Understanding what determines the between-host variability in infection dynamics is a key issue to better control the infection spread. In particular, pathogen clearance is desirable over rebounds for the health of the infected individual and its contact group. In this context, the Porcine Respiratory and Reproductive Syndrome virus (PRRSv) is of particular interest. Numerous studies have shown that pigs similarly infected with this highly ubiquitous virus elicit diverse response profiles. Whilst some manage to clear the virus within a few weeks, others experience prolonged infection with a rebound. Despite much speculation, the underlying mechanisms responsible for this undesirable rebound phenomenon remain unclear.We aimed at identifying immune mechanisms that can reproduce and explain the rebound patterns observed in PRRSv infection using a mathematical modelling approach of the within-host dynamics. As diverse mechanisms were found to influence PRRSv infection, we established a model that details the major mechanisms and their regulations at the between-cell scale. We developed an ABC-like optimisation method to fit our model to an extensive set of experimental data, consisting of non-rebounder and rebounder viremia profiles. We compared, between both profiles, the estimated parameter values, the resulting immune dynamics and the efficacies of the underlying immune mechanisms. Exploring the influence of these mechanisms, we showed that rebound was promoted by high apoptosis, high cell infection and low cytolysis by Cytotoxic T Lymphocytes, while increasing neutralisation was very efficient to prevent rebounds.Our paper provides an original model of the immune response and an appropriate systematic fitting method, whose interest extends beyond PRRS infection. It gives the first mechanistic explanation for emergence of rebounds during PRRSv infection. Moreover, results suggest that vaccines or genetic selection promoting strong neutralising and cytolytic responses, ideally associated with low apoptotic activity and cell permissiveness, would prevent rebound.The online version of this article (10.1186/s12918-018-0666-7) contains supplementary material, which is available to authorized users. Multiphasic infection profiles have been reported for various infections such as Influenza, HIV, Hepatitis B and C, as well as Porcine Respiratory and Reproductive Syndrome (PRRS). They can occur during natural infection , under ion , is of particular interest. It not only constitutes a major concern for the swine industry, responsible for significant economic losses worldwide , 14, butα, IFNα and IFNγ) and immuno-regulatory cytokines, the latter being either pro-cellular (IL12 and IFNγ) or pro-humoral (IL10 and TGFβ); (iii) the orientation of the adaptive response towards the cellular (Cytotoxic T Lymphocytes and IFNγ), humoral (antibodies and IL10), or regulatory (TGFβ and IL10) response ).Furthermore, caution is advised when interpreting the actual estimated model parameter values  immune response alone. This result appears to stand in conflict with previous genetic studies of the PHGC data, which found that viremia rebound was not heritable and which led to the conclusion that rebound was more likely caused by factors related to the virus or the environment , 19, 21.Previous studies proposed a number of alternative mechanisms responsible for the emergence of viremia rebound. These include within-host viral mutations , 12, 19,Insightsα-induced apoptosis should also prevent rebound, but to our knowledge, such vaccines have not yet been explored ), and down-regulations (multiply by κ−) depend on the cytokine concentration (Ck). The higher the cytokine concentration, the stronger the effect. However, there is a limited number of receptors, so the effect saturates above a given cytokine concentration. Cytokine effects are hence classically based on the Michaelis–Menten formalism [Cytokines are synthesised by the immune cells. They are involved in the regulation of most infection and immune processes, including the cytokine syntheses. Up-regulations, whether activations , a synergistic effect by κ±(CkCk′). To reduce the model complexity, we assumed that the regulation parameters km and vm were the same, whatever the cytokine(s) involved. This simplification was based on our sensitivity analyses, where both parameters were found to exhibit a negligible influence on the viral dynamics  and qualitative (shape – for immune components) data from PRRSv experimental studies were collected to define realistic within-host dynamics; (iii) through an extensive exploration of the parameter space, parameter ranges were refined to obtain realistic dynamics. We hence obtained ranges for all model parameters  this criterion. We then looked for not one but several data-compatible parameter sets. To do so, we developed a fitting method that resembles Approximate Bayesian Computation (ABC), but is less computationally costly. This allowed us to specify a full range of data-compatible parameter sets.Fitting criteria. Before calculating the fitting criterion, the simulation profile was determined. A viremia curve was classified as: (i) uniphasic if and only if (i) it exhibited a single peak above the detection threshold within the first 42 days of infection and (ii) the viremia was below the detection threshold at day 42; (ii) biphasic if and only if it exhibited at least two peaks above the detection threshold within the first 42 days of infection.i, the error Δi was defined as the shortest standardised distance between the viral indicator value simulated by the model n=4 for the uniphasic profile, n=9 for the biphasic profile): If the simulated viremia curve matched the expected profile, the corresponding criterion was computed as follows. Both uniphasic and biphasic criteria were based on the viral indicators Fig.  and the Indicator errors were normalised to account for differences in terms of magnitude and ranges. As we aimed for zero-valued errors for all indicators, if some indicator errors had outweighed the others, it could have affected the convergence of the optimisation algorithm. NB: Viral indicators would correspond to the summary statistics in an ABC method. A fitting criterion equal to zero would correspond to an ABC acceptance criterion with: (i) ABC data defined as mean viral indicator values; (ii) ABC tolerance defined as half the viral indicator ranges.If, in contrast, the simulated viremia curve did not match the expected profile, the corresponding fitting criterion Implementation and initialisation. The model simulation and model fitting were conducted in Scilab 5.5.3 [ab 5.5.3 . The minment}C=0 , was perment}C=0  in our cplanor [5.8 iterations on average.In order to thoroughly explore the parameter space, we performed the fitting procedure starting from 625 initial parameter sets that proceeded independently. They were chosen following a fractional factorial design built with the R package planor , in ordement}C=0 , within Selection of representative individuals for both viremia profiles. In order to capture the full range of parameter combinations associated with each profile without sampling bias, we generated a representative set of 35 individuals per viremia profile using a clustering method. Indeed, among the 625 individuals of each profile, some were very close, others quite distinct. Consequently, taking into account the full set would have lead to over- or under-representations of some individuals. We proceeded similarly but independently for both viremia profiles. We used a k-means clustering method (kmeans function of R package stats), which partitions the 625 parameter sets obtained by the fitting procedure into a given number of clusters. The number of clusters has to be big enough in order to represent the variability of the parameter sets but small enough to prevent the over-representation of some parameter sets. To set the number of clusters, various heuristics are employed, such as the “elbow rule”: between-class inertia increasing with the number of clusters, this rule consists in detecting the cluster number for which adding another cluster does not result in a notable inertia increase. We ran the k-means method for all possible numbers of clusters (1 to 625) and used the “elbow rule” to get a rough idea of the appropriate number of clusters (between 20 and 50). We then selected the smallest number corresponding to 80% between-class inertia for both profiles, namely 35 clusters. The representative individual of each cluster was chosen as the parameter set closest to the cluster barycentre. 35 individuals over the 625 represent a sufficiently large number to cover the full range of parameter combinations associated with each profile and to provide sufficient statistical power to detect differences between both profiles.Identification of immune mechanisms discriminating between both viremia profiles. In order to identify the key immunological drivers that lead to either uniphasic or biphasic viremia profiles, we compared, for the 35 individuals selected per profile: (i) the dynamics of the immune components represented in the model, over four time periods ; (ii) the estimated baseline rates of the immune mechanisms; and (iii) the efficacy of key immune mechanisms for the earlier (0 to 20 dpi) and later (21 to 42 dpi) time periods. The efficacy of a particular immune mechanism  was quantified by the ratio of the total number of viral particles or cells mobilised by the mechanism and the total number of viral particles or cells generated, over the time period considered. As an example, the efficacy of viral neutralisation was defined, from Eq. 2, as the total number of viral particles neutralised 4 permutations) and Kolmogorov–Smirnov for the baseline rates and mechanism efficacies (R package Matching).Comparisons were performed by visual inspection and standard uni-variate statistical tests: mean values and standard deviations, permutation tests for the trajectories  to ×103 (resp. 10−3). We performed the simulations on the 35 representative individuals per viremia profile. We focused on mechanisms which directly affect the viral dynamics and are assumed to play a critical role in the infection dynamics [βT), naive target cell apoptosis by TNFα  the values of the parameters driving the efficacy of each mechanism by six gradual levels from ×10  on chromosome 4, denoted WUR SNP, that was found to confer significant differences in cumulative viremia (i.e. area under the viremia curve) within the first 21 days post infection . So we tAdditional file 1Within-host dynamics. Evolution over infection time of the 19 state variables of the model: free viral particles, target cells (APC), natural killers, adaptive effectors including cytotoxic T lymphocytes, plasma cells, neutralising antibodies and cytokines. Comparison between uniphasic and biphasic viremia profiles. (PDF 124 kb)Additional file 2Estimated parameters. Values of the 14 parameters linked to between-host variability: host–virus interactions, viral replication, activation of the adaptive response, cytokine syntheses by activated target cells; for the 35 representative individuals selected for the uniphasic and biphasic viremia profiles. Comparison between uniphasic and biphasic viremia profiles. (PDF 87 kb)Additional file 3α, cytolysis by cytotoxic T lymphocytes, neutralisation by antibodies. Cytolysis by natural killers never resulted in a profile inversion (not shown). (PDF 87 kb)Mechanisms resulting in viremia profile inversion. Percentage of individuals, among the 35 representative individuals selected for the uniphasic and biphasic viremia profiles, that had a profile inversion when boosting or inhibiting (depending on the profile) either mechanism (one at a time): infection, apoptosis by TNFAdditional file 4Smoothed viremia data. Parameters of the Wood’s functions fitted to the PHGC data , for theAdditional file 5Model description & Sensitivity analyses. The file provides a complete description of the dynamic model representing the within-host dynamics induced by a primary PRRSv infection in a naive pig. It specifies the modelling assumptions and includes all model equations. The file also describes the global sensitivity analyses performed to assess the impact of model parameters on the viral dynamics. Corresponding aims, methods and results are presented. (PDF 710 kb)


