Reductions in malaria morbidity have been reported following azithromycin mass drug administration (MDA) for trachoma. The recent Macrolides Oraux pour Reduire les Deces avec un Oeil sur la Resistance (MORDOR) trial reported a reduction in child mortality following biannual azithromycin MDA. Here, we investigate the effects of azithromycin MDA on malaria at the MORDOR-Malawi study site. A cluster-randomized double-blind placebo-controlled trial, with 15 clusters per arm, was conducted. House-to-house census was updated biannually, and azithromycin or placebo syrup was distributed to children aged 1–59 months for a total of four biannual distributions. At baseline, 12-month, and 24-month follow-up visits, a random sample of 1,200 children was assessed for malaria with thick and thin blood smears and hemoglobin measurement. In the community-level analysis, there was no difference in the prevalence of parasitemia , gametocytemia , or anemia  between placebo and azithromycin communities. Further interrogation of the data at the individual level, both per-protocol (including only those who received treatment 6 months previously) and by intention-to-treat, did not identify differences in parasitemia between treatment arms. In contrast to several previous reports, this study did not show an effect of azithromycin MDA on malaria parasitemia at the community or individual levels. Azithromycin is a broad spectrum antibiotic with a relatively long half-life which is used in the treatment of pneumonia and diarrhea but also displays antimalarial activity.3 Field trials of the effects of azithromycin MDA on malaria infection and symptoms have previously reported reductions in malariometric indices; and recent results from the Niger MORDOR site indicate an association between azithromycin MDA and lower parasitemia.7 It is feasible that the mortality benefit seen with azithromycin MDA may be due, at least in part, to a decrease in malaria prevalence or severity.The Macrolides Oraux pour Reduire les Deces avec un Oeil sur la Resistance (MORDOR) trial, conducted in Niger, Malawi, and Tanzania, demonstrated a reduction in child mortality following biannual mass drug administration (MDA) with azithromycin.This study reports malaria parasitemia, parasite density, and gametocytemia data from the MORDOR Malawi study site, aiming to improve our understanding of the effects of azithromycin MDA on malaria infection. Samples were collected from children in villages representative of the MORDOR trial and used the same biannual census updates and cluster-randomized trial structure to make the results as representative as possible of the wider study area and to assess community-level effects of any outcomes. The hypothesis for this study was that azithromycin MDA would reduce the community prevalence of malaria compared with placebo.The randomization unit for the MORDOR trial in Malawi was defined as the catchment area of a health surveillance assistant (HSA), approximately 1,000 total population. Communities with a population < 200 or > 2,000 on a pre-baseline census were excluded. Thirty communities were randomly selected from the pool of communities for the MORDOR trial for follow-up as part of this malaria prevalence study. The randomization was stratified to produce six communities in each of the five geographical zones of Mangochi district for geographical generalizability and for logistical reasons regarding fieldwork. Biannual census updates were performed, and communities received study drug in the same treatment rounds as the MORDOR trial.All children aged 1–59 months and weighing ≥ 3.8 kg were eligible for treatment at each of four biannual mass distributions. At the baseline, 12-month, and 24-month follow-up visits, guardians of a randomly selected sample of 40 children per community were asked to provide written informed consent for finger-prick blood samples. The procedures and study were explained by trained local nursing staff who subsequently collected thick and thin blood smears if consent was obtained. Illiterate guardians provided a thumb print to acknowledge consent.Azithromycin was administered at a dose of 20 mg/kg. Children old enough to stand received an approximate dose estimated from their height, and younger children were weighed. The placebo bottles and suspension appeared identical to azithromycin. Distribution of drug took place after sample collection was complete and was performed by the HSAs and field-workers conducting house-to-house visits. Guardians were asked to inform the HSA of any adverse events that occurred within 7 days of receiving study drug. Health surveillance assistants subsequently informed the study team.The primary prespecified outcome was prevalence of malaria parasites on thick blood smears in children aged 1–59 months. Prespecified secondary outcomes included parasite density, gametocyte prevalence and density, hemoglobin concentration, and presence of anemia (Hb < 11 g/dL). Primary analyses were by intention-to-treat, and per-protocol analyses were secondary.Sample collection took place during the baseline visit (May–July 2015) and at 12-month and 24-month visits , approximately 6 months after the second and fourth treatment rounds. Sample collection involved selected children receiving a finger stick and thick and thin blood smears collected on a single slide ). Slides were labeled with a random number and barcode and scanned using the data collection app to link to census data. The thin smear was fixed with absolute methanol and the slide stained with 8% Giemsa. Parasite density was assessed from the thick smear by two independent slide readers at MORDOR trial laboratories in Mangochi and Blantyre. Parasite density was estimated as parasites per microliter by assessing up to 100 high-power fields and assuming that 500 high-power fields contain the equivalent of one microliter of blood. If after two reads there was a discrepancy of greater than 20%, a third reading was taken as the final read. Thin slides were used to assess malaria species.Fifteen communities per arm, with 40 children sampled from each community (600 children per arm), provided 90% power to detect a reduction of malaria parasitemia from 20% to half that value at each study phase.Study drug was labeled with six letters by the manufacturer , with three letters corresponding to azithromycin and three to placebo. Communities were randomly assigned to one of the six drug letters by the study statistician using the statistical package R . All field and laboratory staff and participants in Malawi were blinded to the treatment code until after all data collection was complete.The main analysis was by intention-to-treat at the community level, in keeping with the cluster-randomized design of the MORDOR trial. Further analyses by both intention-to-treat and per-protocol at the individual level were used to further explore the data.Community-level prevalence of parasitemia, anemia, and gametocytemia by treatment arm were assessed for the 12-month and 24-month visits using mixed-effects linear regression models including fixed effects for baseline prevalence and study phase and a random effect for community.Individual-level parasitemia, parasite density, hemoglobin, and gametocytemia by treatment arm were assessed for the 12-month and 24-month visits using mixed-effects logistic or linear regression models, as appropriate, including fixed effects for age, baseline prevalence, and study phase and nested random effects for individuals within communities. Individual-level analyses were performed by both intention-to-treat and per-protocol, including only those who received study drug as indicated at the previous phase. Intra-class correlation coefficients (ICCs) were derived from the regression models at the level of the randomization unit.t-test. Association between the presence of parasitemia and the hemoglobin level was assessed at the individual level using Student’s t-test.The hemoglobin levels in individuals between the treatment arms were split by the presence of parasitemia and compared using Student’s Ethical approval for morbidity assessments alongside the MORDOR trial was obtained from the College of Medicine, University of Malawi; the London School of Hygiene and Tropical Medicine; and the UCSF Committee on Human Research. Written consent was obtained from the guardians of participants. There were no incentives for participation.Demographic details of sampled children in the 30 study communities are shown in Plasmodium falciparum, and the remainder were Plasmodium malariae. Analyses included both malaria species. The community-level prevalence of malaria parasitemia, anemia, and gametocytemia was similar between placebo and azithromycin groups at baseline . The prevalence of anemia, defined as Hb < 11 g/dL, was not significantly different between treatment groups at the 12- and 24-month follow-up visits: 1.7% lower in azithromycin-treated communities . The prevalence of gametocytemia also remained similar between treatment groups at the follow-up rounds: 0.7% lower in azithromycin-treated communities .Over 98% of malaria species detected were P = 0.67; ICC = 0.06). Parasite density in parasitemic individuals was similar between treatment groups at the 12- and 24-month follow-up visits: 23 parasites/µL lower in individuals in azithromycin-treated communities after adjusting for age, baseline parasitemia, and follow-up phase . Hemoglobin was also similar between treatment groups at 12- and 24-month follow-up visits: 0.08 g/dL lower in individuals in azithromycin-treated communities after adjusting for age, baseline community prevalence of anemia, and follow-up phase .Individual-level unadjusted data are shown by intention-to-treat in P = 0.17; ICC = 0.06). Parasite density in parasitemic individuals was similar between treatment groups at 12- and 24-month visits after adjusting for age, baseline parasitemia, and follow-up phase: 19 parasites/µL lower in azithromycin-treated individuals . Hemoglobin levels were also similar between treatment groups at the 12- and 24-month follow-up visits after adjusting for age, baseline community prevalence of anemia, and follow-up phase: 0.03 g/dL lower in azithromycin-treated individuals , shown in Supplemental Tables 1 and 2.In the per-protocol analysis, the odds ratio for parasitemia in azithromycin- compared with placebo-treated individuals at 12- and 24-month visits after adjusting for age, baseline parasitemia, and follow-up phase was 0.71  and both age and baseline community parasitemia or hemoglobin. The effect of treatment did not vary with age when an interaction term between the treatment arm and age was included in the models.Supplemental Table 3.Hemoglobin levels were approximately 1 g/dL lower in parasitemic individuals at all study phases and in azithromycin and placebo groups, shown in Several morbidity sub-studies were nested within the MORDOR trial to investigate mechanisms through which azithromycin MDA may reduce mortality. This research was not carried out in the MORDOR mortality study communities to reduce the risk of interventions impacting the primary MORDOR outcome of mortality. Morbidity study communities were randomly selected from the same pool as the main MORDOR trial, designed so that results would be representative of the mortality study. This malaria study, nested within MORDOR, did not identify a reduction in malaria parasitemia, gametocytemia, or hemoglobin in azithromycin-treated compared with placebo-treated communities. Secondary analyses at the individual level, by intention-to-treat and per-protocol, including only those who received study drug at the previous phase, approximately 6 months earlier, also did not identify significant differences in the prevalence of parasitemia or parasite density in parasitemic individuals.7 The mechanism through which a reduction in parasitemia over this time period may occur is unclear. Azithromycin displays delayed activity against the malaria parasite, preventing the progeny of antibiotic-treated parasites from fully maturing, and has a long terminal half-life of approximately 68 hours. These are properties that could feasibly contribute to a period of antimalarial activity of days to weeks but not months.8 The studies showing longer term