JP7094520B2 - Protozoan infection testing equipment and methods - Google Patents
Protozoan infection testing equipment and methods Download PDFInfo
- Publication number
- JP7094520B2 JP7094520B2 JP2018171213A JP2018171213A JP7094520B2 JP 7094520 B2 JP7094520 B2 JP 7094520B2 JP 2018171213 A JP2018171213 A JP 2018171213A JP 2018171213 A JP2018171213 A JP 2018171213A JP 7094520 B2 JP7094520 B2 JP 7094520B2
- Authority
- JP
- Japan
- Prior art keywords
- protozoan
- infection
- erythrocytes
- leukocytes
- blood
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000012360 testing method Methods 0.000 title claims description 33
- 208000010362 Protozoan Infections Diseases 0.000 title claims description 31
- 238000000034 method Methods 0.000 title description 22
- 210000003743 erythrocyte Anatomy 0.000 claims description 41
- 208000015181 infectious disease Diseases 0.000 claims description 32
- 239000008280 blood Substances 0.000 claims description 30
- 210000004369 blood Anatomy 0.000 claims description 24
- 201000004792 malaria Diseases 0.000 claims description 21
- 210000000265 leukocyte Anatomy 0.000 claims description 20
- 208000035473 Communicable disease Diseases 0.000 claims description 10
- 201000008680 babesiosis Diseases 0.000 claims description 9
- 241000223836 Babesia Species 0.000 claims description 7
- 241000224016 Plasmodium Species 0.000 claims description 6
- 210000000601 blood cell Anatomy 0.000 claims description 6
- 230000003071 parasitic effect Effects 0.000 claims description 6
- 238000010241 blood sampling Methods 0.000 claims description 5
- 241001470497 Leucocytozoon Species 0.000 claims description 3
- 241000223777 Theileria Species 0.000 claims description 3
- 241000223996 Toxoplasma Species 0.000 claims description 3
- 241000124008 Mammalia Species 0.000 claims description 2
- 238000007689 inspection Methods 0.000 description 15
- 210000002381 plasma Anatomy 0.000 description 11
- 239000006227 byproduct Substances 0.000 description 5
- 210000003936 merozoite Anatomy 0.000 description 5
- 230000002503 metabolic effect Effects 0.000 description 5
- 230000000704 physical effect Effects 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000011120 smear test Methods 0.000 description 4
- 210000003046 sporozoite Anatomy 0.000 description 4
- 241000271566 Aves Species 0.000 description 3
- 201000005485 Toxoplasmosis Diseases 0.000 description 3
- 108010080417 hemozoin Proteins 0.000 description 3
- 230000000394 mitotic effect Effects 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- 208000003322 Coinfection Diseases 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 238000009534 blood test Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 210000003250 oocyst Anatomy 0.000 description 2
- 230000024241 parasitism Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000001850 reproductive effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 201000002311 trypanosomiasis Diseases 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 208000000230 African Trypanosomiasis Diseases 0.000 description 1
- 241000256186 Anopheles <genus> Species 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010037075 Protozoal infections Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000223997 Toxoplasma gondii Species 0.000 description 1
- 241000223104 Trypanosoma Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000005534 hematocrit Methods 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 208000029080 human African trypanosomiasis Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 201000011475 meningoencephalitis Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 208000028172 protozoa infectious disease Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 201000002612 sleeping sickness Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は、原虫感染症の検査装置および検査方法に関するものである。 The present invention relates to an inspection device and an inspection method for protozoan infections.
マラリアはマラリア原虫が赤血球に、トキソプラズマ症はトキソプラズマが白血球を含む有核細胞に寄生することで引き起こされ、トリパノソーマ症はトリパノソーマが血流中で増殖することによって引き起こされる感染症である。 Malaria is caused by malaria protozoa parasitizing red blood cells, toxoplasmosis by parasitizing nucleated cells containing leukocytes, and trypanosomiasis is an infectious disease caused by the growth of trypanosoma in the bloodstream.
