Detection and quantification of Plasmodium falciparum in human blood by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a proof of concept study.

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has revolutionized identification of bacteria and is becoming available in an increasing number of laboratories in malaria-endemic countries. The purpose of this proof-of-concept study was to explore the potential of MALDI-TOF as a diagnostic tool for direct detection and quantification of Plasmodium falciparum in human blood. METHODS: Three different P. falciparum strains (3D7, HB3 and IT4) were cultured and synchronized following standard protocols. Ring-stages were diluted in fresh blood group 0 blood drawn in EDTA from healthy subjects to mimic clinical samples. Samples were treated with saponin and washed in PBS to concentrate protein material. Samples were analysed using a Microflex LT MALDI-TOF and resulting mass spectra were compared using FlexAnalysis software. RESULTS: More than 10 peaks specific for P. falciparum were identified. The identified peaks were consistent among the three genetically unrelated strains. Identification was possible in clinically relevant concentrations of 0.1% infected red blood cells, and a close relationship between peak intensity and the percentage of infected red blood cells was seen. CONCLUSION: The findings indicate that the method has the potential to detect and quantify P. falciparum at clinically relevant infection intensities and provides proof-of-concept for MALDI-TOF-based diagnosis of human malaria. Further research is needed to include other Plasmodium spp., wildtype parasite isolates and to increase sensitivity. MALDI-TOF may be a useful tool for mass-screening purposes and for diagnosis of malaria in settings where it is readily available.

Exploring training needs of newly graduated medical doctors to inform the undergraduate simulation-based curriculum: a national Delphi consensus study.

PURPOSE: Mastering technical procedures is a key component in succeeding as a newly graduated medical doctor and is of critical importance to ensure patient safety. The efficacy of simulation-based education has been demonstrated but medical schools have different requirements for undergraduate curricula. We aimed to identify and prioritize the technical procedures needed by newly graduated medical doctors. METHODS: We conducted a national needs assessment survey using the Delphi technique to gather consensus from key opinion leaders in the field. In the first round, a brainstorm was conducted to identify all potential technical procedures. In the second round, respondents rated the need for simulation-based training of each procedure using the Copenhagen Academy for Medical Education and Simulation Needs Assessment Formula (CAMES-NAF). The third round was a final elimination and prioritization of the procedures. RESULTS: In total, 107 experts from 21 specialties answered the first round: 123 unique technical procedures were suggested. Response rates were 58% and 64% in the second and the third round, respectively. In the third round, 104 procedures were eliminated based on the consensus criterion, and the remaining 19 procedures were included and prioritized. The top five procedures were: (i) insert peripheral intravenous catheter, (ii) put on personal protection equipment, (iii) perform basic airway maneuvers, (iv) perform basic life support, and (v) perform radial artery puncture. CONCLUSION: Based on the Delphi process a final list of 19 technical procedures reached expert consensus to be included in the undergraduate curriculum for simulation-based education.

Performance of a Histidine Rich Protein-2 Based (First Response) and a p-Lactate Dehydrogenase-based (Optimal) Rapid Diagnostic Test for Diagnosis of Malaria in Patients With Pediatric Sickle Cell Disease.

BACKGROUND: Rapid diagnostic tests (RDTs) have been extensively evaluated and play an important role in malaria diagnosis. However, the accuracy of RDTs for malaria diagnosis in patients with sickle cell disease (SCD) is unknown. METHODS: We compared the performance of a histidine rich protein 2 (HRP-2)-based RDT (First Response) and a lactate dehydrogenase (LDH)-based RDT (Optimal) with routine microscopy as reference standard in 445 children with SCD and an acute febrile illness in Accra, Ghana. RESULTS: The overall sensitivity, specificity, and positive and negative predictive values of the HRP-2-based RDTs were 100%, 95.7%, 73.8%, and 100%, respectively. Comparable values for the LDH-based RDTs were 91.7%, 99.5%, 95.7%, and 99.0%, respectively. A total of 423 results were true in both tests, 1 result was false in both tests, 16 results were false in the HRP-2 test only, and 5 were false in the LDH test only (McNemar test, P = .03). At follow-up, 73.7% (28/38), 52.6% (20/38), 48.6% (17/35), and 13.2% (5/38) of study participants were HRP-2 positive on days 14, 28, 35, and 42, respectively, compared with 0%, 2.6% (1/38), 2.9% (1/35), and 2.6% (1/38) for LDH. CONCLUSION: The HRP2-based RDT fulfilled World Health Organization criteria for malaria diagnosis in patients with SCD and may provide diagnostic evidence for treatment to begin in cases in which treatment would otherwise have begun presumptively based on symptoms, whereas LDH-based RDTs may be more suitable as a confirmatory test in low-parasitemic subgroups, such as patients with SCD.