effects were in Niger and the Gambia, in areas with lower background levels of parasitemia, which may be more amenable to reductions in transmission that could last longer than individual-level prophylaxis and treatment. This study assessed malaria gametocytemia as an effect of azithromycin on the sexual stages of the parasite which could explain the longer term community-level reductions in parasitemia as previously reported. Reductions in gametocytemia were not identified, which is consistent with previous evidence.9Previous published reports of the effect of azithromycin MDA on malaria have suggested a reduction in malaria over 6-month duration.1 The best estimates for the odds ratio for the presence of parasitemia in individuals in azithromycin communities compared with placebo ones were 0.89 by intention-to-treat and 0.71 per-protocol, and it is possible that this study of limited size with follow-up restricted to 6 months post-MDA represents type II error. However, while the main MORDOR trial suggested increased child survival benefit in those younger than 6 months, in this study, age and parasitemia were positively associated , further suggesting that the child survival benefits in MORDOR were not best explained through effects on malaria. Further investigation of the effects of azithromycin MDA on malaria severity and mortality, as well as cause-specific mortality for all the major causes of child mortality, is required to improve our understanding of whether the reported mortality reductions may be due to specific anti-pathogen effects or to other mechanisms, such as the immunomodulatory and anti-inflammatory effects of azithromycin.11In the context of the MORDOR trial showing a reduction in child mortality, this study does not provide evidence for an effect of azithromycin MDA on malaria mortality at the Malawi site.13 Malaria was also the commonest inferred cause of death from verbal autopsy in the MORDOR trial in Malawi.14 Parasitemic children had significantly lower mean hemoglobin levels at all follow-up rounds, consistent with known effects of malaria as a cause of anemia in children.17 This analysis provides some validation of the integrity of the data. In addition, the ICCs were low for all analyses, suggesting considerable heterogeneity in malaria infection and hemoglobin levels by community, consistent with usual epidemiological patterns of malaria and similar to the MORDOR Niger site data.7The prevalence of malaria parasitemia in this study was approximately 30% in children aged 1–59 months. Malawi is hyperendemic for malaria with 95% of the population susceptible to infection, and large-scale surveys before and after this study reported malaria parasitemia prevalence of 33% and 24%, respectively.18 At the community level, this may also reduce the reservoir of infectious individuals at the beginning of the following high-transmission period. In Malawi, where despite some seasonality, malaria prevalence remains high year-round, the optimal time to treat, if indeed there is any benefit from the intervention, is unclear.One limitation of this study is that treatment took place at the beginning and end of each dry season for logistical reasons. Mathematical modeling of the effect of azithromycin MDA on malaria in areas of highly seasonal transmission suggests there may be most benefit from treatment during the low-transmission season when treatment of established infection may produce more sustained benefit because of the lower risk of reinfection.7 Any effect of azithromycin MDA on malaria may be impacted by other malaria interventions, such as seasonal malaria chemoprophylaxis (SMC), which was not taking place at the MORDOR-Malawi site. A recent study indicated no additional child mortality benefit when adding azithromycin MDA to SMC in Mali and Burkina Faso.19Monitoring for infection took place during the baseline, 12-month, and 24-month visits, early in the dry season, and approximately 6 months after the previous treatment round for the latter two visits. This seasonality and timing of sample collection may not provide a complete understanding of the effect of the biannual azithromycin MDA on malaria prevalence, which may vary with time lapsed after administration. Finally, while carefully designed to be representative of the MORDOR trial site in Malawi, the data may not be representative of other countries with different malaria prevalence and healthcare interventions, although some data are available from other MORDOR sites.In conclusion, a cluster-randomized placebo-controlled study of the effects of azithromycin MDA on malaria parasitemia was unable to detect a difference in malaria parasitemia at the community or individual levels, providing no evidence that any child survival benefit was mediated by effects on malaria mortality. Further investigation is required to understand the effect of azithromycin MDA on malaria morbidity and mortality and, indeed, to elucidate the mechanisms by which azithromycin MDA may reduce child mortality.Supplemental materials


Manual microscopy remains a widely-used tool for malaria diagnosis and clinical studies, but it has inconsistent quality in the field due to variability in training and field practices. Automated diagnostic systems based on machine learning hold promise to improve quality and reproducibility of field microscopy. The World Health Organization (WHO) has designed a 55-slide set (WHO 55) for their External Competence Assessment of Malaria Microscopists (ECAMM) programme, which can also serve as a valuable benchmark for automated systems. The performance of a fully-automated malaria diagnostic system, EasyScan GO, on a WHO 55 slide set was evaluated.The WHO 55 slide set is designed to evaluate microscopist competence in three areas of malaria diagnosis using Giemsa-stained blood films, focused on crucial field needs: malaria parasite detection, malaria parasite species identification (ID), and malaria parasite quantitation. The EasyScan GO is a fully-automated system that combines scanning of Giemsa-stained blood films with assessment algorithms to deliver malaria diagnoses. This system was tested on a WHO 55 slide set.The EasyScan GO achieved 94.3 % detection accuracy, 82.9 % species ID accuracy, and 50 % quantitation accuracy, corresponding to WHO microscopy competence Levels 1, 2, and 1, respectively. This is, to our knowledge, the best performance of a fully-automated system on a WHO 55 set.EasyScan GO’s expert ratings in detection and quantitation on the WHO 55 slide set point towards its potential value in drug efficacy use-cases, as well as in some case management situations with less stringent species ID needs. Improved runtime may enable use in general case management settings. Microscopic examination of Giemsa-stained blood films continues to be widely used for parasitological confirmation of malaria diagnosis, representing roughly half of all malaria diagnostic tests performed worldwide in 2018 .Malaria microscopy is thus a high-value target for automated image-processing and machine learning (ML) systems because such systems can potentially be widely deployed, mitigating the expert-training bottleneck, and because their results are reproducible. Since a thorough review in 2018 , there hA lack of benchmark datasets hampers the evaluation and comparison of various automated systems , 5. One This paper reports performance of a fully-automated end-to-end malaria diagnostic system, the Motic EasyScan GO , on an EµL, and the median of 24 readings was taken as the reference count. Slides with statistically significant inter- or intra-reader count variation are never selected for the ECAMM sets. The 55-slide set used in the experiments for this study was assembled by WHO WPRO to represent the template for ECAMM slide sets described in [A set of Giemsa-stained blood films consisting of both malaria positive and negative cases and varying parasite densities were obtained from the WHO WPRO  regional malaria slide bank, a collection of reference slides managed by the WHO Collaborating Centre for malaria diagnosis at the Research Institute for Tropical Medicine (RITM) . All sliribed in  and showThe ECAMM slide set is designed to evaluate a microscopist’s competence in three key areas important for malaria diagnosis: 1) malaria parasite detection, (2) malaria parasite species ID, and (3) quantitation of malaria parasites. Accuracy in each of these areas is evaluated using a different subset of the ECAMM slide set . Given the samples in the set provided, the Plasmodium falciparum samples with a parasitaemia below 200 p/µL  and all non-falciparum and mixed-species samples (8 slides rather than 10) were used to evaluate detection. The non-falciparum slides ranged from 164 to 10,184 p/µL with most samples above 1000 p/µL, well above the ideal range of 80–200 p/µL.Ideally, the detection component uses 20 negative samples and 20 positive samples with a parasitaemia ranging from 80 to 200 parasites per microlitre . The same subset of slides, 20 negative and 15 positive, used for Detection were used for Species ID. The provided set also contained only one mixed-species sample, rather than four as called for by the WHO malaria microscopy quality assurance manual .P. falciparum slides with parasitaemias within 200 ≤ p/µL ≤ 2000, plus one or two very high parasitaemia slides. From the provided set, the 20 P. falciparum slides with parasitaemia > 200 p/µL (rather than 15) were used. Of these, 18 slides had 200 ≤ p/µL ≤ 2000, and two slides had ∼200,000 p/µL. An estimated parasitaemia within 25 % of the reference value was considered correct, per the WHO malaria microscopy quality assurance manual [Ideally, the quantitation component uses 15 e manual . The pare manual ).A newer, slightly modified version (“V2”) of the ECAMM slide set template, with detailed protocols on its use during evaluation of microscopists, is found in . “V1” 1 was usedThe malaria detector algorithm was trained and validated using over 500 slides from 11 countries. Sources of training data (with geographic source of the slides in parentheses) included Shoklo Malaria Research Unit (Thailand), Amref Health Africa (Kenya), University of Lagos (Nigeria), Universidad Peruana Cayetano Heredia (Peru), World Wide Antimalarial Resistance Network , Hospital for Tropical Diseases and the London School of Hygiene and Tropical Medicine (UK), Kenya Medical Research Institute (Kenya), Centers for Disease Control and Prevention (USA and other countries), James Cook University (Solomon Islands), and the Defence Services Medical Academy (Myanmar).No slides from RITM were used for training the machine learning algorithm models. A separate set of 48 slides from RITM, also roughly analogous to an official ECAMM set, was used along with the other slides listed above to tune final diagnostic parameters in the patient-level disposition step, prior to any exposure to the ECAMM test set.µm at the sample plane. Each field-of-view (FoV) was captured as a stack of 5 slices with 0.5 µm vertical spacing, to ensure in-focus thumbnails of all objects-of-interest (e.g. parasites), which can lie at different depths due to the thickness of the blood films and/or slight tilting of the microscope slide.The slides, permanently mounted with cover glass, were scanned with a prototype version of the Motic EasyScan GO . The mic2, equivalent to ∼580 fields of view on a manual microscope with 100× oil immersion lens and 20 mm field number eyepiece, yielding ∼3 k to 17 k white blood cells (WBCs).On the thick film, a 20 × 20 grid of FoVs near the centre was manually selected and scanned. This gave an area scanned of roughly 18 mmOn the thin film, a rectangular region near the “feathered edge”, where a monolayer of red blood cells (RBCs) is most often found, was manually selected and scanned, yielding 150 to 192 FoVs and ∼10 k to 50 k non-overlapped RBCs. The area selected was based only on the presence of a monolayer of RBCs and not on whether parasites were visible in the region.