JP2015052505A - Viability method of acanthamoeba (cyst) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a test method or the like that can assay life and death about Acanthamoeba (cyst) easily and reliably.SOLUTION: A test method includes: (1) a cyst culture preparation step of preparing a cyst culture by growing Acanthamoeba on a solid medium for growth obtained by adding a substance serving as bait of trophozoite ameba, and by making a cyst exist by bringing all Acanthamoeba into cyst configuration, with degradation of the breeding condition due to the decrease of the bait; (2) an exposure step of immersing the cyst culture in a test liquid containing a test substance; (3) a trophozoite derivation step of deriving the trophozoite ameba from the cyst configuration, by placing the cyst culture passed through the exposure step on a nutrient agar and culturing it; and (4) an evaluation step of calculating a de-cyst rate by counting a de-cyst count showing the trophozoite ameba grown from the cyst configuration and being the number of de-cyst traces, and a non-de-cyst count being the number of non-de-cysts remaining as the cyst configuration, after a constant period of time elapses from the implementation of the trophozoite derivation step.

Description

  The present invention relates to a method for discriminating the life and death of an acanthamoeba.

  With the spread of contact lenses, corneal infections due to Acanthamoeba are increasing. Acanthamoeba is a small free-living amoeba that inhabits widely in soil and environmental waters. When it is in good growth conditions, it becomes a nutrient, has motility, and divides and proliferates. Nutrients are highly sensitive to drugs due to their high membrane permeability. On the other hand, when the growth conditions deteriorate, Acanthamoeba becomes a cyst having a double wall. The cysted amoeba has resistance to dryness, heat resistance, and chemical resistance and resists various treatments. In addition, when cysts are formed, they wait for changes in the environment while spraying and diffusing in the air, and when they reach a favorable environment, they are decysted and become nutrients.

  When Acanthamoeba invades the cornea, it grows in the corneal epithelium, but when it undergoes an inflammatory reaction, it becomes cystic and escapes from the reaction. report). As an infection route for Acanthamoeba, infection via a soft contact lens is suspected, and the reasons include incomplete disinfection of soft contact lenses and user compliance problems. In the disinfection test of disinfectants for soft contact lenses, ISO / FDIS 14729 (Ophthalmic optics-Contact lens care products-Microbiological requirements and test methods for products and regimens for hygienic management of contact lens) is the standard method for bacteria and fungi. Are known. However, no test method for amoeba has been established.

Many researchers refer to ISO14729 for disinfection test on amoebae. Briefly, the survival rate is calculated after contacting the amoeba with the disinfecting solution for a certain period of time. However, since the amoeba precipitates due to the stationary contact, it is unclear whether the effect of the disinfectant is correctly evaluated. In addition, ISO14729 is designed for organisms that can be easily determined for life and death, such as bacteria and fungi, so it is difficult to determine against cystized amoeba, and it is confirmed that it has been decysted at the time of evaluation. There is a need. Furthermore, since the desiccated amoeba divides and proliferates, it is difficult to make a quantitative evaluation.
In view of such a situation, a measurement method specialized for Acanthamoeba has been developed (Patent Document 1). In this technique, an amoebae of a vegetative body is detected quickly and accurately by using an IgM type monoclonal antibody that specifically reacts with two or more types of Acanthamoeba.

Japanese Patent Laid-Open No. 9-105751

A characteristic of keratitis caused by Acanthamoeba is that there is much damage caused by soft contact lenses. Soft contact lenses are disinfected with a disinfectant when not worn, but this disinfectant is not designed for amoeba although it has disinfection against bacteria and fungi. In particular, there is no effective disinfectant for cystic amoeba.
As described above, since a disinfection effect test method has not been established for Acanthamoeba, it is necessary to construct a method that can be more simply and correctly evaluated.
The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a test method or the like that can evaluate an effective disinfectant for a cyst-type acanthamoeba.

