SG174650A1

SG174650A1 - A method of monitoring parasite development in blood

Info

Publication number: SG174650A1
Application number: SG2010022473A
Authority: SG
Inventors: Laurent Renia; Benoit Malleret
Original assignee: Agency Science Tech & Res
Priority date: 2010-03-31
Filing date: 2010-03-31
Publication date: 2011-10-28
Also published as: WO2011123070A1

Abstract

A METHOD OF MONITORING PARASITE DEVELOPMENT IN BLOODAbstractA method and a kit to detect parasitemia and blood quantification of a blood samplewith several dyes in a flow cytometer allowing quantification of the a percentage ofthe sample containing a parasite in relation to leukocytes and reticulocytes.Figure 3

Description

A METHOD OF MONITORING PARASITE DEVELOPMENT IN BLOOD

Background

[01]. IHaematozoa is a general term that includes blood parasites, mainly protozoans and Helminths (parasitic worms). Well known examples include the protazoan parasites causing malaria, Leishmania and trypanosome. 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 used 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.

[02]. Malaria, which affects some 300 million people a year, may cause miscarriages, stillbirths or underweight, anaemic children 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.

[03]. 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 except the atemisinin 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.

[04]. Flow cytometric 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 or are very complex and time consuming.

[05]. Reticulocytes are immature 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.

[06]. Leukocytes are found in blood. The number of leukosites in the blood is often an indicator of disease. There are normally between 4x10° and 1.1x10'° 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

[07]. 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; a nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid and a dyed antibody capable of selectively binding a leukocyte cell marker; 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 percentage of the sample containing a parasite in relation to leukocytes and reticulocytes,

[08]. 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 dyed antibody capable of selectively binding a leukocyte cell marker.

Brief description of the drawings

Figure 1. Flow cytometric 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)

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

Figure 4. Flow cytometry analysis of the parasitemia in a patient infected with

Plasmodium vivax y

Figure 5. Flow cytometric 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. 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 patient infected with

Plasmodium vivax detected with ultra violet light (A) or a laser in the violet spectrum (B).

Detailed description

[09]. We have developed a technique using flow cytometry to quantify a parasite in whole blood samples. The technique includes a method for determining parasitemia and blood quantification of a blood sample comprising the steps of : 1. adding to the sample a nucleic acid dye that reacts with deoxyribonucleic acid; a nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid; and a dyed antibody capable of selectively binding a leukocyte cell marker; 2. exciting the sample with a light; 3. measuring a light emission pattern from the sample; 4. analysing the light emission pattern to quantify a percentage of the sample containing a parasite in relation to leukocytes and reticulocytes,

[010]. 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 step 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 mimmum and having high reproducibility.

[011]. Parasite detection

[012]. The method can monitor a range or parasites. Particularly, Haematozoa parasites, such as intra-erythrocytic Haematozoa, such as Plasmodia (eg:

Plasmodium falciparum), Babesia, or Bartonella and extra-erythrocytic Haematozoa such as Trypanosoma, Wuchereria bancrofti, Loa loa, Brugia malayi/timori,

Mansonella, Dirofilaria. The technique can quantify a wide range of protozoa and helminths haematozoa parasites known in the art.

[013]. Blood samples

[014]. 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.

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

[016]. 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 I, ethidium bromide, 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.

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

[018]. A nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid may include dihydroethidium, 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.

[019]. dyed antibody capable of selectively binding a leukocyte cell marker

[020]. A dyed antibody capable of selectively binding a leukocyte cell marker may be dyed with any dye known in the art to be capable of binding to an antibody and produce a signature emission pattern under a light stream. In some embodiments, the detectable label (dye) bound to the antibody may be a fluorophore. 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 fluorophores 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. Many of these dyes that can be conjugated to antibodies as well as others known in the art are available commercially from companies such as Molecular Probes, GE Healthcare and the like. In some embodiments, the fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a fluorometer or a photosensor such as CCD camera equipped with appropriate emission filters.

[021]. The specific antibodies useful for detecting the leukocyte cell can also be detectably labeled by coupling to a chemiluminescent compound (dye). The presence of a chemiluminescent- tagged antibody is then determined by detecting the luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound such as a bioluminescent protein may be used to label antibody reagent. Binding is measured by detecting the luminescence. Useful bioluminescent compounds include luciferin, luciferase and aequorin.