Plasmodium-infected blood represents a risk for malaria transmission, a rare but severe event. Several non-endemic countries implement a strategy for the screening of candidate blood donors including questionnaire for the identification of at-risk subjects and laboratory testing of blood samples, often serology-based, with temporary deferral from donation for individuals with a positive result. In Italy, the most recent legislation, issued in November 2015, introduced the use of serological tests for the detection of anti-Plasmodium antibodies.Transfusion with In the absence of a gold standard for malaria serology, the aim of this work was to evaluate five commercial ELISA kits, and to determine their accuracy (sensitivity and specificity) in comparison to immuno-fluorescence antibody test (IFAT), and their agreement (concordance of results). Serum samples from malaria patients or from subjects with malaria history (N = 64), malaria naïve patients with other parasitic infections (N = 15), malaria naïve blood donors (N = 8) and malaria exposed candidate blood donors (N = 36) were tested.The specificity of all ELISA kits was 100%, while sensitivity ranged between 53 and 64% when compared to IFAT on malaria patients samples. When tested on candidate blood donors’ samples, ELISA kits showed highly variable agreement (42–94%) raising the possibility that the same individual could be included or excluded from donation depending on the test in use by the transfusion centre.These preliminary results indicate how the lack of a gold standard for malaria serology must be taken into account in the application and future revision of current legislation. There is need of developing more sensitive serological assays. Moreover, the adoption of a unique serological test at national level is recommended, as well as the development of screening algorithms based on multiple laboratory tests, including molecular assays.The online version of this article (10.1186/s12936-019-2650-0) contains supplementary material, which is available to authorized users. Plasmodium parasites to a recipient. The infection may be responsible for the development of severe clinical symptoms in the recipients, especially in malaria naïve individuals, and may become life threatening [Transfusion-transmitted malaria (TTM) is an alternative accidental route of malaria infection caused by the transfusion of whole blood, or a blood component, from an infected donor harbouring eatening , 2.Plasmodium are currently known to cause malaria in humans with 216 million new cases and 445,000 deaths estimated in 2016 [Plasmodium falciparum, the most diffused species—especially in sub-Saharan Africa (SSA)—is responsible for the vast majority of severe and fatal cases. Plasmodium vivax, mainly present in Central and South America and Southeast Asia can also cause severe disease, although the fatality rate is lower compared to P. falciparum [P. vivax and Plasmodium ovale (the latter being mainly observed in SSA) have a dormant liver stage—hypnozoite—which can relapse months or years after treatment completion; while the worldwide distributed Plasmodium malariae infection can persist indefinitely [Plasmodium knowlesi and Plasmodium simium have also been associated with human malaria cases. The former usually infects macaque monkeys in Southeast Asia [Six species of  in 2016 . Plasmodast Asia , 8, whilast Asia .Plasmodium infection has become an important issue especially with the increased travelling of people and migratory phenomena, leading to the implementation of blood safety policies [Plasmodium antibodies and, in case of a positive result, to be excluded from blood donation for 3 years [In non-endemic countries, the identification of donors at risk of policies . To avoipolicies , 11. In policies , 12. In  3 years .Plasmodium parasites on Giemsa-stained thick and thin blood smears by microscopic examination has been the gold standard for malaria diagnosis for over a century, allowing both the determination of species and that of parasite density. The theoretical limit of detection of microscopy is 5 parasites/µl but sensitivity varies according to the microscopist’s experience in the 5–100 parasites/µl range [In principle, a blood screening test could be based either on direct or indirect methods. Detection of µl range . Rapid dµl range . Nowadayµl range .Plasmodium antibodies include indirect immuno-fluorescence antibody test (IFAT), a well-established technique in several European countries [Plasmodium-specific antibodies has virtually no place in diagnosis of clinically acute malaria cases nor infection, while is among the diagnostic criteria used to determine chronic forms of malaria such as hyper reactive malarial splenomegaly. Nonetheless, serological tests are recommended by the World Health Organization and the European Directorate for the Quality of Medicines for the screening of candidate blood donors considered at risk of carrying an asymptomatic Plasmodium infection [Indirect methods for the detection of anti-ountries  which wanfection , 18. ThiHowever, establishing sensitivity of serology is a challenging task, as the magnitude and longevity of Plasmodium-specific antibody responses vary according to several host , parasite  and environmental (transmission level) factors , 20. SucThe objective of this observational study was to evaluate five commercial ELISA kits as a malaria screening tool of blood donors.Leishmania spp., n = 4 Trypanosoma cruzi, n = 4 Strongyloides stercoralis and n = 4 Schistosoma spp.) were also collected at CTD and included as negative controls.The retrospective study was carried out using archived serum samples from subjects recruited in two Italian laboratories. Serum samples (N = 64) obtained from 38 patients hospitalized between 2014 and 2016 at the Centre for Tropical Diseases (CTD) of the IRCCS Sacro Cuore-Don Calabria Hospital  were used. All subjects were either diagnosed with malaria by microscopy or had a previous history of malaria infection. Samples, previously tested by IFAT, were chosen based on IFAT titre, in order to cover a range of IFAT titres spanning from 0 to 1:10,240 . Samples with IFAT titre = 0 were obtained from patients with ascertained malaria infection at a very early stage of infection, when antibodies are not yet present. However, subsequent tests performed during hospitalization, showed a progressive increase in IFAT titre over time. Serum samples from malaria naïve blood donors (N = 8) and from malaria naïve patients with other parasitic infections  between February 2016 and February 2017, and screened with DRG ELISA (N = 496). The present study included N = 36 sera samples from screened candidate donors: all samples with a positive DRG result (n = 26), as well as random selection of samples with a negative DRG result (n = 10). All serum samples were stored at − 80 °C until use.P. falciparum parasites obtained from in vitro culture on human red cells and coated on slides. According to the manufacturer, the specificity and sensitivity of IFAT compared to another antibody detection method (i.e. electrosyneresis) or clinical symptoms are 98.74% (95% CI 95.43–99.66%) and 98.04% (95% CI 89.55–99.95%), respectively. The test enables the detection of Plasmodium-specific antibodies and samples are considered negative when IFAT titre = 0.IFAT assay  was performed according to the manufacturer’s instructions. The target antigen of the assay consists of Five commercial ELISA kits were employed to test serum samples: (1) Dia.Pro Malaria Ab ; (2) DRG Malaria ELISA ; (3) Novatec NovaLisa malaria ; (4) Euroimmun Anti-Plasmodium ELISA ; (5) BioRad Malaria EIA Test . All kits were obtained as a kind donation from their respective companies.Plasmodium spp. (without species indication) and detects anti-Plasmodium IgG, IgM or IgA. The detection is based on a biotin-streptavidin-HRP system: recombinant Plasmodium spp. proteins are used at the bottom of the plate, for antibody-capture, and in a biotinylated form for the identification of the antigen–antibody complex.The kit is based on recombinant proteins representing immuno-dominant epitopes of P. falciparum and P. vivax to detect IgG and IgM antibodies. Antibodies against P. malariae and P. ovale are also detected due to antigenic similarity.Both these kits use recombinant antigens from Plasmodium spp. most frequently infecting humans. Consequently, it detects IgG against P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi.Among the tested kits, the Euroimmun is the only one based on a mix of antigens from the five Plasmodium proteins for the detection of the antigen–antibody complex, consequently IgG, IgM and IgA anti P. falciparum and P. vivax can be detected. Antibodies against P. malariae and P. ovale can also be detected due to cross-reaction. Alternative names for this test can be found in the literature: Lab21 Healthcare or Lab21 Newmarket, depending on the distributor [The test uses HRP-conjugated sample/ODcutoff × 10), and samples were classified as negative, positive or “grey zone” according to manufacturer’s instructions. Samples with a result falling in the “grey zone” cannot be defined as positive or negative and, according to the kits instructions, the subject should be tested again after a few weeks.Internal positive and negative controls were provided in each kit and samples were manually processed according to the manufacturer’s instructions. The internal validity of ELISA results was evaluated by checking the OD values of the internal control samples against the validity ranges specified in each kit, by building a standard curve using serial dilutions of a pool of hyperimmune sera, and by estimating experimental precision and reproducibility on duplicate samples  from malaria patients and using IFAT as the reference standard. The specificity of the different ELISA kits was further examined by testing serum samples from malaria naïve individuals . The agreement between ELISA and IFAT was further assessed by investigating the distribution of ELISA indexes and the proportion of positive ELISA results according to IFAT titre . In order to further compare ELISA kits with IFAT, and in particular to investigate whether a positive relationship exists, as expected, the association of IFAT titres with ELISA quantitative (indexes) and qualitative (positive/negative) results was assessed by graphical inspection.The agreement between the five different ELISA kits was evaluated through the concordance between results of each pair of kits in samples from candidate blood donors (N = 36), in order to evaluate the probability that the same candidate donor will receive the same response independently of the kit used in the laboratory. The sample size was not formally calculated, as it was constrained by the number of tests supplied free of charge by the distributors.Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC) and GraphPad Prism v7.02 . The accuracy  of the ELISA kits was computed to establish the ability of each test to classify samples (N = 64) as positive or negative, when compared to IFAT results. Samples falling in the “grey zone” were excluded from these calculations. Concordance and Cohen’s k statistics were computed to assess the agreement of qualitative results between each pair of the five ELISA kits in the sample of candidate blood donors. A p-value < 0.05 was considered statistically significant.Statistical analyses were performed in STATA v.15  and thus no false positive results. The sensitivity, instead, varied from 53.6% (BioRad kit) to 64.2% (Dia.Pro kit). Overall, the Dia.Pro showed the best performance, among the tested kits (Table All sera (N = 23) from healthy subjects with no history of malaria exposure (n = 8) as well as from patients suffering from other parasitic diseases (n = 15) resulted negative when tested with each of the five kits, confirming 100% specificity as well as lack of cross-reactivity with non-Plasmodium-specific antibodies.Figure Figure k ranged from poor  to very high . Furthermore, only 12 out of 36 samples (33%) were identically classified as positive or negative by the five kits. The agreement between assays was not affected by the country of birth  of candidate blood donors nor by the result (positive vs negative) of the original screening test (data not shown). Repeatability data for the DRG assay are presented in Additional file The five ELISA kits were used to analyse serum samples from candidate blood donors and to compare their ability in classifying subjects as negative or positive at TTM screening. The concordance among the qualitative results of different ELISA kits is shown in Table Plasmodium parasites and prevent transfusion-transmitted malaria [Plasmodium-specific antibodies [Plasmodium antibodies in both malaria patients and candidate blood donors.Serological methods are currently employed in Italy and other non-endemic countries for the screening of candidate blood donors in order to detect potential asymptomatic carriers of  malaria , 5. Accotibodies . The aimPlasmodium antibodies, due to the high proportion of false negative results [Overall, the five ELISA kits showed unsatisfactory sensitivity (below 65%) for the classification of malaria samples, as compared to the Biomérieux IFAT, as reference test. Although no cross-reactivity with other parasitic infections was observed, such a low sensitivity indicates that these kits are inadequate to achieve an accurate detection of samples positive for the presence of anti- results –27.The sensitivity of ELISA assays compared to IFAT among malaria patients’ samples has been previously assessed by three studies, all evaluating the Cellabs ELISA kit –24. In tWhen assessed on candidate blood donors’ samples, the five kits showed highly variable results, with a percentage agreement varying from poor to high, depending on the pair of kits. It is therefore possible that a candidate blood donor will receive a different response from laboratories using different ELISA kits, with a probability depending on the kits pair, implying that a candidate blood donor could be considered suitable for or excluded from donation, depending on the kit in use in the reference laboratory.The present study is based on a limited sample size, which affects the precision of the estimates, and limits the generalizability of the results. Also, only ELISA kits commercially available in Italy at the time of the study were evaluated, and other products might show different characteristics. However the data presented here should provide evidence that the tested commercial ELISA kits are unsuitable—in particular if employed individually—for the screening of candidate blood donors for TTM risk.Semi-immune individuals represent the biggest challenge for blood safety and TTM screening as they might become asymptomatic carriers with a very low parasite density, which is difficult to detect with current direct diagnostic methods. Seed and colleagues reported that parasite densities as low as 1–10 parasites per blood unit are estimated to be sufficient to generate a malaria infection in a naïve recipient, albeit such levels are far from being detected by current methods, including PCR , 29. TheIn agreement with the literature, the present results highlight the need for the development of more sensitive serological tests, which should also be highly specific and reproducible. Moreover, future revisions, or application guidelines, of the current recommendations should include specific indication regarding which ELISA kit(s) should be adopted by the laboratories for TTM risk screening, in order for serology results and donors screening to be uniform and reproducible at national level.Plasmodium spp. nucleic acids [To ensure blood safety and blood supply, the screening strategy may benefit from the use of multiple tests including more sensitive serological tests and molecular methods for the detection of ic acids . With thic acids . Such anAdditional file 1. Internal validity of ELISA kits. Agreement between results of first and second DRG tests.


Plasmodium falciparum is epileptogenic and in malaria endemic areas, is a leading cause of acute seizures. In these areas, asymptomatic infections are common but considered benign and so, are not treated. The effects of such infections on seizures in patients with epilepsy is unknown. This study examined the relationship between P. falciparum infection and seizure control in children with a unique epilepsy type, the nodding syndrome.P. falciparum, including asymptomatic infections, increases the risk of seizures and impairs seizure control.This cross-sectional study was nested in an ongoing trial ‘Doxycycline for the treatment of nodding syndrome (NCT02850913)’. We hypothesised that, in patients with epilepsy, infection by Participants were Ugandan children with nodding syndrome, age ≥8 years, receiving sodium valproate. All had standardised testing including documentation of the number of seizures in the past month, a rapid malaria test and if positive, the peripheral blood parasite density.The primary outcome was the number of seizures in the past month (30 days).=0.002.A total of 164/240 (68%) had malaria. Asymptomatic infections (without fever) were seen in 160/240 (67%) and symptomatic infections in 4/240 (2.7%). In participants without malaria, the median (IQR) number of seizures in the past month was 2.0 (1.0–4.0) and it was 4.0 (2.0–7.5) in participants with malaria, p=0.017. The number of seizures in asymptomatic persons was 3.0 (IQR 2.0–7.3) and 6.0 (IQR 4.0–10.0) in symptomatic individuals, p=0.024. Additionally, in asymptomatic patients, a positive correlation was observed between the parasite density and number of seizures, r=0.33, pIn patients with nodding syndrome, both asymptomatic and symptomatic malaria are associated with an increased risk of seizures and poorer seizure control. Similar effects should be examined in other epilepsy disorders. Malaria prevention should be strengthened for these patients and chemotreatment and prevention studies considered to improve seizure control. Nodding syndrome is a poorly understood epilepsy disorder not representative of other epilepsies. However, the disease offers the advantage of a uniform population of patients with epilepsy, receiving the same antiepileptic drug, and a similar level of care in Uganda.This was a cross-sectional study that cannot ascribe causality; prospective studies should be conducted to confirm the results.The study also relied on parental recall of the number of convulsive seizures in the past month and could have suffered from the shortfalls of recall bias. Again, a prospective determination of study outcomes will be more appropriate.Plasmodium falciparum is still a major public health problem in tropical countries and especially, in sub-Saharan Africa. Over 200 million cases are reported annually with several thousand deaths majorly among children younger than 5 years and in pregnant women.P. falciparum presents with a spectrum of manifestations from asymptomatic infections , symptomatic but uncomplicated disease, to severe or complicated malaria.et al 2016)14P. falciparum is known to be epileptogenic, and is a leading cause of acute seizures in children living in malaria endemic areas.P. falciparum infections are associated with (1) Poorer seizure control. (2) Seizure control is worse in patients with higher parasitaemia.About 50% of children with acute severe falciparum malaria present with neurological involvement.Onchocerca volvulus have recently been proposed to underlie the pathogenesis.P. falciparum infection and seizure control in nodding syndrome.Nodding syndrome is a poorly understood complex epilepsy disorder that affects children and adolescents in some regions of Africa.P. falciparum malaria infections and seizure control in children with nodding syndrome. The study was nested within and all the participants are enrolled in an ongoing trial ‘Doxycycline for the treatment of nodding syndrome (NCT02850913)’. This trial is testing the hypothesis that nodding syndrome is an O. volvulus induced epileptic encephalopathy with antibodies to the parasite or its symbiotic bacteria, Wolbachia, cross-reacting with and damaging host neuron proteins.This was a cross-sectional study of the relationship between asymptomatic P. falciparum malaria and in 2015 and 2016 experienced a malaria epidemic.The trial is being conducted in the nodding syndrome affected districts of Kitgum, Pader and Lamwo in Northern Uganda. This region is inhabited by the Acholi, a Luo speaking community that is recovering from a decade-old civil war, with high levels of poverty and psychosocial problems. The districts are served by 17 nodding syndrome treatment centres where patients receive clinical care and treatment according to national guidelines.The study recruited all the 240 participants who had been enrolled in the Doxycycline for the Treatment of Nodding Syndrome Trial. The diagnosis of nodding syndrome was made according to the World Health Organisation (WHO) criteria. All were receiving sodium valproate (doses 12–35 mg/kg/day) as antiepileptic therapy plus nutritional, physical and psychological therapy.The Uganda National Council for Science and Technology and the National Drug Authority in Uganda provided regulatory approvals. Consent was obtained from each participant’s carer and assent was obtained from the participants (except for cases with severe cognitive impairment).Most children with nodding syndrome in Uganda live within a few kilometres of the 17 nodding syndrome treatment centres in the country. Patient registers from the selected centres were accessed to identify potential participants. All patients with nodding syndrome in the specific locations were invited to the nearest follow-up centre or a central location in a village by the study field staff. Patients were then screened for eligibility and eligible participants consented. The inclusion criteria included participants with confirmed