Diagnosing Severe Falciparum Malaria in Parasitaemic African Children: A Prospective Evaluation of Plasma PfHRP2 Measurement

Background

In African children, distinguishing severe falciparum malaria from other severe febrile illnesses with coincidental Plasmodium falciparum parasitaemia is a major challenge. P. falciparum histidine-rich protein 2 (PfHRP2) is released by mature sequestered parasites and can be used to estimate the total parasite burden. We investigated the prognostic significance of plasma PfHRP2 and used it to estimate the malaria-attributable fraction in African children diagnosed with severe malaria.

Methods and Findings

Admission plasma PfHRP2 was measured prospectively in African children (from Mozambique, The Gambia, Kenya, Tanzania, Uganda, Rwanda, and the Democratic Republic of the Congo) aged 1 month to 15 years with severe febrile illness and a positive P. falciparum lactate dehydrogenase (pLDH)-based rapid test in a clinical trial comparing parenteral artesunate versus quinine (the AQUAMAT trial, ISRCTN 50258054). In 3,826 severely ill children, Plasmadium falciparum PfHRP2 was higher in patients with coma (p = 0.0209), acidosis (p<0.0001), and severe anaemia (p<0.0001). Admission geometric mean (95%CI) plasma PfHRP2 was 1,611 (1,350–1,922) ng/mL in fatal cases (n = 381) versus 1,046 (991–1,104) ng/mL in survivors (n = 3,445, p<0.0001), without differences in parasitaemia as assessed by microscopy. There was a U-shaped association between log₁₀ plasma PfHRP2 and risk of death. Mortality increased 20% per log₁₀ increase in PfHRP2 above 174 ng/mL (adjusted odds ratio [AOR] 1.21, 95%CI 1.05–1.39, p = 0.009). A mechanistic model assuming a PfHRP2-independent risk of death in non-malaria illness closely fitted the observed data and showed malaria-attributable mortality less than 50% with plasma PfHRP2≤174 ng/mL. The odds ratio (OR) for death in artesunate versus quinine-treated patients was 0.61 (95%CI 0.44–0.83, p = 0.0018) in the highest PfHRP2 tertile, whereas there was no difference in the lowest tertile (OR 1.05; 95%CI 0.69–1.61; p = 0.82). A limitation of the study is that some conclusions are drawn from a mechanistic model, which is inherently dependent on certain assumptions. However, a sensitivity analysis of the model indicated that the results were robust to a plausible range of parameter estimates. Further studies are needed to validate our findings.

Conclusions

Plasma PfHRP2 has prognostic significance in African children with severe falciparum malaria and provides a tool to stratify the risk of “true” severe malaria-attributable disease as opposed to other severe illnesses in parasitaemic African children. Please see later in the article for the Editors'' Summary.     

Nectin4 is an epithelial cell receptor for canine distemper virus and involved in neurovirulence

Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.     

Conversion of capsular polysaccharide, involved in pellicle formation, to extracellular polysaccharide by galE deletion in Acetobacter tropicalis

Acetobacter tropicalis SKU1100 produces a pellicle-forming capsular polysaccharide (CPS), consisting of galactose, glucose, and rhamnose. We cloned the galE gene, a UDP-galactose synthesis gene, from A. tropicalis SKU1100 by PCR. A galE-disruptant was prepared and found not to produce CPS and thus not to form a pellicle under the static condition. Instead, the DeltagalE mutant secreted an extracellular polysaccharide (EPS), which was purified and found to have a unique character, different from the original CPS.     

The Complete Amino Acid Sequence of Green Turtle (Chelonia mydas) Egg White Ribonuclease

Egg white ribonuclease was first found in green turtle eggs. This enzyme has been purified by CM-toyopearl cation exchange. Two isoforms (GTRNase-1 and GTRNase-2) were further separated by RP-HPLC, with the same M.W. (13 kDa) and activity. These isoforms carried one amino acid exchange of Ser and Leu at the position 37. The N-terminal sequence, ETRYEKF, was determined for the transblotted protein. Internal sequences were analyzed by protein sequencer and ESI-Q-TOF mass spectrometry for tryptic peptides (Ts). The overlapping sequences were obtained from chymotryptic peptides, CNBr fragments and ISD-MS/MS analysis. The C-terminal Ile was identified by CPase-Y. The established sequence composed of 119 residues with the molecular mass of 12,942.1 Da for GTRNase-1 and 12,967.8 Da for GTRNase-2. The comparison of sequence with known pancreatic RNases, 27 positions including catalytic residues at the position 11 and 114 were conserved. Also basic residues contributed to phosphate binding residues were conserved with the exception of Lys 66. One insertion at the position 14, and 3 deletions at the position-1, between position 64–65, and 110 and 111 were found. Two Cys residues at position 65 and 72 that form a disulfide bond in mammalian RNase were deleted and exchanged. All these difference in the sequence were similar to reptile pancreatic RNase.Data deposition: The sequence reported in this paper has been submitted to the UniProt Knowledgebase under accession No. P84844.     

