JP2023015426A - Malaria inspection method and malaria inspection device - Google Patents

Abstract

To quantify the number of Plasmodia easily and correctly.SOLUTION: A malaria inspection method comprises: a specimen creation step of creating a specimen in which red blood cell are separated by removing 99.9% of white blood cells and blood platelets from a blood sample and the nucleus of the Plasmodium infecting the red blood cells is dyed; a first image acquisition step of acquiring an image from the specimen; and a quantifying step of quantitatively measuring the number of Plasmodia on the basis of the image acquired in the first image acquisition step.SELECTED DRAWING: Figure 5

Description

The present invention relates to a malaria testing method and a malaria testing device.

Known methods for detecting malaria parasite include a method of extracting the malaria parasite gene from a blood sample and amplifying the malaria parasite gene by polymerase chain reaction (PCR), and a detection method by immunochromatography using antigen-antibody reaction. there is
Of these methods, the method of extracting genes from blood samples and amplifying them by Polymerase Chain Reaction (PCR) takes time to extract genes from blood samples and the amplification reaction, and requires a stable power supply. Use will be restricted in emerging countries that are not well equipped. In addition, immunochromatographic detection using antigen-antibody reaction can provide results in a relatively short period of time of about 15 minutes, but it is a qualitative test such as whether there is a suspicion of being infected with malaria parasites, and quantitative Necessary information cannot be obtained due to lack of sexuality and false positives.
Therefore, a method of microscopically observing a blood sample stained by Giemsa staining has been studied (see, for example, Patent Document 1).

JP 2016-136158 A

However, the method of observing blood samples stained with Giemsa staining under a microscope requires skillful techniques because the examiner directly observes the cells. It is not suitable as a simple screening test.

An object of the present invention is to provide a malaria testing method and a malaria testing device that can easily and accurately quantify the number of malaria parasites.

In order to solve the above problems, the malaria testing method of the present invention is
A first image acquisition step of acquiring an image of a specimen from which 99.9% or more of leukocytes have been removed from a blood sample collected from a subject and from which processing has been performed to visualize malaria parasites present in the blood sample;
A quantification step of quantitatively measuring the number of the malaria parasites based on the image acquired in the first image acquisition step;
characterized by having

In addition, the malaria inspection device of the present invention is
a first image acquisition means for acquiring an image in which 99.9% or more of leukocytes and platelets are removed from a blood sample and processed for visualizing malaria parasites present in the blood sample;
Quantification means for quantitatively measuring the number of the malaria parasites based on the image acquired by the first image acquisition means;
characterized by comprising

According to the present invention, it is possible to easily and accurately quantify the number of malaria parasites.

It is an external view of an inspection apparatus. It is a functional block diagram which shows the control structure of an inspection apparatus. It is a figure which shows an analysis tool. It is the figure which showed the white blood cell count before and behind filter processing. 4 is a flow chart showing the flow of inspection. It is an example of a low resolution image. It is an example of a high resolution image. It is an example of an image showing the morphology of a malaria parasite.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

[composition]
FIG. 1 is an external view of an inspection apparatus 10 according to this embodiment.
As shown in FIG. 1, the inspection apparatus 10 is used while loaded with an analysis tool 20 holding a specimen containing nuclear-stained red blood cells.
The inspection device 10 is a device that inspects the presence or absence of malaria parasites in red blood cells using the above specimen, that is, whether or not malaria parasites invade red blood cells, parasitize in red blood cells, and are infected with malaria. . Moreover, the inspection device 10 is a device for determining the type of malaria parasite when infected.

As shown in FIGS. 1 and 2, the inspection apparatus 10 includes, for example, a housing 11, an insertion section 12, a display section 13, an operation section 14, an image acquisition section 15, a control section 16, a storage section 17, a power supply section 18, and the like. It has

The housing 11 is formed in such a size and shape that a user such as a medical worker can pick it up and perform diagnostic work, and is made of resin, for example. In this embodiment, as shown in FIG. 1, it is formed in a flat rectangular parallelepiped shape.
An insertion portion 12 for inserting the analytical tool 20 into the inside of the housing 11 is provided on the side surface of the housing 11, and a display portion 13 and an operation portion 14 are provided on the upper surface thereof.

