malaria infection status at enrolment using PCR  Fig. . OverallFour studies n = 2739) assessed peripheral malaria infection at delivery by PCR. Two found a heightened risk with a female fetus, while the other two did not, but none of these differences were statistically significant  assessed for placental malaria infection at delivery using PCR . This was the only one out of seven malaria markers that showed a statistically significant result in meta-analysis, but the directionality of effect was similar when placental infection detected by LM at delivery was considered (RR 1.10). At the individual study level, one small cohort study suggested an association with male sex and increased risk of peripheral infection at delivery, but not other malaria markers32. Of the ten individual studies where malaria diagnosed by LM at antenatal enrolment was available, fetal sex was statistically significantly associated with malaria infection in two studies, both conducted in western Kenya in the 1990s when malaria transmission was very high, and there was a trend towards increased risk in another three studies  was associated with an increased incidence of malaria in infancy in male but not female babies14, and IPTp with dihydroartemisinin-piperaquine was found to reduce incidence of malaria in male but not female infants34. These studies suggest that fetal sex might play a role in modulating malaria risk in infancy that is associated with placental malaria.To date, only one study has explored the interaction between fetal sex and malaria risk in pregnancy. The study followed 339 women living in eastern Sudan, where intermittent preventive treatment in pregnancy (IPTp) is not used, and assessed placental malaria by histology. Risk of placental malaria was reported to be 2.55 times higher in women carrying female fetuses36. Several studies found that X chromosome inactivation results in sex-dependent differential gene expression, in both autosomal and sex-linked genes37. This includes upregulation of certain immunological mediators, including TNFα, in female placentas38. Previously, high levels of TNFα have been associated with higher parasite density and slower clearance40. Perhaps this upregulation of TNFα receptors led to an increased sensitivity to TNFα, resulting in slower clearance of parasites and more detectable infections in mothers carrying female fetuses. Asthma studies have shown that female placentas tend to upregulate cortisol levels in response to changes in maternal cortisol levels, which eventually results in increased expression of TNFα41. Possible mechanisms include sex-differential function of placental glucocorticoid receptor isoforms and reductions in placental 11β-hydroxysteroid dehydrogenase type 2 expression42. Further studies are required to confirm if a similar mechanism is observed with malaria infection. Upregulation of receptors used by parasites to sequester in the placenta should also be investigated as a possible mechanism in future studies.The mechanisms underpinning the potential association between female fetuses and malaria infection risk in the mother have yet to be elucidated. The placenta acts as an endocrine organ, secreting hormones and cytokines that might affect the physiology of both the mother and the fetus45. It is plausible that malaria in early gestation increases the risk of male fetal loss, which may manifest in an increased prevalence of malaria in pregnant women carrying female fetuses.The present study, and the preceding study by Adam et al., considered women who had a live born baby only. Male fetuses are at increased risk of stillbirth, and malaria is associated with stillbirth and miscarriage16, the dataset we used was of substantially larger sample size, including 4457 women for whom placental histology was available. The study population was drawn from a wider geographical area, including Papua New Guinea and multiple sub-Saharan African countries. This allows for more generalisation of the findings to P. falciparum-endemic regions. Furthermore, infection was assessed using LM, PCR and histology. The addition of PCR as a diagnostic tool enabled the detection of sub-microscopic infections. However, only four out of 11 studies tested for malaria infection using PCR, and the meta-analysis lacked power to conclusively evaluate the association between fetal sex and submicroscopic infection. Furthermore, it might be that fetal sex contributes to regulation of parasite density, rather than incidence of infection, and this could manifest in differences in infections detectable by microscopy, but not infections detected by PCR. The M3 initiative did not collate information on parasite densities. Lastly, differences in malaria risk, or the apparent lack of an association between fetal sex and malaria risk at birth, may have been the result of interventions started at antenatal enrolment such intermittent preventive treatment or provision of insecticide treated nets. In order to yield more conclusive results, assessing associations between fetal sex and malaria infection may be strengthened by including more studies that screened for infection, e.g., at enrolment using both LM and PCR, and that include pregnancies that resulted in stillbirth or miscarriage. Moreover, as the study population was diverse, other variables such as the maternal age and gravidity could have been confounders.This study had strengths and limitations. Compared to the study by Adam et al.In conclusion, carrying a female fetus may be associated with a higher risk of malaria in pregnancy, as previously reported, but the magnitude of any such risk is modest. Women with a female fetus had a higher risk of peripheral malaria infection at antenatal enrolment when determined by LM. This time point included the largest number of observations, and preceded the initiation of interventions such as IPTp or insecticide-treated bed nets that could have potentially masked sex-differential effects. Individual participant data meta-analysis of more recent interventional and cohort studies, adjusting for potential confounders and using ultrasound dating and including fetal losses, could confirm our findings, while efforts should be made to identify the underlying biological mechanisms that could underpin fetal sex-based differences in the risk of malaria in pregnancy.17, was derived from a dataset gathered by the Maternal Malaria and Malnutrition (M3) initiative that originally included data pooled from 13 studies following 13,898 pregnant women in P. falciparum-endemic areas, including Papua New Guinea (PNG) and several countries in sub-Saharan Africa (see cohort profile)17. Studies included were either prospective cohort studies or randomised controlled trials conducted between 1996 and 2015. Variables such as age, gravidity, and body mass index of the participants were recorded. Participants were also assessed for malaria infection at antenatal clinic enrolment and at delivery, using various diagnostic tools including light microscopy (LM) of blood films and polymerase chain reaction (PCR). Most studies used LM to detect parasitaemia, while some also used PCR to detect sub-microscopic infections. At delivery, some studies assessed the presence of placental malaria using LM, PCR or placental histology17, and each was analysed individually.The study population, summarised in detail elsewhere46.Where available, placental histology results were classified into three categories. Active infection was characterised by presence of parasites, regardless of presence of pigment in fibrin. Past infection was characterised by absence of parasites and presence of fibrin pigment. No infection was characterised by absence of both parasites and fibrin pigment47. Women without data for fetal sex were excluded, as were women who had not been screened for malaria infection by LM, PCR or histology.Univariable meta-analysis was performed using RStudio version 4.0.2. The dataset was reshaped for compatibility with the metafor package in R2. A P-value of less than 0.1 and I2 value of more than 40% were considered significant. Rows with missing values for respective malaria infection indicators were omitted. In meta-analyses by time-point and diagnostic modality, studies that had less than 100 women with data for a respective malaria indicator of interest were also excluded, as were studies that had no women with malaria in at least one of the groups . To account for multiple comparisons (n = 7) a Bonferroni-corrected P-value of < 0.007 was used to denote statistical significance.The exposure of interest was fetal sex, defined as male or female, and the outcome measure was malaria infection, assessed using differing techniques at enrolment or delivery. The association between fetal sex and maternal malaria infection was investigated for peripheral malaria infection detected by LM and PCR at enrolment and delivery, and for placental malaria detected by LM, PCR and placental histology. We calculated risk ratios and sampling variances using log-binomial regression analyses  and fitted them to a random-effects model to calculate the summary estimate and heterogeneity (rma function). Forest plots were generated using the forest function. Heterogeneity was assessed using the P-value of the Chi-square statistic and I17.All studies received approval by their local ethics board and obtained informed consent from all participants. Details of relevant procedures are highlighted in the published cohort profile Supplementary Figures.