The relationship between protein energy malnutrition (PEM) and malaria is controversial. While most studies demonstrate that PEM is associated with greater malaria morbidity, some indicate that PEM may in fact have a protective effect. PEM is differentiated into three subgroups: kwashiorkor (marked protein deficiency), marasmus , and kwashiorkor/marasmus. None of the studies concerning PEM and malaria seem to distinguish between these subgroups, and significant differences in susceptibility to malaria between these subgroups may have been overlooked. Plasmodium parasites and malaria infected erythrocytes are sensitive to oxidative stress. Since kwashiorkor patients seem to display an excess of prooxidants and as serum albumin is an important antioxidant, we hypothesized that patients with different forms of PEM might have different levels of malaria parasitaemia. 72 PEM children older than 6 months admitted to Kwale Family Life Training Programme (Kenya) were included in the study. Mean parasitaemia was significantly lower in the kwashiorkor group than in the marasmus group . There was no correlation between serum albumin and parasitaemia. Our study suggests a protective effect of kwashiorkor against malaria, warranting further studies. The relationship between protein energy malnutrition (PEM) and malaria is controversial. In a review concerning malaria and nutrition most studies demonstrated that PEM is associated with greater malaria morbidity and mortality; on the other hand some studies indicated that PEM may protect against malaria –3. ThereKwashiorkor (K): Protein malnutrition dominant, grossly hypoalbuminaemic with oedemas, and serum albumin <40 g/l.Marasmus (M): Deficiency in calorie intake, hypo- or normoalbuminaemic.Marasmus–Kwashiorkor (K/M): Marked protein deficiency  and marked calorie insufficiency signs present, sometimes referred to as the most severe form of malnutrition.PEM is differentiated into three subgroups:As far as we know none of the studies concerning PEM and malaria distinguish between these three subgroups, and thus significant differences in susceptibility to malaria between these subgroups may have been overlooked. in vitro, we hypothesized that patients with low levels of albumin as seen in kwashiorkor would have lower levels of parasitaemia as compared to other patients with malnutrition  in the coastal province of Kenya were invited to participate in the study consecutively and included after informed oral consent from the mother. Patients were assigned to clinical subgroups based on clinical evaluation and appearance including oedema, weight, weight/length, and weight/age.Exclusion criteria were treatment with antimalarial drug within 1 week before admittance, fever above 37.5°C and hospitalisation or treatment for malaria on the day of admittance, or during the stay. Other criteria for exclusion during the stay were vomiting more than 2-3 times a day and/or diarrhoea more than 5 times a day for more than 2 days during the stay.Seventy-two malnourished children were included in the study . Sex, agSeventeen patients were assigned to the K group based on the presence of pitting oedemas, distended abdomen, and typical dermatitis with normal body weight, and blood for determination of serum albumin was obtained from ten patients.Forty-five patients were assigned to the M group based on a body weight of less than 60% of the expected weight for age, and blood for determination of serum albumin was obtained from thirty of them.Ten patients were assigned to the K/M group based on the presence of symptoms of both kwashiorkor and marasmus, and serum albumin was obtained from nine of them.The study was performed according to the Declaration of Helsinki and was approved by the ethical committee of the provincial health authority. After adequate explanation of the aim of the study informed consent was obtained from the parents of the children. P. falciparum] were counted by experienced laboratory personnel against 1000 WBC and converted to parasites pr microliter blood assuming 8000 WBC/microliter. The analysis of serum albumin was performed at Aalborg Hospital, Denmark by an immunochemical method .The blood smears were stained with a 5% Giemsa solution and the parasites  of protection . Age is In summary, our study indicates a difference between subgroups of protein energy malnutrition (PEM) concerning malaria parasitaemia and suggests a protective effect of kwashiorkor. As far as we know, previous studies have not distinguished between kwashiorkor and marasmus in PEM and more studies are needed to elucidate our hypothesis.


Plasmodium falciparum infected erythrocytes sequestering in placental tissue release Plasmodium lactate dehydrogenase (pLDH) and histidine-rich protein-II (HRP-II). These proteins can be detected in peripheral blood using monoclonal antibody-based rapid diagnostic tests (RDTs). Nevertheless, studies to evaluate the reliability of RDTs in detecting placental malaria compared with microscopy of placental tissue impression smear (PTIS) as the gold standard are scarce.Between August 2013 and January 2015, Giemsa-stained blood smears for peripheral blood smear (Pbs), placental intervillous space (IVS) blood smear and placental tissue impression smear (PTIS)] were prepared from HIV-negative women during delivery at the Marie Reine Medical Health Centre in Yaoundé, Cameroon. RDTs with monoclonal antibodies specific to HRP-II (P.f) or pLDH (Pan) antigens were used to screen maternal peripheral blood samples.The prevalence of malaria was 16%, 7.5%, 11.5%, 8% and 13% for One Step malaria HRP-II and pLDH RDTs, peripheral blood smear, IVS blood and placental tissue impression smears, respectively. The proportion of women positive by One Step malaria pLDH RDT and Pbs increased with parasite density in PTIS, while One Step malaria HRP-II RDT detected high proportion of infected women even with low parasite density. Although the prevalence of malaria infection by both microscopy and RDTs decreased significantly with mother age (0.0008 ≤ p ≤ 0.025), parity seemed to have very little influence. The sensitivity of One Step malaria HRP-II and pLDH RDTs were 96.15% and 61.53%, respectively, compared to 80.76% for Pbs . The specificity of these RDTs was 96.49% and 100%, respectively, compared to 100% for Pbs (p ≥ 0.12). In addition, the positive predictive values were 80.64% and 100% for HRP-II and pLDH-based RDTs, respectively, compared to 100% for Pbs , while the negative predictive values were 99.40% and 94.48%, respectively, compared to 97.16% for Pbs (p ≥ 0.49). The combination of One Step malaria HRP-II RDT and Pbs showed the similar performance as that observed with One Step malaria HRP-II RDT only.P. falciparum infection in pregnant women compared to Giemsa-stained peripheral thick blood smear. This is important for better case management since microscopic examination of PTIS cannot be employed during pregnancy.These results depict One Step malaria HRP-II RDT to be better in detecting placental  Plasmodium falciparum-infected erythrocytes (IE) express a VAR2CSA antigen that mediates binding of IE to the chondroitin sulfate A (CSA) on syncytiotrophoblasts lining the intervillous space (IVS) of the placenta  were primipara, 22% [16.22–27.78%] secondipara and 42% [35.11–48.89%] multipara. The age ranged from 16 to 39 with a median age of 26. The median parasitaemia from peripheral and intervellous space thick blood smear was 4463 and 1306 parasites/µL respectively, while the median percentage of infected red blood cells in placental tissue impression smear was 2.1%. Concerning anaemia, 19% [13.52–24.48%] of women were anaemic. In this study population, 82% [76.64–87.36%] took intermittent preventive treatment in pregnancy-based sulfadoxine-pyrimethamine (IPTp-SP) against malaria and 70% [63.60–76.40%] slept under LLINs during pregnancy  One Step malaria pLDH RDT which was 8% [4.21–11.79%] (p = 0.12), (ii) the microscopic examination of placental tissue impression smear which was 13% [8.30–17.70%] (p = 0.68), (iii) the thick smear of peripheral blood which was 11% [6.63–15.37%] (p = 0.54) and, (iv) the thick smears of IVS blood which was 9% [5.00–13.00%] (p = 0.12)  and One Step malaria pLDH RDT (χ2 = 13.62 and p = 0.0002), but not with One Step malaria HRP-II (χ2 = 0.165 and p = 0.68).In order to investigate whether parasite density in the placenta tissue had an influence on infection detection by microscopy and RDTs in the peripheral blood, the proportion of women with positive thick smear and RDTs performed with peripheral blood was analysed in relation to parasite density in the placental tissue impression smear. As shown in Fig. Although no significant association was found in this study between malaria infection by RDT and parity, the infection frequency by microscopic of IVS blood and placental tissue impression smear was lower in multipara and was 5% 0.31–9.69%] and 8% [2.16–13.84%], respectively) compared to secondipara which was 16% [5.00–26.00%] and 23% [1210.42–35.58%]  and primipara women which was 8% [1.68–14.31%] and 12% [4.44–19.56%]  , but not by that of pLDH based RDT  after adjusting for parity, mother age, IPTp and bed net usage ] while the specificity was not statistically different between the two tests (p = 0.12). In addition, no significant difference was observed with NPV between the two tests while PPV was higher with peripheral blood smear (p < 0.0001). Peripheral blood smear and HRP-II based RDT were both more sensitive than pLDH based RDT . No significant difference was observed with specificity, PPV and NPV between these tests.The susceptibility to pregnancy-associated malaria has been shown to increase in primipara women as well as with parasite density. Therefore, an investigation was performed in order to determine whether parity could influence the performance of RDTs in the diagnosis of placental malaria. Although the results showed significant variations in some parameters with RDTs performance between primipara, secundipara and multipara women Table , these vThe diagnosis of placental malaria in women during pregnancy remains a major challenge. Although the WHO considers microscopic examination of Giemsa-stained peripheral, thick blood smears as the standard method for the detection of malaria parasite in humans , about 2P. falciparum infected erythrocytes has to sequester in the placental tissue through expression of a VAR2CSA antigen, which mediates their binding to the chondroitin sulfate A (CSA) on syncytiotrophoblasts lining the intervellous space of the placenta [In general, the prevalence of malaria infection in the present study was higher with One Step malaria HRP-II RDT (16%) than with One Step malaria pLDH RDT 7.5%), microscopic examination of placental tissue impression smear (13%), thick smear of peripheral blood 11.5%) and thick smear of placental tissue intervillous space blood (IVS) (8%). In addition, the percentage of malaria positive cases with placental tissue impression smear was higher in the present study compared to that of peripheral blood thick smear. This result is similar to the findings of previous studies [%, micros.5% and tP. falciparum can be present in the placenta tissue, but not detectable in the peripheral blood [P. falciparum antigen can persist in circulation for about 2 weeks following parasite clearance [The high prevalence of