JP2020041965A - Device and method for testing for protozoal infections - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection device and an inspection method capable of inspecting a protozoan infection easily, highly accurately and inexpensively, and particularly quickly in the early stage of infection. A protozoan infection by detecting changes in the physical properties of whole blood and / or erythrocytes and / or leukocytes and / or plasma infected with a parasitic protozoan with high sensitivity as changes in electrical properties. Is to be inspected with high accuracy and speed. As an electrical characteristic, it is desirable to measure impedance. [Selection diagram] Fig. 2

Description

  The present invention relates to a protozoan infection inspection apparatus and an inspection method.

  Malaria is caused by malaria parasites in erythrocytes, toxoplasmosis is caused by parasitism of toxoplasma in nucleated cells including leukocytes, and trypanosomiasis is an infection caused by trypanosomes growing in the bloodstream.

  Piroplasmosis is an infection caused by Babesia parasitizing an animal's erythrocytes or Tyleria on an animal's erythrocytes and / or leukocytes.

  Leukocytosonosis is an infection caused by leukocytozone mainly infesting avian red blood cells.

  As of 2016, 216 million people worldwide are infected with malaria annually, of which 44.5 million die (Non-Patent Document 1).

  It is said that one-third of the world's population is infected with Toxoplasma. In healthy adults, infection remains asymptomatic or causes mild cold-like symptoms (opportunistic). Infection). However, when the patient is in an immunosuppressed state, such as a fetus / infant, an organ transplant, or a patient with AIDS, the disease may become severe and lead to death (Non-Patent Document 2).

  Trypanosomiasis is also called `` African sleeping sickness '' because as the disease progresses, it causes meningoencephalitis, eventually falling into a coma and dying, and people at risk of infection are located in sub-Saharan Africa. It is estimated that the number is between 60 and 70 million in 36 countries (Non-Patent Document 3).

  Piroplasmosis, along with trypanosomiasis, has caused great damage to livestock and the like (Non-Patent Document 4).

  Leukocytosonosis is an infectious disease that damages the poultry industry (Non-Patent Document 5).

  For example, a blood smear test method has been widely used as a test method for malaria in the past, and blood is applied to a slide glass and subjected to Giemsa staining, followed by microscopic observation.

  Further, a method of mechanically performing a step corresponding to microscopic observation, such as measuring the intensity of transmitted fluorescence light by fluorescence staining, has been proposed (Patent Document 1).

  In recent years, a simple test kit that detects a specific substance of malaria parasite in blood and shows that the infection has occurred as a red line on a test paper has been used (Non-Patent Document 6).

  For other protozoal infections, the conventional blood smear test method is used.In trypanosomiasis, test methods such as centrifugation of blood and observation of leukocyte parts are used to increase sensitivity. ing.

  Furthermore, in recent years, as a new test method in the early stage of malaria infection, a technology for detecting a change in magnetic properties of hemozoin, which is a metabolic byproduct produced by malaria parasite from hemoglobin in infected red blood cells, has been published (Non-Patent Document 7). ).

JP-A-6-300753

a b c d FORTH Latest News About Malaria (Fact Sheet) (2018) “Toxoplasmosis”, Montoya J, Liesenfeld O Lancet 363 (2004) Modern Media Vol. 57, No. 6, 2011 Journal of the Beast Society of Japan 68 245-252 (2015) Livestock surveillance epidemic, Agricultural Research Institute ABC of Malaria, International Cooperation Agency Human Resources Department NATURE MEDICINE Volume 20 Number 9 (Sep. 2014)

The inspection method described in the background art has the following problems.
(1) The blood smear test method requires each step of preparation, fixation, and staining before microscopic observation, which is troublesome and difficult to detect in the case of low protozoan concentration in the early stage of infection.
(2) Although a method of mechanically performing a process corresponding to microscopic observation has been proposed, the device is extremely expensive, and such a device must be introduced to many infectious disease sites and sites in developing countries. Is not realistic.
(3) The test method using the simple test kit has lower sensitivity and accuracy than the blood smear test method described above. Further, if the test kit is stored in a place where the temperature is high, the accuracy is further deteriorated. Furthermore, in infected areas such as Africa, infections other than malaria, such as trypanosomiasis described later, may be co-infected, but since a malaria test kit can test only malaria, multiple tests are needed to identify infectious diseases. As a result, the amount of blood collected increases, the invasiveness increases, and it takes time. For this reason, it is not enough as a quick and accurate diagnostic method on site or on site.
(4) The technique of detecting a change in magnetic properties cannot detect a protozoan infection that does not produce metabolic by-products such as hemozoin, and the magnetic property change detection device is expensive and difficult to handle.

  SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems, and a protozoan that can easily and accurately test for protozoal infectious diseases at a low cost, and that can detect double infection by a single blood sampling and test and does not produce metabolic by-products It is an object of the present invention to provide an inspection device and an inspection method capable of inspecting an infectious disease.