また、ピロプラズマ症はバベシアが動物の赤血球に、もしくは、タイレリアが動物の赤血球および/または白血球に寄生することによって引き起こされる感染症である。 Piroplasmosis is also an infectious disease caused by Babesia parasitizing animal erythrocytes or Theileria on animal erythrocytes and / or leukocytes.
また、ロイコチトゾーン症はロイコチトゾーンが主に鳥類の赤血球に寄生することによって引き起こされる感染症である。 Leucocytozoonosis is an infectious disease caused by leucocytozoon parasitizing mainly avian erythrocytes.
２０１６年時点で、全世界では年間２．１６億人がマラリアに感染し、うち４４．５万人が死亡している（非特許文献１）。 As of 2016, 216 million people worldwide are infected with malaria annually, of which 445,000 die (Non-Patent Document 1).
また、世界人口の１／３がトキソプラズマに感染しているといわれており、健康な成人の場合には感染しても無徴候に留まるか、軽い風邪のような症状が出る程度である（日和見感染症）。しかし胎児・幼児や臓器移植やエイズの患者など、免疫抑制状態にある場合には重症化して死に至ることもある（非特許文献２）。 In addition, it is said that one-third of the world's population is infected with Toxoplasma gondii, and in the case of healthy adults, even if infected, the symptoms remain asymptomatic or a mild cold-like symptom appears (opportunistic). Infection). However, if the patient is in an immunosuppressed state, such as a fetus / infant, an organ transplant, or an AIDS patient, the condition may become severe and death may occur (Non-Patent Document 2).
また、トリパノソーマ症は病状が進行すると髄膜脳炎を起こし、最終的には昏睡状態に陥って死に至ることから「アフリカ睡眠病」とも呼ばれており、感染リスクを抱える人々はサハラ以南のアフリカ３６カ国で６０００から７０００万人と推定されている（非特許文献３）。 Trypanosomiasis is also called "African sleeping sickness" because it causes meningoencephalitis as the condition progresses and eventually leads to coma and death. People at risk of infection are in sub-Saharan Africa. It is estimated that there are 60 to 70 million people in 36 countries (Non-Patent Document 3).
また、ピロプラズマ症は、トリパノソーマ症とともに家畜等に大きな被害をもたらしている（非特許文献４）。 In addition, piroplasmosis causes great damage to livestock and the like together with trypanosomasis (Non-Patent Document 4).
ロイコチトゾーン症は、養鶏業に被害をもたらす感染症である（非特許文献５）。 Leucocytozoonosis is an infectious disease that causes damage to the poultry industry (Non-Patent Document 5).
例えば、従来から広く用いられているマラリアの検査方法としては血液塗沫検査法があり、血液をスライドガラスに塗りつけギムザ染色を行い顕微鏡観察している。 For example, there is a blood smear test method as a method for testing malaria that has been widely used in the past, and blood is smeared on a slide glass, stained with Giemsa, and observed under a microscope.
また、蛍光染色により蛍光透過光の強度を測定する等、顕微鏡観察に相当する工程を機械的に行う方法も提案されている（特許文献１）。 Further, a method of mechanically performing a step corresponding to microscopic observation, such as measuring the intensity of fluorescent transmitted light by fluorescent staining, has also been proposed (Patent Document 1).
また近年、血液中のマラリア原虫が持つ特異的な物質を感知し、感染したことを試験紙の上に赤い線として現す簡易検査キットが用いられるようになっている（非特許文献６）。 Further, in recent years, a simple test kit that senses a specific substance possessed by Plasmodium malaria in blood and shows the infection as a red line on a test paper has been used (Non-Patent Document 6).
他の原虫感染症については、従来からの血液塗沫検査法が用いられており、トリパノソーマ症においては、感度を高めるために血液を遠心分離して白血球部分を観察する等の検査法が取られている。 For other protozoan infections, the conventional blood smear test method is used, and for trypanosomasis, a test method such as centrifuging the blood and observing the leukocyte portion is taken in order to increase the sensitivity. ing.
