Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Dihydrobiopterin in Adult Falciparum Malaria: Association with Disease Severity,Impaired Microvascular Function and Increased Endothelial Activation

Tetrahydrobiopterin (BH₄) is a co-factor required for catalytic activity of nitric oxide synthase (NOS) and amino acid-monooxygenases, including phenylalanine hydroxylase. BH₄ is unstable: during oxidative stress it is non-enzymatically oxidized to dihydrobiopterin (BH₂), which inhibits NOS. Depending on BH₄ availability, NOS oscillates between NO synthase and NADPH oxidase: as the BH₄/BH₂ ratio decreases, NO production falls and is replaced by superoxide. In African children and Asian adults with severe malaria, NO bioavailability decreases and plasma phenylalanine increases, together suggesting possible BH₄ deficiency. The primary three biopterin metabolites (BH₄, BH₂ and B₀ [biopterin]) and their association with disease severity have not been assessed in falciparum malaria. We measured pterin metabolites in urine of adults with severe falciparum malaria (SM; n=12), moderately-severe malaria (MSM, n=17), severe sepsis (SS; n=5) and healthy subjects (HC; n=20) as controls. In SM, urinary BH₄ was decreased (median 0.16 ¼mol/mmol creatinine) compared to MSM (median 0.27), SS (median 0.54), and HC (median 0.34)]; p<0.001. Conversely, BH₂ was increased in SM (median 0.91 ¼mol/mmol creatinine), compared to MSM (median 0.67), SS (median 0.39), and HC (median 0.52); p<0.001, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4 in severe malaria. Overall, the median BH₄/BH₂ ratio was lowest in SM [0.18 (IQR: 0.04-0.32)] compared to MSM (0.45, IQR 0.27-61), SS (1.03; IQR 0.54-2.38) and controls (0.66; IQR 0.43-1.07); p<0.001. In malaria, a lower BH₄/BH₂ ratio correlated with decreased microvascular reactivity (r=0.41; p=0.03) and increased ICAM-1 (r=-0.52; p=0.005). Decreased BH4 and increased BH₂ in severe malaria (but not in severe sepsis) uncouples NOS, leading to impaired NO bioavailability and potentially increased oxidative stress. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.     

Modeling of biopterin-dependent pathways of eNOS for nitric oxide and superoxide production

Endothelial dysfunction is associated with increase in oxidative stress and low NO bioavailability. The endothelial NO synthase (eNOS) uncoupling is considered an important factor in endothelial cell oxidative stress. Under increased oxidative stress, the eNOS cofactor tetrahydrobiopterin (BH₄) is oxidized to dihydrobiopterin, which competes with BH₄ for binding to eNOS, resulting in eNOS uncoupling and reduction in NO production. The importance of the ratio of BH₄ to oxidized biopterins versus absolute levels of total biopterin in determining the extent of eNOS uncoupling remains to be determined. We have developed a computational model to simulate the kinetics of the biochemical pathways of eNOS for both NO and O₂^•− production to understand the roles of BH₄ availability and total biopterin (TBP) concentration in eNOS uncoupling. The downstream reactions of NO, O₂^•−, ONOO⁻, O₂, CO₂, and BH₄ were also modeled. The model predicted that a lower [BH₄]/[TBP] ratio decreased NO production but increased O₂^•− production from eNOS. The NO and O₂^•− production rates were independent above 1.5 μM [TBP]. The results indicate that eNOS uncoupling is a result of a decrease in [BH₄]/[TBP] ratio, and a supplementation of BH₄ might be effective only when the [BH₄]/[TBP] ratio increases. The results from this study will help us understand the mechanism of endothelial dysfunction.     

An enzymatic method to distinguish tetrahydrobiopterin from oxidized biopterins using UDP-glucose:tetrahydrobiopterin glucosyltransferase

The quantitative determination of tetrahydrobiopterin (BH4) and its oxidized forms (dihydrobiopterin and biopterin) is important in searching for possible markers of neuropsychiatric and cardiovascular disorders as well as in diagnosing BH4 deficiencies. Currently, two high-performance liquid chromatography (HPLC) methods are available, although both have some limitations. We developed an enzymatic method to distinguish BH4 from the oxidized forms by employing BH4:UDP-glucose α-glucosyltransferase (BGluT), which catalyzes glucosyl transfer from UDP-glucose to BH4. The recombinant BGluT isolated from Escherichia coli converted essentially all of the BH4 in a mixture containing oxidized biopterins to the glucoside while leaving the oxidized forms intact. Therefore, acidic iodine oxidation of the reaction mixture followed by single fluorescence HPLC permitted the determination of biopterin and biopterin-glucoside, which represent oxidized biopterins and BH4, respectively. The validity of the method was evaluated using authentic biopterins and animal samples such as human urine, rat plasma, and rat liver. The BGluT-catalyzed reaction not only would reduce the burden of chromatographic separation but also would promise non-HPLC analysis of BH4.     

