Genetic diversity in human erythrocyte pyruvate kinase.

Previously, we have shown that pyruvate kinase, liver and red cell isoform (PKLR) deficiency protects mice in vivo against blood-stage malaria, and observed that reduced PKLR function protects human erythrocytes against Plasmodium falciparum replication ex vivo. Here, we have sequenced the human PKLR gene in 387 individuals from malaria-endemic and other regions in order to assess genetic variability in different geographical regions and ethnic groups. Rich genetic diversity was detected in PKLR, including 59 single-nucleotide polymorphisms and several loss-of-function variants (frequency 1.5%). Haplotype distribution and allele frequency varied considerably with geography. Neutrality testing suggested positive selection of the genein the sub-Saharan African and Pakistan populations. It is possible that such positive selection involves the malarial parasite.

Evidence for two-stage binding by the 175-kD erythrocyte binding antigen of Plasmodium falciparum.

Plasmodium falciparum malaria merozoites invade human erythrocytes bearing sialic acid in a multistage process involving the sialic acid-dependent binding of a malaria molecule, the 175-kD erythrocyte binding antigen (EBA-175). We show here that after the initial interaction of EBA-175 with its sialic acid-containing erythrocyte determinant, endogenous proteases can cleave EBA-175 to 65-kD fragment(s), whose binding to erythrocytes is sialic acid independent. A 65-kD fragment was immunoprecipitated by antibodies against peptides between residues 354 and 1061 but not beyond residue 1062. Binding experiments utilizing combinations of native protein, expression-PCR-synthesized EBA-175 polypeptides, peptide synthesis, and antibodies, demonstrated that sialic acid-independent binding could be further mapped to a small (about 40-amino acid) homologous part of the dimorphic allelic region of EBA-175, residues 898-938 (Camp strain numbering). These data support a two-step binding hypothesis and are discussed in relation to the formation of a junction between the merozoite and the erythrocyte, and the finding that after the interaction of some viruses with specific cellular receptors, they undergo conformational changes or cleavage permitting membrane fusion with the host cell.

Ethnic differences in cardiovascular risk factors in healthy Caucasian and South Asian individuals with the metabolic syndrome.

BACKGROUND: The metabolic syndrome is a cluster of atherothrombotic risk factors that are commonly associated with insulin resistance. OBJECTIVES: The aim of this study was to investigate ethnic differences in insulin resistance and non-traditional cardiovascular risk factors in relation to the International Diabetes Federation (IDF) definition of the metabolic syndrome. PATIENTS AND METHODS: A total of 245 healthy South Asians and 245 age- and sex-matched Caucasians were studied. C-reactive protein (CRP), complement C3, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) were measured and homeostasis model assessment-insulin resistance (HOMA-IR) was calculated from fasting plasma glucose and insulin levels. RESULTS: Fifty Caucasian (20%) and 95 (39%) South Asian subjects had the metabolic syndrome as defined by the IDF. In South Asian subjects, HOMA-IR, CRP, C3, PAI-1 and t-PA were significantly higher in subjects with the metabolic syndrome. In contrast, in Caucasian individuals there was no difference in HOMA-IR or C3 levels and only CRP, PAI-1 and t-PA were higher in subjects with the metabolic syndrome. In a logistic regression model, plasma levels of CRP and PAI-1 were independent predictors of the metabolic syndrome in Caucasians, whereas plasma levels of C3 and t-PA as well as HOMA-IR were independent predictors of the metabolic syndrome in South Asian subjects. CONCLUSIONS: In the cohort of individuals studied, the IDF definition of the metabolic syndrome was associated with insulin resistance in the South Asian but not the Caucasian population. This work also showed ethnic differences in non-traditional cardiovascular risk factors in the presence of the metabolic syndrome.

Solar-powered oxygen delivery: proof of concept.

