Diagnosing severe falciparum malaria in parasitaemic African children: a prospective evaluation of plasma PfHRP2 measurement

Abstract

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 log(10) plasma PfHRP2 and risk of death. Mortality increased 20% per log(10) 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.

Figure 1. Observed and modelled malaria-attributable mortality and morbidity according to plasma PfHRP2 concentrations.

(Top graph) Observed number of patients (grey bars, n = 3,826) and observed probability of death (squares with 95% CI error bars, n = 381) according to PfHRP2 half-log10 strata. The statistical polynomial regression model (dashed line) and the mechanistic model (black line) show the probability of death according to PfHRP2 half-log10 strata. For a detailed description of the mechanistic model see Text S2. (Bottom graph) Malaria-attributable mortality and morbidity according to plasma PfHRP2 concentrations. The curve derived from the mechanistic model (top) describing the relationship between log10 plasma PfHRP2 concentration and probability of death has been deconvoluted in two separate functions: (1) Non[en-dash]malaria-attributable probability of death (dotted line, left axis), which describes the negative exponential probability of dying from non-malaria illness with increasing plasma PfHRP2 concentrations, at a constant PfHRP2 independent case fatality rate of 30%. (2) Malaria-attributable probability of death (thin solid line, left axis), which describes the exponential increase in the probability of death with increasing plasma PfHRP2 concentration, a measure of total parasite burden, in the patient population with “true” severe malaria. From these deconvoluted functions the proportion of the total number of deaths attributable to “true” severe malaria was derived according to PfHRP2 half-log10 strata (diamonds and heavy solid line, malaria-attributable deaths, right axis). Using the “true” severe malaria case fatality rates per PfHRP2 half-log10 strata, the proportion of “true” severe malaria-attributable cases according to PfHRP2 half-log10 strata was derived (circles and dashed line, malaria-attributable cases).

Figure 2. Comparison of circulating parasite burden and total parasite burden between surviving (blue circles, n = 3,070) and fatal (red squares, n = 327) cases.

Circulating parasite burden was calculated from the peripheral blood parasitaemia and the total parasite burden was estimated from plasma PfHRP2, including 3,397 patients with both detectable PfHRP2 and malaria parasites on the peripheral blood smear.

Figure 3. Treatment effect, as odds ratio for death, of artesunate versus quinine.

Effect is measured according to plasma PfHRP2 tertiles and compared to the overall treatment effect observed in the AQUAMAT trial in 5,425 African children and in the similar SEAQUAMAT trial in 1,461 (predominantly) adults in Asia.