Date Published: May 01, 2019
Publisher: The American Society of Tropical Medicine and Hygiene
Author(s): Yutatirat Singhaboot, Srisuda Keayarsa, Nattaporn Piaraksa, Weerapong Phumratanaprapin, Parinya Kunawut, Arjen Dondorp, Kesinee Chotivanich.
Plasmodium falciparum infection causes febrile illness and severe disease with multiple organ failure and death when treatment is delayed. Antipyretic treatment is standard, and inducing hypothermia has been proposed to protect the brain in cerebral malaria. Here, we investigated the temperature dependence of asexual-stage parasite development and parasite multiplication in vitro. Plasmodium falciparum laboratory strain TM267 was incubated for 2 hours (short exposure) or 48 hours (continuous exposure) at different temperatures (32°C, 34°C, 35°C, 38°C, 39°C, and 40°C). The starting parasite developmental stage (ring, trophozoite, or schizont) varied between experiments. The parasite multiplication rate (PMR) was reduced under both hyper- and hypothermic conditions; after continuous exposure, the mean PMR ± SD was 9.1 ± 1.2 at 37°C compared with 2.4 ± 1.8 at 32°C, 2.3 ± 0.4 at 34°C, and 0.4 ± 0.1 at 40°C (P < 0.01). Changes in PMR were not significant after 2-hour exposure at temperatures ranging from 32°C to 40°C. Morphological changes in parasite cytoplasm and nucleus could be observed after long exposure to low or high temperature. After 48-hour incubation, rosette formation (≥ 2 uninfected red blood cells bound to infected red blood cells) was decreased at 34°C or 39°C compared with that at 37°C. In conclusion, both hyper- and hypothermia reduce PMR and delay erythrocytic stage development of P. falciparum, subsequently reducing rosette formation.
Plasmodium falciparum malaria remains a leading cause of death in the tropical world. Among all human malaria species, most cases of severe malaria with multiple organ failure are caused by this parasite.1–4 Fever is the key symptom; the classic description of a regular tertian pattern is observed in 25% of cases. Compared with adult patients, children are more prone to high fever (> 40°C), that is, often accompanied by febrile convulsions. Fever also contributes to nausea and vomiting, which may compromise treatment with oral antimalarial drugs. Because of this, antipyretic therapy with paracetamol or tepid sponging is recommended. However, it has been argued that antipyretic therapy with paracetamol prolongs the parasite clearance time after antimalarial treatment, although this was not confirmed in a more recent study.5,6 To assess the benefit of antipyretic therapy, it is important to determine whether temperature affects the growth and multiplication of asexual-stage P. falciparum parasites because the total body parasite biomass is one of the main determinants of disease severity.7,8 In vivo and in vitro studies suggest that parasites obtained from patients with severe disease have a higher parasite multiplication rate (PMR),9–12 and P. falciparum isolates from patients with severe malaria show higher in vitro PMRs than those with uncomplicated malaria.12 Previous studies have shown that hypothermic conditions (28–32°C) delayed the erythrocytic life cycle development of P. falciparum,13,14 whereas febrile temperature (40°C) inhibited parasite growth15,16 in vitro. In addition, it is important to assess the effect of temperature on parasitized red blood cell (PRBC) adhesion properties affecting microcirculatory blood flow. Microcirculatory impairment is central in the pathogenesis of severe falciparum malaria and is caused by cytoadherence of PRBCs to vascular endothelium (causing sequestration) and to uninfected red blood cells (RBCs) (causing rosette formation).17–19 Furthermore, it has been shown that fever causes PRBCs to cytoadhere earlier in their 48-hour asexual life cycle.20 In this study, we conducted a comprehensive systematic investigation of the impact of hyper- and hypothermic culture conditions on P. falciparum growth and rosette formation.
Blockage of the microcirculation by sequestered PRBCs is the central cause of organ failure in severe falciparum malaria. Other systemic manifestations, such as fever, are attributed to pro-inflammatory cytokines released in response to the parasite, plasmodial DNA, and red cell membrane products.25 Plasmodial DNA is presented through hemozoin produced by the parasite, which interacts with Toll-like receptor 9, leading to the release of pro-inflammatory cytokines that in turn induce cyclooxygenase-2-upregulated prostaglandins, subsequently causing fever.26,27 It should be noted that the pro-inflammatory cytokine response does not only cause fever, but can also contribute to endothelial changes including increased expression of receptors for PRBC adhesion28 and host cell apoptosis.29 In the present study, we show that continuous exposure of P. falciparum in an in vitro culture under hyperthermic conditions reduces the PMR and changes parasite morphology. High fever might contribute to parasite killing during falciparum malaria infection. A previous study has shown inhibition of in vitro growth of P. falciparum at 40°C, and our results support this finding.15 Short exposure to hyperthermia, mimicking a fever spike, did not significantly decrease the PMR in vitro. It had been reported that P. falciparum was least affected when incubated at high temperature for a short period (1–6 hours).16,30 This suggests that P. falciparum can resist short-term, but not long-term, hyperthermia associated with malarial infection. A possible mechanism is the effective heat shock protein (Hsp) response in P. falciparum. Heat shock protein is a 90-kDa protein complex (PfHsp90), which consists of PfHsp70, PfPP5, tubulin, and other proteins.31 PfHsp90 functions as an adenosine triphosphate-dependent molecular chaperone that is responsible for stabilizing misfolded proteins during heat stress. In this way, PfHsp90 protects the parasite when stressed by elevated temperatures.32