Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2

BackgroundIn falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria.ConclusionPlasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes.     

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.     

The Development of Diet-Induced Obesity and Glucose Intolerance in C57Bl/6 Mice on a High-Fat Diet Consists of Distinct Phases

High–fat (HF) diet-induced obesity and insulin insensitivity are associated with inflammation, particularly in white adipose tissue (WAT). However, insulin insensitivity is apparent within days of HF feeding when gains in adiposity and changes in markers of inflammation are relatively minor. To investigate further the effects of HF diet, C57Bl/6J mice were fed either a low (LF) or HF diet for 3 days to 16 weeks, or fed the HF-diet matched to the caloric intake of the LF diet (PF) for 3 days or 1 week, with the time course of glucose tolerance and inflammatory gene expression measured in liver, muscle and WAT. HF fed mice gained adiposity and liver lipid steadily over 16 weeks, but developed glucose intolerance, assessed by intraperitoneal glucose tolerance tests (IPGTT), in two phases. The first phase, after 3 days, resulted in a 50% increase in area under the curve (AUC) for HF and PF mice, which improved to 30% after 1 week and remained stable until 12 weeks. Between 12 and 16 weeks the difference in AUC increased to 60%, when gene markers of inflammation appeared in WAT and muscle but not in liver. Plasma proteomics were used to reveal an acute phase response at day 3. Data from PF mice reveals that glucose intolerance and the acute phase response are the result of the HF composition of the diet and increased caloric intake respectively. Thus, the initial increase in glucose intolerance due to a HF diet occurs concurrently with an acute phase response but these effects are caused by different properties of the diet. The second increase in glucose intolerance occurs between 12 - 16 weeks of HF diet and is correlated with WAT and muscle inflammation. Between these times glucose tolerance remains stable and markers of inflammation are undetectable.     

Defining the In Vivo Phenotype of Artemisinin-Resistant Falciparum Malaria: A Modelling Approach

BackgroundArtemisinin-resistant falciparum malaria has emerged in Southeast Asia, posing a major threat to malaria control. It is characterised by delayed asexual-stage parasite clearance, which is the reference comparator for the molecular marker ‘Kelch 13’ and in vitro sensitivity tests. However, current cut-off values denoting slow clearance based on the proportion of individuals remaining parasitaemic on the third day of treatment (''day-3''), or on peripheral blood parasite half-life, are not well supported. We here explore the parasite clearance distributions in an area of artemisinin resistance with the aim refining the in vivo phenotypic definitions.ConclusionsCharacterisation of overlapping distributions of parasite half-lives provides quantitative insight into the relationship between parasite clearance and artemisinin resistance, as well as the predictive value of the 10% cut-off in ''day-3'' parasitaemia. The findings are important for the interpretation of in vitro sensitivity tests and molecular markers for artemisinin resistance and for contextualising the ‘day 3’ threshold to account for initial parasitaemia and sample size.     

Sequence variation does not confound the measurement of plasma PfHRP2 concentration in African children presenting with severe malaria

ABSTRACT: BACKGROUND: Plasmodium falciparum histidine-rich protein PFHRP2 measurement is used widely for diagnosis, and more recently for severity assessment in falciparum malaria. The Pfhrp2 gene is highly polymorphic, with deletion of the entire gene reported in both laboratory and field isolates. These issues potentially confound the interpretation of PFHRP2 measurements. METHODS: Studies designed to detect deletion of Pfhrp2 and its paralog Pfhrp3 were undertaken with samples from patients in seven countries contributing to the largest hospital-based severe malaria trial (AQUAMAT). The quantitative relationship between sequence polymorphism and PFHRP2 plasma concentration was examined in samples from selected sites in Mozambique and Tanzania. RESULTS: There was no evidence for deletion of either Pfhrp2 or Pfhrp3 in the 77 samples with lowest PFHRP2 plasma concentrations across the seven countries. Pfhrp2 sequence diversity was very high with no haplotypes shared among 66 samples sequenced. There was no correlation between Pfhrp2 sequence length or repeat type and PFHRP2 plasma concentration. CONCLUSIONS: These findings indicate that sequence polymorphism is not a significant cause of variation in PFHRP2 concentration in plasma samples from African children. This justifies the further development of plasma PFHRP2 concentration as a method for assessing African children who may have severe falciparum malaria. The data also add to the existing evidence base supporting the use of rapid diagnostic tests based on PFHRP2 detection.     

Cryptic genetic divergence in <Emphasis Type="Italic">Scolopsis taenioptera</Emphasis> (Perciformes: Nemipteridae) in the western Pacific Ocean

We studied the phylogeny, population structure, and demographic history of Scolopsis taenioptera in the western Pacific Ocean. Using the 80 samples collected from four locations, we obtained the nucleotide sequences of mitochondrial cytochrome c oxidase subunit I and cytochrome b genes. We identified two distinct lineages showing a clear phylogeographic break that was possibly due to the Pleistocene sea-level change. One lineage was distributed in Iloilo (Philippines) and the other in Terengganu (Malaysia), Rayong (Thailand), and Ha Long Bay (Vietnam). The Terengganu and Rayong populations showed clear signs of demographic expansion; the Iloilo and Ha Long Bay populations were relatively stable or spatially expanded as geographically subdivided populations.     

The effect of exercise induced cytokines on insulin stimulated glucose transport in C2C12 cells

Skeletal muscle contractile activity increases the production of the myokines interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-15 (IL-15) and also skeletal muscle glucose transport. Previous work has revealed a role for IL-6 in mediating glucose uptake, while research on the physiological roles of IL-8 and IL-15 is not so abundant. In the present study we investigated the effects of different concentrations and combinations of IL-6, IL-8 and IL-15 on insulin stimulated glucose transport in C2C12 cells. Furthermore, we also measured AMPK Thr172 and Akt Ser473 phosphorylation via Western blotting. Exposure to 20 pg/ml of individual cytokines had no affect on glucose transport while 1 ng/ml enhanced (P<0.05) glucose uptake with IL-6, IL-8 and IL-15, respectively. Moreover, the combinations of IL-8+IL-6 and IL-15+IL-6 at both 20 pg/ml and 1 ng/ml stimulated (P<0.05) glucose transport with IL-8+IL-15 and IL-8+IL-6+IL-15 only increasing (P<0.05) glucose transport at 1 ng/ml, with no affect observed of these combinations at 20 pg/ml. The changes in glucose transport were all associated with an increase (P<0.05) in AMPK Thr172 phosphorylation with no changes in Akt Ser473 phosphorylation. These findings demonstrated that the exercise induced myokines IL-6, IL-8 and IL-15 enhance glucose transport at 1 ng/ml, with changes only seen at 20 pg/ml with certain myokine combinations. Furthermore these changes in insulin stimulated glucose transport were associated with increased AMPK phosphorylation.