nodding syndrome as defined by WHO; age 8 years or older (to avoid doxycycline toxicity) and written consent by the parent or guardian. Women with a positive urinary Human chorionic gonadotrophin (HCG) (pregnancy) test, known hypersensitivity to tetracycline, reported inability to swallow capsules, enrolled into another trial and suspected high likelihood of non-compliance with the study drug and follow-up schedule were excluded.As part of the requirements of the trial, all consenting participants and caregivers were invited to and hospitalised in the Kitgum General Hospital for about a week. During this period, they had detailed history, a full clinical and neurological assessment, an assessment of functioning using the Gross Motor Function Classification System and Modified Rankin Score, cognitive function on psychometric testing and the CogState (a computerised cognitive test), intellectual disability using the Child and Adolescent Intellectual Disability Screening Questionnaire, quality of life with the Quality of Life in Childhood Epilepsy Questionnaire and a diagnostic electroencephalogram test. The types of seizures were described and the burden reported as the number of seizures in the past month.P. falciparum malaria histidine rich protein-2 (HRP2) rapid diagnostic test . Participants testing positive for malaria on the rapid diagnostic test (RDT) had thick Giemsa-stained blood smear slides prepared to determine the parasite density. Each slide was examined by two observers, and any differences were reconciled by a third observer. The number of asexual malaria parasites observed was reported per 200 white blood cells (WBCs) and the parasite density per microlitre of blood estimated assuming 8000 WBC/ul of blood. The laboratory technicians performing these tests were blind to the rest of the clinical information, including the burden of seizures in the past month, which was obtained earlier by the study clinical and nursing staff.All participants had 10 ml of venous blood drawn for a complete blood count, liver and renal function, and study-specific tests. Malaria was tested using the P. falciparum parasitaemia with no history of fever in the past week and axillary temperature <37.5o C.Asymptomatic malaria was defined as P. falciparum parasitaemia with either history of fever in the past week or axillary temperature ≥37.5°C.Symptomatic malaria was defined as Seizure burden was defined as the number of seizures in the past 30 days as reported by the caretaker.Good seizure control was defined as No seizures in the past 30 days as reported by the caretaker.Poor seizure control was defined as one or more seizures in the past 30 days as reported by the caretaker.In this report, only the relevant clinical and laboratory testing, obtained at the enrolment screening before the interventions, is reported.The public was involved in developing the research questions of the overall study, that is, understanding the aetiology and treatment of nodding syndrome but not with the design of this substudy. The main trial is ongoing and in addition to personal contact with participants’ families every 6 months, there are also community meetings every 3–6 months. The study results will be fed back to the community at these meetings and during the biannual local FM radio broadcasts that the study conducts.2 test (proportions), the Student’s t-test for normally distributed data, and the Mann-Whitney test was used for skewed data. Participants were categorised into those with good seizure control (no seizures in the past month) and those poor seizure control (one or more seizures in the past month). A logistic regression model was used to examine the relationship between seizure control and infection with Plasmodium. Variables with a p value of 0.3 or less at bivariate level were considered for multivariate analysis. Variables significantly associated with infection by P. falciparum were further assessed for interaction with Plasmodium infection and seizure control. All variables that were not in the model because of a non-significant p value were assessed for a confounding effect using a 10% difference between the adjusted and unadjusted odds to show confounding. To examine if there is a direct relationship between the malaria parasite density and seizure burden, Spearman’s rank correlation testing was performed between the peripheral blood parasite density (log10 parasites/ul) and the number of seizures in the past month in patients with asymptomatic malaria.Data were entered in a Microsoft Access Database using Epi info V.7.1.5.2. All patient data were then exported and analysed with STATA V.12.0  and GraphPad Prism V.6.01 . Descriptive statistics where used to explore the data and this is reported as proportions, percentages, means (SD) and medians (IQR), as appropriate. Differences between the groups were tested by the χBetween September 2016 and August 2017, a total of 240 patients with nodding syndrome was recruited into the trial. All were included in this study. Of these, 140/240 (58.3%) were male. The mean age was 15.6 (SD 2.0) years. The mean dose of sodium valproate was 22.6 (SD 7.2) mg/kg/day. A total of 164/240 (68.3%) participants tested positive for falciparum malaria, most of whom had asymptomatic infections (160/164 (97.6%)).A total of 159/240 (66.3%) reported experiencing at least one convulsive seizure in the past year of whom 139 (57.9%) experienced at least one such seizure in the past 30 days. In patients without malaria, the median (IQR) number of seizures in the previous 30 days was 2.0 (1.0–4.0) and it was 4.0 (2.0–7.5) in patients with malaria, p=0.017, Mann-Whitney test . There wAmong patients with asymptomatic malaria, the median (IQR) number of seizures experienced in the past month was 3.0 (2.0–7.3). Generalised tonic-clonic and absence seizures were the most common types of seizures described. Other seizure manifestations were infrequent. Also, there were no differences in the manifestations of seizures in the three groups .Participants were categorised into those with good seizure control (no seizures in the past 30 days) and poor seizure control (one or more seizures in the past 30 days). In addition to presence of malaria parasitaemia, sex (AOR 1.96 (95% CI 1.11 to 3.46), p=0.03) and dose of antiepileptic drug (AOR 1.04 (95% CI 1.00 to 1.13), p=0.04) were significantly associated with seizure control . FurtherP. falciparum malaria infection experienced a significantly higher number of seizures in the previous month compared with patients without malaria and there was a direct correlation between the peripheral blood parasite load and the number of seizures. The study would suggest that in patients with seizure disorders on antiepileptic drug treatment, malaria parasitaemia, whether asymptomatic or symptomatic, may increase the risk of seizures and impair seizure control.This study investigated the relationship between asymptomatic malaria infection and seizure control in children with a complex epilepsy disorder, the nodding syndrome. The study found that patients with P. falciparum is even thought to be epileptogenic.The association between acute infections and an increase in seizures in children with epilepsy is a well-recognised phenomenon. In the context of this study, P. falciparum infection is characterised by sequestration of the late stages of the intraerythrocytic cycle, particularly in the brain. In the case of severe malaria, acute seizures may potentially be induced through multiple pathways: (1) Indirectly through biochemical mechanisms associated with hypoglycaemia, hyponatraemia or acidosis.Apart from neurocysticercosis, the epileptogenic mechanisms of parasitic infections are not well understood.Patients with malaria, on average, experienced twice the number of seizures experienced by those without malaria. The higher burden of seizures will impact on the clinical and antiepileptic drug needs of the patients and on physician time, healthcare costs, patient’s productivity, learning and achievements and on quality of life. There are an estimated 10 million people with epilepsy in Africa.The study had some limitations. First, nodding syndrome is a poorly understood complex epilepsy disorder that may not be representative of all other seizure disorders. However, the syndrome offers the advantage of a uniform population with all patients receiving the same antiepileptic drug and a similar level of care in Uganda.In conclusion, in patients with nodding syndrome, both asymptomatic and symptomatic malaria infections are associated with an increased risk of seizures and poorer seizure control. Similar effects should be examined in patients with other epilepsy disorders. Malaria prevention should be strengthened for these patients and chemoprevention studies considered.


Malaria is still a major public health concern in the Democratic Republic of Congo. Its morbidity and mortality challenge the actual strategies of the fight agains malaria. This study was aimed to describe the epidemiology, the clinical caracteristics and the risk factors of death associated to severe malaria in the pediatric population under 5 years at Sendwe Hospital of Lubumbashi.This analytical retrospective study was conducted in Lubumbashi, in the province of Haut-Katanga. All patients under 5 years hospitalized for severe malaria were registered from January 2014 to December 2016.Among the 3,092 patients hospitalised during our study period, 452 (14.6%) were admitted for severe malaria. The average age was 27.04 months, the male sex was the most affected (53.54% with the sex-ratio 1.15). The most frequent forms of gravity noticed were cerebral malaria (48.23%) and severe anemia (46.90%). Death was noted in the evolution in 28.32%. Repeated convulsion , coma  and severe acute malnutrition  were asscociated with a high risk of death.This research shows that severe malaria is still an important cause of morbidity and mortality among young children in Lubumbashi. Neurologic and anemic forms are the most frequent. The predictive signs of death are: repeated convulsions, coma and severe acute malnutrition. Plasmodium falciparum malaria has a rapid evolution from a non-complicated febrile disease generally treated with an oral drug, to a potentially deathly multisystemic disease. The mortality risk associated with non complicated falciparum malaria is estimated to less than 0.1% reaching 1% when treatment fail in the context of antimalaria drugs resistance [Children under 5 years are of the most vulnerable group affected by malaria. Following the recent estimation of the World Health Organization (WHO), about 429,000 death linked to malaria were registered all over the world in 2015 and 70% of this number concerned children under 5 years [sistance . Many fasistance -7, acidosistance ,4,8, ressistance , 8, 9 ansistance ,9. The osistance . Followisistance . It is wst January 2014 to the 31 December 2016. It’s a cross-sectional study concerning children under 5 years admited for severe malaria in the pediatric emergency of this hospital. This health structure offers health cares to a large range of the population of Lubumbashi, with easy accessibility to patients from all areas. Also, chilren can have acces to a specific conslutation of a pediatrician. Children were recruted in the pediatric service. Inclusion citeria were: age under 5 years, having a positive thick blood film test to plasmodium falciparum and showing or having shown