[022]. 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.

[023]. A leukocyte cell marker should be a cell marker that is primarily found on leukocyte cells and not found on red blood cells or reticulocyte cells. Commonly known a leukocyte cell marker may include CD3, CD2, CD4, CDS8, CD11b, CD68 and CD45r. Any other leukocyte cell markers known in the art would also be suitable. In one preferred embodiment the leukocyte cell marker is CD45.

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.

[024]. Antibodies of leukocyte cell markers 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 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.

[025]. 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"); 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.

[026]. The use of multiple specific dyes concurrently in the detection method has the advantage of being able to quantify blood leukocytes and reticulocytes at the same time of parasitemia determination.

[027]. exciting the sample with a light

[028]. The sample may be excited with light such as laser light and there may be a variety of wavelengths used. Preferably the light is emitted from a flow cytometer.

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

[030]. 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 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.

[031]. 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.

[032]. 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.

[033]. 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.

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

[035]. Multiparameter flow cytometry allows one to estimate, with high accuracy, relative quantities of a variety of cell 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.

[036]. analysing the light emission pattern to quantify a percentage of the sample containing a parasite in relation to leukocytes and reticulocytes

[037]. 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.

Staging of the infection

[038]. The method may be further adapted to enrich the samples allowing the development of the parasitic infection to be staged. To enriched the sample it was magnetically sorted using the MACS Miltenyi technology. Late blood stage parasites synthesize high quantity of the pigment hemozoin (Hz), which is rich in the iron metaland thus can be retained by the magnet on the purification column and further enriched 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.

[039]. 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 dyed antibody capable of selectively binding a leukocyte cell marker. Exemplary dyes and markers are listed above. Preferably the kit 1s for use in with flow cytometry as described above.

[040]. The invention will be more fully understood in light of the following examples which are not fo 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 1041]. Malaria Detection method

[042]. 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).

[043]. 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 and 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 leukocytes.

[044]. 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).

[045]. 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. As shown in Fig. 2, we obtained a perfect correlation between the two techniques.

[046]. 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

[047]. 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 retain by the magnet on the purification column and further enriched on late form). This allows another validation of the technique in its ability to detect the different stages of the parasites.

Monitoring of malaria development in human blood by flow cytometry (Mal-Count, Malaria Enumeration Kit) 1. For each sample to be stained, mix 1 pl of whole blood in 100 pl of cold

PBS. 2. Add 1 ul of Hoechst 33342 (Sigma), stock solution concentration 800 nM. 3. Add 1 pl of Dihydroethidium (Sigma), stock solution concentration 500 pg/ml. 4. Add 2 ul of anti-human CD45 APC (Miltenyi). 5. Incubate for 20 minutes at room temperature in the dark. 6. Add 400 pl of cold PBS. 7. Proceed to flow cytometric acquisition.

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

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

[050]. 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

[051]. 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 1s the extra- erythrocytic Haematozoa. The technique can be easily adapted to quantify these parasites.

[052]. List of Haematozoa infecting mammals:

Intra-erythrocytic Haematozoa (protozoa)

[053]. Plasmodia

[054]. Babesia

[055]. Bartonella

Extra-erythrocytic Haematozoa (protozoa/helminths)

[056]. Trypanosoma

[057]. Wuchereria bancrofii

[058]. Loa loa

[059]. Brugia malayi/timori

[060]. Mansonelia

[061]. Dirofilaria

[062]. 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.

[063]. 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.

[064]. 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.

[065]. 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.

[066]. 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.

[067]. 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

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

[069]. 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

1. 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; a nucleic acid dye that reacts with deoxyribonucleic acid and ribonucleic acid and a dyed antibody capable of selectively binding a leukocyte cell marker;

b. exciting the sample with a light;

¢. measuring a light emission pattern from the sample;

d. analysing the light emission pattern to quantify a percentage of the sample containing a parasite in relation to leukocytes and reticulocytes.

2. 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 dyed antibody capable of selectively binding a leukocyte cell marker.