Oxygen gradient ektacytometry does not predict pain in children with sickle cell anaemia.

The loss of red blood cell (RBC) deformability in sickle cell anaemia (SCA) is considered the primary factor responsible for episodes of acute pain and downstream progressive organ dysfunction. Oxygen gradient ektacytometry (Oxygenscan) is a recently commercialised functional assay that aims to describe the deformability of RBCs in SCA at differing oxygen tensions. So far, the Oxygenscan has been evaluated only by a small number of research groups and the validity and clinical value of Oxygenscan-derived biomarkers have not yet been fully established. In this study we examined RBC deformability measured with the Oxygenscan in 91 children with SCA at King's College Hospital in London. We found a significant correlation between Oxygenscan-derived biomarkers and well-recognised modifiers of disease severity in SCA: haemoglobin F and co-inherited α-thalassaemia. We failed, however, to find any independent predictive value of the Oxygenscan in the clinical outcome measure of pain, as well as other important parameters such as hydroxycarbamide treatment. Although the Oxygenscan remains an intriguing tool for basic research, our results question whether it provides any additional information in predicting the clinical course in children with SCA, beyond measuring known markers of disease severity.

The significance of spleen size in children with sickle cell anemia.

It is well established that splenic dysfunction occurs in early childhood in sickle cell anemia (SCA), although the determinants and consequences of splenic injury are not fully understood. In this study, we examined spleen size and splenic function in 100 children with SCA aged 0-16 years at King's College Hospital in London. Spleen size was assessed by abdominal ultrasound (US) and splenic function by pitted red blood cells (PIT counts). In our cohort, 5.6% of children aged 6-10 years and 19.4% of children aged 11-16 years had no visible spleen on US (autosplenectomy). Splenomegaly was common in all age groups, with 28% of children overall having larger spleens than the average for their age. Only one child had a PIT count suggesting preserved splenic function. We found no correlation between hemoglobin F levels and spleen size, nor was there any difference in spleen size between children treated with or without hydroxyurea. Although there was a trend toward increased spleen length in children with co-inherited α-thalassemia, this did not reach statistical significance. Finally, we found a strong association between erythrocyte deformability measured with oxygen gradient ektacytometry, spleen size, and PIT counts. In conclusion, our results do not agree with the general perception that most children with SCA undergo autosplenectomy within the first decade of life and indicate that loss of erythrocyte deformability contributes to loss of splenic filtration capacity in SCA, as well as phenotypical variations in spleen size.

Oxacillinase-181 Carbapenemase-Producing Klebsiella pneumoniae in Neonatal Intensive Care Unit, Ghana, 2017-2019.

We sequenced 29 carbapenemase-producing Klebsiella pneumoniae isolates from a neonatal intensive care unit in Ghana. Twenty-eight isolates were sequence type 17 with blaOXA-181 and differed by 0-32 single-nucleotide polymorphisms. Improved surveillance and infection control are needed to characterize and curb the spread of multidrug-resistant organisms in sub-Saharan Africa.

Critical evaluation of the appetite test for children with severe acute malnutrition.

