Endotoxaemia is common in children with Plasmodium falciparummalaria

Endotoxin is a complex lipopolysaccharide (LPS) present in the cell walls of gram-negative bacteria and in substantial quantities in the bowel, and a potent trigger of innate immunity due to activation of Toll Like Receptor (TLR) signalling [1]. The effects of endotoxin in sepsis are wide ranging, including upregulation of endothelial cytoadherence receptors [2], anaemia and various metabolic disturbances such as acidosis and hypoglycaemia. Endotoxin acts as a potent stimulus for the release of pro-inflammatory cytokines such as TNFα, IL1 and IL6 that have been implicated in the pathophysiology of severe infectious diseases, including malaria. Nevertheless, establishing the relationship between endotoxaemia and infection has been frustrated by the limitations of available endotoxin assays [3]. In adults with critical illness, recent studies have shown that endotoxaemia is associated with mortality, organ failure and prolonged intensive care unit admission [4]. In children, endotoxaemia has been associated with more severe illness requiring admission to intensive care, severity or duration of illness but not mortality [5, 6].

Severe forms of Plasmodium falciparum malaria are characterised be excessive inflammation, organ damage and metabolic dysregulation, a picture similar to that observed in patients suffering from severe bacterial sepsis [7]. Malaria infection strongly predisposes African children to invasive bacterial disease (IBD). Whilst the epidemiological data supporting this causal link are robust the mechanism for this association remains obscure. The leading causes of IBD in P. falciparum infection are non-typhoidal Salmonellae (NTS), E. coli and other enteric organisms with gram-negative organisms (EGNO) becoming increasingly more predominant across the severity spectrum: from those with evidence of recent malaria infection (slide negative, Paracheck-Pf® positive), intercurrent malaria and greatest in those with severe malaria and hyperparasitaemia [8–12]. Although the mechanisms underlying the increased susceptibility are uncertain, they may include disordered gut barrier function secondary to intense sequestration of parasitized red cells in the intestine [13–15] and/or acute severe perfusion injury secondary to severe anaemia (haemoglobin < 5 g/dl) and shock. A recent study in a rodent model of malaria showed that induction of heme oxygenase I in neutrophils during acute malarial anaemia resulted in loss of function and was associated with increased susceptibility of mice to Salmonella typhimurium bacteraemia [16]. These observations have now been corroborated in children with malaria showing that neutrophil dysfunction was associated with parasite biomass and markers of hemolysis [17].

We hypothesised that either translocation of endotoxin alone or translocation of gram-negative bacteria and subsequent release of endotoxin into the blood stream may play a role in pathogenesis of severe malaria and its complications such as shock and organ dysfunction. We prospectively examined endotoxin levels and its relationship to disease severity and cytokine activation in children with malaria at two hospitals in East Africa.

To our knowledge this is the first study to document the prevalence of endotoxaemia in children suffering from malaria. Approximately 30% of children with malaria had detectable levels of endotoxin (LPS) in their blood. Surprisingly, and contrary to findings in other critical illnesses but not all paediatric studies [6], we found no evidence that endotoxaemia or high levels of endotoxaemia (EAA> 0.6) were associated with the degree of disease severity or led to a worse outcome. These findings suggests that endotoxaemia may be important in the transition of an uncomplicated episode of malaria in children living in endemic regions to requiring hospitalisation but does not distinguish those at greatest risk of life-threatening complications or organ dysfunction, as in adults with severe sepsis [4].

In our study, the most notable clinical parameter associated with endotoxaemia was anaemia. The origins of malaria anaemia are multiple. Next to acute vascular haemolysis of infected erythrocytes during acute malaria, both acute and chronic infection cause inflammation resulting in dyserythropoiesis due to changes in iron storage metabolism and cytokine-mediated suppression of the generation of progenitor cells [24–26]. It seems unlikely that impaired oxygen delivery to the bowel as a result of anaemia, directly causes endotoxaemia but rather that inflammation is independently associated with anaemia and increased gut permeability. Additional evidence comes from studies in patients suffering from sepsis and of endotoxaemia induced in human volunteers, which suggested that inflammation resulted in enterocyte death, increased gut permeability or both [27, 28]. I-FABP is particularly highly expressed in cells present on the tips of the villi, the initial site of ischaemia reperfusion injury, indicating that circulating I-FABP is a useful plasma marker for identifying bowel injury secondary to splanchnic hypoperfusion [22]. This association has been demonstrated in both murine and human studies of ischaemia reperfusion injury [23, 29]. We therefore tested the plasma concentration of I-FABP in children with and without endotoxaemia. In our study, I-FABP was detected in a similar proportion of patients with or without endotoxaemia, contrary to the findings in other studies [5]. However, we may have missed a direct association between endotoxaemia and I-FABP because the half-life of I-FABP is estimated to be 11 min [30] and plasma I-FABP concentrations reflect immediate enterocyte damage rather than recent cell damage. By contrast, pathological concentrations of I-FABP were associated with increased plasma concentrations of TNFα, IL6 and IL10 suggesting that at least in a proportion of children, enterocytes and thus the gut barrier function are damaged due to inflammation.