(iii) the total parasite count  given as parasites per 8000 WBCs. This quantitation unit allows direct comparison to standard methods that assume a WBC density of 8000/µL, but also allows for correction when the WBC count is known.The image sets were analysed by both thick film and thin film malaria detector algorithms that detect and evaluate objects of interest. A final synthesis block combined the thick and thin film findings to produce a diagnosis report indicating: (i) whether malaria parasites were detected; (ii) the suspected malaria species; and To assess performance on the ECAMM set, the results of the algorithm on each slide were compared with the reference readings from RITM, and performance was evaluated according to the rubric in the WHO malaria microscopy quality assurance manual  shown i.A schematic representation of the algorithm modules is shown in Fig. Pass1 at the top to Pass6 at the bottom. The term “branch” refers to multiple blocks in Pass3, Pass4 processing , whereas the thick film uses a region-based convolutional neural network (CNN), Faster R-CNN , for WBCFinds candidate parasites in all FoVs at all focus levels, via traditional computer vision techniques. The most in-focus thumbnail of each candidate object is selected for further processing. Weeds out obvious non-parasites (distractors) via traditional feature extraction and a gradient-boosted decision tree classifier .Pass3. In thin film only, a species classifier is applied to late-stage objects in a species branch [ Applies a CNN classifier to thumbnails of all candidate parasite objects that survived the distractor filter of s branch . Arbitrates the appropriate stage type (ring or late) of objects detected by multiple branches, using the highest CNN score.(Disposition) processes all the detected and classified objects to (i) determine whether the blood film (thick or thin) is overall positive for malaria; (ii) estimate the parasitemia if positive; and (iii) determine the species of malaria if positive .Pass3-Pass4 modules are applied to the detected candidate objects. Each branch is trained and tuned to detect a different life-cycle stage of the malaria parasite. The ring-stage branch targets young ring-form trophozoites . The late-stage branch targets late-stage trophozoites, schizonts, and gametocytes, which are common in non-falciparum species and much rarer in P. falciparum. The species branch (thin film only) extracts a set of manually designed features for species ID, then classifies objects by species and stage via a gradient-boosted decision tree classifier.Multiple branches of paired The thin film algorithm used in this study is identical to the one reported in . The thi(i)The algorithm in  was trai(ii)The WBC detector is now based on a CNN object detection architecture, Faster R-CNN , as oppo(iii)Pass3 has been enhanced by doubling the number of discriminative features.The distractor filter of (iv)falciparum ring-stage trophozoites. This helps distinguish P. falciparum from non-falciparum infections solely from the thick film, even if a non-falciparum sample presents few or no late-stages, by recognizing that beyond the youngest ring-stages, non-falciparum and P. falciparum trophozoites differ morphologically.The thick-film algorithm has a new third detection branch targeting non-(v)Pass6 disposition block has an added function that assesses whether a thick film slide may be poorly prepared, based on the presence of very large numbers of distractor objects. If this condition is detected, the disposition block can adjust the diagnosis thresholds to reduce the chance that a malaria-negative but debris-filled slide will be diagnosed as malaria-positive.The The thick and thin film algorithms are independent and can provide conflicting results for the same sample. The synthesis block combines the two findings as follows. It uses the thick film analysis to determine three results:(i)Malaria diagnosis . This matches standard microscopy practice .(ii)µL) [Quantitation of parasites, at all parasitaemias. This differs from microscopy practice, which recommends switching to the thin film when the density on thick films is very high   due to tµL) ).(iii)P. falciparum, non-falciparum, or a mixed infection. This depends on the ratios between the numbers of late-stage, P. falciparum ring-stage, and non-falciparum ring-stage parasites as well as the total ring-stage count. Mixed infections are identified by very high ring-stage counts  combined with sufficiently high late-stage counts .Determination of whether the species is falciparum or mixed species infection. Thin film results then determine the non-falciparum species, as in standard microscopy [falciparum or mixed species and the thin film result is negative, the species is predicted as P. vivax due to P. vivax being the most prevalent non-falciparum species of malaria globally [The synthesis block only uses the thin film results if the thick film algorithm reports a non-croscopy . If the globally .P. falciparum slides, the ring-stage count was used rather than the total parasite count. This is because RITM’s reference readings counted only asexual stages (i.e. non-gametocytes) consistent with malaria microscopy standards [P. falciparum blood films.The final report gives three parasitaemias: ring-stage (immature trophozoite), late-stage , and total. It does not differentiate between the various late-stages. To evaluate quantitation accuracy on ECAMM tandards , and becThe final report lists malaria species, WBC count, RBC count (thin film only), and poor slide quality warnings if indicated. It also includes a mosaic of high-scoring objects’ thumbnails for possible examination by the clinician.The EasyScan GO malaria detector correctly detected whether or not malaria was present in 33 of 35 slides (20 negative slides and 15 positive slides), for 94.3 % accuracy. It achieved 82.9 % accuracy for species ID, correctly labeling 29 of 35 slides. It correctly quantitated 10 of 20 slides, for 50 % accuracy.These results correspond to a Level 1, Level 2, and Level 1 microscopist for diagnosis, species ID, and quantitation, respectively, according to the rubric in  , and 13 of 15 malaria positive slides were classified as positive (86.7 % sensitivity). This yielded an accuracy of 94.3 % . The two false negative samples were P. falciparum samples as negative. Also misclassified were 1 out of 3 Plasmodium malariae samples, both Plasmodium ovale samples, and the single mixed species  sample. A confusion matrix is shown in Table P. falciparum quantitation slides. Of the 20 quantitation slides, 19 were correctly classified as P. falciparum and one was incorrectly classified as Plasmodium vivax.The species was correctly labeled for 29 of the 35 slides (82.9 %) used for evaluating species identification accuracy. This included correctly labelling all 20 (100 %) negative samples, and 9 of 15 (60 %) positive samples, for a balanced accuracy of 80 %. Two errors were due to misclassifying 2 of the 7 P. falciparum versus non-falciparum was higher. When combining all 27 P. falciparum-only samples in the 55-slide set, 24 were classified as P. falciparum, 1 was classified as P. vivax, and 2 were classified as negative (88.9 % accuracy). Of the 7 non-falciparum samples, 1 was classified as P. falciparum and the remaining 6 were classified as non-falciparum species (85.7 % accuracy). The mixed sample was classified as P. vivax.Performance on the simpler task of identifying P. falciparum slides used to evaluate quantitation, the EasyScan GO reported parasitaemias within 25 % of the reference  parasitaemias on 10 samples (50 %). Figure µL P. falciparum samples, and 6 of 8 (75 %) non-falciparum or mixed species samples.Of the 20 The mean time to scan and analyse each slide in the configuration used for this assessment totalled 54.4 minutes: 12.8 and 5.8 minutes for scanning the thick and thin films respectively, and 28.7 and 7.0 minutes for analysing the thick and thin films, respectively. This is well above the 10 minutes per slide allowed in the WHO malaria microscopy quality assurance manual .P. falciparum, the lack of a thin film result did not affect its final disposition or the above analysis of accuracy.The system is designed to reject a film scan if there are not enough in-focus FoVs, too few WBCs (thick film), or too few RBCs (thin film). On all slides both thick and thin films were successfully scanned, with two exceptions. One thick film was rejected because every FoV was out-of-focus, but was re-scanned successfully. One thin film was rejected because all FoVs were reported as out-of-focus. Upon review, the entire thin film had a very dark background whether viewed with the EasyScan GO or with a standard manual microscope. An example FoV is shown in Additional File 2. Re-scanning was unsuccessful, because the malaria detector could not evaluate focus on the dark background. Since this sample’s thick film was reported as The WHO 55 is a template for slide sets used to evaluate malaria microscopist competence (ECAMM) as part of WHO’s quality assurance program. Besides having a consistent composition, it assumes use of existing field infrastructure (i.e. field-prepared Giemsa-stained blood films), and emphasizes (in both content and scoring rubric) the key performance requirements of the most important use-cases. Thus, it may serve as a valuable benchmark dataset to assess and compare automated malaria detection systems. Such benchmarks are currently lacking.In this paper, the performance of a fully-automated malaria diagnosis system, the EasyScan GO, on such a WHO 55 set is presented. The system scanned Giemsa-stained thick and thin films with an EasyScan GO automated microscope, then applied image-processing and ML methods to generate a slide-level diagnosis.There are some limitations to using the WHO 55 template as a benchmark for testing automated systems. Each ECAMM slide set is a unique instance of the template, and performance on the set is only a single snapshot of system performance. The slide set is also fairly small, resulting in low statistical power. Ideally, a system should be tested on several ECAMM-style slide sets and should achieve Level 1 on most or all of them as confirmation of its performance. The ECAMM slide sets are also generally of high quality, and do not represent the high variability in sample preparation possible in the field. Internal evaluations (e.g. ) and fiefalciparum slides had higher-than ideal parasitaemias. However, it has been observed  that late-stage parasites of non-falciparum samples are much easier to detect and distinguish from distractor objects than are P. falciparum ring-stages, and the algorithm has consistently posted lower limits of detection for late-stages than for ring-stages, making non-falciparum samples easier to diagnose at low parasitaemias than P. falciparum samples. It is, therefore, likely that the higher parasitaemias on non-falciparum slides did not impact the system’s sensitivity results.The system achieved Level 1 performance on diagnosis of low-parasitemia slides, with 94.3 % accuracy, 86.7 % sensitivity, and 100 % specificity. In the ECAMM slide set used, the non-P. falciparum samples, indicating its potential for use in new drug evaluation and drug resistance studies where accurate estimation of parasite densities is critical.The system also achieved Level 1 performance on quantitation of the 20 relevant µL), the system’s estimates (done on thick film) were low relative to the reference counts (done on thin films [On the two high parasitaemia samples (∼200 k p/in films ). Given µL for 6  samples, and only one sample was mixed species instead of four. Had the test set matched the template, the system likely would have achieved Level 3 due to poor performance on mixed species samples and perhaps reduced performance on lower parasitaemia non-falciparum samples. The system’s low species ID rating points to two weaknesses:The system achieved Level 2 performance on species ID. However, this score was likely inflated due to the differences in the ECAMM test set used compared to the ideal ECAMM template: there were fewer positive samples (15 vs. 20), the parasitaemia of samples was higher than 200 p/(i)P. falciparum and P. vivax, but contained few P. ovale and P. malariae samples since these are less common in malaria-infected humans. Plasmodium vivax training samples sufficed to ensure strong P. falciparum vs. non-falciparum classification on thick films, which enabled the system to have high accuracy at the level of P. falciparum vs. non-falciparum. But the vivax-ovale-malariae classifier suffered from lack of training data.Machine learning algorithms are often data-hungry , requiri(ii)falciparum samples tend to have lower maximum parasitaemias and P. falciparum late-stage counts tend to be low [P. falciparum gametocyte, any low-parasitaemia sample containing late-stages is classified as non-falciparum. Indeed, the ability of human microscopists to correctly classify such samples is a testament to their skill.The system diagnoses mixed infections by leveraging priors on likely parasitaemias and ring-stage to late-stage ratios of the various species (non-o be low ). It hasP. falciparum to generate parasite clearance curves. Additionally, geographic priors often indicate the likely type of non-falciparum infection. Examples include: (a) In South America the two dominant species are P. vivax and P. falciparum [(b) in West Africa, innate resistance to infection with P. vivax makes it rare compared to P. ovale [Fortunately, fine-grained species ID is a secondary need in some use-cases. One example is drug resistance studies which involve only quantitation of lciparum , and Perlciparum ; and (b)P. ovale . The ECAP. ovale , 16. How(a) P. falciparum infection has a high fatality rate, so high P. falciparum sensitivity is important; and (b) P. vivax and P. ovale develop hypnozoites, which require distinct treatment, so accurate falciparum-malariae vs. vivax-ovale species ID is desirable.Species ID is important for some treatment decisions . ExampleThe system mostly depends on thick film results, for various reasons:(i)falciparum species such as Peru), this is sufficient.In many situations In the field, thin film quality can be highly variable , and thick film-only workflows can be preferable for various reasons .