The insertion portion 12 is an opening corresponding to the shape of the analysis tool 20, into which the analysis tool 20 is inserted.

The display unit 13 is composed of a color liquid crystal display or the like, and displays various screens for displaying captured images and diagnosis results according to display control signals input from the control unit 16 .

The operation unit 14 includes a touch panel provided on the screen of the display unit 13 and various hard keys arranged around the screen of the display unit 13 . When a touch panel or various hard keys are pressed with a finger or a touch pen, the operation unit 14 detects the XY coordinates of the pressed power point as a voltage value, and outputs an operation signal associated with the detected position to the control unit 16. output to

The image acquisition unit 15 acquires image data of the specimen on the analysis tool 20 .
The image acquisition unit 15 is configured including irradiation means, imaging means, imaging means, and the like. The irradiating means is composed of a light source, a filter, etc., and irradiates the sample on the analysis tool 20 inserted into the insertion portion 12 with light. The imaging means is composed of an eyepiece lens, an objective lens, and the like, and forms an image of transmitted light, reflected light, or fluorescence emitted from the specimen on the analysis tool 20 by the irradiated light. The imaging means includes a CCD (Charge Coupled Device) sensor or the like, and generates digital image data by imaging an image formed on an imaging plane by the imaging means.
The image acquisition unit 15 is provided with a bright field unit combining irradiation means and imaging means suitable for bright field observation, and a fluorescence unit combining irradiation means and imaging means suitable for fluorescence observation. By doing so, it is possible to switch between bright field and fluorescence.
In addition, the image acquisition unit 15 can switch the resolution to capture an image. Specifically, low-resolution (enough to measure fluorescent spots, eg, 5M pixels or more) and high-resolution (enough to understand the morphology of the protozoa, eg, 50M pixels or more) images can be taken.

The control unit 16 includes a CPU (Central Processing Unit), a RAM (Random Access Memory), etc., and executes various processes in cooperation with various programs stored in the storage unit 17 to control the inspection apparatus 10. Overall control of operations. For example, the control unit 16 performs image analysis processing in cooperation with a program stored in the storage unit 17, quantitatively measures the number of malaria parasites, and determines the types of malaria parasites. come true.

The storage unit 17 is composed of, for example, an HDD (Hard Disk Drive), a semiconductor non-volatile memory, or the like. Various programs and various data are stored in the storage unit 17 as described above.

The power supply unit 18 supplies power for realizing the operation of the inspection apparatus 10 . Power supply unit 18 is configured by, for example, a rechargeable lithium-ion battery. Also, the power supply section 18 may be configured by a dry battery or an AC power connection section.

3(a) to 3(c) are diagrams showing the analysis tool 20. FIG.
The analysis tool 20 is a tool capable of separating erythrocytes from a blood sample containing human blood as a main component.

The blood sample is, for example, blood collected from a subject diluted with a predetermined diluent and containing a predetermined staining liquid.

As the diluent, for example, a buffer, an isotonic solution, a culture medium, or the like, which does not denature the cells contained in the biological sample, is used. Also, an anticoagulant may be used, such as EDTA. However, heparin-based blood coagulation inhibitors are not preferable because they affect Giemsa staining.

As the staining solution, for example, 4',6-diamidino-2-phenylindole dihydrochlorid (DAPI), acridine orange, Hoechst 33342, etc. are used as fluorescent dyes capable of binding to DNA. Alternatively, SYTO59 (registered trademark) can also be used as a staining solution for malaria parasite DNA in erythrocytes.

Further, when SYTO59 (registered trademark) is used as the staining solution, the nucleic acid of the malaria parasite infected with red blood cells is fluorescently stained, and fluorescence is emitted from this nucleic acid. This staining solution emits fluorescence with a wavelength of 640 nm to 660 nm when excited with excitation light with a wavelength of 600 nm to 635 nm.