malaria infection observed with One Step malaria HRP-II RDT suggest that it can detect great proportion of malaria positive cases undetected by peripheral thick blood smears. In fact, of the five cases of malaria positive placental tissue impression smear with negative peripheral thick blood smears observed in the present study, four were positive to HRP-II based RDT. It is known that al blood , 10. Thulearance , 12. ConP. falciparum infection is usually asymptomatic in pregnancy. Yet parasites may be present in the placenta and contribute to maternal anaemia even in the absence of documented peripheral parasitaemia [In the present study, the relationship between malaria prevalence with parity and mother age was examined. Unlike in previous studies showing significant associations between increased parity and decreasing susceptibility to pregnancy-associated malaria , 16, thesitaemia . Both masitaemia .These observations justify the necessity of several studies carried out in different eco-epidemiological zones for malaria to determine the context or condition in which RDTs will be efficient for diagnosing pregnancy-associated malaria. Among previous studies that assessed the performance of RDTs for the diagnosis of placental malaria, only the study by Leke et al. used placental impression smears as the standard  techniquP. falciparum parasitaemia in pregnant women compared to Giemsa-stained peripheral thick blood smear.Concerning sensitivity and specificity when using placental impression tissue smears as gold standard, the sensitivity of One Step HRP-II was higher and that of pLDH RDT lower compared to that of peripheral blood smears while the specificity of RDTs ranged between 96 and 100% compared to 100% for peripheral blood smears. Concerning the positive and negative predictive values, while the PPV for RDTS ranged between 80.64 and 100% compared to 80.76% for peripheral blood smears; the NPV of the RDTs ranged between 99.39 and 94.4% compared to 97.15% for peripheral blood smears. These results demonstrate that the performance of HRP-II based RDT used in this study for placental malaria diagnosis was in general, higher compared to previous study from Cameroon –23. One P. falciparum infection in women during pregnancy. This suggestion is supported by the fact that the combination of One Step malaria HRP-II RDT and peripheral blood smears showed in this study the similar performance as that observed with One Step malaria HRP-II RDT. Although One Step malaria pLDH RDT showed a good specificity and positive predictive value, its sensitivity was very poor suggesting that this RDT cannot be used alone to accurately diagnose placental malaria. The enzyme pLDH is released from viable parasitized blood cells and is rapidly detected by series of monoclonal antibodies. Since pLDH is the product of viable parasites, the test may be used to monitor effective antimalarial therapy due to the very short half-life in circulation following parasite clearance compared to HRP-II or that negative results can be confirmed with an additional HRP-II based test.To be recommended by the WHO, any RDT need to have a sensitivity and specificity beyond 95% compared to standard method. With a sensitivity of 96.5% and specificity of 96.1% One Step malaria HRP-II based RDTs may be an important tool for detecting placental A previous study reported high sensitivity of HRP-II based RDT in multigravida and secundigravida women compared to primigravida women , suggestP. falciparum parasitaemia in pregnant women compared to Giemsa-stained peripheral thick blood smear. With a sensitivity and specificity higher than those recommended by WHO for the validation of an RDT, HRP-II based RDT appears as an important tool for detecting placental P. falciparum infection in women during pregnancy in Cameroon, since the standard diagnostic method used in this study is not recommended during pregnancy.These results indicate that HRP-II based RDT may be more accurate in detecting placental


The haemolysis associated with clinical episodes of malaria results in the liberation of haem, which activates the enzyme haem oxygenase-1 (HO-1). HO-1 has been shown to reduce neutrophil function and increase susceptibility to invasive bacterial disease. However, the majority of community-associated malaria infections are subclinical, often termed “asymptomatic” and the consequences of low-grade haemolysis during subclinical malaria infection are unknown.Plasmodium falciparum infections (determined by qPCR) were compared with 21 village-matched uninfected control children. Infected children showed evidence of persistent haemolysis over 35 days, with raised plasma haem and HO-1 concentrations. Concentrations of IL-10, which can also directly activate HO-1, were also higher in infected children compared to uninfected children. Regression analysis revealed that HO-1 was associated with haemolysis, but not with parasite density, anaemia or IL-10 concentration.As part of an ongoing study of subclinical malaria in Burkina Faso, 23 children with subclinical P. falciparum malaria infection is associated with sustained haemolysis and the induction of HO-1. Given the association between HO-1, neutrophil dysfunction and increased risk of Salmonella bacteraemia, prolonged HO-1 induction may explain epidemiological associations and geographic overlap between malaria and invasive bacterial disease. Further studies are needed to understand the consequences of persistent subclinical malaria infection, low-grade haemolysis and raised HO-1 on immune cell function and risk of comorbidities.This study reveals that subclinical  Plasmodium falciparum parasites, sometimes present at very low densities, can be detected in the peripheral blood of these individuals either microscopically or by highly sensitive PCR [In malaria endemic areas, the vast majority of malaria infections that are detected in community surveys are ‘asymptomatic’, that is they do not present with fever or other obvious clinical signs that prompt treatment-seeking behaviour. Individuals carrying these infections are often referred to as being ‘clinically immune’. However, tive PCR –3. Asymptive PCR , 5. Moretive PCR , strengtAcquired immunity to malaria develops after repeated exposure but is non-sterilizing; clinically immune individuals harbour frequent, persistent or recurrent infections throughout their lives –11. ThesSalmonella, NTS), malaria infection and anaemia have been noted since 1929 [Salmonella through reduced neutrophil migration into infected tissues [Associations between bacteraemia  and the Ministry of Health in Burkina Faso (reference number 2015-3-033). Venous blood was collected into RNAprotect, heparin and EDTA-containing vacutainers. Whole blood samples were collected for parasitology from the EDTA tube. The blood from the heparin tube was centrifuged, and the plasma layer aspirated, aliquoted and stored at − 80 °C until use. Unfortunately, no leucocytes were collected for functional analysis.A MagNAPure LC automatic extractor (Roche Applied Science) was used to extract total nucleic acids from 100 µL venous blood samples collected in RNAprotect. Parasite density was quantified using 18 s qPCR  and seriHaemoglobin concentrations were determined by self-calibrating Hemocue photometer . Anaemia was defined using WHO guidelines for circulating levels of haemoglobin (g/dL) [Haemoglobin genotypes were determined in 21 of the 23 infected patients. Extracted DNA was amplified using previously published primers (300 nM each) . After aHbA(C) ,HbC ,HbA(S) ,HbS . This methodology is described in detail elsewhere .Enzyme-linked immunosorbent assays (ELISA) were conducted according to the manufacturers’ instructions to measure plasma concentrations of haptoglobin , haemopexin , haem oxygenase-1 , transferrin , and ferritin . Colorimetric determination of haem in plasma samples was conducted according to the manufacturer’s instructions . Samples were diluted prior to testing as follows: 1:50,000 (HPT); 1:1 (haem), 1:40,000 (HPX), 1:10 (Ferritin), 1:6.6 (HO-1), and 1:200 (Tf). Plasma cytokine concentrations were determined by magnetic bead multiplex assay (EPX01A-10228-901 (IFN-γ), EPX01A-10215-901 (IL-10), EPX01A-12001-901 (G-CSF), EPX01A-10213-901 (IL-6), EPX01A-10223-901 (TNFα), EPX01A-10224-901 (IL-1β), EPX01A-10225-901 (IL-4), EPX01A-10204-901 (IL-8), ProcartaPlex Multiplex Immunoassay, Invitrogen, USA) following the manufacturer’s instructions, and analysed on a Luminex 100  running Bioplex Manager software. Samples were diluted 1:2.5. Samples giving values below the limit of detection were arbitrarily assigned a concentration at the limit of detection for the purposes of statistical analyses .Concentrations of cytokines and markers of haemolysis  were compared between uninfected controls and infected (PCR-positive) children at enrollment using the Mann–Whitney test. Correlations and partial correlations were calculated using the combined log-transformed data (day 1 and day 35). For the latter analysis, p-values were calculated using robust standard errors to account for repeated measures and within-individual correlation. Analyses were conducted using GraphPad Prism 7 and Stata version 14.P. falciparum qPCR positive children ranged from 1 to 18967 parasites/µL . IndeedAcute malaria infection induces a robust inflammatory response  and is accompanied by a compensatory (homeostatic) increase in circulating interleukin (IL)-10) –37. To dTo understand the possible causal relationships between parasite infection, haemolysis (haem), inflammation (IFN-γ) and immunoregulation (IL-10), associations between these different factors in infected children were calculated, combining observations from days 1 to 35 if qPCR positive (n = 45). Plasma IFN-γ and IL-10 concentrations were positively correlated   which is sustained for at least 35 days as infections persist. Moreover, haemolysis is accompanied by, and highly correlated with, sustained induction of HO-1. This observation highlights the need for larger and more extensive studies of neutrophil function in subclinically infected children.Clinically, haemolytic anaemia is defined by low haemoglobin concentration accompanied by low HPT and low haemopexin (HPX) . HPT binP. falciparum infections.While haemolysis and liberation of free haem induces HO-1, and these data show a clear correlation between plasma concentrations of haem and HO-1, it is important to consider the possibility that IL-10 may also play a role. Malaria-induced inflammation leads to a compensatory secretion of the anti-inflammatory cytokine IL-10  and IL-1While these preliminary data suggest that subclinical malaria infections induce persistent haemolysis and sustained production of HO-1, the analyses and interpretations are limited by the relatively small sample size and the lack of data on neutrophil function. Moreover, absolute concentrations of HO-1 were lower than previously reported for children recovering from acute febrile malaria , raisingIn this pilot study, children with subclinical malaria infections showed evidence of persistent haemolysis; with raised plasma haem, IL-10 and HO-1. This study supports the growing evidence that these low density malaria infections are not strictly “asymptomatic”. Further work is needed to clarify the impact of these observations on immune status and resilience to co-infection.