OBJECTIVES: The appetite test is used to risk stratify for children with severe acute malnutrition (SAM) in inpatient or outpatient care. The test is recommended in guidelines despite lack of evidence. We evaluated its ability to identify children at risk of a poor treatment outcome. METHODS: We conducted an observational study of children diagnosed with SAM at three health facilities in Ethiopia. The appetite test was done independently, and the result did not affect decisions about hospitalisation and clinical care. Data were analysed using mixed linear and logistic regression models. RESULTS: Appetite was tested in 298 (89%) of 334 children enrolled; 56 (19%) passed. Children failing the appetite test had a 6.6% higher weight gain per day (95% CI: 2.6, 10.8) adjusted for type of treatment, oedema, duration of follow-up and age than children passing the test. We found medical complications in 179 (54%) children. Medical complications were associated with blood markers of metabolic disturbance. Children with medical complications tended to have lower weight gain than those without complications (3.5%, 95% CI: -0.25, 7.0). Neither the appetite test nor medical complications were correlated with bacteraemia or treatment failure. CONCLUSIONS: Our findings question the use of the appetite test to identify children who need inpatient care. An assessment of medical complications alone could be a useful risk indicator but needs to be evaluated in other settings.

OBJECTIF: Le test de l'appétit est utilisé pour stratifier les risques chez les enfants souffrant de malnutrition aiguë sévère (MAS) en soins hospitaliers ou ambulatoires. Le test est recommandé dans les directives malgré le manque d'évidence. Nous avons évalué sa capacité à identifier les enfants à risque de mauvais résultats de traitement. MÉTHODES: Nous avons mené une étude observationnelle chez des enfants diagnostiqués avec une MAS dans trois établissements de santé en Ethiopie. Le test de l'appétit a été effectué indépendamment et le résultat n'a pas affecté les décisions d'hospitalisation et de soins cliniques. Les données ont été analysées à l'aide de modèles de régression linéaire et logistique mixtes. RÉSULTATS: : L'appétit a été testé chez 298 (89%) des 334 enfants inscrits; 56 (19%) ont réussi le test. Les enfants qui échouaient au test de l'appétit avaient un gain de poids de 6,6% plus élevé par jour (IC95%: 2,6 à 10,8) ajusté pour le type de traitement, l'œdème, la durée du suivi et l'âge que les enfants réussissant le test. Nous avons trouvé des complications médicales chez 179 (54%) enfants. Des complications médicales ont été associées à des marqueurs sanguins de troubles métaboliques. Les enfants souffrant de complications médicales avaient tendance à avoir un gain de poids plus faible que ceux sans complications (3,5% ; IC95%: -0,25 à 7,0). Ni le test de l'appétit ni les complications médicales ne corrélaient avec une bactériémie ou à un échec du traitement CONCLUSION: Nos résultats remettent en question l'utilisation du test de l'appétit pour identifier les enfants qui ont besoin de soins hospitaliers. Une évaluation des complications médicales à elle seule pourrait être un indicateur de risque utile, mais doit être évaluée dans d'autres contextes MOTS-CLÉS: malnutrition aiguë sévère, appétit, gestionnaire de communauté, évaluation des risques, aliments thérapeutiques.

High rates of multi-drug resistant gram-negative organisms associated with surgical site infections in a teaching hospital in Ghana.

BACKGROUND: There is limited data to guide the prevention and management of surgical site infections (SSI) in low- and middle-income countries. We prospectively studied aetiological agents associated with SSI and their corresponding antibiotic susceptibility patterns in a tertiary hospital in Ghana. METHODS: As part of a cohort study carried out at the surgical department of the Korle Bu Teaching Hospital (KBTH) from July 2017 to April 2019, wound swabs were collected from patients diagnosed with SSI. Isolates cultured from the wound swabs were identified by MALDI TOF and susceptibility testing was conducted according to EUCAST 2020 guidelines. Clinical data were monitored prospectively. RESULTS: Of 4577 patients, 438 developed an SSI and 352 microbial isolates were cultured. Isolates were predominantly Gram negative (286, 81%), a pattern seen for all kinds of surgery and all wound classes. The most common species included Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Acinetobacter baumannii. The majority of organisms were multi-drug resistant including 86% of E. coli, 52% of A. baumannii and 86% of K. pneumoniae; and 65% (17/26) of the cefotaxime-resistant K. pneumoniae were extended spectrum ß-lactamase producing. One of 139 E. coli, 15 of 49 P. aeruginosa, and 6 of 23 A. baumannii were meropenem resistant, but no clonal pattern was found. There was a 1% (5/428) prevalence of methicillin-resistant S. aureus. CONCLUSIONS: The predominance of Gram-negative organisms and the high level of multi-drug resistance indicate a need to re-evaluate antibiotic prophylaxis and treatment protocols in surgical practice in low- and middle-income countries.