  In addition, it has been made in view of the fact that there is no method for performing high-sensitivity, high-precision, and rapid testing, especially in the early stages of infection, and it has been proposed that the whole blood and / or red blood cells and / or white blood cells and / or plasma due to protozoal infection be detected. It is an object of the present invention to provide a completely novel protozoan infection inspection apparatus and method for detecting a change in physical characteristics as a change in electrical characteristics with high sensitivity.

  According to one aspect of the present invention, in order to solve the above-mentioned problems, the present invention provides a method for altering the physical properties of a test whole blood and / or erythrocyte and / or leukocyte and / or plasma infected with a parasitic protozoan. The present invention relates to an apparatus for detecting protozoal infections, which detects a change in electrical characteristics.

  Further, the present invention relates to a protozoan infection testing apparatus for measuring the impedance of whole blood, red blood cells, white blood cells, or plasma by applying an AC voltage having a predetermined frequency between electrodes as an electrical characteristic.

  Further, the present invention relates to a protozoan infection inspection apparatus that measures impedance while sequentially switching the frequency of an applied AC voltage.

  Further, the present invention relates to a protozoan infection inspection apparatus that sequentially switches the frequency in a range of 0.1 MHz to 300 MHz.

  Furthermore, the present invention relates to a protozoan infection testing apparatus that measures impedance while sequentially switching the frequency of an applied AC voltage, and tests for double infection by one blood collection and test.

  Furthermore, the present invention relates to a protozoan infection testing apparatus that detects malaria parasite and / or babesia in erythrocytes, tyrelia in erythrocytes and / or leukocytes, toxoplasma in leukocytes, and trypanosomes in whole blood in mammals.

  Furthermore, the present invention relates to a protozoan infection testing apparatus for detecting malaria parasite and / or babesia and / or leukotit zone in erythrocytes in birds.

  According to another aspect of the present invention, in order to solve the above-mentioned problems, the present invention provides a physical property of a test whole blood and / or erythrocyte and / or leukocyte and / or plasma infected with a parasitic protozoan. The present invention relates to a method for detecting protozoal infections, which detects a change in the electrical characteristics as a change in electrical characteristics.

  ADVANTAGE OF THE INVENTION According to this invention, the test apparatus and test which can test a protozoan infection easily and highly accurately and at low cost, can test for superinfection by one blood collection and test, and can also test protozoal infection which does not produce metabolic by-products A method can be provided.

  In addition, it has been made in view of the fact that there is no method for performing high-sensitivity, high-precision, and rapid testing, especially in the early stages of infection, and it has been proposed that the whole blood and / or red blood cells and / or white blood cells and / or plasma due to protozoal infection be detected. It is possible to provide a completely novel protozoan infectious disease inspection apparatus and method for detecting a change in physical characteristics as a change in electrical characteristics with high sensitivity.

FIG. 1 shows a container with a counter electrode for measuring the impedance of a sample in an embodiment of the present invention. FIG. 3 shows the results of measuring the resistance and reactance of a specimen having a malaria infection rate of red blood cells of 0.5%, 0.1%, and 0.01% while changing the frequency from 0.1 MHz to 300 MHz in Examples of the present invention. It is.

  Hereinafter, embodiments and examples of the present invention will be described, but embodiments of the present invention are not limited to the embodiments and examples described below.

  Infection with a parasitic protozoa changes the physical properties of the test whole blood, red blood cells, white blood cells or plasma.

  For example, during the life cycle of a malaria parasite, sporozoites, which are salivary gland-infected parasites, are injected into the body of a host when an anopheles infected with the parasite sucks blood. Sporozoites reach the liver in minutes and invade hepatocytes. In a few weeks, it breaks down into tens of thousands of "merozoites" and is released into the blood. Merozoites immediately invade erythrocytes and form "merozoites" through the ring, vegetative bodies, and meiosis. The new merozoites destroy infected red blood cells, infect the next red blood cells and multiply. This cycle is repeated, resulting in fever and anemia. Some of the merozoites differentiate into male and female reproductive mothers (gametocytes), and when they enter the mosquito by blood feeding, they mature into male and female reproductive bodies in the mosquito's midgut and then join. The zygotes become motile okineto and differentiate into oocysts in the midgut basement membrane. Thousands of "sporozoites" created within the oocyst migrate to the salivary glands and become infectious "mature sporozoites". The above cycle is known, and changes occur in the physical properties of red blood cells.

  It has been found that this change in physical properties can be detected as a change in electrical properties.

  In the above description, malaria is caused by parasitism of malaria parasite in erythrocytes. However, it is known that the form of infection varies depending on the type of protozoal infection. For example, toxoplasmosis is caused by toxoplasmosis parasitizing nucleated cells, including leukocytes, trypanosomiasis is caused by the proliferation of trypanosomes in the bloodstream, and pyroplasmosis is caused by Babesia in animal red blood cells, or Theileria is caused by the parasitism of red and / or white blood cells in animals, and leukocytosonosis is an infection caused by the parasitism of leukocytozone mainly in avian red blood cells.