The analysis tool 20 includes, for example, an inlet path 21 through which a blood sample flows, a filter 22 provided in the inlet path 21, a diffusion path 23 through which the blood sample flowing from the inlet path 21 is diffused, and diffusion through the diffusion path 23. and a holding portion 24 for holding the collected blood sample in a uniform state.

An appropriate amount of blood sample is flowed into inlet channel 21 . The entrance path 21 is formed in a cylindrical shape with a diameter of about 5 mm, for example.

The filter 22 removes 99.9% or more (more preferably 99.99% or more) of leukocytes from the blood sample that has flowed through the inlet path 21 .
Specifically, the filter 22 has a non-object trapping structure formed by a plurality of fibrous materials that traps white blood cells.
The fibrous material is, for example, silicon oxide containing silicon oxide as a main component, preferably amorphous silicon dioxide. The thickness of the fibrous substance is about 0.01 μm to 1 μm. The fibrous substances are densely packed so as to be entangled with each other. The fibrous substances are mixed with those branched in irregular directions. Also, the fibrous substances are curved and intertwined.
Since the shortest distance between the fibrous substances is smaller than the trapped substances such as leukocytes, the trapped substances can be effectively removed from the blood sample. That is, leukocytes and those whose maximum diameter is larger than the voids between the fibrous substances are captured by the fibrous substances as trapped substances. On the other hand, the non-object capturing structure can extract red blood cells by allowing red blood cells that can pass through the voids between the fibrous substances to pass through the fibrous substances. Even if the gap between the fibrous substances is narrower than the size of the red blood cells, the red blood cells have deformability that allows them to be easily deformed, so the red blood cells can pass through the gaps and be extracted.

The non-object capturing structure may be made of porous material other than fibrous material. Porous materials are, for example, nitrocellulose, polyvinylidene fluoride (PVDF), agarose. Alternatively, it may be formed by forming a large number of through holes in an inorganic material substrate such as silicon, glass, or ceramic.
Alternatively, the non-target capture structure may comprise fibrous material and the porous material described above.

Although the filter 22 is provided in the inlet path 21 here, the blood sample may be filtered in advance by the filter 22 separate from the analysis tool 20 and the filtered sample may be flowed into the inlet path 21. good.

The diffusion path 23 is provided in communication with the inlet path 21 and has a height of about 2 mm. Since leukocytes are about 2 mm in size, leukocytes in the blood sample that could not be completely removed by filter 22 can be removed by diffusion path 23 .

The holding portion 24 is provided in communication with the diffusion path 23 and has a height of about 5 to 7 μm. Since red blood cells are about 5 to 7 μm in size, the red blood cells in the blood sample are uniformly held in the holding section 24 . However, it is also effective to choose a configuration of 100-1000 μm in order to derive the capillary force necessary for diffusion of the blood sample.
A cyclic olefin polymer (COC, COP, etc.) is provided on the bottom surface of the holding part 24, and has a function of efficiently detecting fluorescence signals.

By using such an analysis tool 20, when a subject is infected with malaria, most white blood cells are removed from the blood sample, and red blood cells parasitized by malaria parasites, which are the causative microorganisms of malaria, are separated from the blood sample. A sample obtained by staining the protozoan DNA with a fluorescent dye is adhered to the holding portion 24 . Since leukocytes have a nucleus, they are stained with a fluorescent dye or the like that can bind to the DNA. Therefore, if the measurement sample contains leukocytes, the nuclei of the leukocytes may be stained and may be detected as malaria parasites, which affects identification of malaria parasites and is a factor in lowering detection sensitivity and quantification.
By using this analysis tool 20, 99.9% or more of leukocytes that cause noise can be removed, the nuclei of leukocytes are stained, and detection as malaria parasites is suppressed, so the detection sensitivity of malaria parasites improves. Further, since the holding portion 24 of the analysis tool 20 has a structure that white blood cells cannot reach, red blood cells can be separated with high accuracy. According to non-patent literature, the detection sensitivity of immunochromatography is equivalent to 200 parasites/μl in terms of the number of malaria parasites per 1 μl, but only 55% of malaria-infected persons can be detected. With this conversion value, 83% of malaria-infected patients can be detected with 20 parasites/μl equivalent, and 95% can be detected with 2 parasites/μl equivalent. When 99.9% or more of the leukocytes are removed, the signal due to the effect of noise can be reduced to 10 parasites/μl or less, so that a test method with high detection sensitivity can be provided. can be reduced to 1 parasite/μl or less, enabling high-sensitivity detection of malaria parasites.
(Non-patent document: Slater et al. Nature volume 528, pagesS94-S101 (2015)).