(iii)Machine learning algorithms are often data hungry, and owing to the greater density of blood on thick films the training sets contained more parasite images from thick films than from thin films.falciparum species using only thick films has not yet been achieved and the algorithm still depends on thin films for this task.However, successfully distinguishing between non-The “V2” ECAMM slide set  has someWhether the current system runtime is acceptable depends on particular field constraints. In some field clinics, fast throughput is essential, and the system would be inappropriate for such scenarios. In other settings, the ability to analyse batches of samples overnight would mitigate long runtime .Since these experiments were run, several changes have been made to the EasyScan GO software to shorten runtime, including:(i)Scan a lower volume of blood, using thresholds on FoVs, WBCs (thick film) and RBCs (thin film), rather than scanning a fixed number of FoVs. This enables the blood volume scanned to more closely match manual microscopy protocols falciparum result.Analyze thick film first and proceed to thin film analysis only when indicated, i.e. only in case of a positive, non-(iii)Perform scanning and analysis in parallel instead of in series, e.g. a second sample can be scanned while the first sample is being analysed.Further experiments are needed to characterize the resulting changes in runtime, and to ensure there is no loss in accuracy due to the decreased scan area. Preliminary experiments indicate typical total time of 18–25 minutes per slide with these changes. To simulate the effect on accuracy of reducing FoVs, the samples were re-analysed using 70 FoVs , selected from the original scanned FoVs, for each thick film. This did not change the detection or species ID results for any sample, however the quantitation results varied by 0.7x to 2.3x compared to the original counts. A detailed discussion is beyond the scope of this report, but these results are included as columns in Additional file Finally, analysis time is dependent on computing power. This study was performed on a computer with specifications matching that provided as part of the EasyScan Go system – a relatively high-end laptop (as of 2019) with an Intel i7-9750H CPU, an NVIDIA GeForce GTX 1650 GPU, and 16 GB RAM.In malaria microscopy, as in other domains, machines have pros and cons relative to human workers:(i)In general, machines can run long hours without getting tired, but conversely they are at risk of mechanical failures.(ii)Once developed, machines can be deployed at scale, without personnel training bottlenecks .(iii)Machine learning algorithms are notoriously brittle in the face of test samples that differ markedly from their training data. In the malaria task, algorithm performance can degrade in deployments to new locations if the clinics’ slide preparations have unfamiliar characteristics . In suchTo develop ML algorithms for malaria is to enjoy ample opportunities to recognize and admire the skill, versatility, rapid learning, and adaptability of human workers. At the same time, EasyScan GO’s strong performance on this benchmark slide set is evidence that fully-automated systems are poised to play a meaningful role in malaria field microscopy.Automated (machine learning-based) systems are a promising way to improve the quality and consistency of malaria microscopy. The WHO 55 slide set, designed to evaluate microscopists’ competence in crucial field use-cases, can serve as a benchmark for evaluating such systems. The fully-automated EasyScan GO, a slide scanning microscope coupled with malaria detection algorithms, was evaluated on a WHO 55 slide set. It achieved Level 1 competence in Diagnosis and Quantitation, and Level 2 in Species ID, the best performance on this benchmark test of any fully-automated system to our knowledge. While runtime and species ID both require improvement for use in general case management settings, its strong results on this slide set indicate its potential value for research use-cases such as drug efficacy monitoring, and possibly for case management use-cases with less stringent species ID requirements.Additional file 1:A full list of samples, reference results, and results of the malaria detector algorithms.Additional file 2:An example Field of View from the unreadable thin film, compared to an acceptable FoV.


Plasmodium falciparum strains with mutations/deletions of the genes encoding the histidine-rich proteins 2/3 (pfhrp2/3) have emerged during the last 10 years leading to false-negative results in HRP2-based rapid diagnostic tests (RDTs). This can lead to unrecognized infections in individuals and to setbacks in malaria control in endemic countries where RDTs are the backbone of malaria diagnostics and control.pfhrp2/3-negative P. falciparum infection acquired in Ethiopia by a 63-year old female traveller is presented. After onset of symptoms during travel, she was first tested negative for malaria, most probably by RDT, at a local hospital in Harar, Ethiopia. Falciparum malaria was finally diagnosed microscopically upon her return to Germany, over 4 weeks after infection. At a parasite density of approximately 5387 parasites/µl, two different high-quality RDTs: Palutop + 4 OPTIMA, NADALRMalaria PF/pan Ag 4 Species, did not respond at their respective P. falciparum test lines. pfhrp2/3 deletion was confirmed by multiplex-PCR. The patient recovered after a complete course of atovaquone and proguanil. According to the travel route, malaria was acquired most likely in the Awash region, Central Ethiopia. This is the first case of imported P. falciparum with confirmed pfhrp2/3 deletion from Ethiopia.Here the detection of a P. falciparum strains may not be recognized by the presently available HRP2-based RDTs. When malaria is suspected, confirmation by microscopy and/or qPCR is necessary in order to detect falciparum malaria, which requires immediate treatment. This case of imported P. falciparum, non-reactive to HRP2-based RDT, possibly underlines the necessity for standardized, nationwide investigations in Ethiopia and should alert clinicians from non-endemic countries to the possibility of false-negative RDT results which may increase in returning travellers with potentially life-threatening infections.HRP2-negative The online version contains supplementary material available at 10.1186/s12936-021-03678-2. Plasmodium antigens present in blood containing infected erythrocytes. RDTs, which focus on the detection of Plasmodium falciparum commonly use the histidine-rich protein 2 (Pf-HRP2) as major target antigen. Tests with other targets, such as P. falciparum-specific or pan-parasite lactate dehydrogenase  and aldolase, are available but their use is associated with lower sensitivity and a reduced stability at high temperatures [Rapid diagnostic tests (RDTs) are lateral flow immunochromatographic assays, which detect eratures .P. falciparum, albeit at much lower concentration [A major component of the HRP2-based RDT is a monoclonal anti-HRP2 mouse antibody. This antibody shows cross-reactivity with the HRP3 antigen, a protein of quite similar composition, which is also present in developing stages of Plasmodium falciparum isolates with deletion of pfhrp2 and pfhrp3 were first described in the Amazonas region of Peru in samples collected in 2005 [P. falciparum isolates with single (pfhrp2- or pfhrp3-) or double (pfhrp2- and pfhrp3-) deletion are present in many countries and on different continents [P. falciparum will not be recognized by HRP2-based RDTs, especially in countries or regions where the P. falciparum transmission rate is low but where a high number of strains with deletions circulate.ntinents . There iP. falciparum in 1.4 to 100% of cases [P. falciparum samples, which had been collected in two different regional hospitals [P. falciparum infection acquired by a traveller in Ethiopia, and the impact of negative RDT results on malaria case management in non-endemic countries is discussed.It is estimated that in sub-Saharan African countries RDTs fail to detect of cases . In Eritospitals . This siA 63-years-old patient presented in the Walk-in-Clinic for Infectious Diseases of the University Hospital in Bonn, Germany, 10 days after returning from a 6-week trip to Ethiopia. The patient and her accompanying husband had received pre-travel advice for repellent administration and bed-net use and were provided with an on-demand dose of anti-malarials (atovaquone/proguanil). Vaccination against hepatitis A/B and yellow fever was performed prior to travel. She had never been diagnosed with malaria before and she reported two short stays in malaria-endemic regions before, in Okavango Delta in 2007 (with chemoprophylaxis) and in Amazon rainforest, Peru, in 2014 (with stand-by medication). There was no longer residency in an endemic country before.The couple arrived in Addis Ababa on 17 January, 2020. Most of the trip took place in the highlands. Data of the travel route and the events were provided by the patient and her husband, . Examinations revealed a newly described systolic murmur and splenomegaly. Initial blood count showed pancytopenia . C-reactive protein was only slightly elevated to 11.22 mg/L. Lactate dehydrogenase was elevated (360 U/L), sodium was reduced to 132 mmol/L. Other laboratory parameters, such as creatinine, transaminases and bilirubin, were in normal range. As the main differential diagnosis, malaria tests were performed (both RDT and microscopy), blood cultures, X-ray of the thorax and tests for dengue and chikungunya due to a current outbreak situation in Eastern Africa.Plasmodium falciparum infection was demonstrated microscopically in Giemsa-stained thin and thick blood smears. Typical ring forms and crescent-shaped gametocytes were visible  were used. Palutop + 4 is a RDT with four bands . The limit of detection for P. falciparum is 100 parasites per microlitre at “Pan” and “Pf” bands. The limit of detection for P. vivax is 200 parasites per microlitre at “Pan” and “Pv” bands. NADAL RDT test has three bands (Pf-HRP2 and pan-Plasmodium LDH). The sensitivity was tested against thick blood smear test: P. falciparum detection: sensitivity 92.6%, specificity 100%. “Pan” malaria detection: sensitivity 95%, specificity 100%.ble Fig. a/b. ParaRMalaria PF/pan Ag 4 species) reaction at the “Pan” test line  to weak positive  was administered. After 3 days in hospital, the patient could be discharged with improved condition. At follow-up