WO2011123070A1 - A method of monitoring parasite development in blood - Google Patents

Abstract

A method and a kit to detect parasitemia and blood quantification of a blood sample with several dyes in a flow cytometer allowing quantification of a proportion of infected cells in the sample containing a parasite in relation to a total number of erythrocytes= in the sample. This method also allows quantification of the proportion of erythrocytes, of leukocytes or reticulocytes in relation to the total number of cells present in the samples.

Description

A METHOD OF MONITORING PARASITE DEVELOPMENT IN BLOOD

CROSS-REFERENCE TO RELATED APPLICATION

[01]. This application claims the benefit of Singapore Patent Application No. 201002247-3 filed on 31 March 2010, the entire contents of which are incorporated herein by reference.

FIELD OF INVENTION

[02]. The invention relates generally to methods and kits for determining parasitemia and blood quantification of a blood sample.

Background

[03]. The following discussion of the background to the invention is intended to facilitate an understanding of the present invention. However, it should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was published, known or part of the common general knowledge in any jurisdiction as at the priority date of the application.

[04]. Haematozoa is a general term that includes blood parasites, mainly protozoans. Well known examples include the protozoan parasites causing malaria, Leishmaniasis and trypanosomasis. Intra-erythrocytic haematozoa results from the multiplication of parasites within erythrocytes (red blood cells^'). Often the parasites have complex life cycles that include vertebrate hosts and invertebrate vectors. These diseases include malaria, filariasis, Chagas disease, leishmaniasis and African sleeping sickness. Some of these diseases cause severe anaemia or blood loss and may require the use of blood products or transfusions in order to save lives. Diagnosis and treatment reduces the need for blood transfusions. The most commonly used method to quantify blood parasites is microscopic examination using a thin blood smear. The major limit of this technique is that the counts obtained vary depending on the expertise of the observer. Moreover, the process is tiring and time consuming. [05]. Malaria, which affects some 300 million people a year, may cause many pathologies such as kidney and liver failure, anemia, miscarriages and stillbirths and is responsible for a high level of morbidity and almost 1 million fatalities (WHO, World Malaria report 2009). Early detection of Malaria and other blood parasites allows effective and timely treatment. Prompt parasitological confirmation of malaria diagnosis is part of effective disease management. The two diagnostic methods recommended by WHO are: optic microscopy to quantify malaria parasites via microscopic examination of Giemsa-stained thin blood smear; or rapid diagnostic tests (RDT) based on lateral flow immunochromatography. The major limit of the optic microscopy technique is that the counts obtained vary depending on the expertise of the observer. Moreover, the process is tiring and time consuming. Implementation of RDT is not widespread due to poor product performance, inadequate methods to determine the quality of products and poor storage for such tests in tropical climates.

[06]. In the past, any possible malaria symptoms were treated with anti-malarial drugs in an unspecific manner often without diagnosing the presence of Plasmodium or with adequate doses, which has led to the situation that resistance has been reported to all classes of anti-malarial, drugs even recently for artemisinin derivatives. The replacement of conventional antimalarial drugs with high-cost, artemisinin-based alternatives has created a gap in the successful management of malaria. This gap reflects an increased need for accurate disease diagnosis that cannot be met by traditional microscopy techniques.

[07]. Flow cytometry techniques are now routinely utilized in clinical hematology particularly in leukaemia diagnosis. Recently, flow cytometry methods of parasites quantification have been developed. These new methods appear to overestimate parasitemia and have not been adapted to quantify parasites in samples from infected patients. Also, often, these techniques are very complex and time consuming.

[08]. The cell composition of mammalian blood predominantly comprises erythrocytes, thrombocytes and leukocytes.

[09]. Mammalian erythrocytes are non-nucleated cells in their mature form. Because of the lack of nuclei and organelles, mature red blood cells do not contain DNA and cannot synthesize any RNA and consequently cannot divide and have limited repair capabilities.

[010]. Reticulocytes are immature erythrocytes (red blood cells). Reticulocytes develop and mature in the bone marrow and then circulate for about a day in the blood stream before developing into mature red blood cells. Like mature red blood cells, reticulocytes do not have a nucleus. They are characterized by a reticular (mesh-like) network of ribosomal RNA. The normal range of values for reticulocytes in the blood is 0.5% to 1.5%. However, if a person has anemia, their reticulocyte percentage is usually higher than normal. A very high number of reticulocytes in the blood can be described as reticulocytosis and this can happen during diverse pathologies such "as hemochromatosis

[011]. Mammalian thrombocytes are small, regularly-shaped clear cells also with no nuclei. Thrombocytes also called platelets are involved in blood clotting.

[012]. Leukocytes are found in blood. Leukocytes comprise granulocytes, lymphocytes and monocytes. The number of leukocytes in the blood is often an indicator of disease. There are normally between 4x l0⁹ and l .l x lO¹⁰ white blood cells in a litre of blood, making up approximately 1% of blood in a healthy adult. An increase in the number of leukocytes is called leukocytosis, and a decrease is called leukopenia.

