JP2008225351A - Malaria microscope, excitation filter for use in the same, and observation method of malaria plasmodia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus and a method which is capable of quickly, easily, and clearly observing malaria plasmodia with naked eyes by using a general optical microscope and applying a malaria rapid diagnosis method employing acridine orange staining. <P>SOLUTION: A malaria microscope uses: an excitation filter which has such spectral characteristics that the transmittance of light is ≤0.1% at least in a range of at least 510 to 720 nm and is ≥80% at least at about 460 nm and 490 nm being excitation wavelengths of acridine orange and rapidly falls in a range of about 490 to 510 nm; and an absorbing filter which completely cuts the light at least at ≤510 nm is used. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microscope for observing malaria parasites using an optical microscope, an excitation filter used therefor, and a method for observing malaria parasites using these.

  Malaria is one of the most prevalent infectious diseases in the world caused by Plasmodium infection. It is a disease that exhibits a variety of pathological conditions in addition to peculiar fever attacks and subsequent anemia and splenomegaly. It is reported that more than 300 million people have been infected in endemic areas such as South America and Southeast Asia, and the number of deaths has been reported to be over 2 million per year, mainly among African children.

  Many research institutions, including WHO, are promoting malaria countermeasures such as the development of malaria vaccines, the development of new antimalarial drugs, and the control of vector mosquitoes, but there are still specific solutions for eradicating malaria. Not found. In addition, major problems remain in the diagnosis of malaria, and even now, the most reliable method for the diagnosis of malaria is to apply the Giemsa staining to blood smears to prove the presence of protozoa. .

  This Giemsa staining method was published by Gustav Giemsa of the Hamburg Institute for Tropical Diseases in 1902 and has been used for a long time. It has disadvantages such as being highly dependent on the skill level of In fact, in malaria endemic areas, there are many facts that people often have to wait for days to know the diagnosis.

  As test methods to compensate for these drawbacks, various immunological diagnostic methods such as IFA / ELISA and diagnostic methods using DNA probes are being developed. However, it is difficult to judge the result of the former because the past infection is also positive, and the latter has problems of processing complexity and diagnostic accuracy, both of which are suitable for application as diagnostic methods in actual malaria epidemic areas. Improvement is necessary, and it has not become an actual daily inspection method in the field.

  Fumihiko Kawa, one of the inventors of the present invention, examined a new staining diagnosis method for protozoal infections using two fluorescent dyes as a new staining method in place of the above-mentioned Giemsa staining method. See Non-Patent Documents 1 and 2 for the results of this study. This new staining diagnosis method uses two fluorescent dyes, DAPI (4 ', 6-diamidino-2-phenylindole) and PI (propidium iodide), and nuclei (and mitochondrial DNA) by DAPI binding to DNA. And then staining with PI that binds to DNA and RNA makes use of the fact that nucleic acids other than DNA, that is, cytoplasmic RNA (cytoplasm), are stained, so that differential staining of the nucleus and cytoplasm becomes possible. When this method is used, various protozoa are stained and observed in a very short time.In particular, the cytoplasm of the schistosome protozoa, such as malaria and trypanosoma protozoa, becomes red, and the outline of the cell becomes clear. Since they are observed as the same form as the Giemsa method, these can be easily identified. In the laboratory observation, it was possible to identify each stage of malaria by this method, and the type discrimination was not so difficult, and almost the same result as that observed by Giemsa staining could be obtained.

  This DAPI / PI method can be applied as a simple and rapid diagnostic method for malaria, but since the excitation wavelength is in the ultraviolet (UV) region, an observation method for observing fluorescently stained images using a normal optical microscope can be used. However, it is impossible to take an expensive fluorescent microscope, but it is practically impossible to provide a large number of expensive fluorescent microscopes in tropical malaria infested areas.

  Fumihiko Kawa has further researched and devised a rapid diagnosis method of malaria by staining with acridine orange (AO). The results of this research are shown in Non-Patent Documents 3 and 4. This new rapid diagnosis method utilizes the property of AO as a dye in the B excitation range, and enables observation with a simple combination of an interference filter and a halogen or LED light source without using an expensive mercury lamp. Is. The preparation of the staining solution and the staining method are described below.

