arXiv:1311.4103v1 [physics.med-ph] 16 Nov 2013 ih irsoyhv enrpre ob approximately be to parasites/ 100 reported cells been have microscopy red light smears. infected blood of in observation and of (RDT) microscopic tests detection the diagnostic antigen-based methods rapid using the diagnostic parasites are main practice two pre- in in- based, currently the and symptom clinical diagnosis, purely for from sumptive a Apart feasible only are diagnosis. malaria, approaches field these of of diagnosis few high-sensitivity at aiming yee yREVT by Typeset 0%o aai cases. malaria laboratories of diagnostic % 50 routine parasites/ 50 most approximately practice, achieve microscope, In high-powered etc.). microscopist, trained film, blood highly (good-quality conditions ideal threshold under detection achieved the time parasites/ 5 Moreover, is smears of intensive. blood endemic of labor inspection in and visual detect common the very to ii) are settings, sensitivity which sufficient infections possess low-level they not affordable, do and parasitemia more presently of measure becoming quantitative a are provide cannot RDTs limitations: inherent although to subject i) are methods these of Both hi ecin(C)asy ups h efrac of performance the surpass assays (PCR) reaction chain 6 neto n muiyDvso,Wle n lz alInst Hall Eliza and Walter Division, Immunity and Infection lhuhteei ltoao mrigtechniques emerging of plethora a is there Although mn oeua ilg-ae ehd,polymerase methods, biology-based molecular Among 1 7 eateto hsc,Bdps nvriyo ehooya Technology of University Budapest Physics, of Department odne atrRsac ru fteHnainAaeyof Academy Hungarian the of Group Research Matter Condensed oteedo h aaielf yl,w eosrt htour that demonstrate we cycle, life parasite rin as the low mostly as of containing densities end first the the to cultures, parasite two Using aiaeti ehiu nlbrtr eie lo sample blood derived laboratory magnetica on their technique via this crystals validate (hemozoin) wh pigment method (MO) malaria magneto-optical of a developed recently have We a ffra ffiin olt td eoonfrain rc h numbers: trace PACS antima formation, new hemozoin to study parasites to of tool susceptibility/resistance efficient test an intracellular offer release trainin to may special solution no lysis blo de inexpensive requires The capillary an tests, for of in-field concentrations. and microliter ppm 50 clinical to for as down little micro-crystals observa as hemozoin microscopic with light performed by performanc achievable be the threshold exceeds the limit with detection This respectively. µ µ mrvn h ffiinyo aai igoi soeo h mai the of one is diagnosis malaria of efficiency the Improving n -0parasites/ 5-50 and L srte hoeia n a nybe only can and theoretical rather is L 3 4 1–4 atradEiaHl nttt fMdclRsac,Parkvi Research, Medical of Institute Hall Eliza and Walter eateto eia ilg,Uiest fMlore Pa Melbourne, of University Biology, Medical of Department E oaigcytlMlraDanss r-lnclvalida Pre-clinical Diagnosis: Malaria Rotating-crystal X 2 .Orb´an,A. eateto rlBooy emlesUiest,H-1089 University, Semmelweis Biology, Oral of Department h eeto iiso D and RDT of limits detection The Introduction 6,8,9 5 n eiieJmsCo nvriy oga uesad481 Queensland Douglas University, Cook James Medicine and uesadToia elhAlac utainInstitute Australian Alliance Health Tropical Queensland .Hill, D. ∼ 1 ,4 3, 0and 40 .Butykai, A. .M¨uller,I. µ µ n eetabout detect and L ,respectively. L, ∼ 