さらに近年、マラリア感染の初期段階の新たな検査法として、感染した赤血球中でマラリア原虫がヘモグロビンから産生する代謝副産物であるヘモゾインの磁気特性変化を検出する技術が発表されている（非特許文献７）。 Furthermore, in recent years, as a new test method in the early stage of malaria infection, a technique for detecting changes in the magnetic properties of hemozoin, which is a metabolic by-product produced by Plasmodium malaria from hemoglobin, has been published in infected erythrocytes (Non-Patent Document 7). ).
背景技術で示した検査方法には、以下のように課題がある。
（１）血液塗沫検査法は、顕微鏡観察の前に標本の作成、固定、染色の各工程を要し、手間がかかるとともに感染初期の低原虫濃度の場合は検出が困難である。
（２）顕微鏡観察に相当する工程を機械的に行う方法も提案されているが、装置が極めて高価であり、発展途上国内の多数の感染症発生現地・現場にこのような装置を導入することは現実的ではない。
（３）簡易検査キットを用いた検査方法は、上述の血液塗沫検査法より感度・精度が悪い。また、検査キットを温度が高いところに保管すると精度がさらに悪化する。さらに、アフリカ等の感染地帯ではマラリア以外の感染症、例えば後述するトリパノソーマ症と重複感染している場合もあるが、マラリア用検査キットではマラリアしか検査できないため、感染症の特定には複数の検査を行うこととなり、結果として採血量が増えて浸潤性が高まるとともに時間もかかる。このため、現地・現場での迅速かつ正確な診断法としては十分ではない。
（４）磁気特性変化を検出する技術では、ヘモゾイン等代謝副産物を産生しない原虫感染症を検知できず、また、磁気特性変化の検出装置は高価格になるとともに取り扱いが難しいという問題もある。
The inspection method shown in the background technique has the following problems.
(1) The blood smear test method requires each step of preparing, fixing, and staining a specimen before microscopic observation, which is time-consuming and difficult to detect in the case of low protozoan concentration in the early stage of infection.
(2) A method of mechanically performing a process equivalent to microscopic observation has also been proposed, but the equipment is extremely expensive, and such equipment should be introduced at many infectious disease outbreak sites / sites in developing countries. Is not realistic.
(3) The test method using the simple test kit is inferior in sensitivity and accuracy to the blood smear test method described above. Further, if the inspection kit is stored in a place with a high temperature, the accuracy is further deteriorated. Furthermore, in infected areas such as Africa, there are cases of co-infection with infectious diseases other than malaria, such as tripanosoma disease, which will be described later. As a result, the amount of blood collected increases, the infectivity increases, and it takes time. Therefore, it is not sufficient as a quick and accurate diagnostic method in the field or on-site.
(4) The technique for detecting changes in magnetic properties cannot detect protozoan infections that do not produce metabolic by-products such as hemozoin, and the devices for detecting changes in magnetic properties are expensive and difficult to handle.
本発明は、上記課題に鑑みてなされたものであり、原虫感染症を簡単・高精度にしかも安価に検査できるとともに、１回の採血および検査により重複感染を検査でき、代謝副産物を産生しない原虫感染症も検査できる検査装置および検査方法を提供することを目的とする。 The present invention has been made in view of the above problems, and can easily, highly accurately and inexpensively test for protozoan infections, can test for multiple infections 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.
また、特に感染初期でも高感度かつ高精度かつ迅速に検査する方法が見当たらないことに鑑みてなされたものであり、原虫感染による被検全血および/または赤血球および/または白血球および/または血漿の物理的特性変化を電気的特性変化として高感度で検知するという全く新規な原虫感染症の検査装置および検査方法を提供することを目的とする。 It was also made in view of the lack of a sensitive, accurate and rapid test, especially in the early stages of infection, for whole blood and / or red blood cells and / or leukocytes and / or plasma tested for protozoan infection. It is an object of the present invention to provide a completely new inspection device and inspection method for protozoan infections in which changes in physical characteristics are detected as changes in electrical characteristics with high sensitivity.
本発明の一つの観点によれば、上記課題を解決するために、本発明は、寄生性原虫が感染した被検全血および/または赤血球および/または白血球および/または血漿の物理的特性の変化を電気的特性の変化として検知する原虫感染症の検査装置に関する。 According to one aspect of the invention, in order to solve the above problems, the present invention presents a change in the physical properties of whole blood and / or erythrocytes and / or leukocytes and / or plasma infected with a parasitic protozoan. The present invention relates to a protozoan infection inspection device that detects changes in electrical characteristics.