<Method for preparing staining solution>
AO is prepared by dissolving Sigma's A-4921 in 0.1 mol / l of sterilized Tris buffer (pH 7.0 to 7.5) so as to be 10 mg / ml, and NaN _{3 in a range} of 0.5 to 1.0. % (W / v) is added to make a stock solution. The buffer may be a phosphate buffer or PBS. This is diluted 100 times with sterile water to make a staining solution. Store both stock solution and staining solution in a cool dark place, or store frozen if not used for a long time.

<Dyeing method>
Peripheral blood thin-film smears are fixed in methanol and air-dried. The staining method requires only 1 second by placing a few drops of the staining solution on a cover glass and inverting it to apply it to the thin-layer smear. Alternatively, a cover glass may be placed on tissue paper, a staining solution may be placed, and the thin-layer smear may be placed upside down for staining. In either case, the AO staining solution is diffused instantaneously, so that uneven dyeing is reduced. In addition, since it is important to observe the edge of the smear for the four types of discrimination, be sure that one drop of the AO solution spreads to the edge of the smear.

  In AO staining, DNA and RNA are stained, the nucleus is stained green to yellowish white, and the cytoplasm is stained red to orange. Note that if the AO staining solution is put too much, the nucleus will also be dyed red, and if it is too little, the cytoplasm will not be dyed red.

  Patent Document 1 discloses a microscope that enables quick and easy observation of malaria parasites, schistosome parasites, and the like using the acridine orange (AO) staining described above. However, in order to clearly observe the malaria parasite, it is known that the characteristics of the excitation filter and the absorption filter used are closely related, and the success or failure of the clear observation of the malaria parasite is determined.

  The inventors of the present invention apply the acridine orange (AO) staining method on the premise of using an ordinary optical microscope exemplified in Patent Document 1 without using an expensive fluorescent microscope, and clarify malaria parasites. The present invention has been completed by studying a quick and simple method that enables easy observation.

Kawamoto F, Kumada N: Fluorescence probes for detection of protozoan parasites. Parasitol Today 3: pp. 284-286, 1987 Fumihiko Kawa: Trial of rapid diagnosis of various protozoal infections using fluorescent dyes, clinical examination 32: pp. 803-806, 1988 Kawamoto F: Rapid diagnosis of malaria by florescence microscopy with light microscope and interference filter. Lancet 337: pp. 200-202, 1992 Kawamoto F, Billingsley PF: Rapid diagnosis of malaria by florescence microscopy. Parasitol Today 8: pp. 69-71, 1992 JP-A-9-179030

  The present invention is an apparatus capable of observing malaria parasites quickly, easily and clearly with the naked eye by applying a rapid diagnosis method of malaria by acridine orange (AO) staining using a general optical microscope. It is an object to provide a method.

  The excitation filter for a malaria microscope according to the present invention has a spectral characteristic in which the transmittance of the light beam is 0.1% or less in the range where the wavelength of the light beam transmitted through the filter is at least 510 to 720 nm, and at least It has a spectral characteristic that transmits light in the vicinity of 460 nm and 490 nm, which are the excitation wavelengths of acridine orange, and preferably has a spectral characteristic in which the light transmittance is 80% or more in the vicinity of 460 nm and 490 nm. It is.

  The excitation filter for a malaria microscope according to the present invention has a spectral characteristic in which the transmittance of the light beam is 0.1% or less in the range where the wavelength of the light beam transmitted through the filter is at least 510 to 720 nm, and at least The light transmittance is about 80% or more in the vicinity of 460 nm and the vicinity of 490 nm, which are the excitation wavelengths of acridine orange, and the transmittance falls sharply in the range from about 490 nm to 510 nm. Is.

  The excitation filter for a malaria microscope according to the present invention has a spectral characteristic in which the transmittance of the light beam is 0.1% or less in the range where the wavelength of the light beam transmitted through the filter is at least 510 to 720 nm, and at least In the range of 400 to 490 nm, the transmittance of the light beam has a spectral characteristic of 80% or more, and the transmittance sharply falls in the range of 490 to 510 nm.

  The malaria microscope according to the present invention uses any one of the above-described excitation filters and an absorption filter that completely cuts the light rays in the range of visible light having a wavelength of at least 510 nm or less. It is what.