clinical signs of severe malaria according with the WHO criteria [This research was carried at Jason Sendwe Hospital, in Lubumbashi city (DRC) from the 1criteria . Childrecriteria . Cerebratest of Yates or Fisher’s exact test to produce odds ratios (OR) and their confidence intervals at 95%. The level of statistical significance was set at 5%. All statistical analysis were performed using Epi Info software version 7.2.Severe anemia form was revealed through a pallid state of the patient with or without any sign of cardiac decompensation and presenting a hemoglobin rate = 5g/dl. The hemoglobinuria form has been defined as the presence of the hemoglobine in urine after a massive intravascular hemolysis . The massive presence of hemoglobin in urine has been defined by a redish or dark aspect or urines. Microscopic hemoglobin is defined as the presence of hemoglobin in urines revealed through urinary bandelet and the presence of red blood cells observed on the microscopic analysis of urines was defined as hematury. Acute renal failure was defined by the dysfonction of renal physiology with an absolute increase of creatinine superior or equal to 0.3mg/dl, a percentage increase in serous creatinine superior or equal to 50% associated or not with oliguria. Oliguria has been defined as a low production of urine documented as inferior to 12 ml/kg per 24 hours. The data were entered into an electronic database using Microsoft Excel 2013. The risk of death during malaria treatment was measured. Patient characteristics were summarized using descriptive statistics. For the investigation of risk factors for death, we used the chi-square Socio-demographic characteristics: On the 3092 children hospitalised aged under 5 years, 452 (14.60%) were diagnosed of severe malaria. It included 242 boys (53.54%) and 210 girls (46.46%). The average age was 27.04 months (extrems: 3 months and 59 months) and 226 (50.00%) and most cases were less than 2 years , asthenia (59.96%), pallor (54.20%), repeated convulsions (47.79%), vomits (34.07%), splenomegalia (32.96%), diarrhea (22.35%), coma (21.46%) and jaundice (11.50%), severe acute malnutrition was detected on 33 children (7.30%). Urines were of dark coloration in 8.19% of cases. The average rate of haemoglobin was 7.74 mg/dl (extrems: 2.90 and 16.80 mg/dl). A rate of heamoglobin inferior to 5 mg/dl was noted in 212 patients (46.90%) and glycemia inferior to 50 mg/dl noted in 8 (1.77%) creatinemia was superior to the normal rate in 3 children (0.66%).Severity criteria: Severity criteria identified in the 452 were: cerebral malaria (48.23%), severe anemia (46.90%), respiratory distress syndrome (9.07%), hemoglobinuria (7.74%), circulation failure (4.65%), hyperparasitemia (3.32%), hypoglycemia (1.77%), renal failure (0.66%), abnormal bleeding (0.66%), and prostration (0.44%) ( (0.44%) .Clinical forms:Predictive signs of death: The predictive factors of death were: repeated convulsion , coma  and severe acute malnutrition  (56-7.06) .Severe malaria represented 14.6% of hospitalisation during our research period. The hospitalisation frequency of children under 5 years changes following the autor, the country, the region and the period. Its variates from 13.3 to 34% in african country where we find a stable transmission rate of malaria -15. The Severe malaria mostly affects children under 5 years, this age group matches with the period when a child has lost maternal antibodies and is gradually bulding a partial immunity against malaria ,17. The While tracing out the determinants of this high mortality, we found out that automedication to antimalaria drugs before hospitalisation was not associated to any significant high risk of death in this study. Even when used in low doses, automedication could limit a fatal out come of severe malaria ,28. It iThe association between cerebral malaria and the death of children affected by severe malaria as registered in our distribution coinside with results from previous research carried out in Africa ,18,30,31Like in previous studies carried out ,27,28,37This research shows that severe malaria is still an important cause of morbidity and mortality among young children in Lubumbashi. Neurologic and anemic forms are the most frequent. The predictive signs of death are: repeated convulsions, coma and severe acute malnutrition. Further studies will be important to assess strategies for the disease control.In the DRC, malaria is a major public health problem with an important rate of morbidity and mortality among children;Mortality due to severe malaria on young children is generally over 10% and increases with age.In our environment, the risk factors of death for severe malaria on children under 5 years are: repeated convulsions, coma and severe acute malnutrition.


Plasmodium vivax generally recognised in medicine was one associated with either a long (8–9 months) incubation period or a similarly long interval between initial illness and the first relapse. Long-latency P. vivax ‘strains’ were the first in which relapse, drug resistance and pre-erythrocytic development were described. They were the infections in which primaquine radical cure dosing was developed. A long-latency ‘strain’ was the first to be fully sequenced. Although long-latency P. vivax is still present in some parts of Asia, North Africa and the Americas, in recent years it has been largely forgotten.Until World War II the only clinical phenotype of  Plasmodium malariae) from a patient from West Africa, and on 21 July they allowed these mosquitoes to bite two patients suffering from general paralysis of the insane.1 Two weeks later the first patient became ill and a few days after that malaria parasites were detected in their peripheral blood. On 8 September they infected their first patient with Plasmodium vivax, which became their preferred infection after the one patient they infected with Plasmodium falciparum developed disease of ‘fulminating character’ and died.1 All their patients, like tens of thousands of others in psychiatric hospitals across the world, were suffering from neurosyphilis, and Yorke and MacFie were hoping to cure them. At the time, neurosyphilis was a uniformly progressive and ultimately lethal condition, but there was recent hope. This had come initially from Paul Ehrlich’s discovery of his ‘magic bullet’, the arsenical ‘Salvarsan’ , but more recently from the studies of the Viennese neuropsychiatrist Julius Wagner-Jauregg on ‘malariotherapy’, for which he would win the Nobel Prize in 1927.2 He reported that recurrent high fevers could alleviate or cure neurosyphilis, so Yorke and many others were eager to emulate these extraordinary claims.3 Wagner-Jauregg used malaria-infected blood to infect his patients, and initially so did Yorke and MacFie, but when the relatives of the patients with neurosyphilis in the Wythenshaw hospital objected, they turned to mosquito infections. They infected the local malaria vectors Anopheles maculipennis and Anopheles bifurcatus (now Anopheles claviger). Malaria parasites appeared in the blood between 13 and 21 days after their patients were bitten by the infected mosquitoes, and the patients became febrile. After a short course of quinine (3 d), relapses occurred after intervals of 3–6 weeks in 4 of the 31 survivors . A total of 41 patients were infected with P. vivax in this way. There was some improvement in several of the patients’ neuropsychiatric conditions, so Yorke and MacFie duly published their results.1 Over the next year, however, something unexpected happened. In more than half the mosquito-infected patients the P. vivax infection recurred, while none of the 61 patients who had been infected by blood had a recurrence of malaria. The recurrences after 2 months  occurred a median of 8 (range 3–100) months after the initial infections.4In 1922, at the Wythenshaw mental hospital, near Manchester, England, Professor Warrington Yorke and Dr John William Scott MacFie began a remarkable series of therapeutic evaluations. They started by infecting local anopheline mosquitoes with quartan malaria parasites (Anopheles maculipennis mosquitoes had been kindly sent (in the Embassy bag) from Rome via Brindisi by his fellow malariologists Grassi, Bignami and Bastianelli, who had fed them originally on one of their patients with vivax malaria in the Ospedale di Santo Spirito. This Italian P. vivax ‘strain’ caused malaria illness in 2 weeks as hoped, providing confirmation of the Ross findings in India.5 Some 9 months later the younger Manson, who had since moved to Aberdeen, began to feel ill with malaise and abdominal discomfort. Two days later he experienced a sudden onset of chills (the ‘paroxysm’) and a pathologist duly confirmed by microscopy of his blood that he was suffering again from vivax malaria.6 These findings (of long-latency relapse) were consistent with other detailed case reports and observations from Europe by Celli in Italy and, in particular, Korteweg in The Netherlands,7 who proposed that malaria in the early summer months was acquired in the late summer or early autumn of the previous year. Korteweg’s detailed prospective individual observations in the village of Wormeveer, allied with Swellengrebel’s detailed entomological studies, showed clearly that the surge of vivax malaria that occurred in the early summer months preceded the abundance of vector mosquitoes.8 They concluded that the early summer malaria could only have been acquired by mosquito bites in the previous year and therefore that these infections had an incubation period of 8–9 months.These were not the first artificial malaria infections of humans in England. In 1900 Patrick Manson wanted to allay widespread scepticism over the recent claim by his protégé Ronald Ross that dapple winged (anopheline) mosquitoes could transmit malaria. In a well-publicized demonstration, Manson tested the ‘mosquito theory’ by allowing malaria-infected anopheline mosquitos to feed on his son Patrick Thurburn Manson  and another volunteer in London. These malaria-infected 11 The latter is probably the single most studied malaria parasite in history. Beginning on 25 May 1925, under the supervision of the UK’s leading malariologist, Sidney Price James, at ‘C Hospital’ , anopheline mosquitoes, which had earlier fed on a seaman of Indian origin whose last port of call was Madagascar, were allowed to bite two women with neurosyphilis.12 So began a remarkable sequence of infections spanning more than 20 y (1739 patients and 24 361 mosquitoes were infected). Although this parasite lineage was called the ‘Madagascar strain’, as Garnham has pointed out, it may well not have originated on the island of Madagascar.13 When transmitted by mosquitoes, this ‘strain’ caused an initial illness, sometimes an early relapse, but more commonly a late relapse about 8–9 months after the original infection . It was also unclear why some infections had a long latency between primary illness and recurrence and others apparently had a long incubation period. Swellengrebel and his colleagues (including his wife) resolved the uncertainty over the incubation period by allowing themselves to be bitten by single mosquitoes infected with a local Netherlands ‘strain’ of 16 together with earlier studies in Lahore, suggested a different pattern of recurrent vivax malaria to that documented in Europe and the USA. The infections ‘relapsed’ frequently at short intervals. Most of the patients studied were soldiers who