Plasmodium protozoa. Immunoglobulin M responses to α-galactose  -containing glycoconjugates have been associated with protection from P. falciparum infection and, as a result, these molecules are under consideration as vaccine targets; however there are limited field studies in endemic populations. We assessed a wide breadth of isotype and subclass antibody response to α-Gal in children from Mozambique (South East Africa) and Ghana (West Africa) by quantitative suspension array technology. We showed that anti-α-Gal IgM, IgG and IgG1–4 levels vary mainly depending on the age of the child, and also differ in magnitude in the two sites. At an individual level, the intensity of malaria exposure to P. falciparum and maternally-transferred antibodies affected the magnitude of α-Gal responses. There was evidence for a possible protective role of anti-α-Gal IgG3 and IgG4 antibodies. However, the most consistent findings were that the magnitude of IgM responses to α-Gal was associated with protection against clinical malaria over a one-year follow up period, especially in the first months of life, while IgG levels correlated with malaria risk.Naturally-acquired antibody responses to malaria parasites are not only directed to protein antigens but also to carbohydrates on the surface of  However, studies of carbohydrate-based vaccines in mice have shown a dominant IgM response2 with some IgG production3. Since the early 1990s, naturally occurring glycoproteins4, glycolipids5, and even protein-free polysaccharides6 have been shown to be important components of the adaptive repertoire and, currently, polysaccharide-based conjugate vaccines are widely used to provide protective immunity against bacterial meningitis7. At present, there are no vaccines in use against complex human parasites, and there is a need to expand the pipeline of targets of protective immunity against malaria and other neglected diseases. The investigation of parasite glycosylation may provide new opportunities for the discovery of novel vaccine candidates against such diseases8.Carbohydrates have not classically been considered to be significantly involved in adaptive immune responses, mostly being described as T cell-independent antigens that fail to induce immunological memory and immunoglobulin (Ig) class-switchingPlasmodium falciparum has been mainly assessed against protein antigens. However, besides glycosylphosphatidylinositol (GPI) anchors9, the immunogenicity of carbohydrates allegedly located in the surface of this parasite is largely underappreciated since the parasite seems to have lost many of the genes required to elaborate complex carbohydrates10. Nevertheless, recent works showed the presence of precursors involved in glycoconjugate biosynthesis12 and identified new glycosylations in the parasite surface15. Some of these sugars modify important antigens in the fight against malaria, such as the circumsporozoite surface protein (CSP), which is the main component of the RTS,S vaccine16 and is O-fucosylated in malaria sporozoites. These post-translational modifications may alter protein antigenicity, being relevant for vaccine design.The immune response against the malaria parasite Plasmodium spp. infection, reducing malaria transmission by Anopheles mosquitoes17. This study showed that levels of IgG and IgM against α-Gal increased with age, and that IgM responses correlated with malaria incidence and were associated with protection against the disease17. On the contrary, IgG responses to α-Gal did not correlate with malaria exposure and were neither associated with protection17. Remarkably, other works have also reported the reduction of antigenicity of blood stage parasitic proteins after α-galactosidase treatment18. However, specific α-galactose containing glycans have never been isolated or structurally characterized in the malaria parasite.It was recently demonstrated that antibodies against the classical α-Gal  epitopes confer protection against 19 are produced against α-Gal epitopes, which are not expressed by humans due to the inactivation of the α-1,3-galactosyltransferase  in ancestral anthropoid primates20. Thus, anti-α-Gal antibodies are largely produced in response to cross-reactive epitopes present in commensal bacteria or food22, being the most abundant natural antibody in humans, constituting 1–5% of circulating IgM and IgG in healthy adults21. Anti α-Gal antibodies may also be produced in response to infection by pathogens expressing α-Gal, like the Gram-negative bacteria Salmonella spp. or the protozoan parasite Trypanosoma spp25. In fact, it has been argued that the selective pressure that removed this glycan from humans was exerted by an infectious agent like a virus, bacteria or protozoan expressing α-Gal epitopes, or an immunologically cross-reactive carbohydrate structure26.Anti-α-Gal antibodiesIn this study we investigated the anti-α-Gal response in children living in malaria-endemic areas of Mozambique (South East Africa) and Ghana (West Africa) who participated in clinical trials of the RTS,S/AS0 vaccine. We examined the effect of age, malaria transmission intensity (MTI) and other variables on anti-α-Gal IgM, IgG, IgG1, IgG2, IgG3 and IgG4 responses, and assessed their association with protection against clinical malaria and the factors affecting it.IgM and IgG against α-Gal were first evaluated in children age 1–4 years from the RTS,S/AS02A phase 2b trial in Mozambique. Responses measured at the first visit (Month 0 [M0]) increased with age by 1.27 MFIs/year for IgM and by 1.48 MFIs/year for IgG  and a lower increase in children (3.77 MFIs/year)  than in infants (1.5–3 months old) (p < 0.001), whereas total IgGs were equal between them (p = 0.58) Fig. . IgG1, Ir) Table , but whe1–4 subclass levels between time-points within each age group, we confirmed a significant increase of IgM from M0 to M3 in both age groups (p < 0.001)  than Kintampo (high MTI)  Fig. . When stP. falciparum at M0, as indicated by antibody surrogate markers, was positively associated with anti-α-Gal IgM levels at M3 in multivariable linear regression models   , IgG1 , IgG3 , IgG4  and IgM   Fig. . When stP. falciparum antibodies . Univariate models showed a protective association of anti-α-Gal IgM , IgG3  and IgG4  with clinical malaria  IgG levels at M0 were similar to levels at M0 in infants, and increased towards M3. These results evidence a significant maternal transfer of anti-α-Gal IgG to the newborn, and a decay of this IgG during the first months of life, followed by an early and rapid increase. This suggests again a continued exposure to the glycan. These results are also similar to reports in European childrenet al. on subjects from 3 months to 25 years of age in Mali17. In that study, this observation was interpreted as indicative of P. falciparum infection failing to induce class switch of the anti-α-Gal Ig antibody response. However a higher IgM response than its correspondent IgG response is also observed against other polysaccharidae antigens, such as Streptococcus pneumoniae33, suggesting that overall the rate of IgM/IgG switching is not as fast for polysaccharide antigens as for protein antigens.Overall, IgM and IgG to-α-Gal increased with age, however IgM reached higher levels than IgG, being the predominant response in children. This result is in agreement with previous works by Yilmaz Plasmodium17, other pathogenic microbes express this glycan, like the protozoan parasites Leishmania spp. and Trypanosoma spp, the Gram-negative bacteria Salmonella spp., and some viruses34. Furthermore, common commensal bacteria in the midgut microbiota such as Escherichia spp., Klebsiella spp. and Serratia spp. also express α-Gal22. Also, exposure to different types of diet in both sites could be associated to the different anti-α-Gal responses. However, another possible explanation is that malaria infection affects the immune response to α-Gal in children living in high MTI. Previous studies show that P. falciparum malaria impairs the antibody response to polysaccharide vaccines (glycan antigens) but not responses to protein-based and whole parasite vaccines in children with malaria35. Young children (<3 years old) have immunologically immature spleens, mainly due to the still ongoing development of the marginal zone (MZ) B cell subset, which is the main responsible for the IgM response to polysaccharide antigens37. During malaria infection, the anatomy of the spleen becomes disorganized, with sometimes a complete dissolution of the MZ39. Accordingly, several studies have found a reduction of peripheral MZ-like B cells in patients with malaria42, which could explain the reduction in the IgM response in children with higher malaria exposure43 that may affect IgM response to α-Gal. However, in spite of the higher anti-α-Gal IgM response observed in the lower MTI site, the multivariable analysis showed that recent/current exposure to Plasmodium was positively associated to the levels of anti-α-Gal IgM, implying that malaria infection in fact induces IgM against α-Gal. These models also showed a negative effect of maternally transferred P. falciparum IgGs on the anti-α-Gal IgG and IgM responses in the offspring, suggesting an interference with anti-α-Gal antibody induction in children. A negative effect of maternal antibodies has been reported in the context of immune responses to vaccines44.Second, we analyzed the effect of malaria endemicity on the anti-α-Gal response. When comparing anti-α-Gal IgM and IgG responses between two areas of high (Kintampo) and low (Manhiça) MTI, we observed that both antibodies were higher at lower MTI. This may suggest that other exposures besides malaria may be more important for their induction. It is known that, besides et al., where anti-α-Gal IgMs were associated to protection in Malian children >4 years old17. Disparity may result from several differences between the studies: (i) different samples sizes ; (ii) separate countries with different levels of α-Gal exposure due to malaria and other pathogens; (iii) different follow up times ; (iv) different age ranges of subjects ; and, specially, due to the different ways to detect and define clinical malaria .Third, we investigated the role of α-Gal antibodies in malaria risk or protection. Remarkably, anti-α-Gal IgM levels were higher in infants who did not subsequently develop any episode of malaria. Interestingly, this association was