Phytoremediation of arsenic in submerged soil by wetland plants

Wetland aquatic plants including Canna glauca L., Colocasia esculenta L. Schott, Cyperus papyrus L. and Typha angustifolia L. were used in the phytoremediation of submerged soil polluted by arsenic (As). Cyperus papyrus L. was noticed as the largest biomass producer which has arsenic accumulation capacity of 130-172 mg As/kg plant. In terms of arsenic removal rate, however, Colocasia esculenta L. was recognized as the largest and fastest arsenic remover in this study. Its arsenic removal rate was 68 mg As/m2/day while those rates of Canna glauca L., Cyperus papyrus L. and Typha angustifolia L. were 61 mg As/m2/day, 56 mg As/m2/day, and 56 mg As/m2/day, respectively. Although the 4 aquatic plants were inferior in arsenic accumulation, their high arsenic removal rates were observed. Phytostabilization should be probable for the application of these plants.     

Novel Mutations Found in Two Genes of Thai Patients with Isolated Methylmalonic Acidemia

Molecular genetic analysis of three patients diagnosed with isolated methylmalonic acidemia (MMA) revealed that one was mut ⁰ MMA, with a mutation in the MUT gene encoding the l-methylmalonyl-CoA mutase (MCM), and two were cblB MMA, with mutations in the MMAB gene required for synthesizing the deoxyadenosylcobalamin cofactor of MCM. The mut ⁰ patient was homozygous for a novel nonsense mutation in MUT, p.R31X (c.167C → T), and heterozygous for three previously described polymorphisms, p.K212K (c.712A → G), p.H532R (c.1671A → G), and p.V671I (c.2087G → A). The new MMAB mutation, p.E152X (c.454G → T), was found to be homozygous in one cblB patient and heterozygous in the other patient, who also had four intron polymorphisms in this gene.     

Osteopontin expression in normal skin and non-melanoma skin tumors.

Osteopontin (OPN) is an adhesive, matricellular glycoprotein, whose expression is elevated in many types of cancer and has been shown to facilitate tumorigenesis in vivo. To understand the role of OPN in human skin cancer, this study is designed to determine whether OPN is expressed in premalignant [solar/actinic keratosis (AK)] and in malignant skin lesions such as squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), as well as in normal skin exposed or not exposed to sunlight. Immunohistochemical analyses showed that OPN is expressed in SCC (20/20 cases) and in AK (16/16 cases), which are precursors to SCC, but is absent or minimally expressed in solid BCC (17 cases). However, positive staining for OPN was observed in those BCC that manifest differentiation toward epidermal appendages such as keratotic BCC. In sunlight-exposed normal skin, OPN is minimally expressed in the basal cell layer, but in contrast to those not exposed to sunlight, OPN is more prominent in the spinous cell layer with increasing intensity toward the granular cell layer. Additionally, OPN is expressed in the hair follicles, sebaceous glands, and sweat glands of normal skin. In conclusion, these data suggest that OPN is associated with keratinocyte differentiation and that it is expressed in AK and SCC, which have metastatic potential, but minimally expressed in solid BCC.     

Diversity of nitrogen-fixing cyanobacteria under various ecosystems of Thailand: population dynamics as affected by environmental factors

Investigation of N₂-fixing cyanobacteria from Thai soil was carried out at 2-month intervals between July 1997 and November 1999 to determine the population number, population dynamics and favourable habitats. Sites were selected in three parts of Thailand; North, Central and Northeast. In each part, various soil ecosystems were used as sampling sites; at highest elevation as on the top of the mountain, in the middle and at the foot of the mountain, as well as in flat areas of agricultural practice and uncultivated areas. Generally, a high population of N₂-fixing cyanobacteria was found in agricultural areas where rice cultivation was practised, rather than in other sites. The population dynamics in the mountain and uncultivated areas were less fluctuating than in agricultural areas. The population densities in agricultural areas increased in the rainy season and decreased during the dry season. Other environmental factors such as temperature, moisture and pH also affected the population densities in different habitats. Cyanobacterial diversity was notably influenced by the type of ecosystem in both dry and rainy seasons. The cultivation area containing rice in rotation with other crops contained the most genetically diverse range of species.     