Prolonged and persistent diarrhoea is not restricted to children with acute malnutrition: an observational study in Ethiopia.

OBJECTIVES: To assess the prevalence of prolonged and persistent diarrhoea, to estimate their co-occurrence with acute malnutrition and association with demographic and clinical factors. METHODS: Case-control study where cases were children under 5 years of age with diarrhoea and controls were children without diarrhoea, frequency-matched weekly by age and district of residency. Controls for cases 0-11 months were recruited from vaccination rooms, and controls for cases 12-59 months were recruited by house visits using random locations in the catchment area of the study sites. Data were analysed by mixed model logistic regression. RESULTS: We enrolled 1134 cases and 946 controls. Among the cases, 967 (85%) had acute diarrhoea (AD), 129 (11%) had ProD and 36 (3.2%) had PD. More cases had acute malnutrition at enrolment (17% vs. 4%, P < 0.0001) and more were born prematurely (5.7% vs. 1.8%, P < 0.0001) than controls. About 75% of ProPD cases did not have acute malnutrition. Cases with AD and ProPD had different symptomatology, even beyond illness duration. CONCLUSIONS: ProPD is common among children presenting with diarrhoea and is not confined to children with acute malnutrition. There is an urgent need for studies assessing causes of ProPD with and without acute malnutrition to develop treatment guidelines for these conditions.

OBJECTIFS: Evaluer la prévalence des diarrhées prolongées et persistantes, estimer leur co-occurrence avec la malnutrition aiguë et leur association avec des facteurs démographiques et cliniques. MÉTHODES: Etude cas-témoins portant sur des enfants de moins de 5 ans souffrant de diarrhée et sur des témoins, des enfants sans diarrhée, appariées toutes les semaines, en fonction de l'âge et du district de résidence. Les témoins pour les cas de 0 à 11 mois ont été recrutés dans les salles de vaccination et les témoins pour les cas de 12 à 59 mois ont été recrutés au cours de visites à domicile en utilisant des endroits aléatoires dans la zone de recrutement des sites d'étude. Les données ont été analysées par la régression logistique de modèle mixte. RÉSULTATS: Nous avons inscrit 1134 cas et 946 témoins. Parmi les cas, 967 (85%) avaient une diarrhée aiguë (DA), 129 (11%) étaient atteints de diarrhées prolongée (ProD) et 36 (3,2%) de diarrhées persistante (DP). La malnutrition aiguë au moment de l'inscription était plus fréquente (17% contre 4%, P < 0,0001) et davantage étaient nés prématurément (5,7% contre 1,8%, P < 0,0001) par rapport aux témoins. 75% des cas de ProPD ne souffraient pas de malnutrition aiguë. Les cas de DA et de ProPD avaient une symptomatologie différente, même au-delà de la durée de la maladie. CONCLUSIONS: La ProPD est fréquente chez les enfants présentant une diarrhée et ne se limitait pas aux enfants souffrant de malnutrition aiguë. Il est urgent que des études évaluant les causes de ProPD avec et sans malnutrition aiguë développent des recommandations de traitement pour ces affections.

Diagnostic accuracy of the 1,3-ß-D-glucan test for pneumocystis pneumonia in a tertiary university hospital in Denmark: A retrospective study.