0prstsprmcoie fbodfrrn n ciotst schizont and ring for blood of microliter per parasites 10 1 3 Dtd oebr1,2013) 19, November (Dated: s Pr¨ohle, Zs. .Schofield, L. 1,5–7 1 tt fMdclRsac,Prvle itra35,Austr 3052, Victoria Parkville, Research, Medical of itute ,5 3, .F¨ul¨op,G. ob qiaett aaiedniyof density parasite a to equivalent be to uigtern tg n h ihs mutduring amount highest the erythrocytes and in stage present with ring hemozoin parasites; the stage of during blood amount the least of depen- the maturity is the content on straight- hemozoin dent not is Intraerythrocytic infections human forward. in density parasite and of the amount into released whole is suspension. the cell parasites lysed the that by provided produced hemozoin blood infected in Tsadlgtmicroscopy. light and RTDs ehdcndtc eooncnetain onto down concentrations hemozoin that diagnostic pg/ hemo- detect 15 demonstrated (MO) can synthetic blood magneto-optical method using in rotating-crystal study suspended the recent crystals Our zoin from derived para- digestion. free malaria detoxify by they as produced sites compound heme crystalline sa lentv agto pia igoi a been has diagnosis optical groups. of several it use by target to proposed alternative and an (hemozoin) pigment as malaria of properties 1 conditions. field in under parasites diagnosis routine few de- for a a has to blood, PCR corresponding real-time limit that contamination.tection conclude to studies prone trained are Recent and highly reagents, personnel and laboratory equipment expensive quire eoon eoddansis hstechnique this diagnostics, Beyond hemozoin. oee,terlto ewe eoonconcentration hemozoin between relation the However, h dat aeavnaeo h nqemagnetic unique the of advantage take to idea The .Karl, S. fteoeao rseicraet,except reagents, specific or operator the of g aildusihbtn eoonformation. hemozoin inhibiting drugs larial frpddansi et n competes and tests diagnostic rapid of e 13,14 µ netdwith infected s ino lo mas u ehdcan method Our smears. blood of tion l nue oaini lo.Hr,we Here, blood. in rotation induced lly tgsadtescn corresponding second the and stages g L. dEoois 11Bdps,Hungary Budapest, 1111 Economics, nd dadi estv otepeec of presence the to sensitive is and od 1 15 6 ie eindt otbeformat portable a to designed vice, c loshg-estvt detection high-sensitivity allows ich ol fcretmlraresearch. malaria current of goals n eetees ti o e rcia method practical a yet not is it nevertheless, .Zelles, T. oe ehdcndtc parasite detect can method novel mzi ieisi h oyand body the in kinetics emozoin hstrsodcnetainwsestimated was concentration threshold This n .K´ezsm´arki I. and cecs 11Bdps,Hungary Budapest, 1111 Sciences, l,Vcoi 02 Australia 3052, Victoria lle, kil,Vcoi,Australia Victoria, rkville, fToia Health Tropical of uaet Hungary Budapest, 2 lsoimfalciparum ,Australia 1, .Forsyth, W. 10,11 15–19 ,7 1, g parasites, age tion oee,te fe re- often they However, eooni micro- a is Hemozoin 3 ≤ 0parasites/ 30 . alia µ µ 12 L L 2 the schizont stage.20–22 Therefore, the hemozoin concen- A 1st life cycle (approx. 48 h) 2nd life cycle tration in blood derived from human infections depends on the parasite stage distribution at the time the blood sample was collected. In P. falciparum (the most lethal parasite species) infections often only the ring and early trophozoite stages are found in the peripheral circula- tion, since the later developmental stages cytoadhere to the vascular endothelium.23