Increased Asymmetric Dimethylarginine in Severe Falciparum Malaria: Association with Impaired Nitric Oxide Bioavailability and Fatal Outcome

Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is a predictor of mortality in critical illness. Severe malaria (SM) is associated with decreased NO bioavailability, but the contribution of ADMA to the pathogenesis of impaired NO bioavailability and adverse outcomes in malaria is unknown. In adults with and without falciparum malaria, we tested the hypotheses that plasma ADMA would be: 1) increased in proportion to disease severity, 2) associated with impaired vascular and pulmonary NO bioavailability and 3) independently associated with increased mortality. We assessed plasma dimethylarginines, exhaled NO concentrations and endothelial function in 49 patients with SM, 78 with moderately severe malaria (MSM) and 19 healthy controls (HC). Repeat ADMA and endothelial function measurements were performed in patients with SM. Multivariable regression was used to assess the effect of ADMA on mortality and NO bioavailability. Plasma ADMA was increased in SM patients (0.85 µM; 95% CI 0.74–0.96) compared to those with MSM (0.54 µM; 95%CI 0.5–0.56) and HCs (0.64 µM; 95%CI 0.58–0.70; p<0.001). ADMA was an independent predictor of mortality in SM patients with each micromolar elevation increasing the odds of death 18 fold (95% CI 2.0–181; p = 0.01). ADMA was independently associated with decreased exhaled NO (r_s = −0.31) and endothelial function (r_s = −0.32) in all malaria patients, and with reduced exhaled NO (r_s = −0.72) in those with SM. ADMA is increased in SM and associated with decreased vascular and pulmonary NO bioavailability. Inhibition of NOS by ADMA may contribute to increased mortality in severe malaria.     

Biopterin

Intraperitoneally injected [¹⁴C]biopterin (B), dihydrobiopterin (BH₂), and tetrahydrobiopterin (BH₄) penetrated the brain rapidly, but in amounts sufficient to represent only a minor source of supply. Unlike isobiopterin, B was rapidly reduced to BH₂ in brain. The distribution of BH₄ and BH₂, but not of B, could be correlated with the tryptophan and tyrosine hydroxylase activity of various cerebral areas. In the whole brain, the sizes of pools were 0.117 for B, 0.204 for BH₂, and 0.341 g/g for BH₄, while the cerebral turnover rates of B, BH₂, and BH₄ were 0.25, 0.43, and 0.71 nmol/g per h, respectively. From birth through development, the cerebral levels of B remained constant, whereas the levels of the reduced biopterins increased. After subcellular fractionation, 65% of the biopterins (B, BH₂, and BH₄) were recovered from the supernatant. Of the organelles, microsomes contained the largest concentration of pterins. About 1/6 of all pterins in the brain was present in the synaptosomes.     

Clinical Factors for Severity of Plasmodium falciparum Malaria in Hospitalized Adults in Thailand

Plasmodium falciparum is a major cause of severe malaria in Southeast Asia, however, there is limited information regarding clinical factors associated with the severity of falciparum malaria from this region. We performed a retrospective case-control study to compare clinical factors and outcomes between patients with severe and non-severe malaria, and to identify clinical factors associated with the requirement for intensive care unit (ICU) admission of patients with severe falciparum malaria among hospitalized adults in Southeast Asia. A total of 255 patients with falciparum malaria in the Hospital for Tropical Diseases in Bangkok, Thailand between 2006 and 2012 were included. We identified 104 patients with severe malaria (cases) and 151 patients with non-severe malaria (controls). Patients with falciparum malaria with following clinical and laboratory characteristics on admission (1) referrals, (2) no prior history of malaria, (3) body temperature of >38.5°C, (4) white blood cell counts >10×10⁹/µL, (5) presence of schizonts in peripheral blood smears, and (6) albumin concentrations of <3.5 g/dL, were more likely to develop severe malaria (P<0.05). Among patients with severe malaria, patients who met ≥3 of the 2010 WHO criteria had sensitivity of 79.2% and specificity of 81.8% for requiring ICU admission. Multivariate analysis identified the following as independent associated factors for severe malaria requiring ICU admission; (1) ethnicity of Thai [odds ratio (OR) = 3.601, 95% confidence interval (CI) = 1.011–12.822] or Myanmar [OR = 3.610, 95% CI = 1.138–11.445]; (2) referrals [OR = 3.571, 95% CI = 1.306–9.762]; (3) no prior history of malaria [OR = 5.887, 95% CI = 1.354–25.594]; and (4) albumin concentrations of <3.5 g/dL [OR = 7.200, 95% CI = 1.802–28.759]. Our findings are important for the clinical management of patients with malaria because it can help early identification of patients that could develop severe malaria and require ICU admission. Early identification and the timely initiation of appropriate treatments may well improve the outcomes and reduce the mortality of these patients.     