only observed in Manhiça in site-stratified analysis. The fact that this association was only observed in infants but not in children contrasts with the results by Yilmaz et al. of a positive correlation of anti-α-Gal IgM and IgG with the lack of Plasmodium infection in individuals from Senegal45. However disparity of results may also be due to differences between the study site, age of participants and the study design.Unlike IgM, anti-α-Gal IgG levels were associated with a higher risk of malaria in children, which suggests that a higher exposure to other pathogenic microbes containing α-Gal may increase the risk of a future malaria episode by, for example, deviating the immune response and/or causing a worst clinical outcome in co-infection. This result contrasts with the recent observation by Cabezas-Cruz 1–4 subclass responses to α-Gal and observed new associations between certain subclasses and malaria protection. Interestingly, the pattern of IgG1–4 subclasses to α-Gal showed predominance of IgG1 and IgG2, followed by IgG3 and IgG4. This is different to the pattern against P. falciparum proteins, where IgG1 and IgG3 predominate and IgG2 and IgG4 are induced at much lower levels. Higher α-Gal IgG3 and IgG4 levels may correlate with malaria protection in children, also contrasting to what has been observed against protein antigens. Previous studies consistently show that cytophilic antibodies (IgG1 and IgG3) to protein antigens correlate more often with protection from malaria disease50. However, this may be different for glycan antigens. For example, IgG4 responses predominate against Schistosoma mansoni51 with many antigenic glycans on its surface52, and this subclass is associated with protection against S. haematobium53. IgG4 has been shown to be a blocking and tolerance-inducing “anti-allergenic” antibody55. Therefore its protective effect could be mediated through a tolerogenic response to the Plasmodium infection. However, IgG3 and IgG4 associations to malaria protection were lost in multivariable analysis and further studies with larger samples sizes are needed to better address this potential protective effect.We also investigated for the first time IgG42, the documented deficient antibody response to polysaccharide vaccines in children with malaria35 and the higher susceptibility of these children to invasive bacterial infections by polysaccharide encapsulated bacteria 57, warrant further investigation.The study had some limitations, mostly related to the fact that it was performed with samples from the RTS,S clinical trial consisting on two age cohorts, forcing some design issues, e.g., the age range and the vaccination. Second, the unfeasibility to determine the exposure to other sources of α-Gal besides malaria, including other pathogens, commensal bacteria or food, which would have been helpful to understand why anti-α-Gal responses were higher in one site vs the other. Nevertheless, the fact the children from high MTI settings showed significantly lower levels of anti-α-Gal IgG and IgM compared to children living in lower MTI settings, might be a sign of an underlying impairment of the immune response to polysaccharide antigens in the context of high MTI. These data along with the observed reduction of MZ-like B cells in chronically exposed individualsAge and site affect the magnitude of anti-α-Gal IgM and IgG responses in African children. Levels of α-Gal IgG3, IgG4 and, particularly, IgM are associated with protection against clinical malaria, while total IgG levels correlate with malaria risk, supporting further investigations of α-Gal as a promising antigen target for future malaria vaccines.58. This pilot was carried out to set up the α-Gal antibody assay and to confirm the age pattern of response previously described in Malian children17. A second larger study was performed to assess IgG subclasses and association between anti-α-Gal antibodies and malaria protection, and factors affecting it. This analysis included plasma and serum samples from 195 subjects enrolled in the RTS,S/AS01 phase 3 trial59 from two younger age groups (1.5–3 months [infants] and 5–17 months [children]), and two different African sites (Manhiça-Mozambique [low MTI] and Kintampo-Ghana [moderate-high MTI]), having 131 RTS,S-vaccinees and 66 comparator-vaccinees.Samples from African children participating in RTS,S/AS0 clinical trials were included in this analysis. First, a pilot study to assess age patterns of anti-α-Gal antibody immunogenicity in individuals age <5 years old was performed with serum samples from 104 Mozambican children of two cohorts exposed to different levels of MTI , vaccinated with RTS,S/AS02 within a phase 2b trialIn both studies, samples were collected at the first study visit  and the second study timepoint three months later . For the detection of clinical malaria cases (fever >37.5 °C with any parasitaemia), children were followed up by PCD starting 14 days after sample collection at M3 for the subsequent 12 months.P. falciparum protein constructs based on pre-erythrocytic  and erythrocytic  stage antigens analyzed as markers of malaria exposure and maternally-transferred antibodies.Antibodies against α-Gal  were measured by quantitative suspension array technology (qSAT) using the Luminex xMAP™ technology . α-Gal was covalently coupled to MagPlex beads and these were blocked with BSA. α-Gal-coupled beads were added to 96-well μClear® flat bottom plates (Greiner Bio-One)  resuspended in 50 µL of PBS, 1% BSA, 0.05% Azide pH 7.4 (PBS-BN). The multiplex antigens panel also contained beads coupled to 32 P. falciparum for the IgM assay)60, negative control  or PBS-BN (Blanks) were added to the wells and incubated with the beads at 4 °C overnight (ON) in a shaker protected from light. Plates were washed three times with 200 µL/well of PBS-Tween 20 0.05% using a manual magnetic washer. 100 µL of biotinylated secondary antibody were added diluted in PBS-BN as described61: anti-human IgG (Sigma), anti-human IgM (Sigma), anti-human IgG1 (Abcam) and anti-human IgG3 (Sigma). For IgG2 and IgG4 assays, secondary antibodies added were unconjugated mouse anti-human IgG4 (Thermo Fisher) and mouse anti-human IgG2 (Thermo Fisher), respectively, followed by biotinylated goat anti-mouse IgG (Sigma) in PBS-BN. All antibody incubations were performed at room temperature (RT) for 60 min, in agitation and protected from light. Next, 100 µL of streptavidin-R-phycoerythrin (Sigma) in PBS-BN were added to all wells and incubated 30 min, at RT, in agitation and protected from light. Plates were washed as before and beads were resuspended in 100 μL/well of PBS-BN. Plates were covered protected from light and stored at 4 °C ON to be read the next day using the Luminex xMAP® 100/200 analyser, and at least 50 microspheres per analyte were acquired per sample.Fifty µL of sample, positive control .Test samples were assayed at 4 dilutions for IgG , IgG1, IgG3  and IgM , and 2 dilutions for IgG2 and IgG4 (50 and 500) to ensure that at least one dilution lie in the linear range of the respective standard curve. For IgG assays, 18 to 22 serial dilutions (1:2) of the positive control starting at 1:50 were used to perform subclass-specific standard curves. For the IgM assay, 18 serial dilutions (1:2) of a pool of samples from ISGlobal repository with high IgM levels against 10-transformed values of the MFI measurements. The positive control standard curve for each isotype/subclass-plate was estimated using the drLumi R package flow62. Standard curves were fitted in a 5-parameter logistic (5-PL) regression model, and data points were weighted by logarithmic variance. If the model did not converge, 4-PL or exponential regressions were fitted. The quality control for each plate was based on the estimation of the % coefficient of variation (CV) of the 3 blank controls. Blanks were also used to establish the antigen-isotype/subclass specific lower limits of quantification (LLOQ) and lower limits of detection (LLOD) calculated as the blanks mean +10 SD and blanks mean +3 SD, respectively63. The characteristics of the standard curves were visually inspected for quality control purposes. To select the sample working dilution (isotype/subclass and plate specific), an algorithm that detects the two points with the highest slope between them in the positive control sigmoidal curve was used. The slope was computed as:To stabilize the variance, the analysis was done on log10 MFI value of the two points was computed, and the nearest log10 MFI of the test sample and the corresponding dilution was selected. For IgG2 and IgG4 assays standard curves did not converge, then the first sample dilution was assigned. The log10 MFI of the selected dilution was corrected multiplying by its corresponding dilution factor. Blank background signal was not subtracted.The mean logDescriptive comparisons of antibody levels between age groups, time points and sites were done by trajectory plots, boxplots representing the median and interquartile range , and dotplots with bars corresponding to the geometric mean and confidence intervals (CI) . The effect of age was also evaluated through scatterplots and regression models and assessing its interaction with site.P. falciparum exposure index, we selected 28 protein antigens in which IgM responses were M3 > M0 and thus acquired with age (e.g. children > infants) and exposure (e.g. Kintampo > Manhiça) (data not shown). Principal component analysis (PCA) was performed to construct the corresponding variables, and the first component (PC1) that explained 63% of the variability was selected to be used as a variable in the models. To define a P. falciparum maternal antibody index in subjects <10 months of age, we selected 17 antigens including two VAR2CSA pregnancy-specific antigen constructs which IgG responses were M0 > M3 and thus declined with age (e.g. infants > children) and were higher in infants from the high MTI site (e.g. Kintampo > Manhiça) (data not shown). We selected the first component that explained 54% of the variability and used that as a variable in the models.The analysis of factors affecting levels of anti-α-Gal Ig at M3 was performed using data from children participating in the RTS,S phase 3 trial and applying multivariable linear regression models . The predictors assessed were: age as continuous variable (weeks), age cohort (children vs infants), sex , site (Manhiça vs Kintampo), baseline