1,3-ß-D-glucan (BG), a cell-wall component of most fungi including Pneumocystis (PC), is recommended by international guidelines for screening for pneumocystis pneumonia (PCP) in hematologic patients. We retrospectively validated the BG test in our tertiary university hospital. Forty-five patients (median age 53 years, 33% female) tested for PC by polymerase chain reaction (PCR) and/or immunoflourescence (IF)-microscopy with a stored blood sample within ±5 days of the PC test were tested by the Fungitell (cutoff <60 and >80 pg/ml). Cases had symptoms and radiology compatible with PCP and positive IF-microscopy (proven PCP, n = 8) or positive PCR (probable PCP, n = 10). Controls had no compatible symptoms/radiology and negative tests for PC on conventional testing (no PCP, n = 24), or positive PCR/IF-microscopy (colonized, n = 3). Median BG-levels were 1108 pg/ml (proven PCP), 612 pg/ml (probable PCP), 29 pg/ml (colonized), and 48 pg/ml (controls, P < 0.001). Compared to the PCP case/control classification, the BG test showed sensitivities of 83-89% and specificities of 64-74%, positive likelihood ratio (LR) of 3.2 and negative LR of 0.23 at recommended cutoff and moderate agreement between tests. Optimal cutoff was ≥73 pg/ml. In PCR-positive cases, the agreement between the BG test and IF-microscopy was 78-89% with fair/moderate agreement. Elevated BG levels were seen in controls with probable invasive fungal infections (n = 4), hemodialysis, bacterial infections and/or betalactams. To conclude, 11% of patients with PCP would be missed if the BG test had been used for diagnosing PCP. Specificity was moderate. Among PCR-positive patients, the BG test identified more cases than IF-microscopy. BG testing is potentially helpful but sensitivity is insufficient to exclude PCP.

Population Pharmacokinetic Estimates Suggest Elevated Clearance and Distribution Volume of Desethylamodiaquine in Pediatric Patients with Sickle Cell Disease Treated with Artesunate-Amodiaquine.

BACKGROUND: There is limited information on the safety or efficacy of currently recommended antimalarial drugs in patients with sickle cell disease (SCD), a population predisposed to worse outcomes if affected by acute malaria. Artesunate-amodiaquine (ASAQ) is the first-line treatment for uncomplicated malaria (UM) in many malaria-endemic countries and is also used for treatment of UM in SCD patients. There is, however, no information to date, on the pharmacokinetics (PK) of amodiaquine or artesunate or the metabolites of these drugs in SCD patients. OBJECTIVES: This study sought to determine the PK of desethylamodiaquine (DEAQ), the main active metabolite of amodiaquine, among paediatric SCD patients with UM treated with artesunate-amodiaquine (ASAQ). METHODS: Plasma concentration-time data (median DEAQ levels) of SCD children (n = 16) was initially compared with those of concurrently recruited non-SCD paediatric patients with acute UM (n = 13). A population PK modelling approach was then used to analyze plasma DEAQ concentrations obtained between 64 and 169 hours after oral administration of ASAQ in paediatric SCD patients with acute UM (n = 16). To improve PK modeling, DEAQ concentration-time data (n = 21) from SCD was merged with DEAQ concentration-time data (n = 169) of a historical paediatric population treated with ASAQ (n = 103) from the same study setting. RESULTS: The median DEAQ concentrations on days 3 and 7 were comparatively lower in the SCD patients compared to the non-SCD patients. A two-compartment model best described the plasma DEAQ concentration-time data of the merged data (current SCD data and historical data). The estimated population clearance of DEAQ was higher in the SCD patients (67 L/h, 21% relative standard error (RSE) compared with the non-SCD population (15.5 L/h, 32% RSE). The central volume of distribution was larger in the SCD patients compared with the non-SCD patients (4400 L, 43% RSE vs. 368 L, 34% RSE). CONCLUSIONS: The data shows a tendency towards lower DEAQ concentration in SCD patients and the exploratory population PK estimates suggest altered DEAQ disposition in SCD patients with acute UM. These findings, which if confirmed, may reflect pathophysiological changes associated with SCD on DEAQ disposition, have implications for therapeutic response to amodiaquine in SCD patients. The limited number of recruited SCD patients and sparse sampling approach however, limits extrapolation of the data, and calls for further studies in a larger population.