[Example of leukocyte removal by filter]
When the leukocyte count was measured with a general cell counter, when the human whole blood sample was treated with the filter, the leukocyte count decreased before and after the filter treatment, as shown in FIG. 4, and fell below the detection limit.

In this case, it is assumed that the blood sample before being poured into the analysis tool 20 contains a predetermined staining solution. A configuration in which nuclear staining is performed is also possible.

[test]
FIG. 5 is a flow chart showing the flow of inspection using the inspection apparatus 10. As shown in FIG.
As shown in FIG. 5, the inspection using the inspection device 10 removes 99.9% or more of the white blood cells from the blood sample, separates the red blood cells, and visualizes the malaria parasites by performing nuclear staining of the malaria parasites infected with the red blood cells. A specimen preparation step (step S1) for preparing a specimen that has been subjected to processing to perform a first image acquisition step (step S2) for obtaining a low-resolution image from the specimen, and based on the low-resolution image, the malaria parasite A quantification step (step S3) of quantitatively measuring the number, and a second image acquisition step (step S4) of acquiring a high-resolution image of the specimen with a resolution higher than that of the image acquired in the first image acquisition step. and a determination step (step S5) of identifying the morphology of the malaria parasite based on the high-resolution image and determining the type of the malaria parasite.

The sample preparation step is performed by a tester preparing a blood sample and pouring it into the analysis tool 20 described above.

The first image acquisition step is executed when the tester inserts the analysis tool 20 holding the sample into the insertion portion 12 of the inspection device 10 and issues an inspection start instruction using the operation unit 14 .
When receiving the start instruction, the control unit 16 causes the image acquisition unit 15 to acquire a low-resolution (eg, 5 M pixels) image of the sample (stained red blood cells).
As low-resolution images, a fluorescence image shown in FIG. 6(a) and a bright-field image (morphological image) shown in FIG. 6(b) are acquired. The fluorescence image is an image in which the nucleic acid of the malaria parasite is represented by fluorescent bright spots. A bright-field image is an image that represents the morphology of red blood cells in a specimen and includes the same range as the fluorescence image.

The quantification step is a step of analyzing the obtained low-resolution image and quantitatively counting the number of malaria parasites.
The control unit 16 acquires the morphology and number of red blood cells from the bright field image (morphological image). In addition, the control unit 16 measures fluorescent labeling signals such as the number of fluorescent bright spots or emission luminance corresponding to the malaria parasite in the fluorescent image.
This makes it possible to obtain a quantified value of the number of malaria parasites in red blood cells.

The second image acquisition step may be executed when the number of malaria parasites in red blood cells is equal to or greater than a predetermined amount as a result of analysis of the low-resolution image.
The control unit 16 uses the image acquisition unit 15 to acquire a high-resolution image of the specimen (enough to understand the morphology of the protozoa).
As the high-resolution image, the fluorescence image shown in FIG. 7A is acquired, but a bright field image can also be acquired as necessary. In addition, by staining the nucleic acid of the malaria parasite using a plurality of fluorescent dyes having different wavelengths, it is possible to obtain a characteristic stained image according to the species of the malaria parasite. can be determined.
The control unit 16 performs predetermined image processing on the acquired fluorescence image (see FIG. 7(b)) to identify the morphology of the malaria parasite.