さらに、本発明は、電気的特性として、電極間に所定の周波数の交流電圧を印加して被検全血または赤血球または白血球または血漿のインピーダンスを測定する原虫感染症の検査装置に関する。 Furthermore, the present invention relates to a protozoan infection testing apparatus that measures the impedance of whole blood or erythrocytes or leukocytes or plasma to be tested by applying an AC voltage of a predetermined frequency between electrodes as an electrical characteristic.
さらに、本発明は、印加される交流電圧の周波数を順次切り替えながらインピーダンスを測定する原虫感染症の検査装置に関する。 Further, the present invention relates to a protozoan infection testing device that measures impedance while sequentially switching the frequency of applied AC voltage.
さらに、本発明は、周波数を０．１ＭＨｚから３００ＭＨｚの範囲で順次切り替える原虫感染症の検査装置に関する。 Furthermore, the present invention relates to a protozoan infection testing device that sequentially switches frequencies in the range of 0.1 MHz to 300 MHz.
さらに、本発明は、印加される交流電圧の周波数を順次切り替えながらインピーダンスを測定し、１回の採血および検査により重複感染症を検査する原虫感染症の検査装置に関する。 Further, the present invention relates to a protozoan infection testing device that measures impedance while sequentially switching the frequency of applied AC voltage and tests for multiple infections by one blood sampling and testing.
さらに、本発明は、哺乳類において、赤血球中のマラリア原虫および/またはバベシア、赤血球および/または白血球中のタイレリア、白血球中のトキソプラズマ、全血中のトリパノソーマを検知する原虫感染症の検査装置に関する。 Furthermore, the present invention relates to a device for testing protozoan infections that detect malaria protozoan and / or Babesia in erythrocytes, theileria in erythrocytes and / or leukocytes, toxoplasma in leukocytes, and tripanosoma in whole blood in mammals.
さらに、本発明は、鳥類において、赤血球中のマラリア原虫および/またはバベシアおよび/またはロイコチトゾーンを検知する原虫感染症の検査装置に関する。 Furthermore, the present invention relates to a device for testing a protozoan infection that detects Plasmodium and / or Babesia and / or Leucocytozoon in erythrocytes in birds.
また、本発明の他の観点によれば、上記課題を解決するために、本発明は、寄生性原虫が感染した被検全血および/または赤血球および/または白血球および/または血漿の物理的特性の変化を電気的特性の変化として検知する原虫感染症の検査方法に関する。 Also, according to another aspect of the invention, in order to solve the above problems, the present invention presents the physical properties of whole blood and / or red blood cells and / or leukocytes and / or plasma infected with a parasitic protozoan. The present invention relates to a method for testing a protozoan infection that detects a change in plasma as a change in electrical characteristics.
本発明によれば、原虫感染症を簡単・高精度にしかも安価に検査できるとともに、１回の採血および検査により重複感染を検査でき、代謝副産物を産生しない原虫感染症も検査できる検査装置および検査方法を提供することができる。 According to the present invention, a testing device and a test capable of testing protozoan infections easily, accurately and inexpensively, testing for coinfection by one blood sampling and testing, and testing for protozoan infections that do not produce metabolic by-products. A method can be provided.
また、特に感染初期でも高感度かつ高精度かつ迅速に検査する方法が見当たらないことに鑑みてなされたものであり、原虫感染による被検全血および/または赤血球および/または白血球および/または血漿の物理的特性変化を電気的特性変化として高感度で検知するという全く新規な原虫感染症の検査装置および検査方法を提供することができる。 It was also made in view of the lack of a sensitive, accurate and rapid test, especially in the early stages of infection, for whole blood and / or red blood cells and / or leukocytes and / or plasma tested for protozoan infection. It is possible to provide a completely novel inspection device and inspection method for protozoan infections, in which changes in physical characteristics are detected as changes in electrical characteristics with high sensitivity.