  The test method for Acanthamoeba according to the invention for achieving the above object is as follows: (1) Growing Acanthamoeba on a solid medium for growth to which a substance serving as a feed for a nutritional amoeba is added, and reducing the feed A cyst medium preparation step for preparing a cyst medium in which cysts are present by causing all Acanthamoeba to have a cyst form as the breeding conditions deteriorate, and (2) the cyst medium in a test solution containing a test substance. (3) a nutrient induction process for inducing a vegetative amoeba from a cyst form by placing and culturing the cyst medium after the exposure process on a nutrient agar medium, and (4) After a certain period of time from the implementation of the nutrient induction process, the number of desist, which is the number of desist marks indicating that the cyst form has become a nutrient amoeba, and the cyst Counting the non-de-cyst number and which is the number of missing de cysts remain state, characterized by comprising an evaluation step of calculating a de-cyst rate.

“Solid medium” means a solid substance used for cultivation of Acanthamoeba. As such a solid medium, an agar medium, a soft contact lens (non-water-containing type, water-containing type, silicone hydrogel type), a hard contact lens, an oxygen-permeable hard contact lens, or the like can be used.
As the “food of amoeba”, living organisms (such as bacteria), dried organisms, artificial nutrients, and the like can be used. In addition, when there is too much amoeba bait, an acanthamoeba may not become a cyst, Therefore It increases / decreases suitably and a preferable condition is set. When an agar medium is used as the solid medium, it is preferable to apply an appropriate amount of bacteria on the agar medium.

“Decysting” means a cyst that has changed from a cyst to a nutrient. Observing the desiccated trace, it is observed as a cyst shell (although no nutrients can be identified).
“Non-deprived cyst” means a cyst that could not be changed into a nutrient. Observing the unprolapsed cysts reveals that it was not a husk but remained a cyst and died and could not become a nutrient amoeba.
The “desisting rate” means the number of surviving (the number of desisting) out of the total number of cysts observed (the number of desisting + the number of non-desisting cysts). When expressed as a percentage, it can be calculated by “100 × number of desiccated / (number of undescended cysts + number of desistated)”.
The second invention according to the present invention is a test kit for carrying out the above test method, which is a cyst medium, a basic buffer for preparing a test solution, and a multi-hole used in an exposure step. And a plate.
The third invention according to the present invention is an anticyst drug exhibiting antibacterial activity against an Acanthamoeba cyst selected by the above test method.

  ADVANTAGE OF THE INVENTION According to this invention, the test substance which examines whether it shows a bactericidal effect with respect to a cyst about the Acanthamoeba which decysts from a cyst and becomes a nutrient can be evaluated quantitatively. In this way, the effect of the medicine containing the soft contact lens disinfectant can be evaluated with a simpler apparatus configuration in a state close to practical use conditions.

It is a figure which shows a mode that an acanthamoeba is mounted on the agar medium which apply | coated bacteria in the cyst medium preparation process. It is the microscope picture figure which observed a mode that an acanthamoeba turns into a cyst from a nutrient body in a cyst medium preparation process. (A) Nutrients are increasing in about 20 hours after the start of culture, (B) A state in which cysts are formed in about one week after the start of culture, (C) Two weeks after the start of culture It is a photograph figure which shows a mode that almost all amoeba became cyst in the grade. It is a photograph figure which shows the mode of an exposure process. (A) The cyst medium formed in the petri dish is cut out to an appropriate size (about 1 cm × 1 cm), (B) The cut cyst medium is enlarged and copied, (C) Each well of the multi-well plate is tested It is a photograph figure which each shows a mode that the liquid was added and the cut cyst culture medium was added, and (D) the cyst culture medium of said (C) was expanded and copied. It is a photograph figure which shows a mode that the cyst culture medium was immersed (a cyst culture medium is recognized at the front-end | tip of the arrow). It is a microscope picture when the state of an amoeba is observed in the evaluation process after culturing at 25 ° C. for 20 hours after performing the nutrient induction process. (A) Photograph when physiological saline (negative control) is used as a test solution in the immersion process, and (B) Photograph showing a state when a drug having an effect on cysts is used as the test liquid in the immersion process. FIG. In the figure, a circle mark “◯” indicates decystation (a trace of the shell), and an asterisk “☆” indicates non-degeneration. 6 is a bar graph showing the results of Test Example 1. 10 is a bar graph showing the results of Test Example 2.