one week later, both RDTs were negative not only at the “Pf” line but also at the “Pan” line. In addition, thick blood films were negative for P. falciparum trophozoites.A complete course of atovaquone and proguanil  in the patient`s blood by an immunochromatographic assay. False-negative RDTs can arise for different reasons, among them very low (< 250 parasites per microlitre) or high parasitaemia, and dysfunctional tests due to non-functioning antibodies or test equipment after improperly stored test kits. Moreover, variants or lack of HRP2 proteins in the parasite result in false-negative results for P. falciparum [pfhrp2 and parasites lacking HRP2 are emerging in malaria-endemic regions, there is much concern that P. falciparum infections remain under-diagnosed and therefore not treated in time [lciparum . HRP2-balciparum . In sett in time .pfhrp2-negative strains in the Amazon region of Peru in 2010 with frequencies of up to 41% [pfhrp2 deletion have since been described worldwide [pfhrp2-negative parasites were described in the Greater Mekong Sub-region in a frequency of 9.4% [pfhrp2- and/or pfhrp3 strains, as reported in the World Health Organization (WHO) Malaria Threat Map [Plasmodium falciparum isolates with deletion of the pfhrp2 and pfhrp3 genes were identified in Ethiopia, shown by a study from the Adama Malaria Diagnostic Centre in Central Ethiopia [After the first published observation of p to 41% , parasitorldwide , Rwanda orldwide , and Demorldwide . HRP2-neorldwide  and alonorldwide . In smalorldwide . Very re of 9.4% . To datereat Map , 17, witEthiopia , but the. [A study in northwestern Ethiopia showed a reduced sensitivity of RDTs (70.8%) when compared to PCR results. This finding may be associated with RDT failures in this region but still lacks confirmation at genome level  as the P.  also desAdding the findings from this case where a traveller was most probably infected in Central Ethiopia close to the Awash Falls, an area known to be endemic for malaria, suggests a wider spread problem in this country than anticipated. Bordering Eritrea, Ethiopia can be considered at high-risk, with strong implications for local malaria RDT testing and control measurements.P. falciparum was confirmed by microscopy and PCR and parasitaemia was counted according to WHO standards [pfhrp2-detecting RDTs. A parasite concentration of 5387 per microlitre is far above the diagnostic cut-off level of both of the applied RDTs . The positive reactions at the “Pan” test line (= common pLDH) of both RDTs could confirm malaria but not define the species involved. In Ethiopia, other species such as P. vivax amount to approximately 40% of all cases, and low-scale infections with P. ovale and P. malariae have been reported [P. falciparum were excluded by PCR.In this traveller’s sample, tandards . The samreported , 21. In pfhrp-deleted strains have reduced fitness and produce a lower parasite density in humans [The detection of gametocytes in the blood smears confirmed the assumed date of infection (7 or 8 February) retrospectively. At presentation, the patient had been suffering from falciparum malaria for approximately 4 weeks. Fortunately, parasitaemia remained moderate and she did not develop severe malaria, according to WHO criteria. Unrecognized uncomplicated falciparum malaria can result in higher parasitaemia and progress to severe malaria in 10% of cases . It is nn humans  might exP. falciparum-specific HRP2 and pan-Plasmodium LDH (pLDH) is widely used, according to the WHO’s country profile database [pfhrp2-negative parasites via pLDH (Pan line) as shown in the laboratory at > 30 days post infection. However, the test’s sensitivity for pLDH is low during recent P. falciparum single infection with low parasitaemia. As a consequence, a complete negative result of the combination RDT is highly likely at the local setting. Advising the patient to repeat malaria testing within a few days would have been recommended. The available stand-by medication was not used, which is in accordance with recommendations of current guidelines of the German Society of Tropical Medicine and Global Health (DTG). An increasing number of cases, like the one reported here, would justify to recommending regular chemoprophylaxis instead of stand-by medication for travellers to countries where deletions in pfhrp2 gene of P. falciparum are known. In this case, chemoprophylaxis would have avoided disease and a false negative malaria test.At the time of early presentation at a local hospital (approximately 19 days after infection), parasitaemia might have been sufficient for microscopic diagnostic in a non-immune patient. In Ethiopia, a combined RDT  which detects database . This tedatabase . Theoretpfhrp2/3 deletions was detected [P. falciparum cases, thus creating a selection pressure on strains with pfhrp2 deletions [Similar cases have occasionally been described in travellers, such as in a French traveller after a visit to Brazil in 2011 . Accordidetected . This sieletions . WHO receletions .P. falciparum strains with certainty. Pan-LDH-only RDTs show promising results when tested against both HRP2 expressing and non-expressing P. falciparum panels [P. falciparum strains with pfhrp2 deletion, only chloroquine resistance is described for P. falciparum strains with pfhrp2 deletion . The paP. falciparum strain in Ethiopia, most probably in the Awash region. This case illustrates that false-negative results by HRP2-based RDTs may lead to delayed diagnosis and treatment during and/or after travel. Travellers should be advised to ask for a microscopic blood examination when travelling in an area which is endemic for HRP2-negative P. falciparum strains. Clinicians in endemic and non-endemic countries should made aware of emerging HRP2-negative strains and the implications for RDT testing in P. falciparum-infected patients.Reported here is the first case of a returning traveller who acquired a HRP2- negative Additional file 1: Fig. S1. Multiplex detection of pfhrp2 and pfhrp3 genes on the patient's sample (green) and a P. falciparum with known sequence with wild type pfhrp2/3 (red) using deletions assay according to [pfhrp3 in patient and control sample. Amplification only in control; CT 23.70; C internal control in patient and control sample. Detection in patient's sample (CT = 24.12) and control sample (CT = 18.68).rding to . A pfhrp


Plasmodium falciparum malaria. The mechanism underlying this protection is poorly understood. Since both malaria and sickle cell disease (SCD) are associated with the destruction of erythrocytes and widespread systemic inflammation, identifying which inflammatory factor(s) mediate susceptibility of individuals with different hemoglobin genotypes to Plasmodium infection could result in the discovery of new predictive markers and interventions against malaria or SCD severity. We hypothesized that hemoglobin genotypes modulate the inflammatory response to Plasmodium infection. We conducted a cross-sectional study in Ghana, West Africa, between 2014 and 2019 to ascertain the relationships between blood inflammatory cytokines, Plasmodium infection, and hemoglobin genotype. A total of 923 volunteers were enrolled in the study. A total of 74, age and sex-matched subjects were identified with various genotypes including HbAS, HbAC, HbSS, HbSC, HbCC, or HbAA. Complete blood counts and serum inflammatory cytokine expression levels were assessed. The results indicate that differential expression of CXCL10, TNF-α, CCL2, IL-8, and IL-6 were tightly linked to hemoglobin genotype and severity of Plasmodium infection and that these cytokine levels may be predictive for susceptibility to severe malaria or SCD severity.In 2018, 228 million cases and 405,000 malaria-associated deaths were reported worldwide with a majority being in Africa. A wide range of factors, including parasitemia, host immunity, inflammatory responses to infection, and host hemoglobin genotype, mediate the severity of malaria. Among the hemoglobinopathies, hemoglobin S (HbS) is caused by a single amino acid substitution of Glutamic Acid replaced by Valine at the sixth position of the beta-globin chain (E6V). Hemoglobin C (HbC) on the other hand, involves a single amino acid substitution of Glutamic Acid by a Lysine (E6K), which has received the most attention. These substitutions alter the stability of Hb leading to wide-ranging hematological disorders. The homozygous state of hemoglobin S (HbSS) results in sickle cell anemia (SCA) whereas the heterozygous state (HbAS) results in sickle cell trait (SCT). Both mutations are reported to mediate the reduction in the severity and fatality of  Plasmodium parasites that are carried by female Anopheles mosquitos , natural killer, monocytes, macrophages, and other cell types to promote inflammation , two  and no (HbAA) copies of the Hb allele variant S and/or C with (+) and without (-) malaria. This exploratory study provides a new opportunity to assess how alterations in cytokine/chemokine profiles by certain comorbidities may contribute to susceptibility to malaria. As well as the potential discovery of new biomarkers for assessing malaria or SCD severity and new interventions.Volunteers from the Greater Accra region in Ghana, West Africa were enrolled from multiple hospitals and polyclinics: Korle-Bu Teaching Hospital, Ghana Institute of Clinical Genetics, Korle-Bu Polyclinics, and district hospitals namely, Princess Marie Louise Children’s Hospital, Mamprobi Polyclinic, Ussher Polyclinic, and La General Hospital. A total of 923 samples were collected between February and November 2014 and June 2017 to July 2019 as part of an ongoing NIH-funded SCD and malaria study at Morehouse School of Medicine, Atlanta GA, USA, and in collaboration with the University of Ghana.This study was approved by the ethics review boards of Morehouse School of Medicine, College of Health Sciences, University of Ghana, and the Noguchi Memorial Institute for Medical Research at University of Ghana. The study was approved at all collaborating institutions before the commencement of the study. Participants over the age of 18 years gave informed consent.Individuals with high Fetal Hb (HbF), pregnant women, HIV+, and individuals with partial or missing information in their files relating to Complete Blood Counts (CBC) were excluded from the study. Samples were numerically coded. A total of 74, age and sex-matched, subjects were randomly selected from the pool of 923 individuals representing all hemoglobin genotypes either positive or negative for malaria. Eight (8) subjects each were selected from four (4) genotype groups  and six (6) subjects each from seven (7) genotype groups . The HbCC+ genotype was extremely rare and was not included in the pool. The groups were then age and gender-matched, with the average age being close to 30 years for all groups.Plasmodium falciparum Rapid Diagnostic Test kits and thick smear microscopy. HIV status was also assessed using HIV Rapid Diagnostic Tests.All blood samples were collected in BD Vacutainer® CPT™ tubes . Mononuclear white blood cells (WBCs), plasma, and red blood cells (RBCs) were separated from the CPT tubes within an 8 h window, according to the manufacturer’s instructions. Hemoglobin status was determined using cellulose acetate membrane electrophoresis at the Department of Hematology at the Korle-Bu Teaching Hospital . HematolPlasma cytokine concentrations were determined using Bio-Plex Pro Human Cytokine Group I Panel (Bio-Rad Cat#:M500KCAF0Y) which targeted 27 cytokines: Interleukin (IL)-1β, IL-1ra, IL-2, IL-4, Il-5, IL-6, IL-7, Il-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, CXCL10, CCL2, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, VEGF, FGF basic, Eotaxin, G-CSF, GM-CSF, and IFN-γ. The plates were analyzed by a multiplexed microsphere cytokine immunoassay procedure (Luminex LX100) at Emory University’s Peds/Winship Flow Cytometry Core.All statistical analyses were done in GraphPad PRISM version 7.04 for Windows  unless otherwise stated. A sample size calculation using preliminary results determined a minimum of 3 samples per group were needed using a 95% confidence interval and power of 80 by using preliminary multiplexed microsphere cytokine immunoassay data. Study samples and hematologic profiles