weight for age Z score (WAZ) and height for age Z score (HAZ), baseline hemoglobin levels (Hb), malaria episodes prior to M3 (yes vs no), malaria transmission season (low vs high), vaccination , baseline α-Gal IgG levels, baseline α-Gal IgM levels, level of malaria exposure and maternally-transferred malaria antibodies. To define a The analysis of the association between anti-α-Gal antibody levels and clinical malaria was based on a case-control design. Univariate logistic regression models  with α-Gal antibody data at M3 as main predictor, including other covariates (same as above) and their interactions, were fitted to identify factors that affected malaria risk when α-Gal antibodies were taken into account. Covariates that were significant in the univariate models were included in the stepwise (forward and backward) multivariable models to remove potential cofounding effects. P-values were adjusted for multiple testing through Benjamini-Hochberg or Holm, depending on the analysis. None of the interactions were significant after adjusting for multiple comparisons, therefore they are not reported in the tables. All models were also performed stratifying by age group, by site, and by age and site at the same time. Significance was defined at the p < 0.05 level and analyses were performed with R.All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).All methods were performed in accordance with the relevant guidelines and regulations. Approval for the study protocol was obtained from the Ethical Committee of the Hospital Clínic in Barcelona , the National Health and Bioethics Committee , and the Ghana Health Service Ethical Review Committee . Written informed consent was obtained from parents or guardians of participating children in accordance with the Declaration of Helsinki.Supplementary Information


In 2016, 10.4 million cases of tuberculosis (TB) were reported globally. Malaria also continues to be a global public health threat. Due to marked epidemiological overlap in the global burden of TB and malaria, co-infection does occur.Mycobacterium tuberculosis in CSF was confirmed on Xpert MTB/RIF Ultra. On day 2, he was initiated on dexamethasone at 0.4mg/kg/day and induction TB-medications were re-commenced  for TBM. He continued to spike high-grade fevers, a peripheral blood smear showedP. falciparum parasites despite a negative malaria rapid diagnostic test (RDT). He received three doses of IV artesunate and then completed 3 days of oral artemether/lumefantrine. To our knowledge this is the first published case of HIV-TBM-malaria co-infection. An HIV-infected, 32-year-old male presented with a two-week history of headache with fevers to Mulago National Referral Hospital, Uganda. Five months prior, he was diagnosed with pulmonary TB. He endorsed poor adherence to anti-tuberculous medications. TBM/malaria co-infection poses a number of management challenges. Due to potential overlap in symptoms between TBM and malaria, it is important to remain vigilant for co-infection. Access to accurate parasitological diagnostics is essential, as RDT use continues to expand, it is essential that clinicians are aware of the potential for false negative results. Anti-malarial therapeutic options are limited due to important drug-drug interactions (DDIs). Rifampicin is a potent enzyme inducer of several hepatic cytochrome P450 enzymes, this induction results in reduced plasma concentrations of several anti-malarial medications. Despite recognition of potential DDIs between rifampicin and artemisinin compounds, and rifampicin and quinine, no treatment guidelines currently exist for managing patients with co-infection. There is both an urgent need for the development of new anti-malarial drugs which do not interact with rifampicin and for pharmacokinetic studies to guide dose modification of existing anti-malarial drugs to inform clinical practice guidelines. Tuberculous meningitis (TBM) accounts for 1–5% of these2. Although TBM can occur in immunocompetent persons, the disease disproportionately affects persons living with HIV and children. Malaria also continues to be a global public health threat. In 2016, an estimated 216 million cases occurred, with 90% of those in Africa3. Due to marked epidemiological overlap in the global burden of TB and malaria, co-infection does occur. In an Angolan retrospective study of 1,906 TB inpatients (37% HIV-infected),Plasmodium falciparum co-infection occurred in 38% during hospitalization4. In 2016, 10.4 million cases of tuberculosis (TB) were reported globally5. Peak enzyme induction due to rifampicin is mounted ~ 2 weeks post rifampicin initiation. This induction alters the pharmacokinetics of drugs metabolized by these pathways, reducing plasma concentrations of several anti-malarial medications, including artemisinin-based drugs, quinine and atovaquone/proguanil. In HIV/TBM/malaria co-infection, there are additional interactions between anti-retroviral therapy (ART) and anti-malarial medications6.TBM/malaria co-infection poses a number of management challenges. Rifampicin, the cornerstone for drug-sensitive TB treatment, is a potent enzyme inducer that increases the expression of several hepatic cytochrome P450 (CYP450) enzymes, including CYP2A6, CYP2B6, CYP2C, and CYP3A isoenzymes, as well as the efflux drug transporter P-glycoproteinHere; we present the case of a hospitalized HIV-infected adult with HIV/TBM/malaria co-infection, highlighting important diagnostic and pharmacokinetic challenges.An HIV-infected 32-year-old male presented to Mulago National Referral Hospital, Uganda with a 2-week history of headache with fevers and a 1-day history of confusion . He had ISRCTN42218549). Whilst awaiting further investigations, he received empiric therapy of ceftriaxone 2 g twice daily for possible bacterial meningitis.On examination, the patient was febrile (38.6°C). His blood pressure was 112/71 mmHg, pulse 94 beats/minute, respiratory rate 48, and oxygen saturation 98%. He was wasted, dehydrated, and had overt rigors. His Glasgow Coma Scale was 14/15 with nuchal rigidity and positive Kernig’s sign. Cranial nerves were intact. He had normal tone and power in all limbs. A clinical diagnosis of HIV-associated meningitis was suspected and he was recruited into the ‘Improving Diagnostics and Neurocognitive Outcomes in HIV/AIDS-related Meningitis’ study  for TBM. The IV ceftriaxone was stopped, his ART was continued. He continued to spike high-grade fevers (39.6°C.) with tachycardia (pulse 118 beats/min). A peripheral blood smear showedP. falciparum parasites (1+ trophozoites), despite a negative malaria histidine rich protein-2 (PfHPR2)-based rapid diagnostic test . Given his ongoing neurological symptoms, which could be compatible with cerebral malaria, the decision was made to treat for severe malaria. Drug-drug interactions (DDIs) between rifampicin and artemisinin compounds, and rifampicin and quinine are recognized (6. He received three doses of IV artesunate (3 mg/kg), after which a repeat peripheral blood smear showed no malaria parasites. He then completed 3 days of oral artemether/lumefantrine. His fevers subsided on day 6. He was discharged on day 8; medication adherence counselling was provided for the patient and his guardian and outpatient follow-up was arranged for the following week.A finger stick cryptococcal antigen lateral flow assay (CrAg LFA)  was negative. Liver and renal function tests were normal. Cerebrospinal fluid (CSF) opening pressure was elevated to 33 cm CSF , CSF white cells 590 /µl, protein 419 mg/dl , CSF lactate 9.5 mmol/L . CSF glucose was unavailable.ognized ; a decis4; however, to our knowledge this is the first published case of HIV-TBM-malaria co-infection.This case demonstrates the diagnostic and treatment challenges encountered when managing patients with advanced HIV and intercurrent infections. Protracted high-grade fevers are not an uncommon feature of TBM, even after appropriate anti-tuberculous treatment has been commenced, and it is important to remain vigilant for co-infections. Co-infection with HIV-TB-malaria is well recognized6. RDTs—which detectPlasmodium antigens such as PfHPR2,Plasmodium lactate dehydrogenase (pLDH) or plasmodial aldolase—are the backbone of expanding access to malaria diagnostics in resource-limited settings. Since their introduction in the late 1990s, the number of RDTs available, and the scale of their use, has increased rapidly. A meta-analysis of 74 studies assessing accuracy of PfHRP-2 RDTs for diagnosis of uncomplicatedP. falciparum malaria in endemic settings reported average sensitivity and specificity (95% CI) of 95.0% (93.5-96.2%) and 95.2% (93.4-99.4%), respectively7. However RDTs do have several limitations: poor sensitivity at low parasite densities; susceptibility to the prozone effect (PfHRP2-detecting RDTs); false-negative results due to PfHRP2 gene deletions; false-positive results caused by other infections, and susceptibility to heat and humidity8. The largest problem historically has been poor manufacturing quality, which the World Health Organization (WHO) malaria product testing programme has addressed. However, Vaxpert does not participate in this WHO quality assurance program. As demonstrated by this case, false negatives can occur.Due to the potential overlap in symptoms and signs between TBM and malaria , access to accurate parasitological diagnostics is essential. Light microscopy (Giemsa stain) remains the gold standard parasitological diagnostic, but the World Health Organization (WHO) recommends immunochromatographic rapid diagnostic testing (RDT) in settings with limited laboratory facilities6. It is recommended that a lipid rich meal is eaten prior to taking lumefantrine to optimise absorption. Despite the recognition of potential DDIs between first-line anti-malarial drugs and rifampicin, no treatment guidelines currently exist for managing patients with TB/malaria co-infection. The WHO states that there is currently a lack of evidence to recommend dosage modifications but advises clinicians of increased risk of recrudescent infections due to DDIs in co-infected patients6.The WHO recommends treatment with an artemisinin derivative for both uncomplicated  for 3 days) and complicated falciparum malaria infections 9. Co-administration of artemether-lumefantrine and rifampicin should therefore be avoided. There are no published studies examining concomitant administration