Plasma mEV levels in Ghanain malaria patients with low parasitaemia are higher than those of healthy controls, raising the potential for parasite markers in mEVs as diagnostic targets

Abstract

This study sought to measure medium-sized extracellular vesicles (mEVs) in plasma, when patients have low Plasmodium falciparum early in infection. We aimed to define the relationship between plasma mEVs and: (i) parasitaemia, (ii) period from onset of malaria symptoms until seeking medical care (patient delay, PD), (iii) age and (iv) gender. In this cross-sectional study, n = 434 patients were analysed and Nanosight Tracking Analysis (NTA) used to quantify mEVs (vesicles of 150-500 nm diameter, isolated at 15,000 × g, β-tubulin-positive and staining for annexin V, but weak or negative for CD81). Overall plasma mEV levels (1.69 × 10¹⁰ mEVs mL^-1) were 2.3-fold higher than for uninfected controls (0.51 × 10¹⁰ mEVs mL^-1). Divided into four age groups, we found a bimodal distribution with 2.5- and 2.1-fold higher mEVs in infected children (<11 years old [yo]) (median:2.11 × 10¹⁰ mEVs mL^-1) and the elderly (>45 yo) (median:1.92 × 10¹⁰ mEVs mL^-1), respectively, compared to uninfected controls; parasite density varied similarly with age groups. There was a positive association between mEVs and parasite density (r = 0.587, p < 0.0001) and mEVs were strongly associated with PD (r = 0.919, p < 0.0001), but gender had no effect on plasma mEV levels (p = 0.667). Parasite density was also exponentially related to patient delay. Gender (p = 0.667) had no effect on plasma mEV levels. During periods of low parasitaemia (PD = 72h), mEVs were 0.93-fold greater than in uninfected controls. As 75% (49/65) of patients had low parasitaemia levels (20-500 parasites µL^-1), close to the detection limits of microscopy of Giemsa-stained thick blood films (5-150 parasites µL^-1), mEV quantification by NTA could potentially have early diagnostic value, and raises the potential of Pf markers in mEVs as early diagnostic targets.

Keywords: Malaria; extracellular vesicles; parasitaemia.

Figure 1.

Plasma mEVs in P. falciparum infected patients.

Figure 2.

Plasma mEV levels are significantly higher, at low parasitaemia levels, in infected young Ghanain children and the elderly.

Figure 3.

Hyperbolic relation between mEVs released and parasitaemia with significantly increased parasitaemia levels in the young and elderly.

Figure 4.

Plasma mEV levels in infected patients are related to patient delay and significantly raised early in infection.

Figure 5.

Median plasma mEV levels in infected patients and uninfected donors show no differences between males and females.