Evaluation of a novel magneto-optical method for the detection of malaria parasites

Improving the efficiency of malaria diagnosis is one of the main goals of current malaria research. We have recently developed a magneto-optical (MO) method which allows high-sensitivity detection of malaria pigment (hemozoin crystals) in blood via the magnetically induced rotational motion of the hemozoin crystals. Here, we evaluate this MO technique for the detection of Plasmodium falciparum in infected erythrocytes using in-vitro parasite cultures covering the entire intraerythrocytic life cycle. Our novel method detected parasite densities as low as approximately 40 parasites per microliter of blood (0.0008% parasitemia) at the ring stage and less than 10 parasites/microL (0.0002% parasitemia) in the case of the later stages. These limits of detection, corresponding to approximately 20 pg/microL of hemozoin produced by the parasites, exceed that of rapid diagnostic tests and compete with the threshold achievable by light microscopic observation of blood smears. The MO diagnosis requires no special training of the operator or specific reagents for parasite detection, except for an inexpensive lysis solution to release intracellular hemozoin. The devices can be designed to a portable format for clinical and in-field tests. Besides testing its diagnostic performance, we also applied the MO technique to investigate the change in hemozoin concentration during parasite maturation. Our preliminary data indicate that this method may offer an efficient tool to determine the amount of hemozoin produced by the different parasite stages in synchronized cultures. Hence, it could eventually be used for testing the susceptibility of parasites to antimalarial drugs

Using the hemozoin conversion rates reported in the literature for the different parasite stages (rings: 3–5%, trophozoites: 15–20%, and schizonts: 50–70%) [20]–[22], [29], [30] and the stage distribution of the parasites (Fig. 1), we estimated the hemozoin content of culture A (ring stage culture) and culture B (schizont stage culture).

<p>The lower and upper values of the hemozoin content correspond to the lower and upper values of the conversion rates quoted above. Note that the cultures have different parasite densities. We also estimated the hemozoin concentration of the two cultures based on MO signal using the conversion factor c_HZ = 1 ng/<i>µ</i>L →  = 1.4% between the hemozoin concentration and the low-frequency (∼1 Hz) MO signal previously determined for artificial hemozoin crystals suspended in blood <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096981#pone.0096981-Butykai1" target="_blank">[15]</a>.</p

Distribution of parasite life cycle stages in the two <i>Plasmodium falciparum</i> cultures used in the present study.

<p>Panel A: The <i>ring stage culture</i> contained early rings, late rings and some early trophozoites of the first generation after synchronization. The <i>schizont stage culture</i> was on the verge of the first and second life cycles where most of the schizont stages have already turned to early ring stages of the second generation following invasion. Therefore, the ring stage culture contained only the hemozoin present in the parasites up to the early trophozoite stage, while the schizont stage culture had the entire hemozoin content formed during one generation of parasites with the largest portion produced by schizonts. Panel B: Light microscopy images of Giemsa stained thin blood films containing infected red blood cells with parasites in different stages of maturity (taken from these two cultures). In both panels the labels ER, LR, ET, LT, ES and LS correspond to early-ring, late-ring, early-trophozoite, late-trophozoite, early-schizont and late-schizont stages, respectively.</p

Magneto-optical (MO) detection of parasitemia in synchronized <i>Plasmodium falciparum</i> cultures.

<p>Panel A: Red and blue curves show the frequency dependent MO signal for samples from the ring and schizont stage cultures, respectively, with various levels of parasite density given in <i>µ</i>L⁻¹ units on the right of the respective curves. The green curves shows the signal from uninfected reference samples. Data plotted with triangles and diamonds are the residual signal from freshly hemolyzed uninfected blood and water, respectively. The frequency scale corresponds to the rotation speed of the magnetic field. Panel B: Red and blue squares in panel B are the MO signal values measured at 20 Hz – indicated by a vertical solid line in panel A – for the dilution series prepared from the original ring and schizont stage cultures, respectively. Solid and open squares correspond to the duplicate samples labeled as samples #1 and samples #2. Triangles indicate the results obtained by remeasuring samples #1 with 24 h delay. The solid lines following the trend of the MO signal at higher parasite densities for ring (red line) and schizont (blue line) samples are guides for the eye. For ring and schizont stage samples with parasite densities lower than 10 parasites/<i>µ</i>L and 1 parasites/<i>µ</i>L, respectively, the MO signal does not further decrease. The green horizontal line shows the residual MO signal of uninfected blood, which is the mean detection limit of our method. The 95% confidence levels of this mean detection limit for the ring and schizont stage samples are indicated by red and blue dashed lines, respectively. Correspondingly, for ring and schizont stage samples with parasite density higher than 40 parasites/<i>µ</i>L and 10 parasites/<i>µ</i>L, respectively, the diagnosis is positive with a confidence of at least 95%. The background signal for freshly hemolyzed uninfected blood and water are also shown by dark and light grey lines. All these horizontal indicators are also shown in panel A for reference. The upper horizontal scale shows the corresponding levels of parasitemia.</p