以下、本発明の実施形態例及び実施例を説明するが、本発明の実施形態は以下に説明する実施形態例及び実施例に限定されない。 Hereinafter, embodiments and examples of the present invention will be described, but the embodiments of the present invention are not limited to the embodiments and examples described below.
寄生性原虫に感染すると、被検全血、赤血球、白血球または血漿の物理的特性が変化する。 Infection with a parasitic protozoan changes the physical properties of the whole blood, red blood cells, white blood cells or plasma to be tested.
例えば、マラリア原虫のライフサイクルにおいて原虫に感染したハマダラカが吸血する際に唾液腺感染型虫体である「スポロゾイト」が宿主の体内に注入される。スポロゾイトは数分で肝臓に達し、肝細胞に侵入する。数週間で数万の「メロゾイト」に分裂し、血液中に放出される。メロゾイトは、ただちに赤血球に侵入し、輪状体、栄養体、分裂体などを経て「メロゾイト」を形成する。新しいメロゾイトは、感染した赤血球を破壊し、次の赤血球に感染し、増殖する。このサイクルがくり返され、発熱や貧血が起こる。メロゾイトの一部は雄雌の生殖母体（ガメトサイト）に分化し、これが吸血により蚊の体内に入ると、蚊の中腸内で雌雄の生殖体に成熟した後、接合する。接合体は運動性のオーキネトとなり、中腸基底膜でオーシストに分化する。オーシスト内でつくられた数千の「スポロゾイト」は唾液腺に移行し、感染型の「成熟スポロゾイト」になる。以上のサイクルが知られており、赤血球の物理的特性に変化が生じる。 For example, in the life cycle of Plasmodium malaria, when the Anopheles mosquito infected with the protozoan sucks blood, "sporozoite", which is a salivary gland-infected insect body, is injected into the host body. Sporozoites reach the liver in minutes and invade hepatocytes. In a few weeks it splits into tens of thousands of "merozoites" and is released into the blood. Merozoite immediately invades erythrocytes and forms "merozoite" through rings, vegetative bodies, and mitotic bodies. The new merozoite destroys the infected red blood cells, infects the next red blood cell, and proliferates. This cycle is repeated, causing fever and anemia. Part of merozoite differentiates into male and female reproductive mothers (gametosites), and when it enters the mosquito's body by sucking blood, it matures into male and female reproductive bodies in the midgut of the mosquito and then joins. The zygotes become motile orchinets and differentiate into oocysts in the basement membrane of the midgut. Thousands of "sporozoites" produced within oocysts 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.
この物理的特性の変化が、電気的特性の変化として検知できることを見出した。 It has been found that this change in physical characteristics can be detected as a change in electrical characteristics.
また、上記において、マラリアはマラリア原虫が赤血球に寄生することで引き起こされる点を説明したが、原虫感染症の種類に応じて感染形態がことなる点が知られている。例えば、トキソプラズマ症はトキソプラズマが白血球を含む有核細胞に寄生することで引き起こされ、トリパノソーマ症はトリパノソーマが血流中で増殖することによって引き起こされ、ピロプラズマ症はバベシアが動物の赤血球に、もしくは、タイレリアが動物の赤血球および/または白血球に寄生することによって引き起こされ、ロイコチトゾーン症はロイコチトゾーンが主に鳥類の赤血球に寄生することによって引き起こされる感染症である。 Further, in the above, the point that malaria is caused by the parasitism of Plasmodium malaria on erythrocytes has been explained, but it is known that the form of infection differs depending on the type of protozoan infection. For example, toxoplasmosis is caused by the parasitism of toxoplasma on nucleated cells containing white blood cells, tripanosoma is caused by the proliferation of tripanosoma in the bloodstream, and pyroplasmosis is caused by Babesia on red blood cells of animals or by Babesia. Tyrelia is caused by parasitizing animal erythrocytes and / or leukocytes, and leukochitozone disease is an infectious disease caused by leukochitozone parasitizing predominantly avian red blood cells.