Summary

[013]. It is desirable to provide methods and kits for determining parasitemia and blood quantification of a blood sample that overcomes, or at least alleviates, one of the above mentioned problems.

[014]. One aspect of the invention provides a method for determining parasitemia and blood quantification of a blood sample comprising the steps of:

a) adding to the sample a nucleic acid dye that reacts with deoxyribonucleic acid (DNA); a nucleic acid dye that reacts with DNA and ribonucleic acid (RNA) and an antibody coupled to a fluorophore capable of selectively binding a marker present on a leukocyte;

b) exciting the sample with a light;

c) measuring a light emission pattern from the sample; d) analysing the light emission pattern to quantify a proportion of infected cells in the sample containing a parasite in relation to a total number of a cell in the blood sample or quantify a proportion of a total number of leukocytes and/or quantify a proportion of a total number of reticulocytes in relation to a total number of the cell in the sample,

[015]. The cells in the blood sample may refer to all the cells in the blood including erythrocytes, thrombocytes and leukocytes or the cells in the blood sample may refer to erythrocyte cells.

[016]. The method may further comprise calculating the number of infected cells and/or leukocytes and/or reticulocytes as a percentage of the total number of the cells in the blood sample.

[017]. Another aspect of the invention provides a kit to detect parasitemia and blood quantification of a blood sample comprising, a nucleic acid dye that reacts with deoxyribonucleic acid; a nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid and a antibody coupled to a flurorophore capable of selectively binding a marker present on all leukocytes.

Brief description of the drawings

[018]. Preferred embodiments of the invention will be described with reference to the following drawings of which:

Figure 1. Flow cytometry analysis of the parasitemia in a C57BL/6 mouse infected with the rodent malaria parasite Plasmodium berghei ANKA.

Figure 2. Correlation between the parasitemia with GFP+ parasites and the quantification with the Dihydroethidium and Hoechst staining (n=22 measurements)

Figure 3. Comparison of the parasitemia between acquisition immediately after the staining and after 24 hours at samples have been maintained at 4°C.

Figure 4. Flow cytometry analysis of the parasitemia in a patient infected with Plasmodium vivax Figure 5. Flow cytometry analysis of the in vitro parasitemia with Plasmodium falciparum (clone 3D7) before and after magnetic sorting. Hz-; young forms; Hz+: old form of the blood stage parasites.

Figure 6. Validation of the technique for drug testing. Plasmodium falciparum (strain 3D7) culture was treated in triplicate during 24 hours with 19 ng/mL of Artesunate (AS) or 512 ng/mL of Chloroquine (CQ). The percentage of inhibition on schizonts development is 83.7% and 79.0% respectively, with these two high doses.

Figure 7. Flow cytometry analysis of the parasitemia in a whole blood samples from two patients infected with Plasmodium vivax detected with ultra violet light (A) or a laser in the violet spectrum (B).

Figure 8. (A) Representative Dot plot of a synchronized culture of P. falciparum (clone 3D7) with the Ethidium/Hoechst double staining before and after 24 hours of culture. The developmental blood stages were sorted by Flow Cytometry and (B) stained with Giemsa. Representatives images of (1) a single ring, (2) a double ring, (3) a trophozoite, (4) an early schizont and (5) a late schizont.

Figure 9. Flow cytometry analysis of the parasitemia in whole blood blood from two patients infected with P. falciparum. Whole blood samples from two patients infected with P. falciparum were stored frozen in nitrogen. On the day of the experiment, blood samples were thawed and analyzed with the tri-color Flow Cytometry method.

Figure 10. Flow cytometry analysis of the parasitemia in a whole blood samples from one patient infected with P. vivax and identification of the different developmental blood stages in the different gates. Whole blood samples from a P. vivax-infected patient was stored frozen in nitrogen. On the day of the experiment, it was thawed and analyzed by the tri-color Flow Cytometry (A). The developmental blood stages were sorted by flow cytometry and stained with Giemsa. (B) The pictures represent the different life cycle stage, (1) rings and (2) late cycle stages (trophozoite, schizont), present in the DNA gate after cell sorting and Giemsa staining. Figure 11. Application of the Tricolor methor to Flow cytometer having two lasers but not UV laser. Our tricolor method is based on the use of a flow cytometer having 3 lasers and in particular a UV laser for excitation of the Hoechst dye. The method was adapted to be used for Flow cytometer having only two lasers (488nm and 635nm). The Hoechst dye was replaced by the SYBR green dye. Flow Cytometry gating strategy for the 2 lasers} method in uninfected blood (A) and blood infected with P. berghei ANKA (B). Validation of the Tricolor method wit two laser Flow cytometers. by Comparison of the two techniques for measurement of parasitemia in whole blood of mice infected by P. berghei .