SETTING: A resource-limited paediatric hospital in Uganda. OBJECTIVE: Pneumonia is a leading cause of child mortality worldwide. Access to life-saving oxygen therapy is limited in many areas. We designed and implemented a solar-powered oxygen delivery system for the treatment of paediatric pneumonia. DESIGN: Proof-of-concept pilot study. A solar-powered oxygen delivery system was designed and piloted in a cohort of children with hypoxaemic illness. RESULTS: The system consisted of 25 × 80 W photovoltaic solar panels (daily output 7.5 kWh [range 3.8-9.7kWh]), 8 × 220 Ah batteries and a 300 W oxygen concentrator (output up to 5 l/min oxygen at 88% [±2%] purity). A series of 28 patients with hypoxaemia were treated with solar-powered oxygen. Immediate improvement in peripheral blood oxygen saturation was documented (median change +12% [range 5-15%], P < 0.0001). Tachypnoea, tachycardia and composite illness severity score improved over the first 24 h of hospitalisation (P < 0.01 for all comparisons). The case fatality rate was 6/28 (21%). The median recovery times to sit, eat, wean oxygen and hospital discharge were respectively 7.5 h, 9.8 h, 44 h and 4 days. CONCLUSION: Solar energy can be used to concentrate oxygen from ambient air and oxygenate children with respiratory distress and hypoxaemia in a resource-limited setting.

Illness in Canadian travellers and migrants from Brazil: CanTravNet surveillance data, 2013-2016.

BACKGROUND: In light of the 2016 summer Olympic games it is anticipated that Canadian practitioners will require information about common illnesses that may affect travellers returning from Brazil. OBJECTIVE: To identify the demographic and travel correlates of illness among recent Canadian travellers and migrants from Brazil attending a network of travel health clinics across Canada. METHODS: Data was analyzed on returned Canadian travellers and migrants presenting to a CanTravNet site for care of an illness between June 2013 and June 2016. RESULTS: During the study period, 7,707 ill travellers and migrants presented to a CanTravNet site and 89 (0.01%) acquired their illness in Brazil. Tourists were most well represented (n=45, 50.6%), followed by those travelling to "visit friends and relatives" (n=14, 15.7%). The median age was 37 years (range <1-78 years), 49 travellers were men (55.1%) and 40 were women (44.9%). Of the 40 women, 26 (65%) were of childbearing age. Nine percent (n=8) of travellers were diagnosed with arboviruses including dengue (n=6), chikungunya (n=1) and Zika virus (n=1), while another 14.6% (n=13) presented for care of non-specific viral syndrome (n=7), non-specific febrile illness (n=1), peripheral neuropathy (n=1) and non-specific rash (n=4), which are four syndromes that may be indicative of Zika virus infection. Ill returned travellers to Brazil were more likely to present for care of arboviral or Zika-like illness than other ill returned travellers to South America (23.6 per 100 travellers versus 10.5 per 100 travellers, respectively [p=0.0024]). INTERPRETATION: An epidemiologic approach to illness among returned Canadian travellers to Brazil can inform Canadian practitioners encountering both prospective and returned travellers to the Olympic games. Analysis showed that vector-borne illnesses such as dengue are common and even in this small group of travellers, both chikungunya and Zika virus were represented. It is extremely important to educate travellers about mosquito-avoidance measures in advance of travel to Brazil.

Alterations in mitochondria and mtTFA in response to LPS-induced differentiation of B-cells.

Stimulation of immune cells results in altered cell function and metabolism, which must be recognized by and coordinated with energy production from mitochondria. Mitochondria contain their own DNA genome encoding 13 polypeptides that combine with nuclear-derived subunits to create functional enzyme complexes of the electron transport chain. Therefore, coordination of mitochondrial and nuclear transcription is necessary to achieve a sustained elevation in mitochondrial ATP production. Pre-B-lymphocytes stimulated with lipopolysaccharide exhibit increased activity levels of the mitochondrial enzymes, succinate dehydrogenase and cytochrome c oxidase. Immunoblot analyses of purified mitochondria indicate an increase in the mitochondrial transcription factor (mtTFA) levels in mitochondria induced by cell stimulation. This increase is consistent with increased mtTFA production in the cytoplasm. In addition, mitochondrial protein extracts indicate an increase in protein binding to a mtTFA-DNA binding site from the mitochondrial genome, subsequent to cell stimulation. These results indicate that mitochondrial activity changes during B-lymphocyte stimulation, and mtTFA may contribute to the coordination of respiration with cellular energy demand.