had already endured multiple relapses . Sinton and his colleagues found that the newly discovered synthetic 8-aminoquinoline antimalarial drug pamaquine , when given together with adequate doses of quinine, could prevent most of these relapses .18 Subsequent deployment of a radical curative malaria treatment regimen throughout the British army in India led to a dramatic reduction in malaria illnesses.19 Sinton also advocated strongly for standard malaria treatment regimens and standard methods of assessment.19 But this was not accepted by the Malaria Commission of the League of Nations. The committee, comprising mainly malaria experts from Europe, were strongly influenced by their experiences with detailed follow-up of neurosyphilis patients in Europe and reports from North America. They did not accept initially that relapse could be effectively and safely prevented by pamaquine.20 They also did not accept that there could or should be standard malaria chemotherapeutic regimens—they thought treatment required tailoring to the individual.20 The committee believed that all P. vivax had the propensity for late relapse to a greater or lesser extent. The more tropical ‘strains’ had an incubation period of a little less than 2 weeks, sometimes relapsed early, but then usually did not relapse again for more than 6 months .In the two decades before World War II, detailed chemotherapy studies were conducted by the Indian Medical Service, mainly in Kasauli, Himachal Pradesh, Northern India . This series of clinical trials, led mainly by John Alexander Sinton,P. vivax could have long-latency relapses persisted until World War II, in which, just as in World War I, soldiers on both sides were badly affected by malaria. The allied research effort was centred in the USA, where there was a desperate search for new drugs to prevent recurrent malaria. The US research programme, under the Office of Scientific Research and Development, was led by James Shannon, who would later become the head of the entire National Institutes of Health. New drugs were evaluated in malaria-infected neurosyphilis patients and later, when larger numbers were required, in prison volunteers.21 Multiple relapses of vivax malaria were a particular problem for the US forces, who, unlike the British, Indian and Australasian forces, did not recommend radical curative treatment with pamaquine. The US authorities may have been ‘spooked’ by the serious outbreak in May 1943 of blackwater fever in imported labourers working in the Panama Canal zone who had all been given pamaquine.22 Safer alternatives were needed.The belief that all P. vivax infections. So he sought help from the Medical Research Unit , Cairns, Australia, where Neil Hamilton-Fairley and his Australian army colleagues were studying the treatment of vivax malaria in soldiers who had acquired it fighting on the island of New Guinea.23 This vivax malaria in Australian soldiers relapsed at 3-week intervals after quinine and approximately 6-week intervals after treatment with the slowly eliminated quinacrine . Fairley wrote: ‘The strain or strains of P. vivax encountered in New Guinea and New Britain differed from the strains encountered in Australian troops in the Middle East and those previously worked-with experimentally in U.K. and U.S.A. inasmuch as the relapses appear within a few weeks of primary fever or the cessation of suppressive drug therapy; though febrile attacks are readily controlled by anti-malarial drugs the subsequent relapse rate is unduly high’.23Shannon regarded relapsing vivax malaria as the main threat to the military in the Pacific theatre. He was frustrated by the need to wait up to 9 months for the pharmacodynamic read-out  in evaluating potential radical curative treatments with the native US long-latency P. vivax malaria could behave in this way, but he recognized the potential of these ‘unusual’ strains for drug testing. Fairley suggested he look for frequent relapsing P. vivax in returned GIs, and so Sargent Chesson, hospitalized in the Harmon General Hospital  with malaria that he had acquired in New Guinea, was asked if he would kindly donate a blood sample containing the malaria parasites that would later bear his name.24 There followed intense studies of the ‘Chesson strain’ in prison volunteers in the Stateville Penitentiary  and also of an animal model, Plasmodium cynomolgi in rhesus monkeys, which seemed very similar to this frequent-relapse Chesson strain of P. vivax in humans.26 With the same pattern as the infections studied by Sinton in India, the Chesson strain of P. vivax relapsed at intervals of 3–6 weeks depending on the elimination profiles of the antimalarial drugs used in treatment  appeared significantly more efficacious and also less toxic than pamaquine.29The war was soon over and so the military imperative for antimalarial drug discovery decreased. Penicillin was gradually supplanting malariotherapy for the treatment of neurosyphilis. There was an epidemic of long-latency P. vivax for, while there was no falciparum malaria in the Koreas, there was plenty of long-latency/long incubation period P. vivax. The United Nations’ soldiers became heavily infected. A surge of malaria infections occurred 9 months after their intense exposure. Then, as had been observed previously in Europe and North America, once the first long-latency relapses occurred, subsequent relapses followed at short intervals 31  after the intense control efforts associated with the global eradication plan in the 1950s and 1960s and the resurgence of malaria in the following decades.37After the Korean War, malaria research declined again as the World Health Organization’s global malaria eradication initiative began. Most of the research on vivax malaria focussed on the Chesson strain, although detailed malariotherapy studies in the USSR provided the most accurate characterization of the different long-latency phenotypes36 Table . The SovP. vivax parasites with the ‘Madagascar’ long-latency phenotype appear to coexist with Chesson types and perhaps a third phenotype with intermediate characteristics and a shorter latency38 . It is easy to miss. Early relapse may occur with the subtropical strains of long-latency P. vivax, and is the norm for second and subsequent relapses . As the first long-latency relapse is isogenic  compared with the initial infection, parasite genotyping in carefully followed patients can readily identify this phenotype . Obviously if a marker is identified, it would facilitate therapeutic assessments. There is some evidence that the radical curative efficacy of primaquine is greater in long-latency infections, but this could also reflect geographic and epidemiological differences independent of latency  from El Salvador.44P. vivax hypnozoites were demonstrated first in liver biopsies from two chimpanzees, one infected with the Chesson strain and the other with a long-latency North Korean strain.45 Despite all this, long-latency P. vivax has dropped off the radar in the past 30 y. A review entitled ‘Key gaps in the knowledge of Plasmodium vivax’ did not even mention long-latency or long incubation period P. vivax.46 Mapping the current geographic distribution of long-latency P. vivax will be important for both control and elimination planning and will also inform the durations of follow-up required in therapeutic assessments.The long-latency strains of


This study seeks to compare the performance of HRP2 (First Response) and pLDH/HRP2 (Combo) RDTs for falciparum malaria against microscopy and PCR in acutely ill febrile children at presentation and follow-up.This is an interventional study that recruited children < 5 years who reported to health facilities with a history of fever within the past 72 h or a documented axillary temperature of 37.5 °C. Using a longitudinal approach, recruitment and follow-up of participants was done between January and May 2012. Based on results of HRP2-RDT screening, the children were grouped into one of the following three categories: (1) tested positive for malaria using RDT and received anti-malarial treatment ; (2) tested negative for malaria using RDT and were given anti-malarial treatment by the admitting physician ; or, (3) tested negative for malaria using RDT and did not receive any anti-malarial treatment . Independent microscopy, PCR and Combo-RDT tests were done for each sample on day 0 and all follow-up days.Mean age of the study participants was 22 months and females accounted for nearly 50%. At the time of diagnosis, the mean body temperature was 37.9 °C (range 35–40.1 °C). Microscopic parasite density ranged between 300 and 99,500 parasites/µL. With microscopy as gold standard, the sensitivity of HRP2 and Combo-RDTs were 95.1 and 96.3%, respectively. The sensitivities, specificities and predictive values for RDTs were relatively higher in microscopy-defined malaria cases than in PCR positive-defined cases. On day 0, participants who initially tested negative for HRP2 were positive by microscopy (n = 2), Combo (n = 1) and PCR (n = 17). On days 1 and 2, five of the children in this group  tested positive by PCR alone. On day 28, four patients who were originally HRP2-negative tested positive for microscopy (n = 2), Combo (n = 2) and PCR (n = 4).The HRP2/pLDH RDTs showed comparable diagnostic accuracy in children presenting with an acute febrile illness to health facilities in a hard-to-reach rural area in Ghana. Nevertheless, discordant results recorded on day 0 and follow-up visits using the recommended RDTs means improved malaria diagnostic capability in malaria-endemic regions is necessary. Malaria remains one of the most common causes of febrile illness among people living in tropical and sub-tropical regions. Globally, an estimated 214 million cases were reported which resulted in about 438,000 malaria mortalities [Plasmodium species identification and quantification of parasite density [Microscopy remains the gold standard for malaria diagnosis because it is inexpensive, has high sensitivity and allows  density , 4, howe density , 6.Plasmodium falciparum infections than other antigens, such as Plasmodium lactate dehydrogenase (pLDH) [Plasmodium falciparum also produces histidine-rich protein 3 (PfHRP3), an antigen highly similar to PfHRP2 and detected by HRP2-based RDTs [Although RDTs have simplified the diagnosis of malaria, WHO and other agencies advocate that countries test the sensitivity and specificity of malaria RDTs before giving approval –9. In ade (pLDH) . Plasmodsed RDTs ; howeverSince their adoption in Ghana, RDTs have contributed to reduction in presumptive treatment  but theyRecent studies in Peru reported field isolates that lack one or both antigens (PfHRP2 and PfHRP3) and that poses a significant problem for diagnosis. Similar findings have also been reported in African countries, including Ghana , 19. ThiThe aim of the study was to evaluate the implications of a negative malaria test outcome in relation to clinical diagnosis, and to demonstrate the implications of caregiver adherence or otherwise to a negative RDT test in a rural setting in an endemic area. To this end, the diagnostic utility of the HRP2 (First Response), pLDH/HRP2 (Combo), microscopy, and PCR were compared in the following three groups of acutely ill febrile children at presentation (day 0) and follow-up: (i) RDT-positive children who received anti-malarials; (ii) RDT-negative children who received anti-malarials; and, (iii) RDT-negative