Detailed description

[019]. We have developed a technique using flow cytometry to quantify a parasite in whole blood samples. The technique includes a a method for determining parasitemia and blood quantification of a blood sample comprising the steps of: e) adding to the sample a nucleic acid dye that reacts with deoxyribonucleic acid (DNA); a nucleic acid dye that reacts with DNA and ribonucleic acid (RNA) and an antibody coupled to a fluorophore capable of selectively binding a marker present on a leukocyte;

f) exciting the sample with a light;

g) measuring a light emission pattern from the sample;

h) analysing the light emission pattern to quantify a proportion of infected cells in the sample containing a parasite in relation to a total number of a cell in the blood sample or quantify a proportion of a total number of leukocytes and/or quantify a proportion of a total number of reticulocytes in relation to a total number of the cell in the sample.

[020]. The cells in the blood sample may refer to all the cells in the blood including erythrocytes, thrombocytes and leukocytes or the cells in the blood sample may refer to erythrocyte cells.

[021]. The method may further comprise calculating the number of infected cells and/or leukocytes and/or reticulocytes as a percentage of the total number of the cells in the blood sample.

[022]. The number of cells containing a parasite and/or leukocytes and/or reticulocytes in relation to the total number of the cells in the blood samples may be measured as a ratio or a fraction. The fraction may have a denominator of 10 or may have a denominator of 100 or may have a denominator of 1000. Preferably the fraction is measured as a percentage of the total number of the cells in the blood sample.

[023]. The method has the advantage of being able to Screen a large number of samples in a short time. As the incubation time is short and no washing steps are required. In terms of accuracy, the counting has the added advantage when it is performed by a Flow cytometer of reducing any variability to a minimum and having high reproducibility.

[024]. In one embodiment the method may include analysing the light emission pattern, being a reflection of the events detected in a Flow Cytometer, in the following way:

• Calculating a first proportion of particles detected from light emitted by both the nucleic acid dye that reacts with DNA and the antibody selectively binding the leukocyte cell marker. This first calculation effectively is an indication of the proportion of leucocytes counted in the blood sample.

[02]. Preferably, the first proportion is calculated as a first percentage of the total number of the cells in the blood sample, the first percentage indicates a number of leukocytes cells as a percentage of the total number of the cells in the blood sample whereby greater than 1% in combination with a positive number for the forth proportion is further indication of parasitemia.

• Calculating a second proportion of particles detected from light emitted by both the nucleic acid dye that reacts with DNA and the nucleic acid dye that reacts with DNA and RNA. The second calculation effectively is an indication of the proportion cells that includes infected red blood cells and leukocytes in the blood sample.

• Calculating a third proportion of particles detected from light emitted by the nucleic acid dye that reacts with DNA and RNA but does not have light emitted from both the nucleic acid dye that reacts with DNA and the antibody selectively binding the leukocyte cell marker. The third calculation is an indication of the proportion of reticulocytes in the blood sample.

[03]. Preferably, the third proportion is calculated as a third percentage of the total number of the cells in the blood sample, the third percentage indicates a number of reticulocyte cells as a percentage of the total number of the cells in the blood sample whereby when the third percentage is greater than 1.5% in combination with a positive number for the forth proportion is further indication of parasitemia.

• Calculating a fourth proportion of infected red blood cells by subtracting the second calculation from the first calculation whereby the fourth calculation indicates a proportion of erythrocytes infected with parasites or parasitemia. The fourth calculation effectively is an indication of the proportion of infected erythrocytes in the blood sample.

[025]. Preferably the method is performed using a Flow Cytometer and the proportion of cells of interest in relation to the cells in the blood sample is determined. The calculations are preferably done in terms of percentage

[026]. Parasite detection

[027]. The method can be adapted to monitor a range or parasites. Particularly, where parasitemia is caused by haematozoans such as Plasmodia and Babesia, mycoplasma such as hemobartonella or other parasites with a blood phase such as Trypanosomas, Wuchereria Bancrofti, Loa loa, Brugia, Mansonella and Dirofilaria. Preferably, the parasite is an intra-erythrocytic parasite such as Plasmodia; babesia and bartonella. In one embodiment the parasite is P. falciparum or other human Plasmodia such as P. vivax, P. ovale, P. malariae , rodent malaria parasites such as P_. berghei, P. yoelii, P. vinckei, P. chabaudi., or primate malaria such as P. knowlesi or P. cynomolgi.

[028]. In an alternative embodiment the Haematozoa parasites, are blood parasites having an extra-erythrocytic blood phase such as Trypanosoma, Wuchereria bancrofti, Loa loa, Brugia malayi/timori, Mansonella, Dirofilaria. The technique can quantify a wide range of protozoans and haematozoan parasites known in the art.

[029]. Blood samples [030]. Blood samples may be taken from any subject including a vertebrate or a mammal either living or dead. Preferable the blood sample is taken from a human subject. The sample may be a fresh sample drawn from a subject and tested immediately or it may be a stored sample. Samples are preferably stored at 4°C in sterile conditions for later detection.

[031]. The method uses blood containing intact cells referred to as an intact blood sample. This allows the calculation of the proportion of erythrocytes actually containing parasites in relation to the cells in the sample. As the technique does not require hemolysis, or the lysis of the blood cells, it reduces the processing steps and thus time of sample processing. Further, only the proportion of erythrocytes actually containing parasites is determined rather than the propotion of the total number of parasites in relation to the volume of the sample, which may result in more cells overestimating parasitemia.