Increased Plasmodium chabaudi malaria mortality in mice with nutritional iron deficiency can be reduced by short-term adjunctive iron supplementation.

BACKGROUND: Iron deficiency is the most widespread nutrient deficiency and an important cause of developmental impairment in children. However, some studies have indicated that iron deficiency can also protect against malaria, which is a leading cause of childhood morbidity and mortality in large parts of the world. This has rendered interventions against iron deficiency in malaria-endemic areas controversial. METHODS: The effect of nutritional iron deficiency on the clinical outcome of Plasmodium chabaudi AS infection in A/J mice and the impact of intravenous iron supplementation with ferric carboxymaltose were studied before and after parasite infection. Plasma levels of the iron status markers hepcidin and fibroblast growth factor 23 were measured in animals surviving and succumbing to malaria, and accompanying tissue pathology in the liver and the spleen was assessed. RESULTS: Nutritional iron deficiency was associated with increased mortality from P. chabaudi malaria. This increased mortality could be partially offset by carefully timed, short-duration adjunctive iron supplementation. Moribund animals were characterized by low levels of hepcidin and high levels of fibroblast growth factor 23. All infected mice had extramedullary splenic haematopoiesis, and iron-supplemented mice had visually detectable intracellular iron stores. CONCLUSIONS: Blood transfusions are the only currently available means to correct severe anaemia in children with malaria. The potential of carefully timed, short-duration adjunctive iron supplementation as a safe alternative should be considered.

Malaria causes long-term effects on markers of iron status in children: a critical assessment of existing clinical and epidemiological tools.

BACKGROUND: Most epidemiological studies on the interplay between iron deficiency and malaria risk classify individuals as iron-deficient or iron-replete based on inflammation-dependent iron markers and adjustment for inflammation by using C-reactive protein (CRP) or α-1-acid glycoprotein (AGP). The validity of this approach and the usefulness of fibroblast growth factor 23 (FGF23) as a proposed inflammation-independent iron marker were tested. METHODS: Conventional iron markers and FGF23 were measured in children with acute falciparum malaria and after 1, 2, 4, and 6 weeks. Children, who were transfused or received iron supplementation in the follow-up period, were excluded, and iron stores were considered to be stable throughout. Ferritin levels 6 weeks after admission were used as a reference for admission iron status and compared with iron markers at different time points. RESULTS: There were long-term perturbations in iron markers during convalescence from acute malaria. None of the tested iron parameters, including FGF23, were independent of inflammation. CRP and AGP normalized faster than ferritin after malaria episodes. CONCLUSION: Malaria may bias epidemiological studies based on inflammation-dependent iron markers. Better markers of iron status during and after inflammation are needed in order to test strategies for iron supplementation in populations at risk of malaria.

Population Pharmacokinetic Characteristics of Amikacin in Suspected Cases of Neonatal Sepsis in a Low-Resource African Setting: A Prospective Nonrandomized Single-Site Study.

BACKGROUND: Amikacin exhibits marked pharmacokinetic (PK) variability and is commonly used in combination with other drugs in the treatment of neonatal sepsis. There is a paucity of amikacin PK information in neonates from low-resource settings. OBJECTIVES: To determine the PK parameters of amikacin, and explore the influence of selected covariates, including coadministration with aminophylline, on amikacin disposition in neonates of African origin. METHODS: Neonates with suspected sepsis admitted to an intensive care unit in Accra, Ghana, and treated with amikacin (15 mg/kg loading followed by 7.5 mg/kg every 12 hours), were recruited. Serum amikacin concentration was measured at specified times after treatment initiation and analyzed using a population PK modeling approach. RESULTS: A total of 419 serum concentrations were available for 247 neonates. Mean (SD) trough amikacin concentration (from samples collected 30 minutes before the fourth dose) among term (n = 25), and preterm (<37 weeks' gestation n = 36) neonates were 6.2 (3.4) and 9.2 (5.7) µg/mL, respectively (P = 0.02). A 1-compartment model best fitted amikacin disposition, and birth weight was the most important predictor of amikacin clearance (CL) and distribution (V). The population CL and V of amikacin were related as CL (L/h) = 0.153 (birth weight/2.5)1.31, V (L) = 2.94 (birth weight/2.5)1.18. There was a high between-subject variability (58.9% and 50.7%) in CL and V, respectively. CL and V were 0.058 L/h/kg and 1.15 L/kg, respectively, for a mean birth weight of 2.1 kg, and the mean half-life (based on 1-compartment model), was 13.7 hours. CONCLUSIONS: The V and half-life of amikacin in this cohort varied from that reported in non-African populations, and the high trough and low peak amikacin concentrations in both term and preterm neonates suggest strategies to optimize amikacin dosing are required in this population.