Moreover, the MO signal recorded by our technique depends not only on the amount but also on the size and morphology of the hemozoin crystals, which can be differ- ent for synthetic and naturally grown crystals.24–26 The MO method is sensitive only to those hemozoin crystals released into suspension, which can be magnetically ro- ‘ring’ culture ‘schizont’ culture tated. Correspondingly, aggregation of crystals or their binding to other components of lysed infected blood, such B ER LR ET as cell membranes, could substantially decrease the sen- sitivity of our technique. Signal loss may be avoided by appropriate lysis and treatment of blood samples prior to measurement.

LT ES LS

In the present study we aimed to address these key issues and to validate the rotating-crystal malaria di- agnosis method using synchronized cultures of P. falci- parum. (For a short description of the detection scheme see Materials and Methods section.) For this purpose, FIG. 1: Distribution of parasite life cycle stages in we investigated its sensitivity and detection threshold the two cultures used in the for two cultures with different maturity distributions present study. Panel A: The ring stage culture contained of the parasites. The first, hereafter referred to as early rings, late rings and some early trophozoites of the first the ring stage culture, contained mostly ring stages and generation after synchronization, while the schizont stage cul- some early trophozoites with a total parasite density of ture corresponded to the end of the first life cycle where most P≈3.1×105 parasites/µL. It is representative to the dis- of the schizont stages have already turned to early ring stages of the second generation after synchronization. Therefore, the tribution of parasite blood stages most often encoun- ring stage culture contained only the hemozoin present in the tered in P. falciparum infections. The second with a 4 parasites up to the early trophozoite stage, while the schizont lower total parasite density of P≈2.8×10 parasites/µL stage culture had the entire hemozoin content formed over corresponds to the end of the parasite life cycle where one generation of parasites with the largest portion produced some of the parasites are still in the schizont form but by schizonts. Panel B: Light microscopy images of Giemsa most of them, following an invasion, already turned to stained thin blood films containing infected red blood cells early-stage rings of the next generation. The distribu- with parasites in different stages of maturity (taken from these tions of the parasites among the different stages – early- two cultures). In both panels the labels ER, LR, ET, LT, ES ring, late-ring, early-trophozoite, late-trophozoite, early- and LS correspond to early-ring, late-ring, early-trophozoite, schizont and late-schizont stages – in the two cultures late-trophozoite, early-schizont and late-schizont stages, re- are displayed in Fig. 1 together with light microscope spectively. images of parasites representative to these stages. Since the early-stage rings do not contain hemozoin, the hemo- zoin content present in the second culture is formed in Results the first cycle before the invasion mostly during the sch- izont stage.20–22 For this reason, hereafter we refer to this The MO signal, the measure of hemozoin content culture as the schizont stage culture. Schizont stage para- within the lysed cell suspension, is shown in Fig. 2 for di- sites are not normally found in the peripheral blood dur- lution series of the ring and schizont stage cultures. The ing human P. falciparum infection due to their seques- 20 serial 2-fold dilutions using uninfected erythrocytes al- tration in small capillary blood vessels.23 However, this lowed for MO signal to be assessed over 6 orders of mag- stage restriction is not present in other non-sequestering nitude of parasitemia. As a general trend in Fig. 2A, the species of human malaria parasites such as P. vivax.27 MO signal varies proportionally to the parasitemia level 3

B Parasitemia [%] A

-5 -4 -3 -2 -1 0

10 10 10 10 10 10 0 0

10 10 -1

Ring / Schizont conc. [ L ]

Ring / Schizont samples:

4

1.4×10

/ samples #1

5

/ samples #1 24h later 1.6×10

/ samples #2 3

-1 3.5×10 -1

10 10

4

1.9×10 Hz [%] Hz

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3 T/T [%] T/T

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schizont 95%

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-3 -3

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ring 95% confidence 7.5×10

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culture residual MO signal,MO

4.3×10

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-4 -4

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water residual

0 1 2 3 4 5

10 100 10 10 10 10 10 10

-1

Frequency [Hz] Parasite density [ L ]