Screening siderophore producing bacteria as potential biological control agent for fungal rice pathogens in Thailand

Rice (Oryza sativa) is a staple food in Thailand and, in addition, feeds around one half of the world’s population. Therefore, diseases of rice are of special concern. Rice is destroyed by 2 main pathogens, Fusarium oxysporum and Pyricularia oryzae the causative agents of root rot and blast in rice respectively. These pathogens result in low grain yield in Thailand and other Southeast Asian countries. Soil samples were taken from paddy fields in Northern Thailand and bacteria were isolated using the soil dilution plate method on Nutrient agar. Isolation yielded 216 bacterial isolates which were subsequently tested for their siderophore production and effectiveness in inhibiting mycelial growth in vitro of the rice pathogenic fungi; Alternaria sp., Fusarium oxysporum, Pyricularia oryzae and Sclerotium sp., the causal agent of leaf spot, root rot, blast and stem rot in rice. It was found that 23% of the bacteria isolated produced siderophore on solid plating medium and liquid medium, In dual culture technique, the siderophore producing rhizobacteria showed a strong antagonistic effect against the Alternaria (35.4%), Fusarium oxysporum (37.5%), Pyricularia oryzae (31.2%) and Sclerotium sp. (10.4%) strains tested. Streptomyces sp. strain A 130 and Pseudomonas sp. strain MW 2.6 in particular showed a significant higher antagonistic effect against Alternaria sp. while Ochrobactrum anthropi D 5.2 exhibited a good antagonistic effect against F. oxysporum. Bacillus firmus D 4.1 inhibited P. oryzae and Kocuria rhizophila 4(2.1.1) strongly inhibited Sclerotium sp. P. aureofaciens AR 1 was the best siderophore producer overall and secreted hydroxamate type siderophore. This strain exhibits an in vitro antagonistic effect against Alternaria sp., F. oxysporum and P. oryzae. Siderophore production in this isolate was maximal after 15 days and at an optimal temperature of 30°C, yielding 99.96 ± 0.46 μg ml^?1 of siderophore. The most effective isolates were identified by biochemical tests and molecular techniques as members of the Genus Bacillus, Pseudomonas and Kocuria including B. firmus D 4.1, P. aureofaciens AR1 and Kocuria rhizophila 4(2.1.1). The study demonstrated antagonistic activity towards the target pathogens discussed and are thus potential agents for biocontrol of soil borne diseases of rice in Thailand and other countries.     

Location of the integrin complex and extracellular matrix molecules at the chicken myotendinous junction

Summary The distribution of several extracellular matrix macromolecules was investigated at the myotendinous junction of adult chicken gastrocnemius muscle. Localization using monoclonal antibodies specific for 3 basal lamina components (type IV collagen, laminin, and a basement membrane form of heparan sulfate proteoglycan) showed strong fluorescent staining of the myotendinous junction for heparan sulfate proteoglycan and laminin, but not for type IV collagen. In addition, a strong fluorescent stain was observed at the myotendinous junction using a monoclonal antibody against the subunit of the chicken integrin complex (antibody JG 22). Neither fibronectin nor tenascin were concentrated at the myotendinous junction, but instead were present in a fibrillar staining pattern throughout the connective tissue which was closely associated with the myotendinous junction. Tenascin also gave bright fluorescent staining of tendon, but no detectable staining of the perimysium or endomysium. Type I collagen was observed throughout the tendon and in the perimysium, but only faintly in the endomysium. In contrast, type III collagen was present brightly in the endomysium and in the perimysium, but could not be detected in the tendon except when associated with blood vessels and in the epitendineum, which stained intensely. Type VI collagen was found throughout the tendon and in all connective tissue partitions of skeletal muscle. The results indicate that one or more molecules of the integrin family may play an important role in the attachment of muscle to the tendon. This interaction does not appear to involve extensive binding to fibronectin or tenascin, but may involve laminin and heparan sulfate proteoglycan.