An automated method for determining the cytoadhesion of Plasmodium falciparum-infected erythrocytes to immobilized cells.

BACKGROUND: Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin. METHODS: Chinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO cells, was quantified by microscopy of Giemsa-stained chamber slides. In the automated assay, binding was quantified spectrophotometrically in microtiter plates after cell lysis using tetramethylbenzidine as peroxidase-catalysed substrate. The relevance of the method for binding studies was assessed using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression and Bland-Altman plots. RESULTS: The manual and automated quantification showed strong, positive correlation (r(2) = 0.959, p <0.001) and with similar detection limit and precision. The automated assay showed the expected dose-dependent reduction in binding to CHO cells when blocking with soluble chondroitin sulfate A or anti-CD36 antibody. Quantification of binding to endothelial cells showed clear distinction between selected vs. non-selected parasite lines. Importantly, the assay was sufficiently sensitive to detect adhesion of field isolates to endothelial cells. CONCLUSIONS: The assay is simple and in a reproducible manner quantifies erythrocyte adhesion to several types of immobilized cells.

Polymorphisms in the Haem Oxygenase-1 promoter are not associated with severity of Plasmodium falciparum malaria in Ghanaian children.

BACKGROUND: Haem oxygenase-1 (HO-1) catabolizes haem and has both cytotoxic and cytoprotective effects. Polymorphisms in the promoter of the Haem oxygenase-1 (HMOX1) gene encoding HO-1 have been associated with several diseases including severe malaria. The objective of this study was to determine the allele and genotype frequencies of two single nucleotide polymorphisms; A(-413)T and G(-1135)A, and a (GT)n repeat length polymorphism in the HMOX1 promoter in paediatric malaria patients and controls to determine possible associations with malaria disease severity. METHODS: Study participants were Ghanaian children (n=296) admitted to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season from June to August in 1995, 1996 and 1997, classified as having uncomplicated malaria (n=101) or severe malaria (n=195; defined as severe anaemia (n=63) or cerebral malaria (n=132)). Furthermore, 287 individuals without a detectable Plasmodium infection or asymptomatic carriers of the parasite were enrolled as controls. Blood samples from participants were extracted for DNA and allele and genotype frequencies were determined with allele-specific PCR, restriction fragment length analysis and microsatellite analysis. RESULTS: The number of (GT)n repeats in the study participants varied between 21 and 46 with the majority of alleles having lengths of 26 (8.1%), 29/30 (13.2/17.9%) and 39/40 (8.0/13.8%) repeats, and was categorized into short, medium and long repeats. The (-413)T allele was very common (69.8%), while the (-1135)A allele was present in only 17.4% of the Ghanaian population. The G(-1135)A locus was excluded from further analysis after failing the Hardy-Weinberg equilibrium test. No significant differences in allele or genotype distribution of the A(-413)T and (GT)n repeat polymorphisms were found between the controls and the malaria patients, or between the disease groups, for any of the analysed polymorphisms and no associations with malaria severity were found. CONCLUSION: These results contribute to the understanding of the role of HMOX1/HO-1. This current study did not find any evidence of association between HMOX1 promoter polymorphisms and malaria susceptibility or severe malaria and hence contradicts previous findings. Further studies are needed to fully elucidate the relationship between HMOX1 polymorphisms and malarial disease.