FIG. 2: Magneto-optical (MO) detection of parasitemia in synchronized Plasmodium falciparum cultures. 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 µL−1 units on the right of the respective curves. For ring stage samples with parasite densities lower than 10 parasites/µL the MO signal does not further decrease. Data plotted with triangles and diamonds are the residual signal from freshly hemolyzed uninfected blood and water, respectively. The frequency scale denotes twice the rotation frequency of the magnetic field, i.e. the frequency of the modulated intensity. 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. The black horizontal line represents the mean residual MO signal for the cultures, while 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/µL and 10 parasites/µL, 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. and the signal for each sample shows a gradual decrease P ≤10 parasites/µL from the ring stage culture, the sig- with increasing frequencies of the rotating magnetic field. nal does not further drop with decreasing parasitemia. This frequency dependence is in agreement with previous The frequency dependence of the MO signal for these ring results obtained when using synthetic hemozoin crystals stage samples also becomes different; the low-frequency suspended in blood and originates from the viscosity of saturation common for higher concentrations does not the lysed cell suspension hindering fast rotations of the hold anymore. These imply a residual MO signal not crystals. related to hemozoin. As schizont stages contain more hemzoin than ring In order to determine the detection limit of our stage-parasites, samples from the diluted schizont stage method, the results obtained for the dilution series of culture exhibited higher signals than ring stage sam- the ring and schizont stage cultures are summarized in ples with comparable parasitemia. Although the over- Fig. 2B, where the MO signal at 20Hz is plotted ver- all frequency dependence is similar for the two cultures, sus the parasitemia (and parasite density). Sequential the decrease in the signal with increasing frequency is measurements performed on the same sample with time more pronounced for schizont stage samples, which is delays less than one hour gave identical results. In several likely due to the larger crystal size in these samples. At cases we checked the reproducibility of the protocol by low levels of parasite density, namely for samples with repeating the measurement for both of the duplicate sam- 4 ples labeled as samples #1 and samples #2 in Fig. 2B. For ring stage samples with P ≤10 parasites/µL, where the MO signal shows no systematic variation with de- A creasing parasite density, we calculated the mean value of the residual MO signal (≈3.6×10−4 %) and its standard deviation (≈1.5×10−4 %). Assuming Gaussian distribu- tion for the residual MO signal values, we found that B E the 95 % confidence level of the mean detection limit for Hz ring stage samples is ∆T/T ≈6.6×10−4 %, which corre- FVM ∼ sponds to a parasite density of 40 parasites/µL. This is * EM equivalent to the parasitemia level of 8×10−4 %. Since the reproducibility is poorer for schizont stage samples, * PM in this case our rough estimate for the 95 % confidence M level of the mean detection limit is considerably higher C F with ∆T/T ≈2×10−3 %, which corresponds to the par- asite density of approximately ∼10 parasites/µL and a EM −4 M parasitemia level of ∼2×10 %. Note that the repro- * PM ducibility between duplicate samples does not signifi- Hz cantly vary with parasite density for dilutions of either * the ring or the schizont stage cultures. * * HbTV The hemozoin content of the two cultures can be D G roughly estimated from the parasite density and the stages of parasite development specified in Fig. 1. Ring FIG. 3: Hemozoin crystals observed by electron mi- and early trophozoite stages up to 24h were reported croscopy in-situ and after isolation. The transmission to convert about 3-15% of the total hemoglobin in 20,21 electron microscopy (TEM) image in panel A and scanning the infected red blood cells to hemozoin, while at electron microscopy (SEM) images in panels B, C and D show the schizont stage this portion is increased to approxi- hemozoin crystals extracted from the samples previously used mately 50-80 %.28–30 According to the 3-15 % hemoglobin for the MO measurements. The granular background of the conversion rate reported for rings and early tropho- SEM images comes from the gold