The determination step is a step of determining the type of the malaria parasite based on the morphology when the morphology of the malaria parasite is obtained in the image acquisition step.
The control unit 16, for example, as shown in FIG. 8, prepares in advance a standard image including a plurality of images simulating different morphologies according to the type of malaria parasite, and the resulting image of the second image acquisition step. The type of malaria parasite is determined by comparison.

In an inspection using such an inspection device 10, the number of malaria parasites is quantitatively counted based on the image, so the number of malaria parasites can be accurately quantified.
In malaria diagnosis, it is not enough to just detect the number of malaria parasites. Since the type is determined, effective treatment strategies can be determined.
In particular, in developed countries such as Africa where malaria is prevalent, the above-described diagnosis can be made by a simple and rapid method without obtaining advanced test information.
Furthermore, the inspection apparatus 10 of the present embodiment is battery-driven and does not require an external power supply, which is more convenient.

[effect]
As described above, according to the malaria testing method of the present embodiment, 99.9% or more of leukocytes are removed from a blood sample collected from a subject, and processing is performed to visualize malaria parasites present in the blood sample. a first image acquisition step (step S2) of acquiring an image of the sample obtained; a quantification step (step S3) of quantitatively measuring the number of malaria parasites based on the image acquired in the first image acquisition step; have
Therefore, it is possible to easily and accurately quantify the number of malaria parasites.
Further, according to the present embodiment, the specimen preparation step ( step S1).
By preparing such a sample, it is possible to easily and accurately quantify the number of malaria parasites.

Further, according to the present embodiment, 99.99% or more of leukocytes are removed from the blood sample in the sample preparation step.
By using such specimens, more accurate quantification of the number of malaria parasites can be performed.
Further, according to the present embodiment, in the specimen preparation step, the malaria parasite is visualized by staining the nucleus of the erythrocyte-infected malaria parasite.
Therefore, the malaria parasite can be easily visualized.

Further, according to the present embodiment, in the sample preparation step, the staining solution used for staining the nucleus of the malaria parasite infected with red blood cells contains a fluorescent dye.
Therefore, it becomes easy to quantify the number of malaria parasites.
Further, according to the present embodiment, the staining solution contains multiple types of fluorescent dyes.
Therefore, it is possible to identify multiple types of malaria parasites.
Also, according to this embodiment, the fluorescent dye is any of 4′,6-diamidino-2-phenylindole dihydrochlorid (DAPI), acridine orange, and Hoechst 33342.
By using such a fluorescent dye, quantification of the number of malaria parasites can be suitably realized.
Further, according to the present embodiment, the staining solution used for staining the nuclei of malaria parasites infected with red blood cells in the specimen preparation step is SYTO59 (registered trademark).
By using such a fluorescent dye, quantification of the number of malaria parasites can be suitably realized.

Further, according to the present embodiment, the second image acquisition step (step 4) of acquiring a high-resolution image with a resolution higher than a predetermined value for the specimen, and the high-resolution image acquired in the second image acquisition step and a determination step (step 5) of discriminating the morphology of the malaria parasite and determining the type of the malaria parasite.
Therefore, since the type of malaria can be known, it becomes possible to determine an effective treatment policy.

Further, according to the present embodiment, the determination step determines the type of malaria parasite by comparing the standard image prepared in advance with the morphology of the malaria parasite identified from the high-resolution image.
Also, the standard image includes a plurality of images simulating different morphologies according to the type of malaria parasite.
For this reason, it is possible to more accurately determine the type of malaria by performing determination by comparison with the standard image.

Further, according to the present embodiment, in the sample preparation step, red blood cells are separated by passing a blood sample through a filter that removes 99.9% or more of white blood cells.
Therefore, the sample can be prepared by a relatively simple method.