Intravenous ferric carboxymaltose accelerates erythropoietic recovery from experimental malarial anemia.

Iron restriction has been proposed as a cause of erythropoietic suppression in malarial anemia; however, the role of iron in malaria remains controversial, because it may increase parasitemia. To investigate the role of iron-restricted erythropoiesis, A/J mice were infected with Plasmodium chabaudi AS, treated with intravenous ferric carboxymaltose at different times, and compared with untreated controls. Iron treatment significantly increased weight and hemoglobin nadirs and provided enhanced reticulocytosis and faster recovery, compared with controls. Our findings challenge the restrictive use of iron therapy in malaria and show the need for trials of intravenous ferric carboxymaltose as an adjunctive treatment for severe malarial anemia.

Erythropoietin treatment alleviates ultrastructural myelin changes induced by murine cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is a severe complication of malaria with considerable mortality. In addition to acute encephalopathy, survivors frequently suffer from neurological sequelae. The pathogenesis is incompletely understood, hampering the development of an effective, adjunctive therapy, which is not available at present. Previously, erythropoietin (EPO) was reported to significantly improve the survival and outcome in a murine CM model. The study objectives were to assess myelin thickness and ultrastructural morphology in the corpus callosum in murine CM and to adress the effects of EPO treatment in this context. METHODS: The study consisted of two groups of Plasmodium berghei-infected mice and two groups of uninfected controls that were either treated with EPO or placebo (n = 4 mice/group). In the terminal phase of murine CM the brains were removed and processed for electron microscopy. Myelin sheaths in the corpus callosum were analysed with transmission electron microscopy and stereology. RESULTS: The infection caused clinical CM, which was counteracted by EPO. The total number of myelinated axons was identical in the four groups and mice with CM did not have reduced mean thickness of the myelin sheaths. Instead, CM mice had significantly increased numbers of abnormal myelin sheaths, whereas EPO-treated mice were indistinguishable from uninfected mice. Furthermore, mice with CM had frequent and severe axonal injury, pseudopodic endothelial cells, perivascular oedemas and intracerebral haemorrhages. CONCLUSIONS: EPO treatment reduced clinical signs of CM and reduced cerebral pathology. Murine CM does not reduce the general thickness of myelin sheaths in the corpus callosum.

Insights into deregulated TNF and IL-10 production in malaria: implications for understanding severe malarial anaemia.

BACKGROUND: Severe malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized by a high TNF/IL-10 ratio. Whether this TNF/IL-10 imbalance results from an intrinsic incapacity of SMA patients to produce IL-10 or from an IL-10 unresponsiveness to infection is unknown. Monocytes and T cells are recognized as the main sources of TNF and IL-10 in vivo, but little is known about the activation status of those cells in SMA patients. METHODS: The IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLA-DR+ and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA) used as surrogate of unspecific monocyte and T cell stimulant. The number of circulating pigmented monocytes was also determined. RESULTS: Monocytes and T cells from SMA and CM patients showed similar activation profiles with a comparable decreased HLA-DR expression on monocytes and increased frequency of CD69+ and HLA-DR+ T cells. In contrast, the acute-phase IL-10 production was markedly decreased in SMA compared to CM (P = .003) and UM (P = .004). Although in SMA the IL-10 response to LPS-stimulation was larger in amplitude than in CM (P = .0082), the absolute levels of IL-10 reached were lower (P = .013). Both the amplitude and levels of TNF produced in response to LPS-stimulation were larger in SMA than CM (P = .019). In response to PHA-stimulation, absolute levels of IL-10 produced in SMA were lower than in CM (P = .005) contrasting with TNF levels, which were higher (P = .001). CONCLUSIONS: These data reveal that SMA patients have the potential to mount efficient IL-10 responses and that the TNF/IL-10 imbalance may reflect a specific monocyte and T cell programming/polarization pattern in response to infection.