of IV artesunate and rifampicin. However, as DHA (the active metabolite of artesunate) is metabolized by CYP450 enzymes  and lumefantrine during rifampicin intake, co-administration with rifampicin resulted in a significantly lower exposure (area under the curve between 0 and 12 hours post-dosing) to artemether (89% lower) and DHA (85% lower)enzymes , there iP<0.001)11. It is recommended that for patients already receiving rifampicin, quinine doses should be increased. However, to date, no guidance on dose-adjustment strategies have been published, and although it is advised that therapeutic drug monitoring may be useful, this is not feasible in most parts of the world where co-infection occurs5.Similarly, the bioavailability of quinine—metabolized almost exclusively via CYP450 (CYP3A4 and CYP2C19) enzymes—is significantly reduced when co-administered with rifampicin and has been associated with a clinically significant reduction in efficacy. Adults treated for uncomplicated falciparum malaria were randomized to receive oral quinine either alone or in combination with rifampin; recrudescence rates were five times higher  in the rifampicin arm than those treated with quinine alone  I would be interested to know about any follow-up post discharge. Was anything done differently, for example support for adherence etc? The conclusion finishes with reiterating the ‘urgent need for the development of new anti-malarial drugs which do not interact with rifampicin and for pharmacokinetic studies to guide dose modification of existing anti-malarial drugs to inform clinical practice guidelines’ – I entirely agree with this statement, but given the challenges and diversity in the population in question which has been described, I would also have liked a couple of sentences in the discussion  about how such studies could best be designed. What would be ideal? Logistically, how could such a population be drawn together to provide meaningful data? Do healthy volunteer studies have a part to play here? Are the authors involved in any existing work to try and address some of these gaps? Are they aware of any other work that is ongoing? Minor points CSF opening pressure should be measured in cm CSF, not cm H20.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This is a well written case report highlighting an important issue for clinicians practicing in areas where malaria, HIV and TB are endemic. Prompted by a case they saw, the authors helpfully summarise the potential DDIs between anti-malarial medications and anti-tuberculous and anti-retroviral medications in common use. This issue deserves publication in its current form with consideration of a few minor issues. I have only minor comments to make. -Was the ceftiaxone stopped as soon as the Xpert was positive? This would be appropriate given the low pre-test probability of bacterial meningitis (2 week history) and the identification of a an alternative definite diagnosis. It might be helpful to remind readers that 'finishing the course' of an antibiotic in this situation is not necessary and can cause harm. -It would be helpful to confirm that the Xpert MTB/RIF Ultra confirmed rifampicin sensitivity as the patient was clearly at risk of DR-TB. -It would be worth pointing out that it takes around 2 weeks for the full effects of enzyme induction by rifampicin to be felt. If malaria is diagnosed at a similar time to TB, as in this case, the initial effects of enzyme induction might be limited, but increase over time which would increase the challenge of finding the appropriate dose. - It is never a bad thing to remind people of the need for a lipid meal before taking lumafantrine in order to achieve adequate absorption, something which is often neglected.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Plasmodium falciparum infection is nontyphoidal salmonella (NTS) infection, in which a typically intestinal infection becomes systemic with serious, often fatal, consequences. In this review, we consider the role of malaria‐induced immunoregulatory responses in tipping the balance from tissue homeostasis during malaria infection to risk of invasive NTS. Also, neutrophils are crucial in the clearance of NTS but their ability to mount an oxidative burst and kill intracellular Salmonella is severely compromised during, and for some time after, an acute malaria infection. Here, we summarize the evidence linking malaria and invasive NTS infections; describe the role of neutrophils in clearing NTS infections; review evidence for neutrophil dysfunction in malaria infections; and explore roles of heme oxygenase‐1, IL‐10, and complement in mediating this dysfunction. Finally, given the epidemiological evidence that low density, subclinical malaria infections pose a risk for invasive NTS infections, we consider whether the high prevalence of such infections might underlie the very high incidence of invasive bacterial disease across much of sub‐Saharan Africa.Invasive bacterial disease is well described in immunocompromised hosts, including those with malaria infection. One bacterial infection frequently observed in children with  Hmox1heme oxygenase‐1 geneHO‐1heme oxygenase‐1NETsneutrophil extracellular trapsNTSSalmonellanontyphoidal PMNpolymorphonuclear cellsROSreactive oxygen speciesSCVSalmonella containing vacuoles1Bloodstream bacterial infections remain a global health concern, with high case fatality rates and the potential for long‐term, life‐changing sequelae. Life‐threatening organ dysfunction resulting from systemic bacterial infection, or more commonly sepsis,Staphylococcus aureus and Escherichia coliS. aureusE. coli are emerging as major causes of nosocomial infections.Salmonella enterica  and Streptococcus pneumoniae as the most commonly isolated organisms.In developed economies, the organisms most frequently isolated from blood include Plasmodium falciparum malaria.Plasmodium, the causative agent of malaria, is transmitted to humans through the bite of the female Anopheles mosquito causing a range of clinical manifestations including anemia, metabolic acidosis, and end‐organ failure.Salmonella bacteremia had concurrent P. falciparum infections.NTS thrives in the intestinal environment where, in otherwise healthy hosts, localized gastroenteritis allows NTS to outcompete the microbiota, causing diarrhea and promoting transmission.2Salmonella can infect a broad host range  causing varying levels of damage, from enteric fever to severe gastroenteritis to asymptomatic carriage, depending on the particular serovar, typically defined by expression of LPS, flagellar, and capsular Vi antigens.Salmonella typhi and Salmonella paratyphi) are associated with systemic infection and carriage in the gallbladderSalmonella containing vacuoles (SCVs) within which they can persist and replicate.Salmonella pathogenicity island‐2 block lysosomal fusion allowing evasion of ROS‐mediated killing.Although phagocytes, such as neutrophils and macrophages, are efficient in their uptake of NTS, the bacteria can disable the antibacterial machinery of macrophages to create 11 new cells emerging from the bone marrow daily. They are typically thought to have a very short lifespan in blood , are terminally differentiated leukocytes with distinctive lobulated nuclei and contain antimicrobial cytoplasmic granules. PMNs are the most abundant white blood cell, with 1 × 10PMNs are professional phagocytes, which use receptor‐mediated phagocytosis to internalize pathogens and debris into phagolysosomes.Salmonella through oxidative stress.Salmonella.Although macrophages can be permissive to NTS growth, inflammatory monocytes and neutrophils are efficient in killing 3E. coli.Clinically, NTS infections in patients in Africa are not associated with overt diarrheal diseaseSalmonella Typhimurium  were 1000–10,000‐fold higher in Plasmodium yoelii‐infected mice than in malaria‐uninfected mice.Whether increased NTS colonization or lymph node dissemination directly increases the risk of systemic spread is not yet clear but major defects in control of systemic NTS are evident in malaria coinfection: bacterial loads in blood, liver, spleen, and bone marrow after intraperitoneal injection of 4Staphylococcus spp., Streptococcus spp., and Clostridium spp.) while other pathogens replicate inside, and ultimately lyse, RBC . In malaria infection, direct destruction of RBC by parasitization is compounded by eryptosis of large numbers of uninfected RBC by processes that are still incompletely understood;hmox1]) is upregulated to prevent heme‐induced pathology and ensure efficient iron recycling.P. falciparum malaria infections in humans, and during acute P. yoelii infection in mice,P. yoelii‐infected mice to control systemic NTS infections.hmox1 induction increases NTS growth in murine macrophages,Anemia is a heterogeneous condition defined as a reduction in circulating hemoglobin, the iron containing metalloprotein abundant in RBC, which ferry oxygen through the blood. One important cause of anemia is direct lysis of RBC (hemolysis) due to autoimmune conditions or infection. Some pathogens secrete hemolysins IL‐10 is a potent anti‐inflammatory cytokine and an important regulator of inflammation‐induced pathology,The anti‐inflammatory properties of IL‐10 include rendering phagocytes refractory to activation and/or directing macrophage polarization to a regulatory “M2" phenotype.6Salmonella infection.2–) following G oxidase assembly on the phagolysosomal membrane.E. coli‐induced oxidative burst in phagocytes is almost entirely dependent on CD88 signaling.Complement proteins play an essential role in orchestrating inflammation and pathogen clearance,Complement activation on the surface of uninfected RBCs presumably increases the rate of turnover of RBCs during malaria infection,7We have provided a rational for considering that hemolysis and inflammation, leading to induction of HO‐1 and IL‐10 and activation of complement, during malaria infections might all contribute to the increased susceptibility to bacterial coinfection. It is likely that these pathways synergize to increase risk of invasive NTS: in malaria‐infected mice, both exogenous IL‐10 and anemia were required for increased bacteremiaThe risk of invasive NTS during malaria in sub‐Saharan Africa is well defined for acute malaria infection. However, the majority of malaria infections in the world are asymptomatic, with chronic, low‐density infections.