For example, as a device for executing the malaria inspection method of the present invention, a device other than the inspection device 10 of the above embodiment can acquire low-resolution images and high-resolution images and can perform determination processing. It can be.

As described above, the present invention has been specifically described based on the embodiments, but the present invention is not limited to the above embodiments, and can be modified without departing from the scope of the invention.
Wear.
In addition to leukocytes, platelets may also be removed from the blood sample. This makes it possible to more accurately identify the number of malaria parasites.

10 inspection device 11 housing 12 insertion unit 13 display unit 14 operation unit 15 image acquisition unit 16 control unit (first image acquisition means, second image acquisition means, quantification means)
17 Storage Unit 18 Power Supply Unit 20 Analysis Tool 21 Inlet Path 22 Filter 23 Diffusion Path 24 Holding Unit

Claims (16)

A first image acquisition step of acquiring an image of a specimen from which 99.9% or more of leukocytes have been removed from a blood sample collected from a subject and from which processing has been performed to visualize malaria parasites present in the blood sample;
A quantification step of quantitatively measuring the number of the malaria parasites based on the image acquired in the first image acquisition step;
A malaria testing method characterized by having 2. The malaria testing method according to claim 1, wherein said specimen is said blood sample from which 99.99% or more of leukocytes have been removed. 3. The malaria testing method according to claim 1, wherein the malaria parasite present in the blood sample is visualized by staining the nucleus. The method further comprises a specimen preparation step of removing 99.9% or more of leukocytes from the blood sample collected from the subject, and performing a process for visualizing malaria parasites present in the blood sample to prepare the specimen. The malaria testing method according to claim 1. 5. The malaria testing method according to claim 4, wherein 99.99% or more of leukocytes are removed from the blood sample in said sample preparing step. 6. The malaria testing method according to claim 4 or 5, wherein, in said specimen preparing step, nuclei of malaria parasites present in said blood sample are stained to visualize malaria parasites. 7. The malaria testing method according to claim 6, wherein, in said specimen preparation step, nuclei of malaria parasites present in said blood sample are stained with a staining agent containing a fluorescent dye to visualize malaria parasites. . 8. The malaria testing method according to claim 7, wherein the staining agent contains a plurality of types of fluorescent dyes having wavelengths different from each other. 9. The malaria testing method according to claim 7, wherein the fluorescent dye is any one of 4',6-diamidino-2-phenylindole dihydrochlorid (DAPI), acridine orange, and Hoechst 33342. 5. The malaria testing method according to claim 4, wherein, in said specimen preparation step, nuclei of malaria parasites present in said blood sample are stained with SYTO59 (registered trademark) to visualize malaria parasites. The first image acquisition step is characterized by acquiring a fluorescence image that represents the presence of malaria parasites with fluorescent bright spots, and a morphological image that represents the morphology of red blood cells in the specimen and includes the same range as the fluorescence image. The malaria testing method according to any one of claims 7 to 10. a second image acquisition step of acquiring a high-resolution image of the specimen, the resolution of which is higher than that of the image acquired in the first image acquisition step;
a determination step of identifying the morphology of the malaria parasite based on the high-resolution image and determining the type of the malaria parasite;
The malaria testing method according to any one of claims 4 to 11, comprising: 13. The method according to claim 12, wherein the determining step determines the type of the malaria parasite by comparing a standard image prepared in advance with the morphology of the malaria parasite identified from the high-resolution image. Malaria test method. 14. The malaria testing method according to claim 13, wherein said standard image includes a plurality of images simulating different morphologies according to the type of malaria parasite. 15. The malaria testing method according to any one of claims 4 to 14, wherein the specimen preparing step passes the blood specimen through a filter that removes leukocytes. a first image acquisition means for acquiring an image of a specimen from which 99.9% or more of leukocytes and platelets have been removed from the blood sample and which has been processed to visualize malaria parasites present in the blood sample;
Quantification means for quantitatively measuring the number of the malaria parasites based on the image acquired by the first image acquisition means;
A malaria testing device comprising:

Images