were stratified by Hb subtypes and normalized by the D’Agostino & Pearson normality test. Significant differences within and between groups were assessed for cytokine levels and CBC levels using a one-way ordinary ANOVA and Tukey’s multiple comparison tests. Cytokine concentrations that remained unchanged and those that fell out of linear range were excluded. An unpaired two-tailed t-test or two-tailed Mann-Whitney test was used to assess significant differences between two groups for parametric or nonparametric data, respectively . StatistAge, WBC, RBC, Hb, and PLT levels were assessed and compared among all hemoglobin genotypes with and without malaria  or uninfected (-) with malaria parasites were analyzed between and among groups . CXCL10 concentration was significantly different between HbAA- vs HbSS- (P = 0.04), HbSC- (P = 0.02), HbAA+ (P = 0.01) (ROC curves and corresponding area under curve (AUC) assessments enable the prediction that CXCL10is independently discriminated between HbAA- and HbAA+ with an AUC = 0.86 and P = 0.02          and TNF-α vs WBC  as well as for HbSC- for IL-6 vs WBC  were significantly correlated  and IL-6 vs Hb  while in HbAC+ for CCL3  and in HbCC- for IL-8  and CXCL10  vs Hb  as well as IL-6 vs RBC  and in HbCC- for CCL3 vs RBC   interactions . Compara–IL-8 mediates neutrophil trafficking and IL-6 has a “pleiotropic effect on inflammation, immune response, and hematopoiesis” as well as has been associated with acute phase response in malaria , 37, 38.Assessment of cytokine concentrations across all groups with or without malaria indicated differential expression of inflammatory cytokines . PrincipIn conclusion, some cytokines are differentially expressed among individuals with different hemoglobin genotypes infected with or without malaria parasites. Circulating CXCL10, CCL3, IL-8, TNF-α, and IL-6 could be used as potential biomarkers for malaria severity among individuals with hemoglobinopathies. CXCL10 and TNF-α could be used to differentiate HbAA from HbSS. It was also determined that malaria outcomes could be altered based on the crosstalk between inflammatory cytokines  and Hb genotypes. Assessing cytokine levels among individuals with different hemoglobinopathies increases our understanding of the pathogenesis of severe malaria in different hemoglobinopathies. Assessing cytokine levels will also allow us to determine potential biomarkers of disease severity in individuals with malaria and hemoglobinopathies. However, the small sample size limited our ability to assess the specificity and the sensitivity of the proposed markers. For that reason, more studies are required on a larger sample size to determine the validity of the markers identified for assessing malaria and sickle cell disease severity. The use of such markers could facilitate a deeper understanding of both disorders and facilitate rapid treatment implementation in malaria patients to decrease disease severity, comorbidities, and recovery time.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Morehouse School of Medicine, College of Health Sciences, University of Ghana, and the Noguchi Memorial Institute for Medical Research at University of Ghana. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.AD, JS, and KH designed the study. AD, MW, YD-A, and FB provided support in subject enrollment and sample collection. KH and AD conducted the experiments. KH, AD, and JS analyzed and interpreted the data. KH and AD wrote the paper. FB, MW, YD-A, AA, JH, JS, and AD edited and approved the final manuscript. All authors contributed to the article and approved the submitted version.The study was supported by National Institutes of Health, grant numbers NIH/FIC 1K01TW010282 , NIH/FIC UJMT Fogarty Global Health Fellows Program #5R25TW009340 , NIH/NIMHD Research Centers in Minority Institutions (RCMI) 5U54MD007602 , National Center for Advancing Translational Sciences of the National Institutes of Health TL1TR002382 , NIH/NINDS 1R01NS091616  and National Institute on Minority Health and Health Disparities G12MD007602 , and NIH 2U54MD007602. The Elsa U. Pardee Foundation , University of Louisville Department of Pharmacology and Toxicology Faculty Start-up funds  and UofL COBRE NIGMS NIH P20GM113226  are also recognized for supporting J. L. Hood.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Delayed cerebral ischemia (DCI) is a dreadful complication present in 30% of subarachnoid hemorrhage (SAH) patients. DCI prediction and prevention are burdensome in poor grade SAH patients (WFNS 4–5). Therefore, defining an optimal neuromonitoring strategy might be cumbersome. Cerebral microdialysis (CMD) offers near-real-time regional metabolic data of the surrounding brain. However, unilateral neuromonitoring strategies obviate the diffuse repercussions of SAH. To assess the utility, indications and therapeutic implications of bilateral CMD in poor grade SAH patients. Poor grade SAH patients eligible for multimodal neuromonitoring were prospectively collected. Aneurysm location and blood volume were assessed on initial Angio-CT scans. CMD probes were bilaterally implanted and maintained, at least, for 48 hours (h). Ischemic events were defined as a Lactate/Pyruvate ratio >40 and Glucose concentration <0.7 mmol/L. 16 patients were monitored for 1725 h, observing ischemic events during 260 h (15.1%). Simultaneous bilateral ischemic events were rare . The established threshold of ≥7 ischemic events displayed a specificity and sensitivity for DCI of 96.2% and 83.3%, respectively. Bilateral CMD is a safe and useful strategy to evaluate areas at risk of suffering DCI in SAH patients. Metabolic crises occur bilaterally but rarely simultaneously. Hence, unilateral neuromonitoring strategies underestimate the risk of infarction and the possibility to offset its consequences. On the other hand, the role of vasospasm in DCI appearance is nowadays controversial. In fact, angiographic vasospasm resolution induced by novel therapies such as clazosentan, does not correlate neither with DCI prevention nor with functional prognosis3. This fact may support the absence of a causal relationship between vasospasm and DCI5.Delayed cerebral ischemia (DCI) is considered one of the main causes of neurological deficits in subarachnoid hemorrhage (SAH) and it may occur in up to 30% of patients. DCI typically occurs between the fourth and tenth day after bleeding but its pathophysiology remains yet unclear. Nevertheless, various pathological phenomena such as oxidative stress, cortical spreading depolarizations, microvascular thrombosis and disbalances in cerebral microcirculation are known to be involved in its development7. However, CMD requires a small surgery, its cost is not despicable and the locally confined nature of the obtained measurements limits their validity to interpret the global state of the brain. One of the major challenges when dealing with CMD is to extrapolate the local and timely delayed measurements it provides. Therefore, determining the location of the catheters is not a trivial issue. CMD catheters are normally placed in the frontal lobe, in the watershed area between the vascular territories of the middle and anterior cerebral arteries (MCA and ACA). In SAH patients, the catheter is consensually placed in the hemisphere where the aneurysm or most of the blood are located. Nevertheless, ischemic events in non-monitored areas will probably be disregarded. Furthermore, it might be difficult to determine which hemisphere is at risk in some ACA aneurysms or diffuse bleeding patterns. Ischemia in the non-monitored hemisphere is not rare and it may represent up to one third of secondary infarctions9.In poor grade SAH patients, CMD monitoring may be useful for early detection of potentially salvageable brain tissue exposed to ischemic risk. CMD monitoring provides semiquantitative metabolic measurements of the brain tissue independently of the initial insult. CMD patterns of ischemia (Glucose <0.7 mmol/l and Lactate pyruvate ratio (LPR) > 40) have been consistently associated with high specificity and sensitivity to detect DCIUsing a “one catheter” monitoring protocol means assuming that the hemisphere hosting more blood is the most susceptible and that information from one side might be extrapolated to identify metabolic changes in remote areas. However, regarding SAH, there is a lack of knowledge on the frequency of silent infarcts and the metabolic response of those contralateral areas to the primary insult. Thus, in the present clinical investigation we sought to assess the utility of a bilateral CMD monitoring protocol in the setting of poor grade SAH patients. Our main objective was to detect bilateral metabolic ischemic events in order to identify silent infarcts. This information may contribute to anticipate DCI diagnosis and may also provide us with therapeutic tools to prevent this feared complication.10.All methods were carried out in accordance with relevant guidelines and regulations. The methodology of this report follows the recommendations of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelinesThe present research was approved by the institutional review board . Moreover, in the absence of conscious patients, first grade relatives signed an informed consent authorizing patient’s inclusion in the bilateral CMD neuromonitoring protocol.Poor grade SAH patients (WFNS 4 or 5 grades) eligible for multimodal neuromonitoring were prospectively collected between January 2017 and January 2019. Included patients were those whose neurological status required maintaining them under sedation and ventilation for at least 48 hours. Patients who survived less than 24 h or in whom CMD monitoring lasted less than 48 h were excluded.CMD catheters were implanted bilaterally in MCA/ACA watershed territory just after the surgical or endovascular exclusion of the aneurysm. Aneurysm exclusion modality was decided consensually by our neurovascular board. CMD catheters were implanted in the operating room using a mini burr hole located at Kocher’s point. In patients with surgically treated aneurysms, ipsilateral CMD catheter was placed under direct vision through a small corticectomy. A computed tomography (CT) was performed in the 24 hours following CMD catheters placement to confirm their correct location and to rule out early ischemic lesions due to aneurysm exclusion or as a response to SAH primary insult.11.Cerebral microdialysis was performed using a 20 kDa catheter  with a membrane length of 10 mm. The intraparenchymal probe was implanted at a depth of 2–3 cm and connected to the perfusion pump . Perfusion fluid  was pumped at a flow rate of 0.3 μl/minute. Conventional microdialysis analysis equipment  was implemented to record extracellular fluid concentrations of glucose, pyruvate, lactate, glycerol, and glutamateSamples were collected hourly. Data recording was stopped if one or both catheters consistently reported errors. Ischemic events were defined as those observations in which Lactate/Piruvate ratio (LPR) was >40 and Glucose concentration was <0.7 mmol/L.All monitored patients underwent a safety protocol in order to spot any issues related to such monitoring. The safety protocol included: (1) Daily revision of catheter wounds to identify any CSF leak or purulent material; (2) Once the monitoring was over, the catheter tip was routinely sent to the lab for culture; (3) A CT scan was routinely performed after catheter placement in order to rule out hematomas -any blood collection over 5cc- or any other complication related to the catheter insertion.12. Aneurysm’s parent vessel and hemisphere were defined on the AngioCT. The CMD probe located in the hemisphere containing more blood was named “a”, while “b” was reserved for the contralateral one. In those cases, in which differences in volumetry between hemispheres were inferior to 5cc, “a” was used for the catheter ipsilateral to the aneurysm . Anterior communicating artery (AcoA) aneurysms were considered left or right depending on the dominant A1 segment.Cerebral blood volumetry in the initial CT was calculated using OSIRIXCerebral magnetic resonance (MRI) was performed prior to patient’s discharge and within 14 days of SAH. This MRI was aimed to find new ischemic lesions not present in the initial CT. Ischemic lesions were classified into two categories: those happening in the brain tissue typically considered at risk and those occurring in silent areas. It was also determined if the ischemic lesions might be related to aneurysm treatment.Functional neurological outcome was assessed at three months after SAH using the modified Rankin scale (mRS). Patients lost during follow up were discarded.p level of 0.05 (2-sided). Sensitivity and specificity of ischemic events detected by means of CMD for DCI diagnosis were calculated. In addition, a receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cutoff value of ischemic events for the prediction of DCI.All Statistical analyses were performed using Stata/IC 13.1 for Mac version. Continuous variables were reported as mean (standard deviation) or median (interquartile range). Metabolic records obtained in each hemisphere were compared with Student t-test. Level of significance was established at a (Table 1). Mean cisternal blood volume (CBV) was 22cc (SD 13.3) in the hemisphere named “a”, and 12.8cc (SD 7.10) in the contralateral one. In three patients a parenchymal hematoma was additionally present. Differences in cerebral blood volumetry between hemispheres were inferior to 5cc in 50% of the patients.Twenty consecutive poor grade aneurysmatic SAH patients admitted to our institution between January 2017 and January 2019 and requiring multimodal monitoring were prospectively recruited for the study. Four patients were excluded: two died within the initial 24 hours and two experienced an accidental removal of the catheters before completing 48 hours of monitoring. The mean patients’ age was 58 years (SD:7.3). Most of the aneurysms were located in the anterior circulation: MCA aneurysm 38% (6/16); ACoA aneurysm 31% (5/16); Pcom aneurysm 13% (2/16); Carotid aneurysm 6% (1/16); AICA aneurysm 6% (1/16) and PICA aneurysm 6% (1/16). All the patients were admitted in poor clinical condition, 56% (9/16) in WFNS 5. The blood assessment of the early CT scan revealed high amount of cisternal and intraventricular blood in most of the patients. The median (IQR) mFisher scale was 4 (0): 88% (14/16) mFisher:4; 6% (1/16) mFisher:3 and 6% (1/16) mFisher:2 .Altogether, patients were monitored using bilateral CMD for 2048 hours, consistently during the first week from SAH onset and right after the occlusion of the aneurysm. Mean monitoring time per patient was 108 hours (SD 45.9). A total of 323 hours (16%) were withdrawn from final analysis due to errors in their acquisition or processing. Out of the overall 1725 hours of bilateral observations, 15% revealed an ischemic event in at least one of the probes. Ischemic events located in hemisphere “a” were recorded for a total time of 188 hours . Meanwhile, ischemic events in the silent hemisphere (named “b”), were detected for 72 hours . During 5 hours, ischemic events were simultaneously detected in both catheters Ischemic lesions not previously present in the initial CT, were identified in the follow-up MRI of 5 patients, 4 in hemisphere “a” and 1 in hemisphere “b”. All the new infarctions detected were related with delayed ischemia and not attributed to the aneurysm repair procedure. Only 50% (8/16) of the patients had achieved a good functional (mRS 0–3) recovery at the three months follow-up. The median (IQR) mRS at 3 months was 3.5 (2–5). None of the patients initially included were lost at 3 months follow up.. With a threshold of 7 or more ischemic events, bilateral CMD monitoring strategy was able to predict the occurrence of delayed cerebral infarctions with high specificity (96%) and sensitivity (83%)  . Figure Table Fig. . With a –1) Fig. . Figure No complications directly related with the catheters were registered. Daily revisions of the catheter’s wounds were performed and no catheter infection was documented. Furthermore, none of the postoperative CT scans showed any catheter-related hemorrhage larger than 5cc.14. The rest of brain infarctions cannot solely be explained by vasospasm, revealing the existence of other mechanisms of brain injury and raising the need of other diagnostic tools. CMD, as part of a multimodal neuromonitoring strategy, might improve delayed cerebral ischemia prediction. CMD offers direct metabolic information. However, this information is locally constrained and is not updated in real-time. Data from CMD probes is highly influenced by their location in the brain parenchyma and it might be misleading to extrapolate this information to interpret what is the real condition of the entire brain15. If SAH is considered a global cerebral disease, the location of the catheter would not be such a critical decision and one CMD probe might be enough to acquaint the metabolic profile of the brain as a whole. Nonetheless, as outlined by other authors, we believe that brain injury related to SAH may differ depending on the susceptibility of the different parenchymal or vascular territories17. Our results support this assumption proving the existence of infarctions in the silent or the DCI low-risk hemisphere thus providing rationale for considering bilateral CMD monitoring.Cerebral infarctions are likely to occur after aneurysmal SAH. These ischemic lesions are located in territories suffering angiographic vasospasms in 25–81% of cases12. Lagares et al. proved that cisternal blood volumes over 20 cc are clearly associated with an increased risk of poor outcome. In our series of 16 patients, the average volume of cisternal blood was 36 cc (SD: 21.7), supporting our intention to select severely affected patients. Despite these massive volumes, in 50% of our patients, interhemispheric difference in blood volume was lower than 5 cc. This condition reveals how difficult might be to initially select which hemisphere is more exposed to delayed injury. Strategies to determine the location of CMD catheters are based on the belief that the hemisphere containing the greatest volume of blood is the most susceptible to suffer deleterious changes. Tholance et al. developed an algorithm to determine the optimal location of CMD catheters in order to early detect DCI occurrence18. They improved the detection of DCI from 54% when the catheter was implanted in the hemisphere with more blood, to 79% when implementing this algorithm instead18. According to these authors, CMD catheters should be placed in the ACA territory for Anterior communicating artery (ACom) aneurysms and in the ipsilateral hemisphere for Middle cerebral artery (MCA), Internal carotid artery (ICA) and Posterior communicating artery (PCom) aneurysms. Nonetheless this algorithm failed to detect at least 20% of DCI. They also noted that territories where DCI was found had previously shown high LPR values. This finding supports the relevance of local measures in SAH. Our results concur with these statements, since CMD catheters from areas developing DCI showed clear differences compared to those located in areas which did not show any delayed brain injury.Subarachnoid blood volume in the initial CT after aneurysmal SAH has been defined as an independent prognostic factor19. Given the scarce therapeutic strategies to prevent DCI, we prioritized CMD to be specific, thus we used a restrictive definition of ischemia. Other scenarios deemed to increase the sensibility of CMD to predict DCI such as mitochondrial dysfunction  or LPR augmentation without glucose decrease, were not considered20. Importantly, our LPR values were comparable to those detailed by other authors in critical patients17.In this clinical research, ischemic events were defined by validated values of LPR and Glucose21. Therefore, a rationale exists for the use of multiple CMD catheters in patients suffering TBI22. However, this practice has not been established nor generalized in SAH. Jacobsen et al. recently reported their experience with multiple CMD catheters in SAH patients; nonetheless, only one hemisphere was monitored, and the detection of contralateral infarctions was not assessed20. Unlike TBI, diffuse and bilateral delayed infarctions in SAH are rare and probably the result of severe ischemic damage taking place within the very first hours. Hence, global DCI might be present in terminal SAH patients without any chance of therapeutic intervention23. Our results support the singularity of diffuse ischemia in SAH, since synchronic bilateral metabolic ischemic events hardly occurred for 5 hours. Therefore, if global infarction in SAH is that rare, the metabolic changes registered with one catheter should be extrapolated to the rest of the brain with extreme caution. The high efficiency of CMD measuring local changes in brain metabolism could be properly extrapolated if multiple catheters would be implemented15. In 5 out of 16 patients (31.25%) we observed cerebral infarctions, in one of them in a pre-considered silent territory. Herein CMD proved its high sensitivity predicting DCI, since every established infarction was preceded by various records of ischemic metabolism.CMD has consistently proven its ability to display metabolic changes in different areas surrounding a focal concussion in the setting of traumatic brain injury (TBI)6. However, the majority of ischemic events do not lead to DCI, either because they do not last long enough or in response to the implementation of therapeutic measures that prevent infarction from being established. Nonetheless, some patients unavoidably develop permanent ischemic lesions despite of specific therapeutic attempts. Several studies have conferred CMD a high positive predictive value on DCI prediction18. In our series, CMD demonstrated a sensitivity and specificity of 96% and 83% respectively, when a threshold of 7 or more ischemic events was used. Despite the small size of our sample, these results are equal or superior to those reported by other authors18.Our bilateral CMD monitoring strategy allowed us to detect that out of the total of ischemic events, 24% occurred in pre-considered silent hemispheres (b). These ischemic events came with few or no changes in the contralateral hemisphere (a). These findings match with the current evidence that defends that brain infarctions also occur in silent territories3) related to probe implantation. Furthermore, our results show that metabolic deficits detected by CMD rarely occur simultaneously and globally in SAH, affecting non-synchronously different brain territories. Nevertheless, risk-benefit balance should be carefully regarded since the rather small individual risk of implantation of multiple probes might occur, resulting in life threatening complications.Summarizing, herein we have exposed a novel neuromonitoring strategy for SAH based on the bilateral use of CMD probes. This strategy allowed translating focal data into global metabolic trends in the brain. As stated in previous reports, CMD proved to be safe and useful. None of our patients suffered infections nor significative hemorrhages . Hence, it is expected that CMD predictive value would decrease in a more heterogeneous sample of patients. Another limitation of this study worth mentioning is the need to deepen the prediction of bilateral CMD monitoring in the case of posterior circulation aneurysms. A future cohort of patients with posterior fossa aneurysms may be necessary to determine the utility of these resources in these selected patients. Finally, despite obtaining sound statistically significant results, the limited number of patients calls for cautious interpretation. Our findings represent an intriguing starting point for further research but might not justify bilateral monitoring on their own.In selected SAH patients, bilateral CMD catheters seem to be safe and potentially useful to predict ischemic events that would have remained undiagnosed with a unilateral strategy. Almost a quarter of metabolic crisis patterns would have been silent if only one hemisphere had been monitored. Actually, less than 1% of ischemic events occurred simultaneously in both hemispheres. These findings suggest that careful extrapolation of data from unilateral monitoring should be considered. In the absence of effective focal therapies, adhering to locally constrained measurements may mislead our therapeutic decisions resulting in harmful consequences for the unmonitored brain.