Insulin resistance and elevated levels of tissue plasminogen activator in first-degree relatives of South Asian patients with ischemic cerebrovascular disease.

BACKGROUND AND PURPOSE: South Asians in the United Kingdom suffer from an increased mortality from cerebrovascular disease compared with whites. Evidence suggests that the relatives of white stroke patients are at increased risk of vascular disease. The aim of this study was to investigate atherothrombotic risk factors in the first-degree relatives of South Asian patients suffering from ischemic cerebrovascular disease and to compare them with South Asian subjects free from clinically detectable cerebrovascular disease. METHODS: We compared 143 relatives of South Asians with ischemic stroke (South Asian relatives group) with 146 South Asian control subjects from West Yorkshire, UK. RESULTS: The ages and ethnic and sex distributions of South Asian relatives and South Asian controls were similar. There were no significant differences in body mass index, waist-hip ratio, number of current smokers, and past medical history of hypertension, diabetes mellitus, or myocardial infarction between the 2 groups. Fasting blood glucose, glycosylated hemoglobin (HbA(1c)), total cholesterol, triglycerides, and HDL cholesterol were similar in the 2 groups. Fasting insulin (South Asian relatives, 12.0; South Asian controls, 8.5 mU/L; P<0.0001) (independent of tissue plasminogen activator) and insulin resistance (derived by Homeostasis Model Assessment) (South Asian relatives, 2.7; South Asian controls, 1.9; P=0.001) were significantly raised in stroke relatives. Stroke relatives showed elevated levels of tissue plasminogen activator (South Asian relatives, 11.6; South Asian controls, 8.4 ng/mL; P<0.0001), which was independent of plasma insulin. There were no differences in plasminogen activator inhibitor antigen or activity between the groups. CONCLUSIONS: South Asians stroke relatives exhibit hyperinsulinemia, increased insulin resistance, and increased tissue plasminogen activator levels. These observations might account for increased susceptibility to atherothrombotic disease in this ethnic group.

Recent developments in amoebiasis:the Gal/GalNAc lectins of Entamoeba histolytica and Entamoeba dispar.

Amoebiasis is responsible for 50000-100000 deaths annually. Invasive amoebic disease begins with the attachment of Entamoeba histolytica trophozoites to colonic mucin, a process mediated by the amoebic Gal/GalNAc lectin. The non-pathogenic counterpart, E. dispar, is morphologically identical but genetically distinct. Investigations comparing the Gal/GalNac lectin from these two organisms are under way.

Impaired fibrinolysis and increased fibrinogen levels in South Asian subjects.

The potential role of haemostatic risk markers is largely unexplored in South Asians, who have increased morbidity and mortality from cardiovascular disease and an increased prevalence of insulin resistance. To investigate differences in thrombotic risk markers between South Asian and White populations, 42 Asian and 50 White males and 96 Asian and 80 White females, clinically free from vascular disease, were recruited. Venous blood samples were taken for measures of haemostasis and determination of blood lipids. South Asian females showed lower fasting blood glucose than White females (4.6 vs. 4.8 mmol/l, P<0.008). In the South Asian population, total cholesterol was lower in females, with a similar trend in males (females 5.0 vs. 5.5 mmol/l, P<0.001; males 5.1 vs. WM 5.5 mmol/l, P=0.09), but no difference in triglyceride levels. South Asian subjects of both genders had markedly higher levels of fibrinogen (females 3.3 vs. 2.8 mg/dl, P<0.0005; males 3.0 vs. 2.5 mg/dl P<0.002) and PAI-1 activity (females 14.6 vs. 8.7 ng/ml, P<0.0005, males 21.3 vs. 12.2 ng/ml, ) P<0.0005). Factor VII:C was lower in both South Asian groups (females 110.9 vs. 122.4%, P<0.005; males 103.3 vs. 125%, P<0.0005). Factor XII was lower in South Asian females and there were no differences in Factor XII levels in male populations. These results suggest that elevated PAI-1 and fibrinogen in Asians of both genders may contribute to the increased vascular risk experienced in this population; however, the role of dyslipidaemia and Factor VII are not clear in these processes.