Evidence against tetrahydrobiopterin depletion of vascular tissue exposed to nitric oxide/superoxide or nitroglycerin

Several cardiovascular disorders, including atherosclerosis and tolerance to the antianginal drug nitroglycerin (GTN), may be associated with the generation of superoxide anions, which react with nitric oxide (NO) to yield peroxynitrite. According to a widely held view, oxidation of tetrahydrobiopterin (BH₄) by peroxynitrite causes uncoupling of endothelial NO synthase (eNOS), resulting in reduced NO bioavailability and endothelial dysfunction under conditions of oxidative stress. In this study we determined the levels of reduced biopterins and endothelial function in cultured cells exposed to peroxynitrite and GTN as well as in blood vessels isolated from GTN-tolerant guinea pigs and rats. BH₄ was rapidly oxidized by peroxynitrite and 3-morpholino sydnonimine (SIN-1) in buffer, but this was prevented by glutathione and not observed in endothelial cells exposed to SIN-1 or GTN. Prolonged treatment of the cells with 0.1 mM GTN caused slow N^G-nitro-l-arginine-sensitive formation of reactive oxygen species without affecting eNOS activity. Endothelial function and BH₄/BH₂ levels were identical in blood vessels of control and GTN-tolerant animals. Our results suggest that peroxynitrite-triggered BH₄ oxidation does not occur in endothelial cells or GTN-exposed blood vessels. GTN seems to trigger minor eNOS uncoupling that is unrelated to BH₄ depletion and without observable consequence on eNOS function.     

Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A₂ mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44^mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH₄) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O₂ ^•–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O₂ ^•– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH₄ level. Insulin and tempol blocked the high D-glucose–increased p42/44^mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O₂ ^•–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.     

Plasma Plasmodium falciparum histidine-rich protein-2 concentrations are associated with malaria severity and mortality in Tanzanian children

Plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) concentrations, a measure of parasite biomass, have been correlated with malaria severity in adults, but not yet in children. We measured plasma PfHRP-2 in Tanzanian children with uncomplicated (n = 61) and cerebral malaria (n = 45; 7 deaths). Median plasma PfHRP-2 concentrations were higher in cerebral malaria (1008 [IQR 342-2572] ng/mL) than in uncomplicated malaria (465 [IQR 36-1426] ng/mL; p = 0.017). In cerebral malaria, natural log plasma PfHRP-2 was associated with coma depth (r = -0.42; p = 0.006) and mortality (OR: 3.0 [95% CI 1.03-8.76]; p = 0.04). In this relatively small cohort study in a mesoendemic transmission area of Africa, plasma PfHRP-2 was associated with pediatric malaria severity and mortality. Further studies among children in areas of Africa with higher malaria transmission and among children with different clinical manifestations of severe malaria will help determine the wider utility of quantitative PfHRP-2 as a measure of parasite biomass and prognosis in sub-Saharan Africa.     

Contraceptive Use and the Risk of Ectopic Pregnancy: A Multi-Center Case-Control Study

Objective

To evaluate the association between the risk of ectopic pregnancy (EP) and the use of common contraceptives during the previous and current conception/menstrual cycle.

Methods

A multi-center case-control study was conducted in Shanghai. Women diagnosed with EP were recruited as the case group (n = 2,411). Women with intrauterine pregnancy (IUP) (n = 2,416) and non-pregnant women (n = 2,419) were matched as controls at a ratio of 1∶1. Information regarding the previous and current use of contraceptives was collected. Multivariate logistic regression analyses were performed to calculate odds ratios (ORs) and the corresponding 95% confidential intervals (CIs).