coating of the glass sub- zoites, the undiluted ring stage culture with total par- strate. Individual crystals in panels C and D are marked with asite density of P=3.1×105 parasites/µL contained ∼9- asterisks. The length of the black bar at the bottom right 48ng/µL hemozoin. The undiluted schizont stage cul- corner of each panel is 500 nm. The typical length of the ture, which had about 10 times lower parasite density extracted hemozoin crystals ranges from 200 nm to 500 nm. 4 For comparison, a TEM image of synthetic hemozoin crystals with P=2.8×10 parasites/µL, contained all the hemo- 15 used in our previous MO study is shown in panel E. These zoin formed during the first cycle. Using the hemoglobin synthetic crystals have more elongated shape with a typical conversion rates quoted above, this corresponds to ∼14- length of 500-900 nm. Panels F and G display TEM images 23ng/µL hemozoin. An independent estimate, based on of intact infected erythrocytes in the schizont stage. Distinct the MO signal yields approximately 6 ng/µL and 9ng/µL components of the parasite and the erythrocyte can be ob- for the undiluted ring and schizont stage cultures, re- served including the erythrocyte membrane (EM), parasite spectively. For this estimate, we used the conversion fac- membrane (PM), food vacuole membrane (FVM), merozoites tor cHz=1ng/µL → ∆T/T =1.4 % between the hemozoin (M), hemoglobin transport vesicles (HbTV), knobs (K) and concentration and the low-frequency (∼1 Hz) MO signal hemozoin (Hz). previously determined for artificial hemozoin crystals sus- pended in blood.15 Note that the 20-fold dilution of the samples prior to the MO measurement needs to be taken The first scenario is supported by the electron mi- into account, since this conversion factor applies for sam- croscopy images in Fig. 3, where hemozoin crystals inside ples with 50 % hematocrit. of infected erythrocytes and as extracted from the cul- We also estimate the hemozoin concentration of the tures are shown. These elongated crystallites are consid- two undiluted cultures based on MO signal using the con- erably smaller (with typical length of ∼200-500nm) than version factor cHz=1ng/µL → ∆T/T =1.4% between the the synthetic ones (∼500-900 nm) previously studied and hemozoin concentration and the low-frequency (∼1 Hz) also displayed in the figure.15 This is also in accordance MO signal previously determined for artificial hemozoin with the weaker frequency dependence of the MO signal crystals suspended in blood15. This yields approximately observed in the present study, which indicates that natu- 6ng/µL and 9ng/µL for the undiluted ring and schizont ral crystals are able to follow the rotation of the magnetic stage cultures, respectively. Note that the 20-fold dilu- field up to higher frequencies than the synthetic ones as a tion of the samples prior to the MO measurement needs possible consequence of their reduced size. Furthermore, to be taken into account, since this conversion factor ap- the comparison between the natural crystals within the plies for samples with 50 % hematocrit. parasites and those extracted from the cultures confirms 5 no major change either in the size or in the morphology In the present study, the detection limit of our of the crystals due to our lysis-sonication protocol. rotating-crystal MO diagnostic device was found to be In order to minimize binding and aggregation of crys- ∼40 parasites/µL and ∼10 parasites/µL for ring and sch- tals we repeated the measurements 24 h later following izont stage parasites, respectively. These detection limits another 30 min of sonication. We found modest but sys- are below the threshold currently achievable with RDTs tematic increase of the MO signal of typically not more (>100 parasites/µL) and lie within the same range as the than 10-30%. On this basis, we expect that most of the limits of conventional optical microscopy for malaria di- hemozoin is successfully released into suspension, likely agnosis (5-50parasites/µL).1 For the present set of blood in the form of individual crystals. samples kept frozen and thawed before the measurement, The presence of a frequency-dependent residual MO the performance of the method was limited by a residual signal indicates that some components of lysed blood MO signal due to some part of the lysed cell suspension. can be magnetically oriented and rotated similarly to the This residual MO signal obscures the genuine MO sig- hemozoin crystals. We have also studied freshly drawn nal of hemozoin at