children who did not receive anti-malarials.This study was conducted in two health facilities  in Asante Akim North District of the Ashanti Region of Ghana. The region is one of ten in the country and is located in the Forest Zone where there are two distinct seasons: a wet season (April to October) when malaria transmission is highest and a dry season (December to March). The district occupies an area of 1462 sq km. The main economic activities of the district are subsistence farming, animal husbandry and petty trading.The hospital provides both general, specialized and referral services to residents in Konongo-Odumase township, surrounding communities and other residents of Ashanti Region. This hospital serves a population of approximately 100,000 with a 50-bed capacity and staff strength of over 250 healthcare providers.The Juansa Health Centre (JHC), located between Konongo-Odumase and Agogo has a 12-bed capacity, is headed by a physician assistant and provides services to over 15,000 people.The study included all children under 5 years who reported to the health facilities with a history of fever within the previous 72 h or a documented axillary temperature of 37.5 °C.The study, conducted between the months of January and May 2012, employed longitudinal methods that included interventional and quantitative approaches. The sampling strategy and procedures are detailed in Fig. Children < 5 years who presented with fever and tested positive for malaria using RDT and received anti-malarial treatment (Group 1).Children < 5 years who presented with fever and tested negative for malaria using RDT and were given anti-malarial treatment by the admitting physician (Group 2).Children < 5 years who presented with fever and tested negative for malaria using RDT and did not receive anti-malarial treatment (Group 3).After obtaining informed consent, the participants were examined by the physician-in-charge, and a study questionnaire was administered to the parent/guardian. This was followed by RDT screening. Based on the results of the RDT and decision of the admitting physician, the children were grouped into one of the following three categories.Temperature, weight and other demographic characteristics of the children were obtained. Finger and venous blood specimens were collected. All sample collection procedures were done under aseptic conditions. In all, 0.5 µL of venous blood and two dried blood spots (DBS) were deposited onto Whatman 903 protein saver cards with about 50 µL of blood for each circle. The DBS were stored at 20 °C.® Malaria Ag HRP2-HRP2 alone  and SD Bioline Malaria Ag Pf/Pan- HRPII and panLDH  were evaluated following manufacturers’ instructions. Briefly, 20 µL of blood from finger stick was used for the RDTs and colour changes observed after 15 min. For each RDT, cassettes were first labelled with the sample number, then 10 µL of whole blood was added to the sample well and the assay buffer completely emptied into the buffer well. The RDT reaction was considered as positive when two colour bands were seen at the control (C) and test (T) labels. The reaction was considered negative when only one band was seen at the control (C) label. The reaction was considered invalid when no bands were seen at both control and test labels and or when a band was seen at the test label but not at the control label. All invalid reactions were repeated to determine results as either positive or negative.The performance of the First ResponseP. falciparum parasites against 200 leukocytes and multiplied by 40, assuming a standard leukocyte count of 8000 leukocytes/µL.Thick and thin blood films were prepared on slides and stained with 10% Giemsa and examined using oil immersion magnification with a light microscope. Two independent microscopists examined slides for asexual parasite stages. Parasite density was quantified in thick films by counting asexual P. falciparum, Plasmodium vivax and Plasmodium malariae were chosen along with the human actin gene control. The primers and probes were as follows.About 100 µL of blood previously blotted on two circles of Whatman 903 Protein Saver cards filter paper were dried and stored at room temperature (20 °C). Five 3-mm diameter punches were processed with a commercial 96-well kit  to extract the DNA from approximately 25 µL of the dried blood into an eluted volume of 200 µL of water. Multiplex PCR from 10 µL  of the extracted DNA volume was performed in real time (qPCR) on a CFX 384 Detection System Thermocycler . The thermocycler machine detects 4 probes, therefore Plasmodium falciparum 18S rRNA- Forward: 5′-CCACATCTAAGGAAGGCAGCAG Reverse: 5′-CCTCCAATTGTTACTCTGGGAAGG Probe-5′CCCACCATTCCAATTACAA-Cy5.Plasmodium vivax AMA1 Forward 5′-ACGCCAAGTTCGGATTATGG Reverse: 5′-CCGTCATTTCTTCTTCATACTGAG Probe-5′TTGATCTGAGGCACTCGCTCCG-TET.Plasmodium malariae plasmepsin Forward: 5′-CCAACAATACATACACATTAGAACC Reverse: 5′-GTAGGATATAAAGCATACACAAAGTG Probe-5′ATCTAGTAATGGCTCC-TX Red Human beta actin For 5′-GTGCTCAGGGCTTCTTGTCC Rev 5′-CCATGTCGTCCCAGTTGGT Probe-5′ACCCATGCCCACCATCACGCCC-FAM.The human actin gene was used as an extraction control and PCR was performed in duplicate from the single extraction of each sample.P. falciparum, 101% for P. malariae and 70% for P. vivax.A cycle count of 34 was used for the cut off to separate positive and negative PCR samples. The efficiencies for the amplifications were 150% for True positives (TP) for RDT were defined as PCR positive and/or microscopy positive. False positives (FP) were cases in which PCR and microscopy negatives were positive for RDT. True negatives (TN) were negative by all three methods. False negatives (FN) were those cases that were negative by RDT but positive for PCR and/or microscopy.Follow-ups were done by registered nurses and was coordinated by research assistants, the biomedical scientist at Konongo, and the Municipal Director of Health Services. Children who tested positive by RDT and received anti-malarials Group 1) were followed up as outpatients on day 4 and day 28 in their homes. Children who tested negative and received anti-malarials (Group 2) were also followed up as outpatients on day 4 and day 28 at their respective homes. Children who tested negative and did not receive anti-malarials (Group 3) were placed under observation overnight. The plan for observing and following up of participants is detailed in Fig.  were folData were entered into spreadsheets using Microsoft Excel and analysed with the Statistical Package for Social Sciences (SPSS) version 17 . Simple descriptive statistics were used to analyse the demographic data. The malaria parasite density was log transformed before analysis. Significant levels were measured at 95% confidence intervals and values were considered significant at P < 0.05. Sensitivity and specificities of the tests were calculated from the TP, TN, FP, and FN test results using the formulae below.Sensitivity = TP/(TP + FN), Specificity = TN/(TN + FP), Positive predictive value = TP/(TP + FP), Negative predictive value = TN/(TN + FN). The values obtained were expressed as percentages by multiplying by 100.A total of 260 children < 5 years reporting with fever were recruited from the Konongo-Odumase Government Hospital and Juansa Health Centre, both in the Asante-Akim North District. The mean age was 22 months and females accounted for nearly 50% (49.8%) of the study participants. At the time of diagnosis, the mean body temperature was 37.9 °C (range 35–40.1 °C).P. falciparum in all blood specimens except three individuals who were positive for P. malariae . None was positive for P. vivax by qPCR; P. falciparum schizonts were observed in one sample. No gametocytes were detected at the microscopic level.Tables There were ten negative samples for qPCR, which were positive for RDTs and microscopy. With microscopy as gold standard, the sensitivity of HRP2 and Combo-RDTs was 95.1 and 96.3%, respectively. The sensitivities, specificities and predictive values for RDTs were relatively higher in microscopy-defined malaria cases than in qPCR positive-defined cases.All febrile children who were initially HRP2-negative (n = 95) and did not receive anti-malarials were followed up. Day-0 results of initially HRP2-negative children found later to be positive were microscopy (n = 2), Combo (n = 1) and PCR (n = 17) Table . On daysIn this group (n = 68) , one child tested positive by microscopy, and nine children tested positive by PCR on day 0.A total of five children in this group tested positive on day 4 for HRP2, microscopy and Combo tests and 10 by PCR. However, on day 28, two children were positive by microscopy and eight by PCR.Malaria remains a major public health problem in many countries. In the quest to effectively manage cases, early diagnosis and prompt treatment with efficacious anti-malarials is advocated . The WHOP. falciparum [P. falciparum. In endemic areas, it is also characteristic for HRP2 antigen to be produced at the asexual and early gametocyte stages of P. falciparum life cycle [The results indicate that both HRP2 and Combo RDTs recorded high sensitivity when microscopy was used as gold standard. These sensitivity rates are comparable with reports from previous studies , but higlciparum . Most cofe cycle , and itsfe cycle , 26. In fe cycle , while pfe cycle . Aside ffe cycle –31.® Malaria Kit) recorded in this study contrasts with that reported by Ndamukong-Nyanga et al. (95 vs 48.5%) in Cameroon. For the Combo RDT, Xiaodong et al. [Moreover, the sensitivity of the HRP2 (First Responseg et al.  reportedg et al.  and for g et al. .When PCR was used as reference, HRP2 and Combo RDTs recorded lower sensitivity, specificity, PPV, and NPV Table . The higSome recent studies have reported an increase in false-positive results of HRP2-based RDTs due to mutations in the antigen , 38, 39.versus microscopy tend to use PCR as a confirmatory test. The sensitivity, specificity and predictive values of the Combo RDT were higher than the HRP2 with PCR as the gold standard. In view of the fact that HRP2-based RDTs are more sensitive than LDH-based RDTs at low parasite densities, the findings are in agreement with the general conclusions that a positive LDH RDT suggests a parasite density above HRP2 detection threshold [In determining which markers are best diagnostic preferences for malaria in RDTs, some studies that compare RDT hreshold , while mThe use of RDTs can reduce overprescribing of anti-malarial drugs, and studies have shown that health workers prescribe anti-malarials to patients with negative RDT results . In endeP. falciparum has shown a sensitivity of about 95.2 and 98.5%, respectively, from previous works [The sensitivity of HRP2 and pLDH as diagnostic markers in us works , 46 and A major limitation to this study is that the authors were unable to perform any genotyping or sequencing on samples collected on various follow-up visits. It is therefore not possible to draw definitive conclusions as to whether seropositivity for malaria parasites, antigens/DNA was due to a persistent infection or to new infections.HRP2- and pLDH-based RDTs showed comparable diagnostic accuracy in children presenting with an acute febrile illness to health facilities in a hard-to-reach rural area in Ghana. However, the presence of discordant results between the recommended diagnostic tests on presentation and during follow-up suggest the need for improving diagnostic capability for febrile illness in malaria-endemic areas.