したがって、寄生性原虫が感染した被検全血および/または赤血球および/または白血球および/または血漿の物理的特性の変化を電気的特性の変化として検知できるが、検知する電気的特性の変化は、必ずしも原虫そのものの存在による被検全血および/または赤血球および/または白血球および/または血漿の変化である必要はなく、マラリアにおけるヘモゾインのように、代謝副産物によりもたらされる電気的特性の変化からも検知できる。 Therefore, changes in the physical properties of whole blood and / or erythrocytes and / or leukocytes and / or plasma infected with the parasitic protozoa can be detected as changes in electrical properties, but the detected changes in electrical properties are It does not necessarily have to be changes in whole blood and / or red blood cells and / or leukocytes and / or plasma due to the presence of the protozoa themselves, but is also detected from changes in electrical properties caused by metabolic by-products, such as hemozoin in malaria. can.
（検体の作製）
マラリア原虫感染マウスの血液をin vitro培養することによって原虫発育ステージを分裂期に同期化し、検体作製用マウスの尾静脈から分裂期マラリア原虫を感染させ、３から６時間後に心臓穿刺により感染血液を採集して１．４％のヘパリンを加え、９５％以上の感染赤血球中の原虫が感染初期のリングステージであることを確認した。
(Preparation of sample)
By in vitro culturing the blood of malaria-infected mice, the protozoal development stage is synchronized with the mitotic phase, and the mitotic malaria protozoa is infected from the tail vein of the sample preparation mouse, and the infected blood is punctured by heart puncture 3 to 6 hours later. After collecting and adding 1.4% heparin, it was confirmed that the protozoa in 95% or more of the infected erythrocytes were in the ring stage in the early stage of infection.
健常マウスの血漿を添加することでヘマトクリット値を４０％に調整し、非感染赤血球を用いた希釈によって感染赤血球/全赤血球の割合が０．５％、０．１％、０．０１％の検体を作製した。 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 using uninfected erythrocytes. Was produced.
（電気的特性の測定）
図１の電極付き小容器に感染赤血球濃度を調整した検体約５００マイクロリットルを注入し、周波数を０．１ＭＨｚから３００ＭＨｚまで変化させながら健常（非感染）マウス血液ならびに０．５％、０．１％、０．０１％の検体のレジスタンスおよびリアクタンスを各検体につき２０秒程度で測定した。
(Measurement of electrical characteristics)
Approximately 500 microliters of a sample adjusted for the infectious erythrocyte concentration was injected into the small container with an electrode of FIG. 1, and healthy (non-infected) mouse blood and 0.5%, 0.1 while changing the frequency from 0.1 MHz to 300 MHz. The resistance and reactance of% and 0.01% of the samples were measured in about 20 seconds for each sample.
図２に非感染血液、感染初期のリングステージ赤血球濃度が０．５％、０．１％、０．０１％のレジスタンスおよびリアクタンスの周波数依存性をプロットした結果を示す。 FIG. 2 shows the results of plotting the frequency dependence of resistance and reactance of uninfected blood and ring stage erythrocyte concentrations at the initial stage of infection of 0.5%, 0.1%, and 0.01%.
非感染血液と比較して、感染初期のリングステージ赤血球は、レジスタンスおよびリアクタンスが変化することを確認した。 It was confirmed that the resistance and reactance of the ring-stage erythrocytes in the early stage of infection were changed as compared with the uninfected blood.
また、赤血球感染率に応じて、レジスタンスおよびリアクタンスが変化することも確認した。 It was also confirmed that the resistance and reactance changed according to the erythrocyte infection rate.
本発明による検査装置および検査方法により、マラリア感染初期リングステージの赤血球数１００００分の１（図２の赤血球感染率０．０１％）でも、レジスタンスおよびリアクタンス特性から高感度でかつ短時間でマラリア感染を検知できることが明らかになった。 According to the inspection device and inspection method according to the present invention, malaria infection is highly sensitive and in a short time due to resistance and reactance characteristics even if the number of red blood cells in the initial ring stage of malaria infection is 1/10000 (red blood cell infection rate 0.01% in FIG. 2). It became clear that it can detect.
