JP3436803B2 - Quantification of fungi in skin - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying fungi in the skin, and more particularly to a method for quantifying fungi capable of accurately quantifying the number of fungi in the skin.

[0002]

2. Description of the Related Art There are 120 superficial mycosis patients in Japan.
It is estimated to be 0 to 18 million. The cause of the large number of patients is considered to be the intensity of fungal transmission, which is thought to be due to the living environment, lifestyle, and lifestyle, and the difficulty of curative treatment due to repeated infection. This is because the development of effective drugs is slow, and the cause is that the growth of such fungi is in the skin, and the pharmacokinetics are complicatedly involved in the onset of drug efficacy, and the fungus in the skin may not be appropriate. It was because there was no such quantitative method. It was not possible to properly evaluate the drug because the fungus in the skin could not be quantified accurately.

Conventionally, the quantification of the fungus in the skin is performed by sampling the skin, cutting it, taking out a plurality of small fragments, planting them in a medium, counting the number of the fungal fragments from this, and measuring the total fragments. The value divided by the number was used as an index. However, in this method, the numerical value is only a multiple of the value obtained by dividing 1 by the number of fragments, so it is discontinuous, and the fragments that grow only in small quantities and the fragments that grow in large quantities are treated in the same way. This method could determine the presence or absence of fungi on the skin pieces, but could not determine the correct amount.

[0004]

Therefore, it is an object of the present invention to provide a method capable of quantitatively measuring fungi in the skin.

[0005]

[Means for Solving the Problems] Based on the above facts, the present inventors have conducted earnest studies as to a method for quantifying the fungus in the skin, and as a result, the number of fungi present in the skin and the skin having the fungus in the medium have been determined. The inventors have completed the invention by discovering that there is a very good correlation between the sizes of colonies obtained by culturing in.

That is, according to the present invention, (a) a small fragment collected from the skin is cultured, the size of a colony grown on the medium is measured, and (b) the number of conidia is used to control the number of bacteria. The fungus was cultivated under the same conditions as (a), the size of the grown colony was measured, and a calibration curve showing the correlation between the number of conidia and the size of the colony was prepared, and then (a) and (b). The present invention provides a method for quantifying the number of fungi in the skin, which comprises quantifying the number of conidia by comparing the size of the colony in (1).

Here, the fungus to be the subject of the assay of the present invention is not particularly limited as long as it is a fungus capable of growing in the skin, but it is basically a superficial mycosis-causing bacterium. To exemplify this specifically, the genus Trichophyto
n), Microsporum, Epidermophyton and other imperfect filamentous fungi, Candida, Malassezia
Incomplete yeast and the like. Any of these can be quantified by this quantification method. Furthermore, the quantification method of the present invention can be applied to these mutant strains shown below. Examples of the mutant strain include a resistant strain that naturally acquired resistance to a drug, a nutrient-dependent mutant strain that has become nutritionally dependent, and an artificial mutant strain that has been artificially mutated by gene transfer. it can.

[0008] In the quantification method of the present invention, a small piece is first collected from the skin, and the skin used includes mammalian skin including human. In order to collect a small piece from the skin, the hair may be removed, if necessary, and the skin having an appropriate size may be cut out, and then cut into 1 × 1 to 20 × 20 mm per piece. This small fragment is preferably washed with a germicide solution such as benzalkonium chloride or hibitene gluconate for the purpose of removing bacteria other than fungi.

The medium used for culturing the obtained small fragments is not particularly limited as long as it is used for normal culture, bacterial isolation, etc., and examples thereof include Sabouraud medium, modified Sabouraud medium, and Czapek agar medium. it can.

The culture is carried out at 10 to 40 ° C., preferably 20 to
Sufficient time for colonies to grow at 40 ° C, eg 1
Static culture may be performed for up to 20 days.

After culturing, the size of the colonies grown on the medium is measured. The size of a colony is measured by the major axis (l)
And the minor axis (s) are measured, and the product (l × s) is obtained,
Contrast with a control is simple and preferable.

Quantification of the number of fungi is carried out by comparison with a calibration curve prepared separately. The calibration curve is a calibration curve showing the correlation between the size of the colony and the number of fungi, culturing a predetermined number of fungi under the same conditions as above, and measuring the size of the grown colonies. Create by. Here, the number of bacteria is measured by separating bacterial cells and conidia and counting the conidia with a hemocytometer or the like, and this is used as a control of the number of bacteria.
According to this method, it is possible to easily and reproducibly control the number of bacteria. The control fungus used here may be a standard strain or a clinical isolate.

[0013]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but it goes without saying that the present invention is not limited to these examples.

Example 1 Measurement Example of Skin Fungus (1) Preparation of Control Series Trichophyton strain TIMM1189 (Trichophyton men
The modified Sabouraud agar slant was inoculated with tagrophytes TIMM1189) and cultured at 27 ° C. for 21 days. After culturing, a sterilized phosphate buffer solution (containing 0.1% by weight of a surfactant (Tween 80)) was added to the slant, and the slant surface was scraped off with a platinum loop. Next, the bacterial solution was passed through sterile gauze to remove hyphae, and conidia were obtained. Conidia are counted on the hemocytometer and 2 x 10 ⁷
Adjusted to cfu / ml. After that, a 10-fold dilution series of the bacterial solution was prepared using the same phosphate buffer. (100 μl each of the conidium diluted solution was inoculated on a Sabouraud agar dish and cultured at 27 ° C., and the viable cell count was calculated on each of the third, fifth and seventh days.) In parallel with this operation, a Sabouraud agar plate was used. It was created. Cycloheximide 100 μg for this Sabouraud agar
/ Ml, sisomycin 50 μg / ml, and chloramphenicol 100 μg / ml. Although these antibiotics were set under conditions that do not hinder the growth of bacteria but hinder the growth of fungi, Sabouraud agar plates may be prepared by combining other antibiotics. Next, use a spatula sterilized with Sabouraud agar to have a width of 2 mm and a length of 10
Engraved mm grooves (the agar in the groove was removed from the plate). 10 μl of the conidia-diluted column solution was placed in each groove and cultured at 27 ° C. for 5 days to measure growing colonies, and the major axis (1) and the minor axis (s) were measured with a caliper. These l and s
The value A value obtained by multiplying by was plotted on the positive logarithm of the semilogarithmic graph, and the number of viable cells inoculated in the groove was plotted on the logarithmic coordinate. This work was repeated four times with the number of inoculated bacteria changed. Two of these were performed with the same inoculum count. These results are shown in Figure 1.
~ 3. All of these are plotted on a straight line, and it is clear that they are highly correlated. Further, it can be seen that the reproducibility is also good.

(2) Quantification of the number of fungi in the skin of guinea pigs Hartley 5-week-old female guinea pigs were shaved at the left and right two back sites with electric hair clippers and stripped with gum tape to expose the dermis. Next, Trichophyton TIMM
1189 strain (Trichophyton mentagrophytes TIMM1189)
Derived conidia were inoculated on the exposed skin (circular shape with a diameter of 2 cm) on the back skin of 1 × 10 ⁶ cfu guinea pig. Guinea pigs were slaughtered on the 13th and 20th days after inoculation, and the skin of the affected area was
It was cut into a circular shape of cm and washed with 1% bactericide (hibiten gluconate), rinsed with sterilized water to remove bacteria on the surface, and the excised skin was divided into 10 equal parts with scissors. The skin section was embedded in another Sabouraud agar plate described in the above section and cultured at 27 ° C. On the 5th day of culture, the A value was calculated for all the sections of the bacterial growth areas of 5 guinea pigs on the left and right of 10 places by the method described in (1). After calculating the average value of 10 intercepts for each part,
The average value (Avg ± SD) with other sites was obtained. On the 13th day of inoculation of the bacteria in guinea pigs, the average growth area was 1.
764 ± 0.231, that of 20th is 0.846 ± 0.
It was 763. A with control cultured at the same time
From the comparison of the values, the number of bacteria per slice was calculated to be 450 cfu.

Example 2 Confirmation of Effect of Keratin What should be taken into consideration when quantifying fungi in the skin is whether or not keratin, which is a constituent protein of skin, adversely affects the quantification of bacteria. Therefore, regarding the work of (1) of Example 1, the correlation with and without keratin was examined. The results are shown in Table 1. From this, the quantification method of the present invention has an extremely good correlation between the number of conidia planted and the size of colonies produced, regardless of the presence or absence of keratin. Therefore, it can be seen that the quantification method of the present invention can quantify fungi in the skin without being affected by skin constituent proteins.

[0017]

[Table 1]

Example 3 Examination with Candida albicans In the same manner as in Example 1, the strain was replaced with Candida albicans and the same examination was carried out. The results are shown in Fig. 4. From this, even if the strain is replaced by Candida albicans,
It can be seen that the colony area increases in proportion to the number of conidia and that the fungi in the skin can be quantified.

Example 4 Examination with Aspergillus fumigatus Similar to Examples 1 and 3, the same examination was carried out by changing the strain to Aspergillus fumigatus. However, the antibiotics (cycloheximide 10) used in the medium in Examples 1 and 3 were used.
0 μg / ml, sisomycin 50 μg / ml, chloramphenicol 100 μg / ml) were not used because they show antibacterial activity against Aspergillus. Results are shown in FIG.
From this, the same results were obtained with Aspergillus fumigatus as with Candida albicans and Trichophyton. From this, the quantification method of fungus in the skin of the present invention,
It is clear that it can be used regardless of the type of fungus.

Example 5 Investigation with Aspergillus niger As in Example 4, an examination was carried out by replacing Aspergillus fumigatus with Aspergillus niger. The result is shown in Fig. 6.
Shown in. From this result, it is understood that the method for quantifying the fungus in the skin of the present invention can be applied to Aspergillus niger.

[0021]

According to the method for quantifying fungi in the skin of the present invention,
The number of bacteria in the skin can be quantitatively measured.

[Brief description of drawings]

FIG. 1 is a diagram showing the correlation between the viable cell count (conidia count) seeded in Example 1 and the A value.

FIG. 2 is a diagram showing the correlation between the viable cell count (conidia count) seeded in Example 1 and the A value.

FIG. 3 is a diagram showing the correlation between the viable cell count (conidia count) seeded in Example 1 and the A value.

FIG. 4 is a diagram showing a correlation between the viable cell count (conidia count) seeded with Candida albicans and the A value.

FIG. 5 is a diagram showing the correlation between the viable cell count (conidia count) seeded with Aspergillus fumigatus and the A value.

FIG. 6 is a diagram showing the correlation between the live cell count (conidia count) seeded with Aspergillus niger and the A value.

─────────────────────────────────────────────────── ─── Continuation of the front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12Q 1/00-1/70 BIOSIS / MEDLINE / WPIDS (STN) JISST file (JOIS)

Claims (4)

(57) [Claims] 1. A small fragment collected from skin is cultured in a medium, and the size of a colony grown on the medium is measured, while (b) a fungus whose number is controlled by the number of conidia is selected. After culturing under the same conditions as in (a) and measuring the size of grown colonies, a calibration curve showing the correlation between the number of conidia and the size of colonies was prepared, and then the calibration curves of (a) and (b) were prepared. A method for quantifying the number of fungi in the skin, which comprises quantifying the number of conidia by comparing the size of colonies. 2. The quantification method according to claim 1, wherein the fungus whose number is controlled by the number of conidia is a standard strain or a clinically isolated strain, which is separated into bacterial cells and conidia, and conidia are counted. 3. The quantification method according to claim 1, wherein the fungus is a superficial mycosis-causing bacterium or a mutant strain thereof. 4. The quantification method according to any one of claims 1 to 3, wherein the superficial fungal pathogen is one or more selected from incomplete filamentous fungi or incomplete yeast.