Low birth weight (LBW) and preterm birth (PTB) are major contributors to infant mortality and chronic childhood morbidity. Understanding factors that contribute to or protect against these adverse birth outcomes is an important global health priority. Anaemia and iron deficiency are common in malaria-endemic regions, but there are concerns regarding the value of iron supplementation among pregnant women in malaria-endemic areas due to reports that iron supplementation may increase the risk of malaria. There is a lack of evidence on the impact of iron deficiency on pregnancy outcomes in malaria-endemic regions.We determined iron deficiency in a cohort of 279 pregnant women in a malaria-endemic area of Papua New Guinea. Associations with birth weight, LBW and PTB were estimated using linear and logistic regression. A causal model using sequential mediation analyses was constructed to assess the association between iron deficiency and LBW, either independently or mediated through malaria and/or anaemia.p < 0.001), and reduced odds of LBW  and PTB . Magnitudes of effect were greatest in primigravidae . Sequential mediation analyses indicated that the protective association of iron deficiency on LBW was mainly mediated through mechanisms independent of malaria or anaemia.Iron deficiency in pregnant women was common (71% at enrolment) and associated with higher mean birth weights  contains supplementary material, which is available to authorized users. Low birth weight  and preterm birth  are major contributors to infant mortality and chronic childhood morbidity. LBW is associated with >80% of infant deaths . UnderstDespite the high prevalence of undernutrition and anaemia among pregnant women in resource-limited and malaria-endemic settings, there is limited knowledge on the contribution of iron deficiency and other specific preventable deficiencies to LBW and poor pregnancy outcomes. To reduce the burden of anaemia and poor pregnancy outcomes, the WHO recommends that all pregnant women receive daily iron and folate supplements , in addition to “measures to prevent, diagnose and treat malaria” in malaria-endemic areas . HoweverPlasmodium falciparum parasitaemia, associated clinical symptoms of malaria and reduced placental P. falciparum infection in case–control and cross-sectional studies of pregnant women living in malaria-endemic areas  and placental P. falciparum infection .Exposures of interest were iron stores (ferritin) and iron deficiency (ferritin < 15 μg/L) at enrolment. Birth outcomes of interest were birth weight (grams), LBW (<2500 g) and PTB (<37 weeks gestation). Malaria outcomes of interest were peripheral Plasmodium spp. infection was determined by light microscopy of Giemsa-stained thick and thin blood films, and PCR, as previously described  were adjusted for in multivariable regression models. Red blood cell genetic polymorphisms  were not included in the models, as we have previously shown in this cohort that they are not associated with risk of Plasmodium infection or birth weight [p values for interactions were derived from likelihood ratio tests comparing models including and excluding interaction terms. We performed sequential mediation analyses to estimate marginal natural direct and indirect effects for the association between iron deficiency and LBW mediated through: (1) peripheral infection detected by light microscopy or PCR, (2) placental P. falciparum infection detected by light microscopy or histology, and (3) anaemia, by calculating potential outcomes from logistic models with inverse probability weighting to achieve balance in the iron deficiency groups in terms of the confounders . We obtained 95% confidence intervals (CIs) for marginal natural direct effects and marginal natural indirect effects by bootstrapping using the percentile method and 1000 replications. All statistical analyses were performed using Stata Version 13 . Changes in h weight . NewbornPlasmodium spp. infection at enrolment was common; 35% and 66% of women had peripheral Plasmodium spp. parasitaemia detectable by light microscopy and PCR, respectively. The majority (95%) of infections were P. falciparum.At enrolment , the median maternal age was 24 years , the mean estimated gestational age was 25 weeks  and 62% of women were multigravidae. Reported use of a bed net  during the night before enrolment was 76%. For clinical symptoms, 24% of women had a history of fever, headache or chills within the 7 days prior to enrolment, and 19% had a palpable spleen (Table p < 0.001). When potentially misclassified women were excluded (women with raised CRP and ferritin > 15), the prevalence of iron deficiency was 85%. Ferritin and CRP levels were only weakly correlated . There was no correlation between haemoglobin at enrolment and ferritin at enrolment , and no association between iron deficiency and anaemia at enrolment, suggesting that multiple factors contributed to anaemia, aside from iron deficiency. We found that 96% (77/80) of iron-replete and 95% (189/199) of iron-deficient women were anaemic (p = 0.648), 69% (55/80) of iron-replete and 58% (116/199) of iron-deficient women had moderate anaemia (p = 0.105), and 9% (7/80) of iron-replete and 13% (26/199) of iron-deficient women had severe anaemia (p = 0.313).At enrolment, haemoglobin levels were generally very low  and the prevalence of anaemia was very high. Overall, 95% (266/279) of women had anaemia (Hb < 11 g/dL), 61% (171/279) had moderate anaemia (Hb < 9 g/dL) and 12% (33/279) had severe anaemia (Hb < 7 g/dL). Furthermore, ferritin concentrations were low  and iron deficiency (defined as ferritin < 15 μg/L according to the WHO) was present in 71% of women at enrolment (Table p < 0.001), and mean haemoglobin was 9.2 g/dL (SD 1.7), a significant increase from enrolment, with a mean difference of −0.66   , and iron-deficient women gave birth to newborns that were 230 g heavier than newborns of iron-replete women , whereas among multigravidae, birth weight was 125 g higher among iron-deficient women  and median (IQR) gestational age was 38 weeks (37–40 weeks). Poor pregnancy outcomes were common, with 17% being LBW and 22% of babies born preterm (<37 weeks estimated gestation) (Table p = 0.001), and a 43% reduction in the odds of preterm birth, although the association with PTB had weak statistical significance . An analysis of effect modification also showed that the magnitudes of effect between iron deficiency and LBW were highly significant and greater in primigravidae  than in multigravidae  compared to multigravidae , reflective of multiple contributors to anaemia in the study population. Iron deficiency was also associated with a 56% reduction in peripheral blood parasitaemia at enrolment as detected by light microscopy  and a 67% reduction in PCR-detected infection , but no association was found with placental malaria . However, these associations were confounded by women potentially misclassified as iron replete. After excluding these potentially misclassified women, there were no significant protective associations between iron deficiency and anaemia , peripheral parasitaemia  or placental malaria .To investigate the causes of the association between iron deficiency and LBW further, we explored the association between iron deficiency and anaemia (Hb < 9 g/dL), peripheral malaria infection and placental malaria and whether these variables mediated the association between iron deficiency and LBW. Iron deficiency was associated with a 48% reduction in moderate anaemia at enrolment   Table . The mede) Table . The mede) Table . In sequP. falciparum malaria and only 12% was mediated through pathways that included peripheral malaria infection, which included indirect pathways via anaemia and placental malaria . In contrast, only 7% of the association between iron deficiency and LBW was mediated through placental ia Table . Similaria Table . FurtherLBW and PTB are major risk factors for infant morbidity and mortality and identifying factors that contribute to these risks, or protect against them, is crucial for advancing child health globally. Furthermore, concerns regarding potentially harmful malaria–iron interactions have raised questions about the appropriateness of antenatal iron supplementation for anaemia in malaria-endemic populations . Here, wP. falciparum infection of women [We demonstrated that the associations between iron deficiency and malaria infection were confounded by the presence of an acute phase response (raised CRP). Iron deficiency was associated with reduced malaria infection when the analysis included all women. However, when women who were potentially misclassified as iron replete (ferritin> 15 but raised CRP levels) were excluded, no protective association was found. Women with malaria are more likely to have raised CRP and therefore, more likely to be misclassified as iron replete. As a result, the analyses would more likely demonstrate an apparent protective effect of iron deficiency unless these effects are considered in analyses. Previously published analyses of cross-sectional and case–control studies of associations between iron deficiency and peripheral and placental of women  have incof women –23, impaof women , or incrof women , 26. HowPlasmodium spp. infection and placental P. falciparum infection, we found that iron deficiency was still associated directly with a >50% reduction in the odds of LBW. Host iron is essential to a range of other bacteria, parasites and viruses, and iron deficiency may also inhibit the development of these pathogens [A strength of our study is that we used sequential mediation analysis as a valuable approach to estimate the contribution of potential causal relationships. This is the first analysis to quantify the mediation of the effect of iron deficiency on birth outcomes through malaria  and anaemia. This is also the first mediation analysis of the effect of iron deficiency on birth outcomes to account for both exposure-outcome and mediator-outcome confounders, which is a source of bias in traditional mediation analysis . Importaathogens –30. Therathogens , that maathogens , which mathogens . FurtherP. falciparum and P. vivax are endemic. The majority of infections (>95%) in our study were P. falciparum and therefore, our findings may be more broadly applicable to other high P. falciparum transmission areas, such as sub-Saharan Africa. However further studies are required to determine the consistency of our findings and the effect size across areas of varying prevalence of iron deficiency, malaria and other infections and in areas where the relative contribution of the multiple factors that cause iron deficiency differs. Research that identifies populations or settings where iron deficiency associations with birth weight are strongest would be valuable in guiding public health policy. In our study, we experienced a ~40% loss to follow-up; however, given that the characteristics of the women lost to follow-up did not differ from those of the included women  . FerritiOur findings show that iron deficiency in pregnancy is associated with a reduced risk of LBW and PTB that is not simply explained by a potential protective effect against malaria. The mechanism(s) underlying this paradox in malaria-endemic and resource-limited settings with high burdens of infectious diseases and poor nutrition are not yet well understood but are essential to elucidate given the importance and scale of this global public health issue and that LBW and PTB are important determinants of poor infant outcomes. Our results highlight that it is essential to provide both supplementation for anaemia and effective malaria prophylaxis during pregnancy, and future implementation trials to improve the uptake of these interventions as well as trials of integrated interventions for nutrition, malaria and other infections linked with LBW and PTB are warranted. Integration of screening and prophylaxis for other infectious diseases and poor nutrition, alongside current interventions in antenatal care, may be needed to achieve substantial improvements in birth and infant outcomes.Additional file 1:Supplementary methods and analyses. (DOCX 142 kb)