[032]. Nucleic acid dye that reacts with deoxyribonucleic acid

[033]. A nucleic acid dye that reacts with deoxyribonucleic acid (DNA) may include any DNA fluorochromes such as Hoeschst 33342, Ethidium Bromide Hoeschst 33258, DAPI, SYTOX Blue, Chromomycin A3, SYTOX Green, mithramycin, 7-AAD, TO-PRO-3, propidium iodide, SYBR green 1, thiazole orange (TO) and its derivatives, and propidium iodide (PI) or any other dyes known to those skilled in the art to react with DNA. Many of these dyes are commercially available. Effectively, such dyes can be used detect parasites and leukocytes.

[034]. nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid

[035]. A nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid may include dihydro ethidium, acridine orange, pyronin Y (PY), oxazine 1, LDS 751, 7-AAD, SYTOX Orange, or any other dyes known to those skilled in the art to react with both DNA and RNA. Effectively, such dyes can be used detect parasites leukocytes and reticulocytes. This allows quantification of reticulocytes.

[036]. Antibody coupled to a fiuorophore capable of selectively binding a leukocyte cell marker

[037]. A an antibody coupled to a fiuorophore capable of selectively binding a leukocyte cell marker present on all leukocyte populations may be coupled with any flurorophore known in the art to be capable of binding to an antibody and produce a signature emission pattern under a light stream. When the fluorescently labeled antibody is exposed to light of a proper wave length, its presence can then be detected due to fluorescence of the fluorophore. Among the most commonly used fiuorophores are fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin, phycocyanin, allophycocyanin (APC), o-phthaldehyde, sulforhodamine 101 acid chloride (Texas Red), fluorescamine or fluorescence-emitting metals such as 152 Eu or other lanthanides. These metals are attached to antibodies using metal chelators. In some embodiments, the fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a fiuorometer or a photosensor such as CCD camera equipped with appropriate emission filters. Effectively, such antibodies can be used detect leukocytes.

[038]. One type of label commonly used is fluorescent and may be attached to molecules on the outside or inside of cells. The labels generally are light emitting fluorescent tags such as phycoerythrin, fluorescein (green light) or rhodamine (red light). The fluorescent label often is conjugated to a binding agent, such as an antibody, capable of binding to a component of the cell surface. Cell fluorescence is indicative of the presence of the binding partner, such as an antigen, of the binding agent on the cell surface. The intensity of the fluorescence is a function of the number of fluorescent labels bound per cell, and is thus related to the number of binding partners available on the surface of the cell.

[039]. A leukocyte cell marker should be a cell marker that is found on all leukocyte cells and not found on red blood cells or reticulocyte cells. Commonly known leukocyte cell marker present on all leukocyte populations is CD45. Hence, the leukocyte cell marker is CD45. The protein sequences of leukocyte cell marker CD45maybe quickly obtained by a person skilled in the art at the swissprot/uniprot database at accession number Q64224. Based on the sequence obtained the protein can be manufactured using techniques well known in the art such as being synthesized chemically using known techniques such as liquid phase synthesis or solid phase synthesis such as t-BOC or Fmoc, or BOP SPPS or any chemical synthesis method known to those in the art. Alternatively the peptide may be made biologically within a cell or vector designed to use the machinery of translation and/or transcription for the leukocyte cell marker synthesis. The cell surface marker recognizing all leukocytes can be bought commercially for use in making fluorophore-coupled antibodies capable of selectively binding to the specific cell surface marker required.

[040]. Compatibility to a leukocyte cell marker will be specific for the leukocyte cell marker. Thus, the present invention also provides polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, which are capable of specifically binding to the leukocyte cell polypeptides and fragments thereof.

[041]. Antibodies against the leukocyte cell marker, CD45, may be bought commercially from any of the known suppliers such as Abnova, Sigma, Biorad or other such companies or they may be made according to any of the methods known in the art. Exemplary methods include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, Fab expression library, humanized, bispecific, and heteroconjugate antibodies. According to the invention, a polypeptide of a leukocyte cell marker produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as an immunogen to generate antibodies that recognize the leukocyte cell marker polypeptide.

[042]. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, as well as antigen binding portions of antibodies, including Fab, F(ab')₂ and F(v) (including single chain antibodies). Accordingly, the phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule containing the antibody combining site. An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.

[043]. The use of multiple specific dyes together with the Flurophore-coupled antibody to CD45 concurrently in the detection method has the advantage of being able to quantify blood leukocytes and reticulocytes at the same time of parasitemia determination. This can be done quickly in a single step of one pass through the flow cytometer.

[044]. exciting the sample with a light

[045], The sample may be excited with light such as laser light and there may be a variety of wavelengths used. The light may be in a UV wavelength where ethidium bromide is used as the nucleic acid dye that reacts with deoxyribonucleic acid (DNA). An argon laser (488 nm) may be employed as the light source. Preferably the light is emitted from a flow cytometer. In one embodiment two lasers cytometer are used at488-nm and 633 nm to detect Ethidium bromide and sybr green respectively.

