The Importance of Pathogen Load

It seems obvious that the number of pathogens should be important in the pathogenesis of an infectious disease [1,2]. The relationship between the pathogen load and severity is one of the most fundamental questions, and yet, strangely, one of the most difficult to answer [3]. One reason for this is that it is often rather difficult to determine the total pathogen load in an infected host, particularly in an infected human. Pathogens can be distributed, non-uniformly, throughout multiple different cell, tissue, or organ compartments of the body, many of which are difficult to sample. For this reason, we are often constrained by measuring pathogen load in the samples that are readily accessible such as blood, urine, and sputum, and we must assume that these are representative of total pathogen load. The success of this approach is borne out by the usefulness of these measurements to guide clinical management of patients with some infections [4,5], but it is also well recognised that these methods are imperfect [6,7]. Variations in both pathogen and host can alter distribution of the pathogen and the likelihood that a given pathogen burden will cause disease [1,2,8–11]. Imperfect estimation of pathogen load becomes a particularly important problem when trying to understand host responses to infection, and their role in pathogenesis. In order to interpret whether a host response is insufficient, appropriate, or excessive, it needs to be assessed in relation to the pathogen load that triggered it. In this issue of PLOS Pathogens, Nicholas Anstey and colleagues present an analysis of host responses in relation to pathogen load in both Plasmodium vivax and P. falciparum malaria [12]. These authors have previously been instrumental in demonstrating that along with P. falciparum, P. vivax is an important cause of severe malaria, morbidity, and mortality [13,14]. Their current findings help us to understand why and illustrate the importance of trying to determine pathogen load and distribution. Measurement of pathogen load in malaria might seem simple because illness only occurs during the phase of asexual parasite replication within red blood cells. Thus, malaria is often diagnosed by microscopic examination of a blood sample, and the simplest metric of pathogen load is assessment of parasitemia—the percentage of infected red blood cells (iRBCs). However, anemia is a common consequence of malaria, and so the pathogen burden in blood is more accurately quantified by calculation of the absolute parasite density in blood, taking account of the number of red blood cells per mL. Even this refinement can be very misleading with certain species of Plasmodium (notably P. falciparum and the common ‘‘mouse model’’ parasite P. berghei ANKA) because iRBCs can adhere to the endothelium, which lines small blood vessels (sequestration), resulting in their under-representation in circulating blood [15]. Methods have been developed to estimate total parasite load (parasite biomass) in P. falciparum malaria by measuring the plasma concentration of parasite molecules, which are released into the host circulation, and demonstrate that this is a much better predictor of severity than measurement of the circulating parasites alone [15–18]. In addition, the proportion of parasites that are sequestered is particularly high in some manifestations of severe malaria [11,15,16,19]. Anstey and colleagues propose a method to approximate the total parasite biomass in P. vivax malaria using the plasma concentration of parasite lactate dehydrogenase (LDH) [12]. Similar to the established finding in P. falciparum malaria, total P. vivax parasite biomass appears higher in severe than uncomplicated malaria, and also appears to be underestimated by counting parasites in the peripheral blood. Since P. vivax iRBCs exhibit much less endothelial adhesion than P. falciparum iRBCs [20], the authors propose that P. vivax may accumulate outside of the endothelium-lined compartments of the blood, possibly in the slow open circulation of the spleen. This explanation is appealing, but there are some important caveats. First, P. vivax biomass is assumed to be approximately proportional to the plasma concentration of parasite LDH, but this will only be true if this molecule is released evenly throughout the parasite lifecycle or if there is an even distribution of life cycle stages at any point in time (i.e., the infection is totally asynchronous). Whilst all subjects had over-representation of younger parasite stages in the blood, there was no difference in this distribution between subjects with severe and uncomplicated P. vivax malaria, meaning that comparison of parasite LDH between groups should be valid. Second, the rate of production and clearance of parasite LDH could vary between subjects with severe and uncomplicated malaria, resulting in differences in plasma concentration that are not solely due to parasite biomass. Thus, parasite LDH probably only gives a semi-quantitative estimate of P. vivax biomass. Despite these limitations, Anstey and colleagues are to be congratulated for applying this methodology to produce a unique assessment of the relationship between parasite biomass and the major determinants of severe malaria pathogenesis: inflammation, sequestration, and vascular endothelial dysfunction [15,19]. Their data are all the more remarkable because they compare large numbers of healthy controls and subjects with both P. falciparum and P. vivax malaria [12]. Whilst there appear to be many similarities between severe disease caused by both parasite species, it is only when parasite