L-arginine is an essential amino acid in pregnancy and an immediate precursor in the biosynthesis of nitric oxide (NO), but there are limited data on the impact of MIP on NO biogenesis. We hypothesized that hypoarginemia contributes to the pathophysiology of MIP and that L-arginine supplementation would improve birth outcomes. In a prospective study of pregnant Malawian women, we show that MIP was associated with lower concentrations of L- arginine and higher concentrations of endogenous inhibitors of NO biosynthesis, asymmetric and symmetric dimethylarginine, which were associated with adverse birth outcomes. In a model of experimental MIP, L-arginine supplementation in dams improved birth outcomes (decreased stillbirth and increased birth weight) compared with controls. The mechanism of action was via normalized angiogenic pathways and enhanced placental vascular development, as visualized by placental microcomputerized tomography imaging. These data define a role for dysregulation of NO biosynthetic pathways in the pathogenesis of MIP and support the evaluation of interventions to enhance L-arginine bioavailability as strategies to improve birth outcomes.Reducing adverse birth outcomes due to malaria in pregnancy (MIP) is a global health priority. However, there are few safe and effective interventions.  Plasmodium falciparum malaria  via a family of nitric oxide synthase (NOS) enzymes  has been reported in children and adults with severe malaria  in a preclinical model of MIP by regulating angiogenesis and promoting placental vascular development.There is considerable evidence that reduced bioavailable NO contributes to the pathophysiology of severe malaria  in a randomized clinical trial (n = 91 of 379) of women positive by microscopy and 57% (n = 217 of 381) of women positive by polymerase chain reaction (PCR). Submicroscopic infections were common, with 48.1% (n = 137 of 285) of smear-negative women testing positive by PCR. Smear-positive malaria at enrollment was associated with a significant reduction in birth weight compared to women who tested smear-negative for malaria . Submicroscopic malaria infections were not associated with changes in birth weight (P = 0.73). At delivery, 48% (n = 171 of 356) of women had histological evidence of malaria in the placenta (active or past infection). Placental malaria was associated with adverse birth outcomes, with 60.3% of infants born SGA positive for malaria by histology compared to 44.6% of appropriate-for-gestational age (AGA) infants (P = 0.01). There was no difference in the proportion of women with histologically defined placental malaria according to treatment arm (P = 0.48), and treatment arm was not associated with adverse birth outcomes  or birth weight {mean (SD), intermittent-presumptive treatment in pregnancy [sulfadoxine pyrimethamine (IPTp-SP)], 2773 (461) g versus intermittent screening and treatment in pregnancy [dihydroartemisinin-piperaquine (ISTp-DP)], 2790 (388) g; P = 0.69}.We assessed the L-arginine concentrations and microscopy-defined malaria at enrollment. Women with PCR-defined malaria or submicroscopic malaria had significantly higher concentrations of SDMA (P < 0.001) and ADMA (P < 0.05) and a lower L-arginine/ADMA ratio (P < 0.01) than women with PCR-negative malaria  or middle-upper arm circumference (MUAC)] by regression analysis  women had an adverse birth outcome consisting of PTB or SGA. Adverse birth outcomes (PTB or SGA as a composite outcome) were associated with increased ADMA at enrollment compared to normal birth outcome . Using log-binomial regression, ADMA was associated with an increased relative risk of having an SGA infant  after adjustment for maternal age, gestational age at enrollment, BMI, socioeconomic status, smear-positive malaria at enrollment, and treatment group . We observed an increase in plasma concentrations of ADMA . There was no change in concentrations of L-arginine (L-arginine/ADMA (P > 0.05 for both outcomes).To evaluate the kinetics of the  of ADMA  and SDMA of ADMA  over thearginine  or L-argine/ADMA  during pn = 57 of 93) of these women were positive for malaria by PCR and 23.1% (n = 21 of 91) were positive by microscopy. Of the infections detected, 53.6% (n = 37 of 69) were submicroscopic (PCR-positive and microscopy-negative), and 51.1% (n = 45 of 88) of women had histologically defined placental malaria at delivery (2(1) = 4.38, P < 0.04]; however, there were no differences in concentrations of ADMA [χ 2(P > 0.65] or L-arginine  during pregnancy based on malaria status at enrollment (2(1) = 7.70, P = 0.006] and lower L-arginine   = 8.76, P < 0.02]. The groups converged over time, as demonstrated by the addition of the interaction term between SGA and gestational age . Thus, increased ADMA early in pregnancy (reflecting reduced NO biosynthesis) is associated with SGA, but the effect diminishes over the course of gestation, and those with lower baseline concentrations of ADMA show increases later in pregnancy.Because increased ADMA at enrollment was associated with increased relative risk of SGA, we explored this relationship further, comparing ADMA concentrations over the course of pregnancy . We usedL-arginine and NO biosynthesis in humans, we next explored the mechanism and interventions in an experimental MIP (EMIP) mouse model. EMIP recapitulates several features of human MIP, including placental parasite accumulation, damage to the syncytiotrophoblast, an LBW phenotype  or litter size (table S2). L-arginine supplementation did not alter birth weight or fetal viability in offspring from uninfected, control litters . L-arginine supplementation during EMIP was associated with a 2.5-fold decrease in the relative risk of delivering a nonviable pup .On the basis of the findings of altered pathways of  litters . HoweverL-arginine, ADMA, and SDMA in malaria-infected and uninfected pregnant dams. At G19 (day 6 of infection), concentrations of L-arginine were significantly reduced in malaria-infected dams  or litter size (table S2). L-arginine supplementation did not alter birth weight or fetal viability in offspring from uninfected, control litters  imaging of placentas collected before the onset of the LBW and stillbirth phenotypes  are low in dietary L-arginine  and conditions that enhance inflammation while impairing L-arginine bioavailability and intracellular influx (L-arginine pathway was most evident in PCR-detectable infections at enrollment (16 to 28 weeks of pregnancy) and affected more than half of the women enrolled in this study. These changes occurred relatively early in gestation and could Our findings are supported by previous studies reporting reduced concentrations of ADMA in association with L-arginine supplementation , as well as inflammatory cell adhesion molecules (Icam-1). Increased concentrations of these inflammatory factors and mediators of endothelial dysfunction have previously been linked with adverse birth outcomes in other conditions in pregnancy . These small terminal capillaries are the primary sites of vascular remodeling later in pregnancy , which took place between 2011 and 2013, as previously described . In case the data were not normally distributed, we adjusted our sample size upward by 15% to generate a final minimum sample size of 372 women.L-arginine, ADMA, or SDMA using high-pressure liquid chromatography electrospray tandem mass spectrometry, as described below. The coefficients of variation for arginine testing were 5.2% for L-arginine, 2.0% for SDMA, and 1.4% for ADMA. Concentrations of L-arginine, ADMA, and SDMA were quantified as nanograms per milliliter, and the ratios are expressed as L-arginine/ADMA, L-arginine/SDMA, and ADMA/SDMA  were included in the model. To compare the association between markers of NO biosynthesis and nutritional status , we used linear regression, adjusting for maternal age and gestational age at enrollment. For longitudinal analysis, we used linear mixed-effects modeling with the lme4  in malaria-infected dams, as well as the impact of L-arginine supplementation on placental vascular development. The EMIP model used in this study is a validated murine model of MIP, which replicates key pathogenic factors of human MIP (P. berghei ANKA (PbA)–infected erythrocytes in RPMI 1640 (Gibco) via injection into the lateral tail vein. Control pregnant females were injected on G13 with RPMI 1640 alone. Thin blood smears were taken daily and stained with Giemsa stain  to monitor parasitemia. Investigators were not blinded to the experimental group during treatment because the investigators had to prepare the inoculum and L-arginine–supplemented water. However, investigators were blinded during sample processing and outcome assessment, including tissue collection , processing of samples [placental tissue for reverse transcription PCR (RT-PCR) and serum for mass spectrometry], and assessment of vascular development by micro-CT. All experimental protocols were approved by the University Health Network Animal Care Committee and performed in accordance with current institutional regulations.The objectives of the studies using the EMIP model were to examine the impact of L-arginine in drinking water . Mice received L-arginine–supplemented drinking water (or vehicle control) beginning before pregnancy and a minimum of 13 days before malaria infection (depending on what day they became pregnant after pairing). A dose of 1.2% was selected because it represents about twice the daily intake of L-arginine in regular chow , based on the assumption that mice drink 5 to 6 ml of water per day (60 mg/day intake via supplemented water). There was no difference in the daily intake of water between dams receiving the vehicle control and L-arginine–supplemented water at a dose of 1.2% L-arginine. All mice received treatment via ad libitum access to bottled drinking water throughout pregnancy. All supplementation treatments were given in autoclaved water and water bottles.On the day of pairing, mice were randomly assigned to one of the following treatment groups: (i) vehicle control (regular drinking water) or (ii) 1.2% L-arginine–deficient chow were placed on a diet of exclusively deficient chow (Harlan Laboratories) beginning at G9 (confirmation of pregnancy) until tissue collection. Dams were kept on their regular chow (Harlan Teklad) diet until this time (G9) to minimize disruptions to their environment (change in diet) during pairing and early pregnancy. Mice were assigned to the treatment groups, as defined above.Dams that received The EMIP model followed the protocol outlined above. Dams were sacrificed at G19 using carbon dioxide inhalation, yolk sacs were dissected from uteri, fetuses were removed and weighed, and placentas were snap-frozen and stored at −80°C until analysis. Fetal viability was determined by assessing pedal withdrawal reflex. Nonviable fetuses  were considered stillbirths. All fetuses were weighed at this time. RNA extraction was performed on snap-frozen fetal placenta tissue collected at G19. Serum from mice was collected from cardiac punch and stored at −80°C until analysis.Ct (cycle threshold) compared to the standard curve of mouse genomic DNA included on each plate by Light Cycler 480 software (Roche) and was normalized to the geometric average of the expression of the housekeeping genes Gapdh and Hrpt.Only placentas collected from viable fetuses were used in the transcript analysis. Tissue was homogenized in TRIzol  according to the manufacturer’s protocol, and RNA was extracted. Extracted RNA (2 µg per sample) was then treated with DNase (deoxyribonuclease) I (Ambion) and reverse-transcribed to complementary DNA (cDNA) with SuperScript III (Invitrogen) in the presence of oligo(dT)18 primers (primer sequences in table S5) (Fermentas). Residual RNA was degraded with RNase (ribonuclease) H (Invitrogen). Sample cDNA was amplified in triplicate with SYBR Green Master mix (Roche) in the presence of forward and reverse primers (1 µ M both) in a Light Cycler 480 (Roche). Transcript number was calculated on the basis of L-arginine, ADMA, and SDMA were assayed by mass spectrometry, as previously described (Concentrations of n = 7) and malaria-exposed (n = 8)] offspring of control (nonsupplemented) dams and unexposed (n = 7) and malaria-exposed (n = 7) offspring of L-arginine–supplemented dams. Each group contained a minimum of three dams per group and one to three specimens per litter.Detailed methods for preparing the fetoplacental vasculature for micro-CT imaging have been described previously  , post test (Tukey test), independent samples t test, χ2 test, and relative risk were used to examine the statistical significance of differences between experimental groups. Analysis of the cumulative distribution of vessel diameters for each placenta was fit with a natural spline with eight degrees of freedom. A two-way ANOVA was conducted to determine whether there was an effect of treatment group on the spline parameters. There was a significant interaction between spline coefficient and group (P < 0.001), and therefore, a post hoc analysis was performed to compare pairs of treatment groups. Post tests on all groups were conducted using Dunn’s multiple comparison test (P < 0.05).Statistical analysis was performed using Stata v14 (StataCorp), R v3.2.1 , and GraphPad Prism v6 (GraphPad Software Inc.). Student’s www.sciencetranslationalmedicine.org/cgi/content/full/10/431/eaan6007/DC1Table S1. Linear mixed-effects modeling of longitudinal changes in ADMA and SGA.Table S2. Dams’ peripheral parasitemia at G19 and litter size from all cohorts.Table S3. Fetal and placental weight by treatment group.Table S4. Fetal viability by treatment group.Table S5. RT-PCR primer sequences (5′ to 3′).Click here for additional data file.