  A malaria microscope according to the present invention includes a mirror tube that reflects light through a condenser lens tube that collects light from a light source at a desired angle, a stage through which the reflected light passes, an objective lens, and an eyepiece lens. An optical filter in which an excitation filter is disposed in the condenser section below the stage and an absorption filter is disposed in the spectacle tube section in a switchable manner, and the excitation filter is any one of the above excitation filters, In addition, an absorption filter that completely cuts off the light beam in the wavelength range of at least 510 nm or less is used.

  In the method for observing the Plasmodium according to the present invention, a light source is passed through the excitation filter to generate a light beam having an excitation wavelength of acridine orange, and the light smear is applied to a blood smear stained with an acridine orange solution. In the method of generating an excitation emission line having a fluorescence wavelength, passing the excitation emission line through the absorption filter, and observing the malaria parasite by the light beam that has passed through the absorption filter, the excitation filter is any one of the above An excitation filter is used, and as an absorption filter, an absorption filter that completely cuts off the light beam in the wavelength range of at least 510 nm or less is used.

  According to the present invention, compared to conventional methods, it is possible to observe malaria parasites simply, quickly and clearly, and to provide an inexpensive device for that purpose. It makes a significant contribution to medical care, especially malaria control and medical care in developing countries.

[Example 1]
FIG. 1 is a diagram showing an entire configuration of an optical microscope 1 used for carrying out the present invention and a light source used therefor. As a light source of the microscope, a halogen lamp or an LED lamp was incorporated, and a light source unit 2 separate from the microscope was used. A condensing lens cylinder 4 having a condensing lens 3 is attached to the side surface of the light source unit 2. The optical microscope 1 is provided at its lower part with a mirror cylinder 6 having a mirror 5 that reflects light from the condenser lens cylinder 3, and at the upper part thereof is a condenser that allows the reflected light from the mirror cylinder 6 to pass therethrough. The lens 7 is provided below the stage 8 on which the specimen is placed, and further includes an eyepiece tube portion in which an objective lens 9, a monocular or binocular eyeglass tube portion 10, and an eyepiece lens 11 are housed. In the optical microscope having the above configuration, the excitation filter 12 is disposed below the condenser lens 7, and the absorption filter 13 is disposed below the spectacle barrel 10 so as to be replaceable.

  As the absorption filter, a filter having a transmittance of about 0.001% in the range of visible light having a wavelength of at least 510 nm or less is used. The excitation filter has a spectral characteristic of a transmittance of 0.1% or less in the range of 510 to 720 nm, and a spectral characteristic of a transmittance of 80% or more in the range of 400 to 490 nm. And having a transmittance that falls sharply within a range of 490 nm to 510 nm. FIG. 2-1 shows the spectral characteristics of the absorption filter according to the present invention and the comparative example, and FIG. 2-2 shows these spectral characteristics on a logarithmic scale.

  A cover glass coated with an appropriate amount of acridine orange (AO) staining solution is placed on and closely adhered to a smear sample slide glass coated with blood collected from a patient, and the sample blood is fluorescently stained to prepare a specimen. Next, this specimen is fixed at a predetermined position on the stage 8 of the optical microscope.

  Next, the light source unit 2 is turned on to emit light from the light source, and this light is condensed by the condenser lens tube 4 and guided to the mirror tube 6 of the optical microscope 1. The light reflected by the reflecting mirror 5 in the mirror cylinder 6 passes through the excitation filter 12, so that the light beam in the range of 510 to 720 nm is cut to a transmittance of 0.1% or less and irradiated to the specimen. As a result, the nuclear DNA of the malaria parasite in the sample is excited and emits light in the vicinity of 520 nm, and emits fluorescence that is observed in yellow with the naked eye. In addition, due to the irradiation, the cytoplasmic RNA of the malaria parasite in the sample is excited and emits light near 630 nm, and emits fluorescence that is observed in red with the naked eye.

  The light that has passed through the excitation filter is condensed by the condenser lens 7, passes through the objective lens 9, and reaches the absorption filter 13. When passing through the absorption filter 13, visible light having a wavelength of 510 nm or less has a transmittance of 0.001%. Absorbed to a degree. The above-mentioned fluorescent light from plasmodium is extremely weak, so when observing with the naked eye, if the light of other wavelengths as background is strong, these fluorescent light cannot be observed, and nuclear DNA and cytoplasm of plasmodium Since the blue color causing the B excitation in RNA is also in the way, they are all cut by an absorption filter.