また、レジスタンスおよびリアクタンス特性から、感染率も推定できることが明らかになった。 It was also clarified that the infection rate can be estimated from the resistance and reactance characteristics.
なお、本発明による原虫感染症の検査に供する検体は、測定に数十秒程度と時間を要しないため採血した全血そのままでも良いが、ヘパリン等耐凝固剤の添加や成分分離のためにクエン酸等を加えて遠心分離する等、特に血液の処理方法について制限はないが、例えば、遠心分離で成分分離をすることで、赤血球、白血球、血漿等、特定の成分の電気的特性の変化を測定できるので、より高精度に原虫感染症を検知できる。 The sample used for the test for protozoal infection according to the present invention may be whole blood collected as it is because it does not take several tens of seconds to measure, but it is used for adding a coagulant such as heparin and separating components. There are no particular restrictions on the blood treatment method, such as adding acid or the like to centrifuge, but for example, by centrifuging the components, changes in the electrical properties of specific components such as red blood cells, leukocytes, and plasma can be obtained. Since it can be measured, protozoal infections can be detected with higher accuracy.
また、原虫感染症の種類に応じて、電気的特性の変化が異なることは明らかである。したがって、１回の採血および検査により測定した電気的特性の変化から各種の原虫感染症の検知が可能であり、重複感染症も検査することができる。 It is also clear that the changes in electrical properties differ depending on the type of protozoan infection. Therefore, various protozoan infections can be detected from changes in electrical characteristics measured by a single blood sampling and test, and multiple infections can also be tested.
また、電気的特性として、インピーダンス（レジスタンスおよびリアクタンス）を測定したが、他の電気的特性であるインダクタンス、キャパシタンスなどでもよく、原虫感染症の検査が可能な範囲ならば、如何なる電気的特性でもよい。 Impedance (resistance and reactance) was measured as the electrical characteristics, but other electrical characteristics such as inductance and capacitance may be used, and any electrical characteristics may be used as long as the protozoan infection can be inspected. ..
本発明は、原虫感染症の検査装置および検査方法として産業上利用可能である。 INDUSTRIAL APPLICABILITY The present invention can be industrially used as an inspection device and an inspection method for protozoan infections.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018171213A JP7094520B2 (en) | 2018-09-13 | 2018-09-13 | Protozoan infection testing equipment and methods |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018171213A JP7094520B2 (en) | 2018-09-13 | 2018-09-13 | Protozoan infection testing equipment and methods |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2020041965A JP2020041965A (en) | 2020-03-19 |
| JP7094520B2 true JP7094520B2 (en) | 2022-07-04 |
Family
ID=69798092
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2018171213A Active JP7094520B2 (en) | 2018-09-13 | 2018-09-13 | Protozoan infection testing equipment and methods |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7094520B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111781248A (en) * | 2020-07-15 | 2020-10-16 | 浙江师范大学 | A malaria detection device |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007525674A (en) | 2004-02-27 | 2007-09-06 | ベックマン コールター，インコーポレイティド | How to detect malaria parasites and other parasitic infections |
| JP2008076143A (en) | 2006-09-20 | 2008-04-03 | Citizen Holdings Co Ltd | Instrument for measuring hemoglobin concentration |
| JP2012052944A (en) | 2010-09-02 | 2012-03-15 | Nihon Univ | Erythrocyte monitor and erythrocyte monitoring method |
| US20130183243A1 (en) | 2010-07-12 | 2013-07-18 | Arizona State University | Methods and Device for Tuning Multiplexed Markers for Disease Assay |
| JP2015508506A (en) | 2012-01-27 | 2015-03-19 | ユニバーシティ オブ テネシー リサーチ ファウンデーション | Method and apparatus for biomarker detection by AC electrokinetics |
| JP2017071648A (en) | 2010-12-16 | 2017-04-13 | バクスアルタ ゲーエムベーハー | Real time monitoring and control of protein production process using impedance spectroscopy |
-
2018
- 2018-09-13 JP JP2018171213A patent/JP7094520B2/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007525674A (en) | 2004-02-27 | 2007-09-06 | ベックマン コールター，インコーポレイティド | How to detect malaria parasites and other parasitic infections |
| JP2008076143A (en) | 2006-09-20 | 2008-04-03 | Citizen Holdings Co Ltd | Instrument for measuring hemoglobin concentration |
| US20130183243A1 (en) | 2010-07-12 | 2013-07-18 | Arizona State University | Methods and Device for Tuning Multiplexed Markers for Disease Assay |