  Therefore, a change in the physical properties of the test whole blood and / or red blood cells and / or white blood cells and / or plasma infected with the parasitic protozoan can be detected as a change in the electrical properties. Not necessarily a change in test whole blood and / or red blood cells and / or white blood cells and / or plasma due to the presence of the protozoan itself, but also a change in electrical properties caused by metabolic by-products, such as hemozoin in malaria it can.

(Preparation of specimen)
In vitro culture of malaria parasite-infected mice synchronizes the protozoan developmental stage with the mitotic phase by in vitro cultivation, infects the mitotic malaria parasite through the tail vein of the specimen preparation mouse, and 3 to 6 hours later, the infected blood is collected by cardiac puncture. After collection, 1.4% heparin was added, and it was confirmed that 95% or more of the protozoa in the infected erythrocytes were in the ring stage in the early stage of infection.

  The hematocrit value was adjusted to 40% by adding plasma from healthy mice, and the ratio of infected erythrocytes / total erythrocytes was 0.5%, 0.1%, and 0.01% by dilution with uninfected erythrocytes. Was prepared.

(Measurement of electrical characteristics)
Approximately 500 microliters of a sample whose infected erythrocyte concentration was adjusted was injected into the small container with electrodes shown in FIG. 1, and while changing the frequency from 0.1 MHz to 300 MHz, healthy (uninfected) mouse blood and 0.5%, 0.1% % And 0.01% of the sample were measured for resistance and reactance in about 20 seconds.

  FIG. 2 shows the results of plotting the frequency dependence of the resistance and reactance of uninfected blood and ring stage red blood cell concentrations of 0.5%, 0.1% and 0.01% at the initial stage of infection.

  Compared with uninfected blood, ring stage erythrocytes at the early stage of infection were found to have altered resistance and reactance.

  It was also confirmed that the resistance and the reactance changed according to the erythrocyte infection rate.

  According to the test apparatus and test method of the present invention, even if the number of red blood cells in the initial ring stage of malaria infection is 1 / 10,000 (red blood cell infection rate of 0.01% in FIG. 2), malaria infection can be performed in a short time with high sensitivity based on resistance and reactance characteristics. It has been found that can be detected.

  In addition, it became clear that the infection rate could be estimated from the resistance and reactance characteristics.

  The sample to be used for the protozoan infection test according to the present invention does not require a time period of about several tens of seconds for measurement, and may be the whole blood as it is, but may be used for the addition of anticoagulant such as heparin or separation of components. There is no particular limitation on the method of treating blood, such as centrifugation by adding an acid, etc., for example, by separating components by centrifugation, changes in the electrical characteristics of specific components such as red blood cells, white blood cells, plasma, etc. Since it can be measured, protozoal infections can be detected with higher accuracy.

  In addition, it is clear that changes in electrical characteristics vary depending on the type of protozoan infection. Therefore, various protozoal infections can be detected from a change in electrical characteristics measured by one blood sampling and test, and a double infection can also be tested.

  In addition, impedance (resistance and reactance) was measured as an electrical property, but other electrical properties such as inductance and capacitance may be used, and any electrical property may be used as long as a test for protozoal infection can be performed. .

  INDUSTRIAL APPLICABILITY The present invention is industrially applicable as a protozoan infection inspection apparatus and an inspection method.

Claims (8)

  A protozoan infection testing apparatus that detects a change in physical properties of a test whole blood and / or red blood cells and / or white blood cells and / or plasma infected with a parasitic protozoan as a change in electrical properties.   2. The protozoan infection testing apparatus according to claim 1, wherein an AC voltage having a predetermined frequency is applied between the electrodes to measure the impedance of the test whole blood, red blood cells, white blood cells, or plasma.   3. The protozoan infection inspection apparatus according to claim 2, wherein the impedance is measured while sequentially switching the frequency of the applied AC voltage.   The inspection apparatus for protozoal infections according to claim 3, wherein the frequency is sequentially switched within a range from 0.1 MHz to 300 MHz.   The protozoan infection testing apparatus according to claim 2, wherein the impedance is measured while sequentially switching the frequency of the applied AC voltage, and the double infection is tested by one blood sampling and test.   6. The protozoan infection according to any one of claims 1 to 5, wherein malaria parasites and / or babesia in erythrocytes, tyrelia in erythrocytes and / or leukocytes, toxoplasma in leukocytes, and trypanosomes in whole blood are detected in mammals. Inspection device for illness.   The inspection apparatus for protozoal infection according to any one of claims 1 to 5, wherein malaria parasites and / or babesia and / or leucocytozone in red blood cells are detected in birds.   A method for examining a protozoan infection in which a change in physical properties of a test whole blood and / or red blood cells and / or white blood cells and / or plasma infected with a parasitic protozoan is detected as a change in electrical properties.