Results

Previous use of intrauterine devices (IUDs) was associated with a slight risk of ectopic pregnancy (AOR₁ = 1.87 [95% CI: 1.48–2.37]; AOR₂ = 1.84 [1.49–2.27]), and the risk increased with the duration of previous use (P₁ for trend <10⁻⁴, P₂ for trend <10⁻⁴). The current use of most contraceptives reduced the risk of both unwanted IUP (condom: AOR = 0.04 [0.03–0.05]; withdrawal method: AOR = 0.10 [0.07–0.13]; calendar rhythm method: AOR = 0.54 [0.40–0.73]; oral contraceptive pills [OCPs]: AOR = 0.03 [0.02–0.08]; levonorgestrel emergency contraception [LNG-EC]: AOR = 0.22 [0.16–0.30]; IUDs: AOR = 0.01 [0.005–0.012]; tubal sterilization: AOR = 0.01 [0.001–0.022]) and unwanted EP (condom: AOR₁ = 0.05 [0.04–0.06]; withdrawal method: AOR₁ = 0.13 [0.09–0.19]; calendar rhythm method: AOR₁ = 0.66 [0.48–0.91]; OCPs: AOR₁ = 0.14 [0.07–0.26]; IUDs: AOR₁ = 0.17 [0.13–0.22]; tubal sterilization: AOR₁ = 0.04 [0.02–0.08]). However, when contraception failed and pregnancy occurred, current use of OCPs (AOR₂ = 4.06 [1.64–10.07]), LNG-EC (AOR₂ = 4.87 [3.88–6.10]), IUDs (AOR₂ = 21.08 [13.44–33.07]), and tubal sterilization (AOR₂ = 7.68 [1.69–34.80]) increased the risk of EP compared with the non-use of contraceptives.

Conclusion

Current use of most contraceptives reduce the risk of both IUP and EP. However, if the contraceptive method fails, the proportions of EP may be higher than those of non-users. In the case of contraceptive failure in the current cycle, EP cases should be differentiated according to current use of OCPs, LNG-EC, IUDs, and tubal sterilization. In addition, attention should be paid to women with previous long-term use of IUDs.     

Analysis of Plasma Biopterin Levels in Psychiatric Disorders Suggests a Common BH<Subscript>4</Subscript> Deficit in Schizophrenia and Schizoaffective Disorder

Tetrahydrobiopterin (BH₄) is an essential cofactor for amine neurotransmitter synthesis. BH₄ also stimulates and modulates the glutamatergic system, and regulates the synthesis of nitric oxide by nitric oxide synthases. A connection between BH₄ deficiencies and psychiatric disorders has been previously reported; major depression and obsessive-compulsive disorder have been found in subjects with a BH₄ deficiency disorder and more recently we have observed a robust plasma deficit of biopterin (a measure of BH₄), in a large group of schizophrenic patients compared to control subjects. To extend our previous finding in schizophrenia, we analyzed plasma biopterin levels from patients with schizoaffective and bipolar disorders. A significant difference in biopterin was seen among the diagnostic groups (P < 0.0001). Post hoc analyses indicated significant biopterin deficits relative to the normal control group for the schizoaffective group, who had biopterin levels comparable to the schizophrenic group. Bipolar disorder subjects had plasma biopterin levels that were higher that the schizoaffective disorder group and significantly higher than the schizophrenic group. The demonstrated significant biopterin deficit in both schizophrenia and schizoaffective disorder, may suggest an etiological role of a BH₄ deficit in these two disorders, via dysregulation of neurotransmitter systems.     

Plasma Uric Acid Levels Correlate with Inflammation and Disease Severity in Malian Children with Plasmodium falciparum Malaria

Background

Plasmodium falciparum elicits host inflammatory responses that cause the symptoms and severe manifestations of malaria. One proposed mechanism involves formation of immunostimulatory uric acid (UA) precipitates, which are released from sequestered schizonts into microvessels. Another involves hypoxanthine and xanthine, which accumulate in parasitized red blood cells (RBCs) and may be converted by plasma xanthine oxidase to UA at schizont rupture. These two forms of ‘parasite-derived’ UA stimulate immune cells to produce inflammatory cytokines in vitro.