[046]. measuring a light emission pattern from the sample;

[047]. Preferably the light emission pattern is detected and measured in a flow cytometer. Flow cytometry is an analytical method that allows both the rapid measurement of scattered light for particle size determination and measurement of fluorescence emission produced by suitably illuminated particles. The particles are suspended in liquid and produce signals when they pass through a beam of light individually. Because measurements of each particle are made separately, the results are a correlated set of each individual particle's characteristics. An important analytical feature of flow cytometry is its ability to measure multiple particle parameters such as scattered light and fluorescence emission. Scattered light collected in the same direction as the incident light (FSL) reflects cell size and granularity where as the fluorescence is dependent upon the presence of fluorochromes on particles. Thus, a combination of light scattering and fluorescence is a powerful approach to detect multiple targets in one sample without the need for a separation step.

[048]. Preferably the method is carried out using flow cytometry. The terms "flow cytometry" and "flow cytometric" and "cell sorter" refer to the instruments and procedures whereby a population of cells in a liquid suspension is directed through a fine liquid stream passing by the cytometer's laser into a device capable of measuring the physical and/or chemical characteristics of cells based on the light quanta emitted. The light passing through each cell is measured individually and can represent physical characteristics of cells that are unlabeled and also cells that have the various labels/dyes of the invention attached.

[049]. The cell sorter exposes the cells moving through the liquid stream to light, usually a specific wavelength, known as the excitation wavelength that corresponds to the fluorescent label used. In response to excitation wavelengths, the fluorescent label fluoresces and emits light. The cell sorter detects and records the emission of light, both the number of discrete occurrences and the intensity of the light emitted. Optionally, the cell sorter can momentarily divert the cell stream so as to separate fluorescing cells from non-fluorescing cells, or separate cells having different wavelength fluorescence, or those having fluorescence exceeding a certain predetermined minimum intensity from the rest of the cells in the population. The light quanta emitted is measured for each cell individually and presented as such in data representations.

[050]. A very large spectrum of light emission and detection can be used with this method. Advantageously, in contrast to the majority of other flow cytometry methods, staining method using the FL1 (green channel) is compatible with this protocol.

[051]. Thus, a population of cells can be characterized by parameters such as percentage of fluorescing cells, percentage of non- fluorescing cells, and population profiles of cells having various fluorescence intensities (See Figures 1).

[052]. Multiparameter flow cytometry allows one to estimate, with high accuracy, relative quantities of a variety of cells simultaneously. When the measurements are recorded in a list mode, it is possible to attribute each of the several measured features to a particular cell and thus to obtain correlated measurements of these features on a cell by cell basis. Cellular heterogeneity can thus be estimated and subpopulations with distinct characteristics can be discriminated. Thus,

multiparameter flow cytometry offers improved opportunities to describe the complex relationships in a sample.

[053]. analysing the light emission pattern to quantify a percentage of the sample containing a parasite in relation to leukocytes and reticulocytes [054]. The analysis can be done in terms of percentage or total numbers. Generally the expression profile may include at least one component of the forward scatter, the side scatter and the fluorescence signature. Preferably the expression profile includes all three components.

[055] . Parasitemia analysis is done on the basis of determining the percentage (%) of infected red blood cells per total red blood cells. It is calculated as the percentage of cells positive for the nucleic acid dye that reacts with deoxyribonucleic acid and the nucleic acid dye that reacts ribonucleic acid (containing both infected red blood cells and leukocytes) minus the percentage of cells positive for both the nucleic acid dye that reacts deoxyribonucleic acid and the antibody selectively binding the leukocyte cell marker (corresponding to the leukocytes).

[056]. The technology can differentiate the reticulocytes from the leukocytes or other nucleated cells because the reticulocytes are positive for the nucleic acid dye that reacts with ribonucleic acid and is negative for the nucleic acid dye that reacts with deoxyribonucleic acid and the antibody selectively binding the leukocyte cell marker while the leukocytes are positive for all three of the dyes i.e., the nucleic acid dye that reacts with ribonucleic acid; and the nucleic acid dye that reacts deoxyribonucleic acid; and the antibody selectively binding the leukocyte marker recognizing all leukocyte populations.

[057].

Staging of the infection

[058]. The method may also allow determining parasite developmental stages. Because the different developmental stages possess different quantity of DNA and RNA, they can be identified by this method.

[059]. A cell sorting experiment may be used to confirm that the parasite stages detected by the methods of the invention are indeed the real one (ascertained by Giemsa-stained blood smears). Cell sorting is done by the step of enriching the blood sample with magnetic sorting. To enriched the sample it may be magnetically sorted using the MACS Miltenyi technology or other magnetic sorting techniques such as dyna beads by dynal or any other method that sorts heme containing cells magnetically. Late blood stage parasites synthesize high quantity of the pigment hemozoin (Hz), which is rich in the iron metal and thus can be retained by the magnet on the purification column more easily, particularly of late stage infection. This allows the technique the ability to detect the different stages of the parasites. The early and late developing stages of the parasites can be identified.