Despite the importance of epidemiological studies in the development of effective control strategies and provision of basic health services for refugees and internally displaced persons (IDPs), data on the prevalence of malaria are limited. Thus, this study was conducted to estimate the molecular prevalence of malaria amongst the displaced population in Ardamata IDP camp in Al-Geneina City, Sudan.Plasmodium genus and species.A cross-sectional study was conducted from July 2018 to December 2018 to estimate malaria prevalence amongst the displaced population in Ardamata IDP camp in Al-Geneina City, Sudan. A total of 380 patients with suspected malaria were recruited. Nested polymerase chain reaction (nPCR) assays were performed to detect the Plasmodium falciparum was the only prevalent species detected amongst the study population. nPCR analysis revealed that none of the samples had Plasmodium vivax, Plasmodium ovale or Plasmodium malariae. The malaria prevalence rate was higher amongst males (67.1%) than in females (56.8%), and gender was the only risk factor that was significantly associated with malaria infection (p = .042).Of 380 patients, 232 (61.1%) were positive for malaria. Despite control programmes, malaria remains a significant cause of illness amongst a displaced population. The high prevalence of malaria infection in this study indicates that additional health facilities and control strategies should be implemented in displaced camps and the surrounding areas. Plasmodium species have been recognised as the causative agents of malaria that can infect humans: Plasmodium falciparum , Plasmodium vivax (P. vivax), Plasmodium ovale , Plasmodium malariae  and Plasmodium knowlesi (P. knowlesi). Of these five species, P. falciparum and P. vivax are the most common in Sudan.Malaria is a fatal vector-borne tropical disease that remains one of the leading causes of death in many developing countries . The disPlasmodium infections in patients with high levels of parasitaemia [Plasmodium infections amongst children, pregnant women and non-pregnant adults [Plasmodium species, and various sensitivities and specificities have been reported [Plasmodium species will allow better documentation of malaria epidemiology [Blood film microscopy and rapid diagnostic tests are the mainstay of malaria diagnosis that can adequately detect sitaemia , 4. Howesitaemia , 6. Givet adults . Thus, hreported . Amongstreported . PCR hasreported , 9. The emiology  and overThis study was carried out in Ardamata IDP camp established in Al-Geneina City, which is the capital city of West Darfur State, the western part of Sudan. It is located in the latitude of 13° 27′ 15′′ and longitude of 22° 26′ 8′′. Al-Geneina is approximately 1200 km from the capital city of Khartoum.This study was a cross-sectional study that recruited patients presenting clinical symptoms of malaria and visiting the health centre in the study site. A total of 380 patients were recruited for this study between July and December 2018.The sample size was estimated using formula for single proportion to estimate the prevalence of malaria.z = 1.96 (for 0.05 level of significance), margin of error (m) = 0.05, p = 0.575 and 0.33 for P. falciparum and P. vivax respectively [P. falciparum and P. vivax respectively. Hence, the largest sample size based on P. falciparum (376) was used. After adding 5% dropout rate, the adjusted samples size was estimated as 396.The parameters used were ectively . The calAbout 3–5 drops of blood from each enrolled participant were collected on Whatman No. 1 filter paper. The blood samples were allowed to dry, kept in individual plastic bags with desiccant and stored at room temperature. An informed consent questionnaire was used to collect individual socio-demographic data.DNA was extracted from three 3 mm punches of dried blood spot (DBS) following the protocol of Bereczky et al. . DNA wasPlasmodium genus determination. The PCR mixture was prepared in a total volume of 20 μl, containing 10 μl of MyTaq™ mix , 0.4 μM of each primer and 2.5 μl of extracted DNA. PCR was performed under the following conditions: 94 °C for 5 min as the initial denaturation step; 25 cycles at 94 °C for 45 s, 58 °C for 45 s and 72 °C for 1 min; and a final extension step at 72 °C for 5 min. The amplified PCR product (1 μl) was used as a template for the second PCR round for Plasmodium species identification using  primers [Plasmodium positive samples and a negative control sample without DNA template was used in all the reactions as positive and negative control respectively.Nested PCR (nPCR) was performed as described previously  in a two primers . The reaPreliminary data analysis was conducted for data exploration and cleaning to check for missing values and erroneous data entry. The statistical analysis applied in the current study was descriptive analysis and logistic regression analysis. Descriptive analysis focused on frequency, percentages, mean and standard deviation. Logistic regression analysis was conducted to identify significant factors associated with the outcome of malaria infection. All statistical analyses were conducted using SPSS 24.n = 365) of the participants were residents of Ardamata IDP camp. Amongst the study participants, 41.6% (n = 158) were males and 58.4% (n = 222) were females. Their ages ranged from 1 to 80 years, and the mean age was 21.7 years (SD = 14.1). The majority of the participants  belonged to the < 20 age group. More than half of the participants were single . Majority of them  reported receiving primary school education, whereas 34.7% (n = 132) were illiterate. The monthly income was < 20 USD for 14.5% (n = 55), 20–25 USD for 46.6% (n = 177), 25–30 USD for 20% (n = 76) and more than 30 USD for 18.9% (n = 72). Approximately 97.9% (n = 372) of the patients with suspected malaria had mosquito nets.Table P. falciparum was the only prevalent species found amongst the study population  than in females (56.8%). Gender had a statistically significant association with malaria infection , indicating that the males were 1.5 times more likely to have malaria infection than the females. None of the remaining factors demonstrated any significant association with malaria infection .Molecular analysis showed that approximately 61.1% (232 out of 380) of analysed samples were positive for malaria  reported that 50.2% was P. falciparum, 43.8% was P. vivax, 04.9% was P. ovale and 1.1% of the cases was P. malariae; no mixed infection was observed [P. vivax malaria and making P. vivax the second important species in the country [P. vivax malaria is high amongst suspected malaria cases, with a prevalence of 26.6% [In endemic areas, determination of the epidemiological pattern of the malaria infection is crucial for intervention programmes and treatment purposes. Accordingly, all the main malaria species that infect humans have been previously reported in the country , with a reported . The resresearch . None ofobserved . Recentl country . Anothers, Ageep 13 reportof 26.6% .p = .042), and none of the remaining factors had any significant influence . No associations were found in the present study between malaria infection and the use of insecticide-treated bed net (ITNs). Similarly, education status and marital status did not show any significant association with malaria infection.In malaria-endemic areas, protective immunity is always correlated with age. A low prevalence of malaria and low incidence of clinical symptoms are frequently observed amongst adults and older children. This concept is in line with the observations of this study, which showed that the odds of being positive for malaria decreased by 11% amongst those who are ≥ 21 years compared to the ≤ 20 years (These findings were in contrast to other work, which showed that the use of ITNs and many socio-demographic factors are significantly associated with malaria. Recruiting only patients with suspected clinically symptomatic malaria possibly affected the results of this study. Further comprehensive surveys are required to identify the factors associated with malaria infection that were not addressed in this study.In conclusion, results of this study indicated a high prevalence of malaria amongst the displaced participants. This study further emphasises the necessity to strengthen malaria control strategies and establish additional health facilities.