Methods and Findings

We measured plasma levels of soluble UA and inflammatory cytokines and chemokines (IL-6, IL-10, sTNFRII, MCP-1, IL-8, TNFα, IP-10, IFNγ, GM-CSF, IL-1β) in 470 Malian children presenting with uncomplicated malaria (UM), non-cerebral severe malaria (NCSM) or cerebral malaria (CM). UA levels were elevated in children with NCSM (median 5.74 mg/dl, 1.21-fold increase, 95% CI 1.09–1.35, n = 23, p = 0.0007) and CM (median 5.69 mg/dl, 1.19-fold increase, 95% CI 0.97–1.41, n = 9, p = 0.0890) compared to those with UM (median 4.60 mg/dl, n = 438). In children with UM, parasite density and plasma creatinine levels correlated with UA levels. These UA levels correlated with the levels of seven cytokines [IL-6 (r = 0.259, p<0.00001), IL-10 (r = 0.242, p<0.00001), sTNFRII (r = 0.221, p<0.00001), MCP-1 (r = 0.220, p<0.00001), IL-8 (r = 0.147, p = 0.002), TNFα (r = 0.132, p = 0.006) and IP-10 (r = 0.120, p = 0.012)]. In 39 children, UA levels were 1.49-fold (95% CI 1.34–1.65; p<0.0001) higher during their malaria episode [geometric mean titer (GMT) 4.67 mg/dl] than when they were previously healthy and aparasitemic (GMT 3.14 mg/dl).

Conclusions

Elevated UA levels may contribute to the pathogenesis of P. falciparum malaria by activating immune cells to produce inflammatory cytokines. While this study cannot identify the cause of elevated UA levels, their association with parasite density and creatinine levels suggest that parasite-derived UA and renal function may be involved. Defining pathogenic roles for parasite-derived UA precipitates, which we have not directly studied here, requires further investigation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00669084","term_id":"NCT00669084"}}NCT00669084     

Plasmodium vivax Malaria in Pregnant Women in the Brazilian Amazon and the Risk Factors Associated with Prematurity and Low Birth Weight: A Descriptive Study

Introduction

Plasmodium vivax is the most prevalent malaria species in the American region. Brazil accounts for the higher number of the malaria cases reported in pregnant women in the Americas. This study aims to describe the characteristics of pregnant women with malaria in an endemic area of the Brazilian Amazon and the risk factors associated with prematurity and low birth weight (LBW).

Methods/Principal Findings

Between December 2005 and March 2008, 503 pregnant women with malaria that attended a tertiary health centre were enrolled and followed up until delivery and reported a total of 1016 malaria episodes. More than half of study women (54%) were between 20–29 years old, and almost a third were adolescents. The prevalence of anaemia at enrolment was 59%. Most women (286/503) reported more than one malaria episode and most malaria episodes (84.5%, 846/1001) were due to P. vivax infection. Among women with only P. vivax malaria, the risk of preterm birth and low birth weight decreased in multigravidae (OR, 0.36 [95% CI, 0.16–0.82]; p = 0.015 and OR 0.24 [95% CI, 0.10–0.58]; p = 0.001, respectively). The risk of preterm birth decreased with higher maternal age (OR 0.43 [95% CI, 0.19–0.95]; p = 0.037) and among those women who reported higher antenatal care (ANC) attendance (OR, 0.32 [95% CI, 0.15–0.70]; p = 0.005).

Conclusion

This study shows that P. vivax is the prevailing species among pregnant women with malaria in the region and shows that vivax clinical malaria may represent harmful consequences for the health of the mother and their offsprings particularly on specific groups such as adolescents, primigravidae and those women with lower ANC attendance.     

Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages

Generation of nitric oxide (NO.), an autacoid with vasorelaxant and cytotoxic properties, requires at least three cytosolic components in mouse macrophages besides L-arginine and NADPH. One or more components appear after induction by immunologic stimuli; two or more are present in both activated and non-activated macrophages. The constitutive factors can be separated on a Mr approximately 30,000 cut-off filter into high Mr fraction (HF) and low Mr fraction (LF) (Stuehr, D. J., Kwon, N. S., Gross, S. S., Thiel, B. A., Levi, R., and Nathan, C. F. (1989) Biochem. Biophys. Res. Commun. 161, 420-426). Herein we characterize the major active component in LF. The active component was dialyzable (Mr less than approximately 1,000), water soluble, and cationic at acidic to neutral pH. Fractionation on a C18 column in an acetonitrile/water gradient yielded one broad peak of activity, most of which corresponded to a fluorophore with the excitation/emission spectra of biopterins. Gas chromatography isolated a species in this peak with the mass spectrum of biopterin. Of 14 pteridines tested, only 7,8-dihydrobiopterin (H2biopterin) or 5,6,7,8-tetrahydrobiopterin (H4biopterin) could replace LF in synergizing with HF and the inducible component(s) to generate NO-2 and NO-3, the accumulating oxidation products of NO.. Half-maximal activity required 20-30 nM reduced biopterins. LFs from three cell lines were active in proportion to their content of biopterins; addition of reduced biopterins restored activity to LF from biopterin-deficient cells. Enhancement of NO-2 generation in the presence of H2biopterin but not H4biopterin was abolished by methotrexate and aminopterin, inhibitors of dihydrofolate reductase. These findings implicate a redox cycle in which the generation of NO. is facilitated by catalytic amounts of H4biopterin.     

Helminthic Infections Rates and Malaria in HIV-Infected Pregnant Women on Anti-Retroviral Therapy in Rwanda

Background

Within sub-Saharan Africa, helminth and malaria infections cause considerable morbidity in HIV-positive pregnant women and their offspring. Helminth infections are also associated with a higher risk of mother-to-child HIV transmission. The aim of this study was to determine the prevalence of, and the protective and risk factors for helminth and malaria infections in pregnant HIV-positive Rwandan women receiving anti-retroviral therapy (ART).

Methodology and principle findings

Pregnant females (n = 980) were recruited from health centres in rural and peri-urban locations in the central and eastern provinces of Rwanda. Helminth infection was diagnosed using the Kato Katz method whilst the presence of Plasmodium falciparum was identified from blood smears. The prevalence of helminth infections was 34.3%; of malaria 13.3%, and of co-infections 6.6%. Helminth infections were more common in rural (43.1%) than peri-urban (18.0%; p<0.0005) sites. A CD4 count ≤350 cells/mm³ was associated with a higher risk of helminth infections (odds ratio, 3.39; 95% CIs, 2.16–5.33; p<0.0005) and malaria (3.37 [2.11–5.38]; p<0.0005) whilst helminth infection was a risk factor for malaria infection and vice versa. Education and employment reduced the risk of all types of infection whilst hand washing protected against helminth infection (0.29 [0.19–0.46]; p<0.0005);). The TDF-3TC-NVP (3.47 [2.21–5.45]; p<0.0005), D4T-3TC-NVP (2.47 [1.27–4.80]; p<0.05) and AZT-NVP (2.60 [1.33–5.08]; p<0.05) regimens each yielded higher helminth infection rates than the AZT-3TC-NVP regimen. Anti-retroviral therapy had no effect on the risk of malaria.

Conclusion/significance

HIV-positive pregnant women would benefit from the scaling up of de-worming programs alongside health education and hygiene interventions. The differential effect of certain ART combinations (as observed here most strongly with AZT-3TC-NVP) possibly protecting against helminth infection warrants further investigation.     

Neurocognitive outcomes in Malawian children exposed to malaria during pregnancy: An observational birth cohort study

BackgroundAnnually 125 million pregnancies are at risk of malaria infection. However, the impact of exposure to malaria in pregnancy on neurodevelopment in children is not well understood. We hypothesized that malaria in pregnancy and associated maternal immune activation result in neurodevelopmental delay in exposed offspring.Methods and findingsBetween April 2014 and April 2015, we followed 421 Malawian mother–baby dyads (median [IQR] maternal age: 21 [19, 28] years) who were previously enrolled (median [IQR] gestational age at enrollment: 19.7 [17.9, 22.1] weeks) in a randomized controlled malaria prevention trial with 5 or 6 scheduled assessments of antenatal malaria infection by PCR. Children were evaluated at 12, 18, and/or 24 months of age with cognitive tests previously validated in Malawi: the Malawi Developmental Assessment Tool (MDAT) and the MacArthur–Bates Communicative Development Inventories (MCAB-CDI). We assessed the impact of antenatal malaria (n [%] positive: 240 [57.3]), placental malaria (n [%] positive: 112 [29.6]), and maternal immune activation on neurocognitive development in children. Linear mixed-effects analysis showed that children exposed to antenatal malaria between 33 and 37 weeks gestation had delayed language development across the 2-year follow-up, as measured by MCAB-CDI (adjusted beta estimate [95% CI], −7.53 [−13.04, −2.02], p = 0.008). Maternal immune activation, characterized by increased maternal sTNFRII concentration, between 33 and 37 weeks was associated with lower MCAB-CDI language score (adjusted beta estimate [95% CI], −8.57 [−13.09, −4.06], p < 0.001). Main limitations of this study include a relatively short length of follow-up and a potential for residual confounding that is characteristic of observational studies.ConclusionsThis mother–baby cohort presents evidence of a relationship between malaria in pregnancy and neurodevelopmental delay in offspring. Malaria in pregnancy may be a modifiable risk factor for neurodevelopmental injury independent of birth weight or prematurity. Successful interventions to prevent malaria during pregnancy may reduce the risk of neurocognitive delay in children.