Characterization of peroxynitrite-oxidized low density lipoprotein binding to human CD36.

Peroxynitrite-mediated oxidation may be an important physiological mechanism for oxidation of low density lipoprotein (LDL), however, the molecular basis for the interaction of peroxynitrite oxidized LDL (OxLDL) with scavenger receptors such as CD36, has not been characterized. In this study, we compared the biochemical characteristics and receptor binding of LDL that was oxidized using: (1) Cu2+, a standard method of oxidizing LDL in vitro; and (2) 3-morpholinosydnonimine (SIN-1), a source of peroxynitrite. Both methods of oxidation caused an increase in electrophoretic migration of LDL, but greater mobility was observed with Cu2+-OxLDL. In addition, greater fragmentation of apolipoprotein B was observed following Cu2+ oxidation than after SIN-1 oxidation. The levels of lipid peroxides and thiobarbituric acid reactive substances were similar after 20 h of oxidation by both methods, although the time-course was distinct. Cu2+ and SIN-1-OxLDL bound specifically to the macrophage scavenger receptor CD36 with high affinity. Binding of the 20 h SIN-1 treated LDL to CD36 was comparable to a 4 h Cu2+ modified LDL. The binding of Cu2+ and SIN-1-OxLDL to CD36 was similar under different biochemical conditions and modifications of the receptor, suggesting that OxLDL particles, generated by either method, bind to the same domain of CD36. The results demonstrate that SIN-1 produced an oxidized LDL particle that binds specifically to CD36 and suggests that peroxynitrite OxLDL may represent a more physiologically relevant model than Cu2+-OxLDL for studying the interactions of OxLDL with cells and lipoprotein receptors in vitro.

Expression polymerase chain reaction for the in vitro synthesis and epitope mapping of autoantigen. Application to the human thyrotropin receptor.

The clinical applicability of a newly described polymerase chain reaction directed protein expression system was assessed for the in vitro synthesis and partial epitope mapping of large radiolabeled human thyrotropin receptor (hTSH-R) protein segments. PCR amplification of targeted regions within the hTSH-R cDNA followed by in vitro transcription and translation permitted rapid synthesis of protein segments ranging in size from 18 to 62 kDa. Initial epitope mapping was directed at a 52 amino acid segment unique to the hTSH-R compared to otherwise homologous glycoprotein hormone receptors. Sera from Graves' disease patients known to have autoantibodies against the hTSH-R were used to immunoprecipitate two protein fragments differing only by the presence of the unique region in the larger fragment (E5) but not in the smaller fragment (E4). Dense precipitation bands were obtained using Graves' sera to immunoprecipitate E5 whereas little or no specific immunoprecipitation of E4 occurred. Normal sera gave only weak immunoprecipitation bands of E5. The technique provides significant advantages over conventional cloning methods and should have general applicability in the study of other protein targets of autoimmune disease.

Single-strand conformational polymorphism analysis differentiates Plasmodium falciparum treatment failures from re-infections.

Paired primary and recrudescent Plasmodium falciparum isolates were collected from treatment failures identified during the course of antimalarial drug studies on the Thai-Cambodian border. Ten paired samples were subjected to PCR-restriction fragment length polymorphism (RFLP) and PCR-single-strand conformational polymorphism (PCR-SSCP) analysis of the MSP-2 gene. PCR-SSCP analysis of paired samples demonstrated that each recrudescent isolate was identical to, or a subpopulation of, its matched primary isolate and was distinct from all unrelated isolates. This method represents a field applicable method to distinguish re-infections from treatment failures when antimalarial drug studies are performed in malaria-endemic areas.

Two alleles of the 175-kilodalton Plasmodium falciparum erythrocyte binding antigen.