  Thus, with the naked eye viewed through the eyepiece, yellow fluorescence emitted by excitation of malaria parasite nuclear DNA and red fluorescence emitted by excitation of malaria parasite cytoplasmic RNA clearly appear in the dark field. The presence of malaria parasites can be clearly confirmed.

  FIG. 3 is an observation photograph of the malaria parasite observed according to the present invention, and FIG. 4 is an observation photograph of the comparative example, in which three malaria parasites that are brightly lit are projected.

  In the first embodiment described above, the light source having a halogen lamp or LED lamp incorporated therein is used. However, the present invention is not limited to this, and solar direct light, tungsten light source, or the like can also be used.

  The present invention can be used in industrial fields such as medical devices and medical-related research devices.

1 is an overall configuration diagram of an optical microscope loaded with an excitation filter and an absorption filter according to the present invention and a light source used therefor. It is the figure which showed the spectral characteristic of the absorption filter which concerns on this invention, and a comparative example. The spectral characteristic of the absorption filter which concerns on this invention, and a comparative example is shown using especially the logarithmic scale. It is an observation photograph of the malaria parasite observed by the present invention. It is an observation photograph of the malaria parasite observed when the filter of a comparative example is used.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Optical microscope 2 Light source part 3 Condensing lens 4 Condensing lens cylinder 5 Reflecting mirror 6 Mirror cylinder 7 Condenser lens 8 Stage 9 Objective lens 10 Eyeglass cylinder part 11 Eyepiece 12 Excitation filter 13 Absorption filter

Claims (7)

  When the wavelength of light passing through the filter is in the range of at least 510 to 720 nm, the transmittance of the light has a spectral characteristic of 0.1% or less, and at least the excitation wavelength of acridine orange is around 460 nm and around 490 nm. Then, an excitation filter for a malaria microscope characterized by having a spectral characteristic that transmits light.   When the wavelength of light passing through the filter is in the range of at least 510 to 720 nm, the transmittance of the light has a spectral characteristic of 0.1% or less, and at least the excitation wavelength of acridine orange is around 460 nm and around 490 nm. Then, an excitation filter for a malaria microscope characterized by having a spectral characteristic of a light transmittance of 80% or more.   When the wavelength of light passing through the filter is in the range of at least 510 to 720 nm, the transmittance of the light has a spectral characteristic of 0.1% or less, and at least the excitation wavelength of acridine orange is around 460 nm and around 490 nm. Then, an excitation filter for a malaria microscope, characterized in that the light transmittance has a spectral characteristic of 80% or more, and the transmittance sharply falls within a range from about 490 nm to 510 nm.   When the wavelength of light passing through the filter is at least in the range of 510 to 720 nm, the light transmittance is 0.1% or less, and in the range of at least 400 to 490 nm, the transmittance of the light is obtained. Excitation filter for a malaria microscope, characterized by having a spectral characteristic of 80% or more and having a sharp drop in transmittance in the range of 490 nm to 510 nm.   The excitation filter according to any one of claims 1 to 4 and an absorption filter that completely cuts off the light beam in the range of visible light having a wavelength of at least 510 nm or less. A malaria microscope characterized by that. A mirror cylinder that reflects light from a light source through a condenser lens cylinder that collects light at a desired angle; a stage that transmits the reflected light; an objective lens; and an eyepiece; In an optical microscope in which an excitation filter is arranged in the condenser part and an absorption filter is arranged in the eyeglass tube part in a switchable manner,
The excitation filter is the excitation filter according to any one of claims 1 to 4, and the light beam transmitted through the absorption filter is completely at least within a wavelength range of 510 nm or less. A malaria microscope characterized by using an absorption filter for cutting. A light source passes through the excitation filter to generate a light beam having an excitation wavelength of acridine orange, and the light is applied to a smear of blood stained with an acridine orange solution to generate an excitation emission line having a fluorescence wavelength. In a method of observing protozoan malaria by light passing through the absorption filter and passing through the absorption filter,
The excitation filter according to any one of claims 1 to 4 is used as the excitation filter, and the light beam passing through the filter as the absorption filter is at least 510 nm or less in the wavelength range. A method of observing malaria parasites, characterized by using an absorption filter that completely cuts out the material.

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