Andrea Weckman and co-workers study associations between children’s neurodevelopmental outcomes and malaria in pregnancy.     

Polymorphisms in the RNASE3 gene are associated with susceptibility to cerebral malaria in Ghanaian children

Background

Cerebral malaria (CM) is the most severe outcome of Plasmodium falciparum infection and a major cause of death in children from 2 to 4 years of age. A hospital based study in Ghana showed that P. falciparum induces eosinophilia and found a significantly higher serum level of eosinophil cationic protein (ECP) in CM patients than in uncomplicated malaria (UM) and severe malaria anemia (SA) patients. Single nucleotide polymorphisms (SNPs) have been described in the ECP encoding-gene (RNASE3) of which the c.371G>C polymorphism (rs2073342) results in an arginine to threonine amino acid substitution p.R124T in the polypeptide and abolishes the cytotoxicity of ECP. The present study aimed to investigate the potential association between polymorphisms in RNASE3 and CM.

Methodology/Principal Findings

The RNASE3 gene and flanking regions were sequenced in 206 Ghanaian children enrolled in a hospital based malaria study. An association study was carried out to assess the significance of five SNPs in CM (n = 45) and SA (n = 56) cases, respectively. The two severe case groups (CM and SA) were compared with the non-severe control group comprising children suffering from UM (n = 105). The 371G allele was significantly associated with CM (p = 0.00945, OR = 2.29, 95% CI = 1.22–4.32) but not with SA. Linkage disequilibrium analysis demonstrated significant linkage between three SNPs and the haplotype combination 371G/*16G/*94A was strongly associated with susceptibility to CM (p  = 0.000913, OR = 4.14, 95% CI = 1.79–9.56), thus, defining a risk haplotype. The RNASE3 371GG genotype was found to be under frequency-dependent selection.

Conclusions/Significance

The 371G allele of RNASE3 is associated with susceptibility to CM and forms part of a risk associated haplotype GGA defined by the markers: rs2073342 (G-allele), rs2233860 (G-allele) and rs8019343 (A-allele) respectively. Collectively, these results suggest a hitherto unrecognized role for eosinophils in CM pathogenesis.     

Time delays in treatment of snakebite patients in rural Sri Lanka and the need for rapid diagnostic tests

Delays in treatment seeking and antivenom administration remain problematic for snake envenoming. We aimed to describe the treatment seeking pattern and delays in admission to hospital and administration of antivenom in a cohort of authenticated snakebite patients. Adults (> 16 years), who presented with a confirmed snakebite from August 2013 to October 2014 were recruited from Anuradhapura Hospital. Demographic data, information on the circumstances of the bite, first aid, health-seeking behaviour, hospital admission, clinical features, outcomes and antivenom treatment were documented prospectively. There were 742 snakebite patients [median age: 40 years (IQR:27–51; males: 476 (64%)]. One hundred and five (14%) patients intentionally delayed treatment by a median of 45min (IQR:20-120min). Antivenom was administered a median of 230min (IQR:180–360min) post-bite, which didn’t differ between directly admitted and transferred patients; 21 (8%) receiving antivenom within 2h and 141 (55%) within 4h of the bite. However, transferred patients received antivenom sooner after admission to Anuradhapura hospital than those directly admitted (60min [IQR:30-120min] versus 120min [IQR:52-265min; p<0.0001]). A significantly greater proportion of transferred patients had features of systemic envenoming on admission compared to those directly admitted (166/212 [78%] versus 5/43 [12%]; p<0.0001), and had positive clotting tests on admission (123/212 [58%] versus 10/43 [23%]; p<0.0001). Sri Lankan snakebite patients present early to hospital, but there remains a delay until antivenom administration. This delay reflects a delay in the appearance of observable or measurable features of envenoming and a lack of reliable early diagnostic tests. Improved early antivenom treatment will require reliable, rapid diagnostics for systemic envenoming.     