EBA-175, erythrocyte binding antigen 175, is a 175-kDa antigen of Plasmodium falciparum which has been shown to be involved in the recognition of erythrocytes by merozoites and may be involved in the process of erythrocyte invasion. Invasion of erythrocytes by Camp strain merozoites is inhibited by pre-treatment of red blood cells by EBA-175 from the heterologous strain, FCR-3. The sequence of the Camp strain has been published and we report here the sequence of the FCR-3 strain. The sequences are nearly identical except for a 423-bp segment in the FCR-3 strain, F-segment, that is not found in the Camp strain and a 342-bp segment, C-segment, present in the Camp strain but not in the FCR-3 strain. The locations of these two segments are different in Camp and FCR-3 EBA-175 genes and there is little DNA or amino acid sequence homology between them. The essentially dimorphic alleles, F-segment and C-segment, are conserved in all isolates examined to date. Evidence of genetic cross-over between the FCR-3 and the Camp EBA-175 genes was not observed in the analysis of a limited number of wild isolates. The continued study of the biological relevance of these sequence divergences in EBA-175 may further elucidate the sequence of events resulting in merozoite invasion of erythrocytes.

Universal promoter for gene expression without cloning: expression-PCR.

We present a rapid and simple system called expression-PCR (E-PCR) for in vitro synthesis of functional protein from genomic or plasmid DNA. A universal promoter was developed containing an untranslated leader sequence from alfalfa mosaic virus directly downstream from the T7 bacteriophage promoter. When this universal promoter is spliced to a DNA segment, it produces a suitable template for in vitro transcription and translation. The DNA to be expressed is first amplified by the PCR using a 5'-primer that incorporates an area homologous to the 3'-end of the universal promoter. The universal promoter and this DNA fragment are mixed and re-amplified in a reaction analogous to splicing by overlap extension, generating a recombinant DNA template that can be transcribed and translated in vitro without further processing. Unlike standard methods for in vitro transcription and translation, E-PCR is not dependent upon specialized transcription vectors, cloning, plasmid isolation and purification, or restriction enzyme sites. This approach has been used to synthesize and examine the biological activity of malaria proteins that are vaccine candidates for Plasmodium falciparum. E-PCR represents a significant improvement over current in vitro expression systems, most notably in its time savings, versatility of gene expression and its compatibility with rapid PCR-based site-directed mutagenesis procedures.

Malaria in travelers. Epidemiology, disease, and prevention.

The combination of increases in international travel and escalating drug resistance has resulted in a growing number of travelers contracting malaria. Preventing malaria-associated morbidity and mortality will require improved health information for travelers about the risk of malaria and appropriate preventive measures, improved recognition of infection by physicians, rapid and accurate laboratory diagnosis, and prompt initiation of effective therapy.

Response of Plasmodium vivax variants to chloroquine as determined by microscopy and quantitative polymerase chain reaction.

Genotypic heterogeneity in the repetitive portion of the circumsporozoite (CS) protein of Plasmodium vivax has been reported from many P. vivax-endemic areas. The objective of this study was to determine if the VK210 and VK247 CS variants of P. vivax differed in their clearance rates following chloroquine (CQ) therapy. One hundred seventy-one cases of P. vivax infection occurring in patients presenting to a research treatment center in Thailand were analyzed. Finger-prick blood samples were collected for microscopy and spotted onto filter paper at presentation and on each of five days of observation through supervised CQ therapy. A portion of the CS gene was amplified from filter paper samples by the polymerase chain reaction (PCR) and genotyped by oligoprobes specific for the VK210 and VK247 CS repeat regions. The mean time to clear parasitemia as determined by thick blood smear was significantly longer for pure VK210 infections (51 hr; 95% confidence interval [CI] 47.4-54, P = 0.006) and mixed infections (53 hr; 95% CI 49.2-56.7, P = 0.0009) as compared with VK247 infections (44 hr; 95% CI 39.8-47.9). Five patients matched for parasitemia, age, sex, and previous malaria experience were selected from each of the three genotype groups in the larger study for further analysis by quantitative PCR of P. vivax genotype-specific DNA during a treatment course. The mean time to clear parasite DNA, as determined by PCR, was significantly slower for VK210 parasites (65 hr; 95% CI 51-79) than for VK247 parasites (47 hr; 95% CI 30-63, P = 0.045).(ABSTRACT TRUNCATED AT 250 WORDS)

Distinguishing Plasmodium falciparum treatment failures from reinfections by restrictions fragment length polymorphism and polymerase chain reaction genotyping.

