A primate model of severe malarial anaemia: a comparative pathogenesis study

Severe malarial anaemia (SMA) is the most common life-threatening complication of Plasmodium falciparum infection in African children. SMA is characterised by haemolysis and inadequate erythropoiesis, and is associated with dysregulated inflammatory responses and reduced complement regulatory protein levels (including CD35). However, a deeper mechanistic understanding of the pathogenesis requires improved animal models. In this comparative study of two closely related macaque species, we interrogated potential causal factors for their differential and temporal relationships to onset of SMA. We found that rhesus macaques inoculated with blood-stage Plasmodium coatneyi developed SMA within 2 weeks, with no other severe outcomes, whereas infected cynomolgus macaques experienced only mild/ moderate anaemia. The abrupt drop in haematocrit in rhesus was accompanied by consumption of haptoglobin (haemolysis) and poor reticulocyte production. Rhesus developed a greater inflammatory response than cynomolgus macaques, and had lower baseline levels of CD35 on red blood cells (RBCs) leading to a significant reduction in the proportion of CD35+ RBCs during infection. Overall, severe anaemia in rhesus macaques infected with P. coatneyi has similar features to SMA in children. Our comparisons are consistent with an association of low baseline CD35 levels on RBCs and of early inflammatory responses with the pathogenesis of SMA.

Changes in monocyte subsets are associated with clinical outcomes in severe malarial anaemia and cerebral malaria

Monocytes are plastic heterogeneous immune cells involved in host-parasite interactions critical for malaria pathogenesis. Human monocytes have been subdivided into three populations based on surface expression of CD14 and CD16. We hypothesised that proportions and phenotypes of circulating monocyte subsets can be markers of severity or fatality in children with malaria. To address this question, we compared monocytes sampled in children with uncomplicated malaria, severe malarial anaemia, or cerebral malaria. Flow cytometry was used to distinguish and phenotype monocyte subsets through CD14, CD16, CD36 and TLR2 expression. Data were first analysed by univariate analysis to evaluate their link to severity and death. Second, multinomial logistic regression was used to measure the specific effect of monocyte proportions and phenotypes on severity and death, after adjustments for other variables unrelated to monocytes. Multivariate analysis demonstrated that decreased percentages of non-classical monocytes were associated with death, suggesting that this monocyte subset has a role in resolving malaria. Using univariate analysis, we also showed that the role of non-classical monocytes involves a mostly anti-inflammatory profile and the expression of CD16. Further studies are needed to decipher the functions of this sub-population during severe malaria episodes, and understand the underlying mechanisms.

Health Care Seeking Behavior among Caregivers of Sick Children Who Had Severe Malarial Anaemia

Aims: The western region in Kenya is holoendemic to malaria and experience stable P. falciparum malaria transmission. The use of health care options has a direct influence on the outcome of severe malaria. As such, the current study will assess the health care seeking behavior among caregivers of sick children who had severe malarial anaemia (SMA) in western Kenya. Study Design: Cross section study. Place and Duration of Study: The study was conducted at Jaramogi Odinga Oginga Teaching and Referral Hospital (JOOTRH) between September 2014 to July 2015. Methodology: It was open to all children ≤10 years (n=271) admitted and diagnosed with SMA (hemoglobin <5.0 g/dl and any density of P. falciparum. Caregivers were interviewed on the health care options before seeking care at a heath facility, when the child started to get sick, if they took child to another health centre/dispensary/private hospital before coming to JOOTRH Results: Majority of the caregivers interviewed, 80.07% (217) had attained Primary education. Majority of the caregivers were in the age category of 19-24 75(27.67%) years and 25-29 years 75 (27.67%). 74.90% (203) of their children were below five years and 25.09% (68) were above 5 years. 61.62% (167. Majority of the caregivers gave some remainder drugs before presenting to a heath facility 32.5% (88). A good number bought drugs at drug stores/pharmacies 27.7% (75). None visited a traditional healer. A minority used herbs 10% (27). There were no statistically significant differences between most of the pre-hospitalization measures taken s with regard to patient’s gender and age, and caretaker’s level of education. Caregivers who chose to give herbs to their sick children took longer in deciding to take their children to hospital. This was however statistically significant between those who used herbs and those who bought drugs only (median 4 days vs. 3 days, respectively, p = 0.0063). There was no significant difference in the delay of child admission at JOOTRH between caregivers who had had primary education only and those with a minimum of secondary education (p = 0.9842). Conclusion: Self-medication is a common practice before seeking care at a heath facility. There is need for community awareness for correct and comprehensive information about drawbacks associated with self-medication practices. Since safety continues to be a major issue with the use of herbal remedies, it becomes imperative, therefore, that relevant regulatory authorities put in place to ensure that all herbal medicines are safe and of suitable quality.

Collider bias and the apparent protective effect of glucose-6-phosphate dehydrogenase deficiency on cerebral malaria

Case fatality rates in severe falciparum malaria depend on the pattern and degree of vital organ dysfunction. Recent large-scale case-control analyses of pooled severe malaria data reported that glucose-6-phosphate dehydrogenase deficiency (G6PDd) was protective against cerebral malaria but increased the risk of severe malarial anaemia. A novel formulation of the balancing selection hypothesis was proposed as an explanation for these findings, whereby the selective advantage is driven by the competing risks of death from cerebral malaria and death from severe malarial anaemia. We re-analysed these claims using causal diagrams and showed that they are subject to collider bias. A simulation based sensitivity analysis, varying the strength of the known effect of G6PDd on anaemia, showed that this bias is sufficient to explain all of the observed association. Future genetic epidemiology studies in severe malaria would benefit from the use of causal reasoning.

Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is believed to confer protection against Plasmodium falciparum malaria, but the precise nature of the protective effect has proved difficult to define as G6PD deficiency has multiple allelic variants with different effects in males and females, and it has heterogeneous effects on the clinical outcome of P. falciparum infection. Here we report an analysis of multiple allelic forms of G6PD deficiency in a large multi-centre case-control study of severe malaria, using the WHO classification of G6PD mutations to estimate each individual’s level of enzyme activity from their genotype. Aggregated across all genotypes, we find that increasing levels of G6PD deficiency are associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia. Models of balancing selection based on these findings indicate that an evolutionary trade-off between different clinical outcomes of P. falciparum infection could have been a major cause of the high levels of G6PD polymorphism seen in human populations.