parasite densities lower than the lim- blood following the same lysis protocol. The residual its quoted above. Preliminary results indicate that for MO signal observed in this case was considerably lower measurements on freshly lysed blood samples, which is than found for either the ring or schizont stage sam- the condition relevant to instant diagnosis, the detection ples (see Fig. 2), which implies better detection thresh- limit of our rotating crystal MO platform could be further old for instant diagnosis. We suspect that due to the improved. Thus, this methodology has the potential to freeze-thaw-lysis procedure applied to the present set of yield portable tools for instant diagnosis with detection samples, some portion of the hemoglobin may have been limits approaching that of PCR-based platforms. transformed to an aggregated or polymerized form – sim- Limitations of our diagnostic technique include i) the ilar to the intracellular non-covalent polymerization of 31–33 possibility of false positive detections due to the pres- hemoglobin previously observed in sickle cell disease ence of hemozoin in the blood, e.g. contained within –, which may produce the residual MO signal. These white blood cells,42 for extended periods of time after points, requiring additional systematic studies, stress the an infection has been cleared and ii) the possibility of crucial role of an appropriate blood treatment prior to di- false negative results in case an infection only contains agnosis. very early ring stage parasites with little or no hemo- The noise floor of our equipment, determined using zoin. Furthermore, methods targeting only hemozoin as pure water, is roughly frequency independent and about a marker for infection are thought to have limitations in one order of magnitude smaller than the residual signal their diagnostic capacity as they cannot distinguish be- from fresh blood. This enables further improvement of tween different malaria species. The specificity of our the detection limit provided that the residual MO signal MO diagnostic scheme, owing to variations in the typi- from blood can be reduced by optimizing blood treat- cal size and morphology of hemozoin crystals produced ment. Optimizing the properties of the lysis solution may by different species, needs to be tested by a compara- also help to dissociate the crystals without the need for tive study on various Plasmodium strains. We emphasize sonication. that only studies on field isolates will be able to elucidate the impact of these possible confounding factors and the present study is the basis for such field-based trials. Discussion It is currently believed that without active case detec- tion of asymptomatic malaria infections, malaria erad- The potential of exploiting hemozoin as a magnetic ication will be impossible or very difficult to achieve.6 biomarker for malaria diagnosis has stimulated extended However, there are no diagnostic tools for rapidly screen- research over the last few decades. Taking advantage ing hundreds of people per day, on-site and with high of the paramagnetic nature of hemozoin, several ap- sensitivity.43 The rotating-crystal MO diagnostic device proaches have been proposed to improve the sensitiv- has the potential to fulfill these requirements as it is cost- ity of existing methods by the magnetic separation of effective, rapid, highly sensitive, portable and easy to ap- malaria infected erythrocytes from whole blood prior to ply. the diagnosis.16,34–36 More recently, new techniques have been emerging, which directly use hemozoin as a tar- Besides on-site diagnosis, the present methodology get material of magnetic diagnosis. These techniques provides an efficient in-vitro laboratory tool to test the include detection of depolarized side-scatter in flow susceptibility of the parasites to new antimalarial drugs cytometry,37 electrochemical magneto immunoassays,38 by monitoring the effect of treatment on the rate of hemo- magnetically enriched surface enhanced resonance Ra- zoin formation. The scope of the technique described man spectroscopy39,40 and magneto-optical detection us- here may also cover the study or diagnosis of other human ing polarized light.17,18,41 Among them, to the best of our diseases, such as schistosomiasis, which are also caused by knowledge, our rotating crystal MO diagnostic device is blood-feeding organisms producing hemozoin similarly to the first realized in a cost-effective portable format with malaria parasites.44–47 excellent sensitivity. 6