The inability to distinguish recrudescent Plasmodium falciparum infections (treatment failures) from reinfections (new infections) is an important impediment to the evaluation of antimalarial treatment regimens. Ten paired primary and recrudescent isolates collected near the Thai-Cambodian border were analyzed by restriction fragment length polymorphism (RFLP) and by polymerase chain reaction (PCR) genotyping of the genes encoding the following proteins: circumsporozite (CS) protein, erythrocyte binding antigen (EBA)-175, ring-infected erythrocyte surface antigen (RESA), merozoite surface protein-1 (MSP-1), and MSP-2. Both methods demonstrated that the fingerprint pattern of each recrudescent isolate was identical to or was contained within the pattern of the primary isolate. Each recrudescent isolate was unique when compared with the other nine primary isolates. Typing by PCR was more sensitive for the detection of multiclone infections and could be performed with small volumes of whole blood. The PCR genotyping could be a practical method for distinguishing a recrudescent from a new infection when treatment studies are conducted in areas with active malaria transmission.

Parasight F test compared with the polymerase chain reaction and microscopy for the diagnosis of Plasmodium falciparum malaria in travelers.

Imported malaria is an increasing problem worldwide. A rapid and accurate test for Plasmodium falciparum infection would facilitate the diagnosis of malaria in the returned traveler. The ParaSight F antigen capture assay (dipstick test) is a new diagnostic test for P. falciparum based on detection of circulating histidine-rich protein-2 antigen. We performed a blinded evaluation of this assay compared with microscopy and the polymerase chain reaction (PCR) for the detection of P. falciparum infection in 151 febrile travelers. Compared with the PCR, the dipstick test had a sensitivity of 88% and a specificity of 97%. The ability of the dipstick test to detect P. falciparum was similar with that of microscopy (88% versus 83%) since the species of Plasmodium in 14 of 133 malaria-infected patients could not be determined by microscopy due to low parasite numbers. The dipstick test was 40% sensitive for infections with < 50 parasites/microliter, 89% with 50-100 parasites/microliter, and > or = 93% with > 100 parasites/microliter. Circulating antigen was detectable in 68% of the patients seven days after initiation of treatment and in 27% at day 28. The dipstick test represents a simple and accurate test for the diagnosis of P. falciparum infection in the returned traveler.

Plasmodium vivax infections in U.S. Army troops: failure of primaquine to prevent relapse in studies from Somalia.

Different strains of Plasmodium vivax vary in their sensitivity to primaquine, the only drug that prevents relapses. Described are the clinical data and relapse pattern for 75 soldiers treated for vivax malaria since returning from Somalia. Following their initial attack of malaria, 60 of the 75 cases received a standard course of primaquine (15 mg base daily for 14 days). Twenty-six of the 60 soldiers subsequently relapsed for a failure rate of 43%. Eight soldiers had a second relapse following primaquine therapy after both the primary attack and first relapse. Three of these soldiers had received a higher dosage of primaquine (30 mg base daily for 14 days) after their second attack. The apparent ineffectiveness of primaquine therapy in preventing relapses suggests the presence of primaquine-resistant P. vivax strains in Somalia.

Different prevalences of Plasmodium vivax phenotypes VK210 and VK247 associated with the distribution of Anopheles albimanus and Anopheles pseudopunctipennis in Mexico.

The geographic distribution of Plasmodium vivax circumsporozoite protein phenotypes from patient blood used to infect colonized Anopheles albimanus and An. pseudopunctipennis was investigated in southern Mexico. Parasite phenotype types were determined in blood samples by a polymerase chain reaction and oligoprobe hybridization or by immunofluorescent assay of sporozoites. The proportion of infected mosquitoes and the number of oocysts per mosquito confirmed previous in vitro observations indicating that An. albimanus is more susceptible to VK210 and that An. pseudopunctipennis is more susceptible to VK247. All patients living on the coast were infected with VK210 and most patients living above 170 meters above sea level had VK247. Both phenotypes infected patients from intermediate altitudes. These results concur with the distribution of the anophelines, indicating that An. albimanus is the main vector of the phenotype VK210, but that An. pseudopunctipennis transmits both phenotypes. These conditions have direct implications on parasite transmission rates and malaria epidemiology in Mexico.