Next, embodiments of the present invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited by these embodiments, and various forms can be made without changing the gist of the invention. Can be implemented.
<Test method for Acanthamoeba>
1. Cyst medium preparation process Various things, such as an agar medium, a soft contact lens (a non-water-containing type, a water-containing type, a silicone hydrogel type), a hard contact lens, an oxygen-permeable hard contact lens, can be used as a solid medium. Here, a case where an agar medium is used will be described. As the agar medium, a medium generally used for culturing bacteria or the like (a medium solidified at an agar concentration of about 1.5%) can be used. As the cyst medium used in the present invention, an agar medium using a petri dish having a large surface area is preferably used because a place where the Acanthamoeba forms cysts (the surface of the medium or near the surface) may be used. About manufacture of an agar medium, it carries out in accordance with a normal method.
Appropriate amounts of bacteria (such as Escherichia coli) are applied to the surface of the agar medium as a nutrient amoeba food.

  As shown in FIG. 1, an agar medium 2 coated with bacteria is formed in a petri dish 1, and an Acanthamoeba is placed thereon. The amoeba to be placed may be either a nutrient body or a cyst medium. During the experiment, it is time-consuming to maintain the nutrients, so it is preferable to use a cyst medium. FIG. 1 shows a state in which a sponge 4 embedded with a plurality of cyst agar 3 stored in advance is used as an acanthamoeba.

As shown in FIG. 2, when the agar medium is cultured under appropriate conditions (temperature 20 ° C. to 40 ° C. (preferably 20 ° C. to 30 ° C.)), nutrients grow (FIG. 2 (A)). When nutrients proliferate and feed decreases, Acanthamoeba changes to cysts as the breeding conditions worsen (depending on conditions, about 5 to about 10 days from the start of culture. FIG. 2 (B )). Eventually, all Acanthamoeba become cysts (although depending on conditions, about 10 days to about 3 weeks from the start of the culture, FIG. 2 (C)). Thus, a cyst medium in which cysts are present can be prepared.
When a cyst medium is prepared using a petri dish, it can be stored as it is. Also, considering the convenience of use in the next exposure step, the cyst agar (the surface) is a hexahedron with an appropriate size (for example, about 0.5 cm to 1.5 cm x 0.5 cm to 1.5 cm). ) Is preferably cut out (see FIGS. 3A and 3B).

2. Exposure process In the exposure process, it is preferable to use a multi-hole plate (for example, 12 holes, 24 holes, 48 holes) in view of conditions such as the size of cyst agar, the volume of the test solution, the number of test substances, and the like. The cyst agar cut out to an appropriate size is immersed in a test solution containing the test substance (see FIGS. 3C, 3D, and 4). Although the immersion time depends on conditions such as the property / concentration and temperature of the test substance, it can be immersed for several hours to several tens of hours (about 1,2,4,6,8,12,24,48 hours).
In addition, after passing through an exposure process, it is preferable to wash cyst agar in order to wash away a test substance (medium washing process).

3. Nutrient body induction step Next, cyst agar is placed on a nutrient agar medium and cultured. As the nutrient agar medium, in addition to the agar medium used in the cyst medium preparation step, an agar medium (preferably a stable synthetic medium) with increased nutrient components can be used. If the nutritional composition of the nutrient agar medium is appropriate by culturing under appropriate conditions (for example, about 20 ° C to 40 ° C (preferably 20 ° C to 30 ° C), about 15 hours to 24 hours) The cysts that have not been induced are desiccated at a rate of almost 100% and become nutrients.

4). Evaluation process After the nutrient induction process, using a magnifier (microscope, etc.), the number of desist on the medium (the number of desist from traces of cysts that became nutrient amoebae) and the number of undescended cysts (cyst form) The number of cysts). In the case of the non-depleted cyst, the cyst is raised and the shadow is clear, but when decysted, the contents are detached and the shadow becomes unclear, so that a so-called shell state is obtained (see FIG. 5). For this reason, it is easy to distinguish between the undescended cyst and the removed cyst. In FIG. 5, a circle mark “◯” indicates decystation (a trace of the shell), and an asterisk “☆” indicates non-degeneration cyst.
The percentage of desistation is calculated as a percentage from the number of desiccations and the number of undescended cysts [100 × number of desistation / (number of desistation + number of undesisted cysts)], and the effects of the test substance are compared.