This is followed by cell sorting using Flow cytometry (see Figure 8). The sorting by Flow cytometry is then followed by identification of the parasites on Giemsa-stained slides staining as known in the art.

[060]. Detection kits

[061]. A kit to detect parasitemia and blood quantification of a blood sample comprising, a nucleic acid dye that reacts with deoxyribonucleic acid; a nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid and a fluorophore- coupled antibody capable of selectively binding a marker recognizing all leukocyte populations. Exemplary dyes and markers are listed above. Preferably the kit is for use in with flow cytometry as described above.

[062], The kit to specifically detect parasitemia and blood quantification of a blood sample comprising, a nucleic acid dye that reacts with deoxyribonucleic acid (DNA); a nucleic acid dye that reacts with deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and a fluorophore-coupled capable of selectively binding a marker recognizing all leukocyte populations.

[063]. The nucleic acid dye that reacts with DNA of the kit comprises Hoeschst 33342, Ethidium Bromide Hoeschst 33258, DAPI, SYTOX Blue, Chromomycin A3, SYTOX Green, mithramycin, 7-AAD, TO-PRO-3, propidium iodide SYBR green 1, thiazole orange (TO) and its derivatives, or propidium iodide (PI). In one preferred embodiment the kit may comprises Hoeschst 33342 as the nucleic acid dye that reacts with DNA. In another embodiment the kit may comprises SYBR green as the nucleic acid dye that reacts with DNA.

[064], The nucleic acid dye that reacts with DNA and RNA of the kit may comprise dihydroethidium, acridine orange, pyronin Y (PY), oxazine 1, LDS 751, 7-AAD, or SYTOX Orange. In one preferred embodiment the kit may comprises

dihydroethidium as the nucleic acid dye that reacts with DNA and RNA. [065]. Preferably the dyed antibody capable of selectively binding a leukocyte cell marker of the kit comprises a detectable label capable of producing a signature emission as described above. The antibody of the kit may comprise antibodies capable of selectively binding a leukocyte cell marker described above such as CD45 .

[066]. The kit may further comprising magnetic beads suitable for cell sorting.

[067]. The invention will be more fully understood in light of the following examples which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

Preferred embodiments

[068]. Malaria Detection method

[069]. The parasites are stained using two dyes, dihydroethidium (Sigma) and Hoeschst 33342 (Sigma), which react with parasite DNA. Since red blood cells have no DNA, only parasite DNA is stained. We also detect leukocytes in the same sample using an antibody against the CD45 molecules coupled to allophycocyanine (APC) (Miltenyi). This marker is expresses on all leukocytes but not on red blood cells. The whole procedure requires 20 minutes to be performed at room temperature and no washing step. Acquisition of the data is performed using a flow cytometer with Blue and U.V lasers (See Fig 1).

[070]. Procedures

1. Dot plots represent: (left) forward-scatter/side-scatter (FSC-A/SSC-A) scatter gram of representative whole blood sample Events (representing blood cells) are acquired and their size (FSC-A) and granularity (SSC-A) recorded. 2. A secondary analysis allows excluding duplets (FSC-A/SSC-W).

Duplets represents aggregated cells and may bias the analysis.

3. Samples are then analyzed using the Hoechst 33342 dye and/or Ethidium bromide (which bind parasite DNA located inside the infected red blood cells arid blood leukocyte DNA). Normal non-infected red blood cells do not have DNA and thus are negative (not stained) by Flow cytometry). Use of antibody against the CD45 marker allows characterizing all leukocyte populations.

[071]. The parasitemia (% of infected red blood cells/total red blood cells) is calculated as the percentage of cell positive for Hoechst and Ethidium (containing both infected red blood cells and leukocytes) minus the percentage of cells positive for Hoechst and anti-CD45 (corresponding to the leukocytes).

[072]. We have validated this technique using the P. berghei ANKA parasite expressing the fluorescent GFP molecules. GFP parasites can be monitored for parasite development by flow cytometry and measuring the percentage of infected cells in the FL1 channel. As shown in Fig. 2, we obtained a perfect correlation between the two techniques.

[073]. We also tested if detection can be performed 24h after the blood was collected and stored at 4°C. Identical results were obtained. This technique was also tested on whole blood from patients infected with human parasites. Further, this technique was also efficient to detect P. falciparum (Figure 5) from human blood samples. The procedures used for the human parasites were the same used for the P. berghei (Figure 1). We observed that we could detect accurately low parasitemia in the blood of infected patients (Figure 4).

Staging of Malaria infection

[074]. In this experiment, we enriched in the late developing stages of P. falciparum by magnetic sorting using the MACS Miltenyi technology. Late blood stage parasites synthesize high quantity of the pigment hemozoin (Hz), which is rich in the iron metal (and thus can be retained by the magnet on the purification column and further enriched on late form). This allows us to validate of the technique in its ability to detect the different stages of the parasites. Monitoring of malaria development in human blood by flow cytometry

(Mai-Count, Malaria Enumeration Kit)

1. For each sample to be stained, mix 1 μΐ of whole blood in 100 μΐ of cold PBS.

2. Add 1 μΐ of Hoechst 33342 (Sigma), stock solution concentration 800 μΜ.

3. Add 1 μΐ of Dihydroethidium (Sigma), stock solution concentration 500 μ^ηιΐ.

4. Add 2 μΐ of anti-human CD45 APC (Miltenyi).

5. Incubate for 20 minutes at room temperature in the dark.

6. Add 400 μΐ of cold PBS.

7. Proceed to flow cytometric acquisition.

[075]. The commercial application is the development of a kit for the parasitemia quantification in mammalian blood such as in human blood.

[076]. The Hoechst can be excited also with a violet laser or a U.V. laser. The UV laser is preferable. For the ethidium (dihydroethidium), the excitation can be done with a green laser.

[077]. The technology can differentiate the reticulocytes from the leukocytes because the reticulocytes are positive for the Dihydroethidum staining and negative for the Hoechst and CD45 staining while the leukocytes are positive for the Dihydroethidum, Hoechst and CD45 staining.

Parasite detection

[078]. The kit can be extended to monitor the Haematozoa (see the list below) parasites, grouped in two different subsets. The first one is the intra-erythrocytic Haematozoa, such as Plasmodium falciparum, and the second one is the extra- erythrocytic blood Haematozoans or mycoplasma. The technique can be easily adapted to quantify these parasites. [079]. List of Haematozoa infecting mammals:

Intra-erythrocytic Haematozoa (protozoa)

[080]. Plasmodia

[081]. Babesia

[082].

Extra-erythrocytic Haematozoa (protozoa/metazoa )

[083]. Trypanosoma

[084]. Wuchereria bancrofti

[085]. Loa loa

[086]. Brugia malayi/timori

[087]. Mansonella

[088]. Dirofilaria

Extra-erythrocytic mycoplasma

[089]. Hemobartonella

[090]. Validation of the gating strategy by characterization of P. falciparum developmental stages by flow cytometry sorting

[091]. Figure 8 shows Representative Dot plot of a synchronized culture of P. falciparum (clone 3D7) with the Ethidium/Hoechst double staining before and after 24 hours of culture. The developmental blood stages were sorted by flow cytometry and stained with Giemsa (1) single ring, (2) double ring, (3) trophozoite, (4) early schizont and (5) late schizont.

[092]. Detection of P. falciparum from stored infected whole blood

[093]. Whole blood samples from two patients infected with P. falciparum were stored frozen in nitrogen. On the day of the experiment, they were thawed and analyzed by the tri-color Flow Cytometry (Figure 9). The tri-color method

CD45/Hoechst staining and Ethidium/Hoechst staining was used in this experiment.

[094]. Detection of P. vivax from stored infected whole blood

[095]. Whole blood samples from a patient infected with P. vivaz was stored frozen in nitrogen. On the day of the experiment, the blood samples were thawed and analyzed by the tri-color Flow Cytometry. The developmental blood stages were sorted by flow cytometry and stained with Giemsa (Figure 10 (A). The pictures represent the different life cycle stage, (1) rings and (2) late cycle stages

(trophozoite, schizont), present in the DNA gate after cell sorting and Giemsa staining (Figure 10 (B).

[096]. Development of an alternative method suitable for a 2 lasers cytometer (488-nm and 633 nm lasers)

[097]. Our tricolor method was based on the use of a flow cytometer having a UV laser. The method was adapted to be used for Flow cytometer having two lasers. The Hoechst dye was replaced by the SYBR green dye (Figure 11). Flow Cytometry gating strategy for the 2 lasers method is shown in Figure 11 in both uninfected blood (A) and in blood samples infected with P. berghei ANKA. Validation of the method is made by comparison of the 3 lasers method (Hoechst) with the 2 lasers method (SybrGreen) using whole blood of mice infected by P. Berghei (Figure 11 (C)).

[098]. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[099]. Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[0100], Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0101]. The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0102]. The invention described herein may include one or more range of values (eg size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

[0103]. Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as

"comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that

[0104]. are found in the prior art or that affect a basic or novel characteristic of the invention.

[0105]. Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

Claims

A method for determining parasitemia and blood quantification of a blood sample comprising the steps of:

a. adding to the sample a nucleic acid dye that reacts with

deoxyribonucleic acid (DNA); a nucleic acid dye that reacts with DNA and ribonucleic acid (RNA) and an antibody coupled to a fluorophore capable of selectively binding a marker present on a leukocyte;

b. exciting the sample with a light;

c. measuring a light emission pattern from the sample;

d. analysing the light emission pattern to quantify a proportion of infected cells in the sample containing a parasite in relation to a total number of a cell in the blood sample or quantify a proportion of a total number of leukocytes and/or quantify a proportion of a total number of reticulocytes in relation to a total number of the cell in the sample.

The method as claimed in claim 1 whereby the cell in the blood sample is a erythrocyte.