Materials and Methods NaOH) was confirmed not to cause noticeable degrada- tion of hemozoin when added to synthetic malaria pig- Parasite culture ment suspended in lysed blood. Note that the final con- centration of NaOH in the samples was only 2.5 mM. The P. falciparum parasites (laboratory adapted strain samples were subjected to 30 min of ultrasonication to 3D7) were cultured following the method of Trager and dissociate potential aggregates of hemozoin and ensure Jensen with modifications48. The culture medium was an unhindered motion of crystals in the fluid. Owing ∼ RPMI 1640 with L-glutamine (GIBCO cat # 31800) to this treatment a transmittance of 30-40% could be achieved with no substantial light scattering observed supplemented with 2 mg/mL NaHCO3 (Merck, cat # 106329), 25mg/L gentamicin (Pfizer, cat # 61022027), from the blood samples. MO signals were recorded on 50mg/L hypoxanthine (Calbiochem, cat # 4010), 25 mM 1 mL volumes taken from the samples subsequent to the HEPES (SAFC, cat # 90909C) and 10% pooled O+ hu- preparation process and reproducibility was confirmed in several cases after one day of storage of the samples at man serum (mixed blood groups, Australian Red Cross o Blood Service). Cultures were maintained at 4 % hema- 4 C and by carrying out the measurement on both of the tocrit with changes of culture medium every 48 h and duplicate samples. diluted with uninfected O+ red blood cells when the parasitemia exceeded 5 %. Parasites were maintained in Magneto-optical measurements an atmosphere of 5% CO2 and 1% O2 in N2. Para- site cultures were kept in stage synchrony by applying the 5 % Sorbitol method, first described by Lambros and MO measurements were performed with the prototype Vanderberg49. Hemozoin liberated from late stage par- of the rotating magnet setup described in our previous asites by the synchronization process was removed by study15. We utilize a permanent magnetic ring, which washing the cells in RPMI medium after the Sorbitol in- produces a B=1 T magnetic field at the sample position duced cell lysis and before re-establishment of the cul- and can be rotated with adjustable frequency. Polarized ture. light from a laser diode is transmitted through the sample Parasite densities for the the ring and schizont stage in the direction perpendicular to the plane of the rotat- cultures described above were estimated by counting the ing magnetic field. Owing to the magnetic alignment of number of parasites contained in 5000 red blood cells on the freely rotating and dichroic hemozoin crystals present Giemsa stained thin blood films. The commonly used in infected blood, magnetically induced linear dichroism conversion from parasitemia to parasite density based on can be observed and quantified as the difference in trans- the assumption that 1 µL of blood contains 5×106 red mission for light polarized along and perpendicular to blood cells at 50% hematocrit was applied1. For both, the magnetic field direction (∆T ) divided by the average the ring and schizont stage cultures, 2-fold dilution series transmission (T ), i.e. ∆T/T 15. Due to the synchronous were prepared in duplicate in uninfected human erythro- periodic rotation of the crystals the linear dichroism gives cytes (Red Cross blood bank, Royal Melbourne Hospital, rise to a periodic change in the transmitted intensity os- VIC, Australia). The duplicate dilution series prepared cillating with twice the magnet rotation frequency. This for ring and schizont cultures contained 21 and 20 di- second harmonic intensity component (I2f ) can be effec- lutions, respectively, where each of the dilutions had a tively filtered by a lock-in technique. The MO signal is volume of 200 µL at a hematocrit of 50 %. These dilu- then obtained as the ratio of the modulated light inten- tions were immediately frozen and thawed twice to create sity and the average intensity (Idc); ∆T/T = I2f /4Idc. lysates. These lysates, hereafter referred to as ring and This detection scheme for malaria diagnosis provides schizont stage samples, were subsequently frozen at -80oC the best signal-to-noise ratio in the rotation frequency and kept in a frozen state until they were thawed immedi- regime of 10-30 Hz. In order to exclude baseline artifacts ately prior to measurement. The personnel carrying out emerging from mechanical vibration or improper optical the MO measurements were blinded against the contents alignment of the system, the baseline is checked with pure of each of the samples to reduce potential observer bias. distilled water prior to the measurement of each sample. This water baseline – due to electronic and mechanical noise – was generally found to be almost two orders of Blood treatment prior MO diagnosis magnitudes lower than the signal from the samples with the lowest hemozoin concentration. Blood samples prepared for MO measurement were thawed at room temperature and were diluted 20-fold with distilled water, resulting in a total volume of 4 mL Electron microscopy per specimen. Additionally, 100 µL of a special red cell lysis buffer, hereafter referred to as clearing solution, was For transmission electron microscopy (TEM), para- added to the specimens in order to disperse the rem- site samples were fixed in resin blocks and 70-120 nm nants of the lysed RBCs. In preliminary experiments, thin sections were cut using a Leica EM UC6 micro- this clearing solution (2.5 V/V% Triton X-100 in 0.1 M tome (Leica Microsystems, North Ryde, NSW, Aus- 7 tralia) and brought onto carbon coated copper TEM to the gold surface. grids (ProSciTech, Thuringowa, Qld., Australia). The Acknowledgements. The authors acknowledge TEM grids were then stained with 5% uranyl acetate fruitful discussions with T. Hanscheid and assistance for 15 min and Reynold’s lead citrate solution for 5 min. with electron microscopy by L. Kyriliak, M. Saunders, J. TEM was conducted on a JEOL 2100 TEM (JEOL Inc., Shaw and facilities of the Centre of Microscopy, Char- Tokyo, Japan). For details of the method see the Sup- acterization and Analysis at The University of West- porting Information. ern Australia. This work was supported by Hungar- For scanning electron microscopy (SEM), the samples ian Research Funds OTKA K108918, TAMOP-4.2.1.B-´ giving the highest MO signal were used and hemozoin 09/1/KMR-2010-0001, by NHMRC grants GNT1021544 crystals were extracted following the method of Chen and GNT1043345 awarded to IM and by NHMRC grants 44 and coworkers . The dark brown pellet obtained by GNT637406 and GNT1058665 awarded to LS. SK is sup- this method was resuspended in 80 µL water. For SEM ported through a NHMRC early career research fellow- imaging small droplets of the suspension containing the ship (GNT1052760). hemozoin crystals were applied to gold coated glass slides without further purification or treatment. The droplets were dried overnight at room temperature. The SEM Correspondence: Istvan Kezsmarki, Department images were acquired on a LEO 1540XB electron micro- of Physics, Budapest University of Technology and scope using the in-lens detector. The accelerating voltage Economics, 1111-Budapest, Hungary; e-mail: kezs- was set to 3 kV and the viewing angle was perpendicular [email protected].

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