Hypophosphatemia is common in severe infections including malaria. Previous studies suggested that serum phosphate concentrations correlate with temperature, but it is unclear whether the type of infection and other factors occurring during infection influence this association. Here relationships were investigated between serum phosphate levels, cause of fever, demographic, clinical and laboratory parameters.Anonymized data were analysed from 633 adults with malaria or other febrile illness admitted to Northwick Park Hospital, London, UK. Univariable and multivariable generalized linear model analyses were performed to examine associations with serum phosphate levels. Interaction terms were included to investigate whether cause of fever , malaria parasite species, or malaria severity influenced the association of other variables with phosphate.Hypophosphatemia was common in subjects with malaria (211/542 (39%)), and in other febrile illnesses (24/91 (26%)), however median phosphate levels did not differ significantly by diagnostic group, parasite species or severity of malaria. In all analyses, there were highly significant negative associations between serum phosphate and axillary temperature, and positive associations between serum phosphate and platelet count. There were no significant interactions between these variables and cause of fever, parasite species or severity of illness. Sodium and potassium concentrations were associated with serum phosphate in subjects with malaria and when data from all subjects was combined.Serum phosphate is consistently associated with temperature and platelet count in adults with diverse causes of fever. This may be a consequence of phosphate shifts from plasma into cells to support ATP generation for thermogenesis and platelet activation. Plasmodium vivax malaria [Abnormal serum phosphate levels are a common finding in seriously ill patients, and hypophosphatemia  is commonly seen in patients with severe infections –9. Assoc malaria .Serum phosphate concentration is usually maintained in the range 0.8–1.5 mmol/L in adults , 13. PhoMild hypophosphatemia is often asymptomatic, but with more severe hypophosphatemia symptoms such as weakness, malaise, anorexia, and bone pain, develop, and in extreme cases haemolysis, rhabdomyolysis, coma, and death have been reported , 16. ManHypophosphatemia may be caused by reduced intake or absorption from the gut, increased loss of phosphate , or redistribution through transcellular shifts . The conPlasmodium falciparum vs non-P. falciparum malaria) would also influence phosphate concentrations, and that lower phosphate concentrations would be associated with more severe disease.In the present study, the associations between serum phosphate concentrations and temperature, other clinical variables, and other laboratory variables, were investigated in a large cohort of subjects with malaria and a comparison group of subjects with other causes of febrile illness. It was hypothesized that temperature would influence phosphate concentrations, as observed in a previous study , but thaP. falciparum, P. vivax, Plasmodium ovale or Plasmodium malariae and included both severe and uncomplicated infections. Severe malaria was defined as reported previously using World Health Organization (WHO) criteria [Anonymized data were extracted from an audit database of febrile patients admitted to the infectious disease ward of Northwick Park Hospital from April 1991 through to May 2006  and frocriteria . The othPatients with a clinician diagnosis of renal failure, or blood creatinine level ≥ 265 μmol/L  were excP < 0.05 was considered to be significant. Categorical and continuous data were compared between groups using the Chi squared and unpaired Mann–Whitney tests, respectively. Four variables  were log transformed before regression analyses in order to normalize their distributions. Univariable and multivariable generalized linear model (glm) analyses were performed using the R statistical software. Correlation plots were generated with the ggplot2 package and regression lines with 95% confidence intervals were constructed with the linear model method. Variables with significant associations (P < 0.05) with phosphate in univariable analysis were included in multivariable models. Forward and backward selection were used to determine the optimal multivariable model in which the Akaike information criterion was minimized whilst all variables were significantly (P < 0.05) associated with phosphate.All statistical analyses used two-sided tests and P = 0.002).633 subjects were included in the study, 542 with malaria and 91 with other febrile illnesses  non-P. falciparum infections (P. vivax (n = 120), P. ovale (n = 10), and P. malariae (n = 8)). 60 (11%) of the malaria infections  were severe. There were no significant differences between the two groups in terms of gender or age. As expected, the proportion of African patients was greater in the P. falciparum group and the proportion of Asian patients was greater the non-P. falciparum group  vs 57/138 (41%), respectively; hyperphosphatemia in 37/404 (9%) vs 22/138 (16%), respectively; normal phosphate in 213/404 (53%) vs 59/138 (43%), respectively; Chi squared test P = 0.036).Amongst the subjects with malaria, 404 (75%) had  and 138 5% non-P. and 138 5% non-P.10(platelet count), corrected calcium, sodium, potassium and log10(creatinine) were all significantly associated with serum phosphate (Table 10(platelet count), sodium and potassium. A strong negative association was observed between temperature and serum phosphate, with a 0.082 mmol/L decrease in serum phosphate associated with each 1 °C rise in temperature in the multivariable model. Significant positive associations were observed between serum phosphate and log10(platelet count), sodium and potassium. Although malaria was not associated with phosphate concentration in the univariable analyses, it was considered that malaria status may influence the relationships between phosphate and temperature or platelets. However, there were no significant statistical interactions between malaria status and these variables in the multivariable model.Associations between serum phosphate levels and demographic, clinical, and laboratory parameters were evaluated in univariable analyses using the combined dataset of 633 subjects with malaria and other febrile illnesses. Temperature, log10(platelet count), corrected calcium, sodium, potassium and log10(creatinine). This was consistent with the findings in the combined dataset. Neither disease severity, nor parasite species, were significantly associated with phosphate concentration in univariable analysis (Table 10(platelet count), sodium and potassium. A strong negative association was again observed between temperature and serum phosphate, while significant positive associations were observed with platelet count, sodium and potassium (Table 10(platelet count), corrected calcium, and potassium. In multivariable analysis, the best model included only temperature and log10(platelet count). Serum phosphate was negatively associated with temperature and positively associated with log10(platelet count)  Table .Table 4AP. vivax malaria [P. falciparum malaria and non-P. falciparum malaria. Multivariable models were used to identify variables with the strongest independent associations with serum phosphate levels.The causes and effects of hypophosphatemia during infection are not fully understood, although it is plausible that severe hypophosphatemia contributes to both symptoms and adverse outcomes. Previous work has suggested that hypophosphatemia is strongly related to fever, and that there is a strong correlation between body temperature and serum phosphate in subjects with  malaria . This stP. falciparum and non-P. falciparum malaria, but there were small differences in distribution. Consistent with a previous study [Median levels of serum phosphate in the subjects in this study did not differ between those with malaria and those with other febrile illnesses, however there were differences in the distribution of phosphate levels, with malaria subjects more likely to have either hypo- or hyperphosphatemia. Similarly, there were no significant differences in median phosphate levels between us study  a strongThis observational study does not establish whether the observed associations are the causes or effects of changes in serum phosphate, or indirectly mediated by a common unmeasured factor with a direct relationship with phosphate levels. However existing literature indicates plausible mechanisms relating all four variables identified in these multivariable models to serum phosphate concentrations. Both sodium and potassium are known to influence phosphate transport , and so The consistent association between temperature and phosphate across subgroups, and without evidence of any influence of the cause of fever or disease severity, suggests that this relationship is direct. Either temperature may increase cellular phosphate uptake or renal excretion, or phosphate transport into cells may play a causal role in changes in body temperature. Experiments in anesthetized dogs under neuromuscular blockade indicated that exogenously increasing body temperature alone did not cause a decrease in serum phosphate , making An association between platelet count and serum phosphate levels has been previously reported . During This study has several limitations. Phosphate levels, temperature and other variables were only analysed at a single point in time , so the sequence of events cannot be determined, most importantly whether changes in phosphate concentration preceded or followed changes in temperature, platelet count and other variables. Baseline values of phosphate and other variables in each individual before they became unwell were not known, and the change from baseline may be more important than absolute values. Urinary phosphate levels, acid–base status, blood lactate, and inflammatory cytokines, were not measured, but might have been helpful to support hypotheses about mechanisms of causality. There were significant differences in ethnicity between subjects with malaria and those with non-malarial febrile illness, which might explain some of the differences in phosphate levels and associations with other variables which were observed between these groups. Within the heterogeneous group of patients with non-malarial febrile illness, further subdivision by etiology was not performed because resulting groups would be too small to undertake additional meaningful analysis. Despite these limitations, the study did have the strength of complete data on a large number of subjects for the variables of most interest.The clinical consequences of infection-induced changes in serum phosphate are uncertain. Whilst hypophosphatemia can cause a variety of clinical features which are frequently seen in individuals with severe infections, such as weakness, malaise and alterations in mental status , 16, it To further understand the biological mechanisms underlying hypophosphatemia in infection, longitudinal studies in individuals at high risk of infection may be useful to establish the sequence of changes in phosphate, temperature and platelets. Addressing outstanding questions about the mechanisms relating these variables to serum phosphate may have relevance for therapeutics. If phosphate transport into cells is critical for pyrogenesis, transient inhibition might be a novel antipyretic approach. Similarly, inhibition of phosphate transport into platelets might be used to limit platelet activation.Increased body temperature and reduced platelet count are consistently associated with reductions in serum phosphate concentration in subjects with malaria and in subjects with other causes of fever. At present the cause and effect relationships, and therapeutic applications, remain uncertain.Additional file 1. Diagnoses in subjects with other febrile illness.Additional file 2. Dataset containing all analysed data from all study subjects.