Next, the present invention will be described in more detail by describing examples.
<Test Example 1 Effect of Paromomycin Sulfate and Polyhexanide Hydrochloride>
According to the test method described above, paromomycin sulfate (PS) and polyhexanide hydrochloride (PH) were used as test substances in the exposure process, and the effects on cysts were examined.
PS 0.01% + PH 10ppm, PS 0.01% + PH 100ppm, PS 0.01%, PS 0.1%, PH 10ppm, PH 100ppm and PH 1000ppm were used as test substances, respectively. Each test substance is dissolved in KCM solution (KCl 0.7g, CaCl ₂ 0.8g, MgSO ₄ · 7H ₂ O 0.8g dissolved in purified water to 1000mL, adjusted to pH 7.0 with HEPES-KOH). A test solution was obtained. A KCM solution was used as a negative control.
Using cyst agar prepared in advance (about 1cm x 1cm), in the exposure process, cyst agar was immersed in each test solution for 24 hours, followed by a nutrient induction process (25 ° C, 20 hours). Undecysted and decysted were counted. From each data, the desist rate (%) was calculated.
The results are shown in FIG. As is clear from the figure, PS alone showed a cyst bactericidal effect (desisting rate: 11%) at 0.1%. In addition, with PH alone, a bactericidal effect was observed from 10 ppm (desisting rate: 75%), and 1000 ppm completely suppressed desisting (desisting rate: 0%). In addition, when PS and PH were used in combination, a synergistic effect was observed.

<Test Example 2 Effect of Commercial Contact Lens Cleaning Solution>
According to the test method described above, a commercially available contact lens cleaning solution was used as a test substance in the exposure process, and the effect on cysts was examined.
Four types of contact lens cleaning solutions (each sample A to sample D) were used as test substances, and the test solution was used as the concentration during contact lens cleaning. In addition, KCM solution was used as a negative control (control), and paromomycin 0.1% (PS 0.1%) was used as a positive control.
Using cyst agar (about 1cm x 1cm) prepared in advance, in the exposure process, cyst agar is immersed in each test solution for 2.5 hours, and then a nutrient induction process (25 ° C, 20 hours) is performed. Undecysted and decysted were counted. From each data, the desist rate (%) was calculated.
The results are shown in FIG. As is apparent from the figure, the bactericidal effect against cyst was hardly observed in sample A to sample C. Sample D was completely suppressed from desisting (desisting rate: 0%). This was considered to be because sample D was of the type used with a neutralizing agent after adding 3.2 W / V% hydrogen peroxide disinfectant.

  As described above, according to the present embodiment, it was possible to quantitatively evaluate the test substance that examines whether or not the Acanthamoeba that is decysted from the cyst and becomes a nutritional body exhibits a bactericidal effect on the cyst. Thus, it was possible to provide an evaluation method capable of evaluating the effect of a drug containing a soft contact disinfectant with a simpler apparatus configuration in a state close to practical use conditions.

Claims (3)

A method that can easily and reliably quantify life and death of Acanthamoeba (・ cyst),
(1) By growing Acanthamoeba on a solid medium for growth to which a substance serving as a feed for nutrient amoeba is added, and with the deterioration of the breeding conditions due to the decrease in the feed, all Acanthamoeba are made into cyst form A cyst medium preparation step for preparing a cyst medium in which cysts are present,
(2) an exposure step of immersing the cyst medium in a test solution containing a test substance;
(3) a nutrient induction process for inducing a nutrient amoeba from a cyst form by placing and culturing the cyst medium subjected to the exposure process on a nutrient agar medium; and (4) execution of the nutrient induction process. After a lapse of a certain period of time, the number of cysts that are the number of cysts that indicate that the cyst form has become a nutrient amoeba, and the number of uncested cysts that are still in the cyst form A test method for Acanthamoeba, comprising an evaluation step of counting and calculating a desisting rate. The test method for Acanthamoeba according to claim 1, wherein the solid medium is one selected from the group consisting of an agar medium, a soft contact lens, a silicone hydrogel lens, and a hard contact lens. An anti-cyst drug which is antibacterial against an Acanthamoeba cyst and is selected by the test method according to claim 1 or 2.