The method as claimed in claim 1 or 2 whereby analysis of the light emission pattern comprises:

a. Calculating a first proportion of particles detected from light emitted by both the nucleic acid dye that reacts with DNA and the antibody selectively binding the leukocyte cell marker;

b. Calculating a second proportion of particles detected from light emitted by both the nucleic acid dye that reacts with DNAand the nucleic acid dye that reacts with DNA and RNA;

c. Calculating a third proportion of particles detected from light emitted by the nucleic acid dye that reacts with DNA and RNA but does not have light emitted from both the nucleic acid dye that reacts with DNA and the antibody selectively binding the leukocyte cell marker; and

d. Calculating a fourth proportion of infected red blood cells by subtracting the second calculation from the first calculation whereby the fourth calculation indicates a proportion of erythrocytes infected with parasites or parasitemia.

4. The method as claimed in any one of claims 1 to 3 whereby the method is performed in a in a flow cytometer.

5. The method as claimed in any one of claims 1 to 4 whereby the first proportion is calculated as a first percentage of the total number of the cells in the blood sample, the first percentage indicates a number of leukocytes cells as a percentage of the total cells whereby greater than 1% in combination with a positive number for the fourth calculation is further indication of parasitemia.

6. The method as claimed in any one of claims 1 to 4 whereby the third proportion is calculated as a third percentage of the total number of the cells in the blood sample, the third percentage indicates a number of reticulocytes cells as a percentage of the total blood cells whereby when the third percentage is greater than 1.5% in combination with a positive number for the fourth calculation is further indication of parasitemia.

7. The method as claimed in any one of the preceding claims wherein parasitemia is caused by a haematozoa.

8. The method as claimed in claim 7 wherein the haematozoa are selected from Plasmodia; Babesia, mycoplasma, Trypanosome, Wuchereria Bancrofti, Loa loa, Brugia, Mansonella and Dirofilaria

9. The method as claimed in claim 7 wherein the haematozoa is an intra- erythrocyte haematozoa

10. The method as claimed in claim 9 wherein the intra-erythrocyte

haematozoa are selected from Plasmodia; babesia and bartonella

11. The method as claimed in claim 9 or claim 10 wherein the intra- erythrocyte haematozoa are selected from Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae , Plasmodium yoelii, Plasmodium vinckei, Plasmodium chabaudi, Plasmodium knowlesi, Plasmodium cynomolgi, and Plasmodium berghei.

12. The method as claimed in any one of the preceding claims wherein the nucleic acid dye that reacts with DNA comprises Hoeschst 33342, Ethidium Bromide Hoeschst 33258, DAPI, SYTOX Blue, Chromomycin A3, SYTOX Green, mithramycin, 7-AAD, TO-PRO-3, propidium iodide SYBR green 1, thiazole orange (TO) and its derivatives, or propidium iodide (PI).

13. The method as claimed in any one of claims 1 to 11 wherein the nucleic acid dye that reacts with DNA comprises Hoeschst 33342.

14. The method as claimed in any one of claims 1 to 1 1 wherein the nucleic acid dye that reacts with DNA comprises SYBR green.

15. The method as claimed in any one of the preceding claims wherein the nucleic acid dye that reacts with DNA and RNA comprises

dihydroethidium, acridine orange, pyronin Y (PY), oxazine 1, LDS 751, 7-AAD, or SYTOX Orange.

16. The method as claimed in any one of claims 1 to 14 wherein the nucleic acid dye that reacts with DNA and RNA comprises dihydroethidium.

17. The method as claimed in any one of the preceding claims wherein the leukocyte cell marker comprises CD45.

18. The method as claimed in any one of the preceding claims further

comprising the step of enriching the blood sample with magnetic sorting.

19. A kit to detect parasitemia and blood quantification of a blood sample comprising, a nucleic acid dye that reacts with deoxyribonucleic acid DNA); a nucleic acid dye that reacts with deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and an antibody coupled to a flurophore capable of selectively binding a marker present on a leukocyte.

20. The kit as claimed in claim 19 wherein the nucleic acid dye that reacts with DNA comprises Hoeschst 33342, Ethidium Bromide Hoeschst 33258, DAPI, SYTOX Blue, Chromomycin A3, SYTOX Green, mithramycin, 7-AAD, TO-PRO-3, propidium iodide SYBR green 1, thiazole orange (TO) and its derivatives, or propidium iodide (PI).

21. The kit as claimed in claim 19 wherein the nucleic acid dye that reacts with DNA comprises Hoeschst 33342.

22. The kit as claimed in claim 18 wherein the nucleic acid dye that reacts with DNA comprises SYBR green.

23. The kit as claimed in any one of claims 19 to 22 wherein the nucleic acid dye that reacts with DNA and RNA comprises dihydroethidium, acridine orange, pyronin Y (PY), oxazine 1, LDS 751, 7-AAD, or SYTOX Orange.

24. The kit as claimed in any one of claims 19 to 22 wherein the nucleic acid dye that reacts with DNA and RNA comprises dihydroethidium.

25. The kit as claimed in any one of claims 19 to 24 wherein the antibody capable of selectively binding a leukocyte cell marker comprises CD45.

26. The kit as claimed in any one of claims 19 to 25 further comprising magnetic beads suitable for cell sorting.