Impact of Malaria Control on Mortality and Anemia among Tanzanian Children Less than Five Years of Age, 1999–2010

Background

Mainland Tanzania scaled up multiple malaria control interventions between 1999 and 2010. We evaluated whether, and to what extent, reductions in all-cause under-five child mortality (U5CM) tracked with malaria control intensification during this period.

Methods

Four nationally representative household surveys permitted trend analysis for malaria intervention coverage, severe anemia (hemoglobin <8 g/dL) prevalence (SAP) among children 6–59 months, and U5CM rates stratified by background characteristics, age, and malaria endemicity. Prevalence of contextual factors (e.g., vaccination, nutrition) likely to influence U5CM were also assessed. Population attributable risk percentage (PAR%) estimates for malaria interventions and contextual factors that changed over time were used to estimate magnitude of impact on U5CM.

Results

Household ownership of insecticide-treated nets (ITNs) rose from near zero in 1999 to 64% (95% CI, 61.7–65.2) in 2010. Intermittent preventive treatment of malaria in pregnancy reached 26% (95% CI, 23.6–28.0) by 2010. Sulfadoxine-pyrimethamine replaced chloroquine in 2002 and artemisinin-based combination therapy was introduced in 2007. SAP among children 6–59 months declined 50% between 2005 (11.1%; 95% CI, 10.0–12.3%) and 2010 (5.5%; 95% CI, 4.7–6.4%) and U5CM declined by 45% between baseline (1995–9) and endpoint (2005–9), from 148 to 81 deaths/1000 live births, respectively. Mortality declined 55% among children 1–23 months of age in higher malaria endemicity areas. A large reduction in U5CM was attributable to ITNs (PAR% = 11) with other malaria interventions adding further gains. Multiple contextual factors also contributed to survival gains.

Conclusion

Marked declines in U5CM occurred in Tanzania between 1999 and 2010 with high impact from ITNs and ACTs. High-risk children (1–24 months of age in high malaria endemicity) experienced the greatest declines in mortality and SAP. Malaria control should remain a policy priority to sustain and further accelerate progress in child survival.     

CXCL10 Gene Promoter Polymorphism -1447A>G Correlates with Plasma CXCL10 Levels and is Associated with Male Susceptibility to Cerebral Malaria

The risk factors for cerebral malaria (CM) and the wide variation in clinical manifestations of malaria are poorly understood. Recent studies indicate that interferon gamma inducible chemokine, CXCL10, is a strong predictor of both human and experimental cerebral malaria. Increased plasma and cerebrospinal fluid levels of CXCL10 were tightly associated with fatal CM in Indian and Ghanaian patients. In the present study, we hypothesized that in a subset of malaria patients, CM susceptibility is associated with variation in CXCL10 expression. We determined whether polymorphisms in the CXCL10 gene promoter region played a role in the clinical status of malaria patients and addressed the genetic basis of CXCL10 expression during malaria infection. Following extensive bioinformatics analyses, two reported single nucleotide polymorphisms in the CXCL10 promoter (−135G>A [rs56061981] and −1447A>G [rs4508917]) were identified among 66 CM and 69 non-CM Indian patients using PCR-restriction fragment length polymorphism assay. Individuals with the −1447(A/G) genotype were susceptible to CM (adjusted odds ratio [AOR] = 2.60, 95% CI = 1.51–5.85, p = 0.021). In addition, individuals with the −1447(A/G) genotype had significantly higher plasma CXCL10 levels than individuals with the −1447(A/A) genotype. Stratifying patients according to gender, the observed association of CM with over expression of CXCL10 were more pronounced in males than in female patients (AOR = 5.47, 95% CI = 1.34–22.29, p = 0.018). Furthermore, −135G>A polymorphism conferred a decreased risk of CM among males (AOR = 0.19, 95% CI = 0.05–0.78, p = 0.021). Polymorphisms in the CXCL10 gene promoter sequence were associated with increased CXCL10 production, which is linked to severity of CM. These results suggest that the −1447A>G polymorphism in CXCL10 gene promoter could be partly responsible for the reported variation underlying severity of CM outcomes particularly in males.