We hypothesised that the most likely source of endotoxaemia is the gastrointestinal tract. Gastro-intestinal symptoms are common in malaria yet there has been little recent interest in gut barrier dysfunction despite evidence provided over 2–3 decades ago demonstrating histopathological changes and impaired absorption [13, 14, 31, 32]. Histopathological studies have demonstrated intense sequestration of P. falciparum-infected erythrocytes within the endothelial bed of the gut, particularly at the tip of intestinal villi [13, 14] and the presence of small bowel intussusceptions in children with severe malaria [15] has been reported. The central pathological processes of falciparum malaria include increased rigidity (or non-deformability) of non-parasitized red cells [33] and intense sequestration of late stage infected erythrocytes in venules and capillaries- both of which compromise microcirculatory flow to vital organs and alter endothelial cell function [34]. Sequestration may favour direct translocation of gram-negative bacteria or, through induction of inflammation, lead to injury and an impaired gut barrier function with the transfer of endotoxin and/or pathogenic bacteria into the blood stream.

Sequestration is mediated by parasite molecules expressed on the surface of infected erythrocytes that mediate their adhesion to receptor expressed on endothelial cells [35]. Interestingly, infected erythrocytes from children suffering from severe anaemia usually show reduced adhesion to these receptors [36, 37] which may increase the release of inflammatory cytokines in these patients [38] providing another link between inflammation and anaemia. In addition, during acute blood stage malaria TLR agonists such as parasite DNA and GPI are released [39, 40] and may increase responsiveness of leukocytes to secondary challenge with TLR ligands, at least in uncomplicated malaria [41, 42]. Both factors independently increase inflammation and may lead to inflammation-induced anaemia and loss of gut barrier function resulting in the translocation of endotoxin and/or bacteria into the blood stream.

Together these data suggest that parasite-mediated sequestration and inflammation contribute to both anaemia and changes in gut permeability. However, particularly TLR-mediated induction of inflammation leads to altered responsiveness of leukocytes to additional signals such as endotoxin-tolerance. In our study, endotoxaemia was associated with decreased plasma concentrations of TNFα, IL6 and IL10 compared to those without endotoxaemia [43]. In sepsis, it has been shown that patients may enter a phase of immune-paralysis signified by control of inflammation, alternate signalling pathways in leukocytes and enhanced apoptosis of immune effector cells [44]. Although the control of inflammatory responses reduces organ damage and early mortality, it comes at the expense of increased vulnerability to infection due to either activation of latent viruses or secondary infection with pathogens and increased risk of mortality [44–46]. Thus, endotoxin-mediated depression of inflammatory immune responses may contribute to decreased ability of malaria patients to control secondary bacterial infection.

There is strong evidence that recent or current infection with malaria increases the risk of systemic bacterial infection with high associated mortality rates in several sub-Saharan countries [8, 9, 11, 47, 48]. Of note, a study in Tanzanian children with recent or current malaria demonstrated that one of the key bedside predictors of IBD (largely NTS and other Gram-negative organisms) was severe anaemia [12]. Recent studies suggested that that the association between NTS and anaemia may be due to changes in neutrophil function as a result of changes in iron storage metabolism and subsequently inefficient control of pathogenic bacteria once they have entered the blood stream [17].

Our study is limited by the absence of blood culture data and parasite density data, which were available only for a very small number of children. All but one child with blood culture data were negative for gram-negative organisms suggesting that endotoxaemia resulted from endotoxin translocation due to gut barrier dysfunction. Despite the small number of children with blood culture data, this appears plausible since the prevalence of endotoxin observed in this study was much higher than previous reports of bacterial co-infection, which are in the order of 5-12% [8–12]; but this is confounded by the low sensitivity of blood culture and preadmission antibiotic use. Furthermore, we measured endotoxaemia and plasma cytokines only at admission of children with acute disease and therefore can only speculate on the link between endotoxaemia, inflammation and subsequent immune-paralysis. Repeat sampling from the early stages of infection through to acute illness and resolution of malaria would be required to elucidate the complex interactions of inflammatory pathways and their regulation.

We suggest the following hypothesis: A proportion of children suffering from malaria experience loss of the barrier function of the gut due to parasite sequestration (and reduced microcirculatory flow), inflammation or both. This could be augmented by other pathophysiological processes, specific to falciparum malaria including severe anaemia and those leading to microvascular obstruction. In some children, loss of gut barrier function might be sufficient to allow translocation of endotoxin and translocation of gram-negative bacteria with subsequent release of endotoxin into the bloodstream. Endotoxin and other TLR–ligands expressed by gram-negative bacteria could further increase malaria-induced inflammation followed by regulatory immune responses that result in temporary immune paralysis as has been described for sepsis. Then children, having entered a phase of immune paralysis, might not be able to mount an adequate immune response controlling bacteraemia. This hypothesis is testable through investigation of gut-barrier function in children suffering from malaria, characterisation of the cytoadhesion profile of clinical parasite isolates from children with malaria and endotoxaemia and by analysing differences in the innate and adaptive immune response pathways in children with severe malaria in the presence or absence of endotoxaemia.