| JP2012052944A (en) | 2010-09-02 | 2012-03-15 | Nihon Univ | Erythrocyte monitor and erythrocyte monitoring method |
| JP2017071648A (en) | 2010-12-16 | 2017-04-13 | バクスアルタ ゲーエムベーハー | Real time monitoring and control of protein production process using impedance spectroscopy |
| JP2015508506A (en) | 2012-01-27 | 2015-03-19 | ユニバーシティ オブ テネシー リサーチ ファウンデーション | Method and apparatus for biomarker detection by AC electrokinetics |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2020041965A (en) | 2020-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Spielman et al. | Malaria diagnosis by direct observation of centrifuged samples of blood | |
| Küttel et al. | Label-free detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer | |
| Martin et al. | The effect of common anticoagulants in detection and quantification of malaria parasitemia in human red blood cells by ATR-FTIR spectroscopy | |
| Janse et al. | Flow cytometry in malaria detection | |
| Schallig et al. | Development of a fast agglutination screening test (FAST) for the detection of anti-Leishmania antibodies in dogs | |
| Jang et al. | Flow cytometric enumeration of parasitemia in cultures of Plasmodium falciparum stained with SYBR Green I and CD235A | |
| Ertabaklar et al. | Serological and entomological survey in a zoonotic visceral leishmaniasis focus of North Central Anatolia, Turkey: Corum province | |
| JP7094520B2 (en) | Protozoan infection testing equipment and methods | |
| Cursino‐Santos et al. | A novel flow cytometric application discriminates among the effects of chemical inhibitors on various phases of Babesia divergens intraerythrocytic cycle | |
| Hull et al. | Haemogregarines in snakes: the incidence and identity of the erythrocytic stages | |
| Xie et al. | Development and validation of flow cytometric measurement for parasitaemia using autofluorescence and YOYO-1 in rodent malaria | |
| Sunita et al. | Specific antibody detection in serum, urine and saliva samples for the diagnosis of cystic echinococcosis | |
| Akinbo et al. | Prevalence of intestinal parasites in relation to CD4 counts and anaemia among HIV-infected patients in Benin City, Edo State, Nigeria | |
| Noedl et al. | Malaria drug-susceptibility testing. HRP2-based assays: current data, future perspectives. | |
| Saghrouni et al. | Asymptomatic carriage of Leishmania in family members of patients with visceral leishmaniasis in Central Tunisia | |
| Liu et al. | Optimization of in vitro trophoblast assay for real-time impedimetric sensing of trophoblast-erythrocyte interactions in Plasmodium falciparum malaria | |
| Ashankyty | Seroprevalence of Toxoplasma gondii among pregnant women visiting maternity hospital in Hail, KSA | |
| Alsheikly et al. | Prevalence and Serological Diagnosis of Toxoplasmosis in Two regions (Karkh and Rusafa) of Baghdad Province | |
| Shaher Banu et al. | An analysis of the attitude of Patients towards Laboratory Services for Malaria Disease in Hyderabad India | |
| de Lacerda et al. | In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes | |
| Kerboeuf et al. | Flow-cytometry analysis of sheep-nematode egg populations | |
| CN107860913B (en) | Serum marker for distinguishing aortic aneurysm/aortic dissection and acute pulmonary embolism and application thereof | |
| Coêlho et al. | Willis method modification with a view to improving routine coproparasitological diagnosis | |
| Twomey et al. | Eimeria ivitaensis in British alpacas | |
| Gurung et al. | Evaluation of falcivax against quantitative buffy coat (QBC) for the diagnosis of malaria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20210730 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20220304 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220315 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220506 |
|
| RD03 | Notification of appointment of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7423 Effective date: 20220506 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20220509 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20220614 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20220614 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7094520 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |