JPH09127097A - Microplate for testing sensitivity to antifungal agent, test method for sensitivity and kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To test the sensitivity to an antifungal agent simply and with high reproducibility by a method wherein the antifungal agent is changed into a solid phase on a microplate by a reduced-pressure drying method. SOLUTION: An agent solution which is to be changed into a solid phase on a microplate is prepared in such a way that the agent is dissolved in pure water (UHP), for agent preparetion, which is sterlized in high-pressure steam or that the agent is diluted properly by using the pure water. In the case, in which, for example, amphotericin B, 5-fluorocytosine, fluoconasol or the like is used the an agent, the solution is prepared so that the agent becomes a final concentration. By using the prepared agent solution, a twice-dilution series in a proper concentration range is prepared with sterilized distilled water, the solution is dispensed to a plate in an amount of 40-220μl/well (e.g. 10μl/well). and it is dried under a reduced pressure for 8 to 24 hours (preferably 24 hours) so as to be changed into a soild phase. The prepared plate can be preserved at 4 deg.C.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antifungal drug susceptibility test.

[0002]

2. Description of the Related Art In recent years, an increase in deep-seated mycosis caused by yeast fungi has become a problem, and a proper treatment method for this infectious disease is indispensable. In addition, in recent years, for various antifungal agents, since strains having single-agent or multi-drug resistance have begun to be isolated,
Development of a clinically useful drug susceptibility test method is desired. However, as a drug susceptibility test for antifungal agents, a standard test method has not been established yet. Broth dilution method, agar
dilution method, disk diffusion method, turbidimetric method, r
Applying drug sensitivity tests for general bacteria such as adiometric method, drug sensitivity tests for antifungal agents were conducted from the facility's own method. But as Calhoun et al. Reported (J. Clin. Microbiol. 23: 298-301, 1
986), the test method being implemented at each facility,
It has been pointed out that the reproducibility in the facility is high, but the compatibility between the facilities is poor. Against this background, the United States
National Committee for Clinical Laboratory Standa
rds (NCCLS) summarized NCCLS M27-P as a first proposal in December 1992 (NCCLS: Reference Method for Broth Dilutio) for the purpose of establishing a standardized test method that is compatible between facilities.
n Antifungal Susceptibility Testing of Yeast; Prop
osed Standard M27-P, Pensylvania, NCCLS, 1992). The proposed test method is a macrodilution method with a medium volume of 1 ml, and the medium is morpholinopropanesulfonic acid (MOPS) buffered RPMI 1640.
Inoculation amount 0.5-2.5 × 10 ³ cells / mL using medium
After culturing in normal air for 46 to 50 hours (70 to 74 hours for Cryptococcus), the end point of bacterial growth can be read.

[0003]

According to the results of the joint evaluation conducted by the inventor of the present invention at a plurality of facilities, 76.1% of the minimum inhibitory concentration (MIC) of the standard strain for quality control was NC.
Since CLS M27-P is within the acceptable range and MICs of clinical isolates are distributed in a wide drug concentration range, development of this type of test method has been desired. NCCL
The biggest difficulty in adopting SM27-P in clinical examination sites is that this test method is a macro liquid dilution method, and that the bacterial growth end point (80% bacterial growth inhibition) with an azole drug is macroscopic. It is difficult to read. That is, in order to directly introduce this method into the clinical examination site, the procedure is complicated, and it is necessary to have sufficient proficiency in interpreting the bacterial growth end point. Already a microdilution
Although some applications to law have been reported (Radetsky et
al., J. Clin. Microbiol. 24: 600-606, 1986; Espine
l-Ingroff et al., J. Clin. Microbiol. 29: 1089-109
4, 1991; Espinel-Ingroff et al., J. Clin. Microbio
l. 30: 3138-3145, 1992), as conditions to be adopted for clinical examinations, it is necessary to solve two problems: supply of drug-fixed dry microplates and more highly reproducible determination of bacterial growth end point. There is. At present, two methods have been attempted for the interpretation of the bacterial growth end point, and the colorimetric method (Pfal
ler et al., J. Clin. Microbiol. 32: 506-509, 1994;
Pfaller et al., J. Clin. Microbiol. 32: 1625-1628,
1994), the spectrophotometoric method (Pfaller et a
L., J. Clin. Microbiol. 33: 1094-1097, 1995) is being considered.

[0004]

Means for Solving the Problems The present inventor has prepared a microplate in which an antifungal drug is immobilized on a microplate by a reduced-pressure drying method, and a microvolume liquid dilution method using the microplate, in which a medium is used. Colorimetric broth micr with redox dye, resazurin
The odilution method (hereinafter referred to as the microplate method) was developed and it was found that these problems can be solved by these methods. That is, the present invention relates to an antifungal drug susceptibility test microplate in which an antifungal drug is immobilized on a microplate by a vacuum drying method. The present invention also provides (1) placing a sample containing a fungus in a microplate on which an antifungal drug is immobilized by a vacuum drying method, (2)
The present invention also relates to an antifungal drug susceptibility test method, which comprises the step of incubating the above microplate and (3) determining the growth of a fungus by a change in color tone of a redox indicator. Furthermore, the present invention also relates to an antifungal drug susceptibility test kit comprising a microplate on which an antifungal drug is immobilized by a vacuum drying method, a fungal growth medium, and a redox indicator. The production of the microplate of the present invention and the antifungal drug susceptibility test using the same can be carried out as follows.

[0005] 1. Method for producing drug-immobilized microplate A drug solution for immobilizing on a microplate can be prepared as follows. Pure water (UHP) for pharmaceuticals sterilized by high pressure steam (121 ° C, 15 minutes)
Or dissolve appropriately in it. For example, as a drug, amphotericin B (Amphotericin B, A
MPH), 5-Fluorocytosine,
5-FC), fluconazole (Fluconazole, FLC)
Z), miconazole (MCZ) or itraconazole (Itraconazole, ItCZ), these drugs have final concentrations of 16 μg / ml and 64 μg / ml, respectively.
The solution is prepared so as to be ml, 64 μg / ml, 16 μg / ml, 16 μg / ml. Using the prepared drug solution, make a 2-fold dilution series in an appropriate concentration range with sterile distilled water, and
Dispense the plate in an amount of 20 μl / well (for example, 100 μl / well) for 8 to 24 hours (preferably 24 hours),
Dry and solidify under reduced pressure. The concentration range of the immobilized drug is, for example, AMPH: 16 to 0.03 μg / ml,
5-FC: 64-0.125 μg / ml, FLCZ: 64-
0.125 μg / ml, MCZ: 16-0.03 μg / ml, I
tCZ: a range such as 16 to 0.03 μg / ml can be mentioned. The prepared plate can be stored at 4 ° C.

[0006] 2. Fungal drug susceptibility test using drug-immobilized plate 1) Medium and reagents As a medium for fungal growth, RPMI 1640, YNB
(Yeast Nitrogen Base Solution), SAAMF (Syntheti
c amino acid media for fungi) medium and the like. For example, when using MOPS buffer RPMI 1640 medium, UHP 800 ml is added to RPMI 1640 medium.
Powder medium (Kyokuto Pharmaceutical Co., Ltd.) 10.4 g, NaH
CO ₃ 2.0 g and MOPS 34.53 g were added and dissolved, and the pH was adjusted to 7.0 with 1 N NaOH (about 60 to
65 ml). Adjust the total volume to 1,000 ml and filter sterilize. The medium thus prepared can be stored at 4 ° C.

The medium for preparing inoculum is prepared, for example, as follows. Resazurin 17.5mg UHP 10ml
Dissolved in water, sterilized by filtration, and resazurin stock solution (1.75 mg / m
l). This stock solution can be stored at 4 ° C.
1.1 ml of resazurin stock solution was added to MOPS buffer RPMI 1
Mix with 108.9 ml of a medium such as 640 medium to prepare a medium for preparing inoculum. Since the medium mixed with resazurin becomes unstable by light, it can be stored at 4 ° C. in the dark.

[0008] Sterile physiological saline for preparation of bacterial solution is UHP1,
Dissolve 8.5 g of NaCl in 000 ml (0.85% N
aCl) and can be prepared by sterilizing in an autoclave (121 ° C., 15 minutes).

2) Strains Candida albicans , Ca are applicable strains to which this method is applied.
ndida parapsilosis , Candida toropicalis , Torulops
Candida genus such as is glabrata and its related yeast fungi, especially on Sabouraud dextrose agar
Strains that fully grow within 5 hours at 5 ° C are targeted.

As a strain for quality control, Candida albica
ns (ATCC 90028, ATCC 90029, A
TCC 24433, ATCC 76615), Candid
a parapsilosis (ATCC 90018, ATCC 2
2019), Torulopsis glabrata (ATCC 900)
30), Candida tropicails (ATCC 750), Ca
ndida krusei (ATCC 6258), Saccharomyces
cerevisiae (ATCC 9763) and the like are preferably used.

3) Test Method The colorimetric broth microdilution (microplate method) of the present invention can be carried out as follows, for example. (1) A colony of a freshly isolated strain (within 24 hours of culture) that has grown on Sabouraud dextrose agar medium is used for the test. When the preserved strain is used, it is precultured twice on Sabouraud dextrose agar before use.

(2) Using this bacterial colony, McFarlan
d # Prepare a 0.5 turbidity bacterial suspension. (2) -1. Inoculate 3 to 5 bacterial colonies into a test tube containing 5 ml of sterile physiological saline and suspend. After suspending, stir well with a vortex mixer until uniform. (2) -2. Adjust the bacterial concentration to the turbidity corresponding to McFarland # 0.5. (3) 20 μl of the bacterial solution prepared in (2) was collected with a micropipette, and the MOPS buffer RPMI 16 for bacterial solution preparation was collected.
After adding 2 ml of 40 medium and thoroughly stirring with a vortex mixer, 0.5 ml of the mixture was sampled with a micropipette and used as a medium for inoculum preparation (MOPS containing a redox indicator).
Buffer RPMI 1640 medium) (9.5 ml) and sufficiently stirred with a vortex mixer.

(4) The bacterial solution prepared in (3) was added to each well of a drug-containing solid phase microplate containing a control in an amount of 10
Dispense 0 μl each. Inoculate the bacterial solution within 15 minutes after preparation (if it cannot be inoculated, it can be stored at 4 ° C for up to 2 hours). (5) After the plate is covered, it is shielded from light with an aluminum zipper bag or aluminum foil, and aerobically cultured at 35 ± 1 ° C.

4) Judgment Method Judgment is performed twice, 24 hours and 48 hours. (1) The AMPH is determined by taking the minimum concentration that does not change color (blue) as the minimum inhibitory concentration (MIC). (2) For 5-FC, FLCZ, MCZ, and ItCZ, the minimum density showing an intermediate color (being changing from blue to red) is MI.
Judge as C.

3. Composition of Kit The kit of the present invention contains a microplate on which an antifungal drug is immobilized, a fungal growth medium, and a redox indicator, which are produced by the vacuum drying method as described above.
That is, as an example of the kit of the present invention, AMPH,
Microplate on which a suitable antifungal drug such as 5-FC, FLCZ, MCZ, ItCZ is immobilized, MOPS buffer RPMI 1640 medium as a culture medium for preparing a bacterial solution, an inoculum preparation medium containing resazurin, and a fungus An antifungal drug susceptibility test kit containing sterile saline for liquid preparation is included.

[0016]

EXAMPLES Test Method A. Strains used Candida (C.) Parapsilosis , four strains listed in NCCLS M27-P with MIC tolerance for the purpose of quality control
ATCC 90018, C. albicans ATCC 90
028, C. albicans ATCC 90029, Torulo
psis (T.) glabrata ATCC 90030 was used.

For the examination with clinical isolates, strains of the genus Candida and its related yeasts, which are sufficiently fungi to grow within 24 hours on Sabouraud dextrose agar, 10
0 strain was used. The breakdown is T. glabrata 35 strains, C.
28 strains of albicans , 16 strains of C. tropicalis , C. krusei
6 strains, 5 C. parapsilosis strains, Trichosporon (Tr.) Beig
el ii 5 strains, Saccharomyces (S.) cerevisiae 3 strains, C. lip
olytica 2 strain.

Each strain was subcultured twice on Sabouraud dextrose agar, and then tested using bacterial colonies within 24 hours of culture.

B. Test Microplate Three test agents, AMPH, 5-FC, and FLCZ, were used as test targets. According to NCCLS M27-P, after dissolving the drug substance,
0.03 to 16 μg / ml for MPH, 5-FC and FL
For CZ, a 2-fold dilution series was prepared in the concentration range of 0.125 to 64 μg / ml. In the microplate method, a 2-fold dilution series of the drug is prepared in advance with sterile distilled water, and 100 μl is dispensed into each well of the microplate, and then 24
It was dried under reduced pressure for an hour to prepare a drug-fixed dry microplate. Two rows of the same dilution series were prepared on one microplate, and duplicate measurements were performed.

C. Test method (1) Microplate method of the present invention (1) -1. Preparation and culture of inoculated bacterial solution Using a bacterial colony within 24 hours of cultivation, a bacterial suspension was prepared with 5 ml of sterile physiological saline. Using the McFarland # 0.5 turbidity standard solution, the turbidity of the bacterial suspension was accurately adjusted to the McFarland # 0.5 concentration from the absorbance at a wavelength of 530 nm.
10 μl of the prepared bacterial suspension was added to 2 ml of MOPS buffer RPMI 1640 medium for preparing a bacterial solution, and the mixture was thoroughly stirred,
0.5 ml of it was added to a medium for preparation of inoculum containing 0.15 mM M containing 17.5 mg / L of resazurin sodium salt (Sigma).
OPS buffer RPMI 1640 medium, pH 7.0) 9.5
It was added to ml and thoroughly stirred again with a vortex mixer. The diluted inoculum bacterial solution was prepared in B above.
100 μl was dispensed to each well of a drug-fixed dry microplate having a concentration gradient of a double dilution series. After covering the inoculated microplate with a lid, the microplate was placed in a light-shielding bag with an aluminum chuck and cultured at 35 ± 1 ° C. in a normal atmosphere.

(1) -2. Judgment Method Judgment was performed twice for 24 hours and 48 hours. AMP
In H, the wells that were not discolored at all (blue) were designated as bacterial growth negative, and the minimum inhibitory concentration showing negative bacterial growth was set to MI.
Recorded as C. On the other hand, for 5-FC and FLCZ, it is blue or purple (between blue and pink)
The wells discolored to be visually read as the bacterial growth end point,
MIC.

(2) NCCLS M27-P Recommended Test Method As a comparative example, the MCC-buffered RPMI 1640 medium was used, and the NCCLS M27-P recommended test method based on the measurement principle of the macro liquid dilution method of 1 ml culture system was exactly followed. Measured.

D. Statistical analysis method Significant test of MIC judged by different test methods and different culture time was signed by Wilcoxon with log ₂ converted value.
The Hodges-Lehmann estimate was used to estimate the median value of the two differences and to estimate the confidence intervals.

Results A. Results with quality control strains NC when 4 ATCC standard strains were repeatedly tested
The results of the 24-hour and 48-hour determinations by the CLS M27-P macro liquid dilution method and the microplate method of the present invention are summarized in Table 1.

[0025]

[Table 1]

The NCCLS M27-P macro liquid dilution method yielded an acceptable MIC in 57 (68%) of the 84 total tests. The deviation frequency fluctuates depending on the combination of a specific strain and drug, and C. albicans ATCC 900
In 29, there was only one deviation from 21 trials with 3 agents, but C. parapsilosis / 5-FC and FLCZ, C. albicans ATCC 90028 / 5-F
With the C, T. glabrata / FLCZ combination, the frequency of obtaining an acceptable MIC was 0 to 29%. However, the reproducibility of MIC measured repeatedly was high, and all MICs were distributed within 3 log ₂ .

In the microplate method of the present invention, 67% (113/168) in the 24-hour judgment and 80% (135/168) in the 48-hour judgment were NCCLS M27-P.
It was within the MIC tolerance range proposed by. Examples that deviate from the permissible range are almost consistent with the macro liquid dilution method, especially C. para.
In 5-FC is all of the tests in psilosis ≦ 0.125μ
Determined to be g / ml, and its permissible range is 0.25 to 1.0 μg / ml
No results were obtained that were consistent with. Systematic biases were observed for certain strains and drug combinations compared to the proposed tolerance, but MIs obtained by this method were also obtained.
The reproducibility of C is high, and the frequencies distributed within 3 log _{2 in} the 24-hour judgment and the 48-hour judgment are 96% and 9%, respectively.
3%.

B. Results in clinical isolates NCCLS M27-P MIC in macro liquid dilution method as reference result, 24 in microplate method of the invention
The results obtained by comparing the time judgments are summarized in Table 2.

[0029]

[Table 2]

Of the 600 test results in total, 370 (61.7%) were diluted with ± 1 log ₂ and 487 (8
MICs of 1.2%) were obtained at ± 2 log ₂ dilutions. Especially in 5-FC, the concordance rate is high, 93.5% (1
87/200) was matched at ± 1 log ₂ dilution. On the other hand, in AMPH and FLCZ, the MIC in the microplate method of the present invention was low as a whole (P <0.01), and this tendency was particularly remarkable in FLCZ. Statistically 8
AMPH 2.8-3.4 times, 5-F with 0% confidence interval
A difference of 1 to 1.2 times in C and 2.8 to 4.0 times in FLCZ was observed between the macro liquid dilution method.

Table 3 summarizes the results of 48-hour judgment by the microplate method of the present invention.

[0032]

[Table 3]

Compared with the 24-hour judgment, the agreement rate with the macro liquid dilution method was higher, and 51 out of 600 test results were obtained.
Consistent MICs were obtained with 7 (86.2%) ± 1 log ₂ dilutions and 585 (97.5%) ± 2 log ₂ dilutions. Statistically, except for 5-FC, the macro liquid dilution method tended to show a slightly higher MIC, but the difference was extremely small, and the difference between the two was 80% confidence interval.
1.2-1.4 times, 5-FC 1 times, FLCZ 1.
It was 4-1.7 times.

Two rows of the same dilution series were prepared on one microplate, and their reproducibility was evaluated. 3 in FIGS.
Two MICs obtained by combining drugs and 100 strains
The judgment agreement rate is summarized from (dilution rate). 2 obtained
The two MICs showed a high concordance rate over the entire set drug concentration range, with 100% for 24 hours and 99% for 48 hours.
The results were in agreement with the percentage of ± 1 log ₂ dilution.

FIGS. 3, 4 and 5 show the MI of the three drugs examined.
The C cumulative distribution is plotted. In AMPH, all MICs were distributed between 0.06 and 2.0 μg / ml, the 24-hour microplate method was the most sensitive, and the macro liquid dilution method and the 48-hour microplate method showed almost the same cumulative distribution. It was This tendency is the same in 5-FC and FLCZ, but in 5-FC, most of the target strains showed a relatively low MIC, and therefore little difference between the methods was observed. On the other hand, in FLCZ, ≦ 0.
Widely dispersed in the concentration range of 125-> 64 μg / ml, AMPH
Similarly to the above, the 24-hour determination microplate method showed the most sensitivity, and the macro liquid dilution method and the 48-hour determination microplate method showed almost the same cumulative distribution.

Table 4 summarizes 50% MIC and 80% MIC for the 8 bacterial species used in this study.

[0037]

[Table 4]

Comparing the values of MIC ₅₀ and MIC ₈₀ , N
The results of the 48-hour judgment of the CCLS M27-P macro liquid dilution method and the microplate method were almost in agreement, and the 24-hour judgment showed a tendency of being slightly biased in sensitivity. Also MIC
Comparing the ratios of ₅₀ and MIC ₈₀ between drugs, the difference is almost the same or double in AMPH, and C. parapsilos in 5-FC .
Although it is similar except for is, FLCZ shows almost the same value within the same bacterial species, but a 4- to 8-fold difference was observed in the value for all strains. When analyzed by bacterial type, AMPH was 0.125 for both MIC ₅₀ and MIC ₈₀ for all bacterial types.
It was 1.0 μg / ml, and there was no particular difference between the bacterial species.
Most of the bacterial species showed a relatively low MIC in 5-FC, but MIC ₅₀ , M in C. krusei and C. lipolytica.
IC ₈₀ was 16-32 μg / ml. Differences among bacterial species were also observed in FLCZ , such as T. glabrata , C. krusei ,
MIC is high in C. lipolytica , C. albicans , C. t
A lower tendency was observed in ropicalis , C. parapsilosis and S. cerevisiae .

[0039]

EFFECT OF THE INVENTION According to the trace amount liquid dilution method using the microplate of the present invention, a sensitivity test for an antifungal drug or the like can be carried out easily and with high reproducibility.

[Brief description of the drawings]

FIG. 1 is a diagram showing the reproducibility of the method of the present invention, which was determined by incubation for 24 hours.

FIG. 2 is a diagram showing the reproducibility of the method of the present invention determined by 48-hour incubation.

FIG. 3 is a diagram showing the MIC cumulative distribution of fungi against AMPH.

FIG. 4 is a diagram showing the MIC cumulative distribution of fungi against 5-FC.

FIG. 5 is a diagram showing the MIC cumulative distribution of fungi on FLCZ.

Claims (3)

[Claims] 1. A microplate for an antifungal drug susceptibility test, wherein an antifungal drug is immobilized on a microplate by a vacuum drying method. 2. An antifungal drug susceptibility test method comprising the following steps: (1) A sample containing a fungus is placed in a microplate on which an antifungal drug is immobilized by a vacuum drying method, and (2) above. The microplate is incubated, and (3) the growth of the fungus is judged by the color change of the redox indicator. 3. An antifungal drug susceptibility test kit comprising a microplate on which an antifungal drug is immobilized by a vacuum drying method, a fungal growth medium, and a redox indicator.