WO1998053096A1

WO1998053096A1 - Reagents for counting mycobacteria, method therefor, and method for testing drug sensitivity of mycobacteria

Info

Publication number: WO1998053096A1
Application number: PCT/JP1998/002184
Authority: WO
Inventors: Naoki Sato; Yutaka Okazawa; Kazunobu Tanno; Noriaki Hattori
Original assignee: Kyokuto Pharmaceutical Industrial Co., Ltd.; Kikkoman Corporation
Priority date: 1997-05-20
Filing date: 1998-05-19
Publication date: 1998-11-26
Also published as: JPH1132792A

Abstract

A technique for counting mycobacteria by which changes in mycobacterial count during culture can be monitored with the passage of time so that drug sensitivity tests can be completed within a short period of time. Specifically, reagents for counting mycobacteria characterized by containing an ingredient which reacts with ATP and thus emits light, a method involving the step of adding the above reagent to a sample containing mycobacteria and measuring the light emission, and an apparatus for effecting this method.

Description

Description Reagent for measuring the number of mycobacteria, method therefor, and method for testing drug sensitivity of mycobacteria

The present invention relates to a reagent for determining the number of mycobacteria in a sample, a method and an apparatus thereof, and a method and an apparatus for testing the sensitivity of a drug to mycobacteria. Background art

The number of mycobacterial infections, such as Mycobacterium tuberculosis infections caused by Mycobacterium tuberculosis, has been steadily decreasing in Japan due to the development of therapeutic agents and improvement of nutritional status. The decrease is showing a tendency to slow down, and many patients are still newly registered. Therefore, there is a need for the development of therapeutic agents that are more effective against such mycobacteria and the development of a rapid and accurate drug sensitivity test method. In the drug sensitivity test for acid-fast bacilli in Japan, the fixed concentration method using Ogawa medium containing chicken eggs as a main component is mainly adopted. In this fixed concentration method, the inoculum is inoculated on a test medium containing the target drug and a control medium without the target drug, and after culturing, the numbers of acid-fast bacteria are compared to determine the sensitivity of the acid-fast bacterium to the target drug. That is.

However, the growth of mycobacteria is much slower than that of other bacteria, so it usually takes 2 to 4 weeks for the culture to be detectable by this method. Therefore, in order to conduct a drug susceptibility test in a short period of time, there is a need for a technique for measuring the number of acid-fast bacteria capable of detecting changes with time in the number of acid-fast bacteria in culture. Disclosure of the invention

The present invention relates to a reagent for measuring the number of acid-fast bacteria, which contains a component that emits light when reacted with ATP. Here, for example, the components are luciferin, luciferase and magnesium ion.

Further, the present invention relates to a method for measuring the number of acid-fast bacteria, comprising a step of adding the reagent for counting the number of acid-fast bacteria to a sample containing acid-fast bacteria and measuring the amount of emitted light. In particular, it is preferable to heat-treat the sample containing the acid-fast bacterium before adding the reagent, and it is more preferable to add an AΡ extract reagent to the sample before the heat treatment.

Further, the present invention provides a method for testing drug susceptibility of acid-fast bacilli,

A first step of adding a drug to the sample containing the acid-fast bacterium and culturing it for a certain period of time, and then adding the reagent for measuring the number of acid-fast bacilli to measure the amount of luminescence;

After culturing for the same time as the first step without adding the drug to the same sample as the first step, a second step of adding the reagent and measuring the luminescence amount; and

A third step of determining whether or not the drug is susceptible to the acid-fast bacterium based on the luminescence amounts obtained in the first step and the second step

. Here, it is preferable to heat-treat the acid-fast bacterium-containing sample before adding the reagent in the first step and the second step, and it is more preferable to add an ATP extraction reagent to the sample before the heat treatment.

Further, it is preferable that the determination in the third step is made based on the magnitude of the ratio of the light emission amounts obtained in the first step and the second step.

Step A of adding the drug to the acid-fast bacterium-containing sample and culturing the mixture, and then adding the acid-fast bacterium counting reagent and measuring the amount of luminescence over time; After culturing the same sample as in step A without adding the drug, and then, with time, adding the reagent and measuring the luminophore; and

A step C of judging whether or not the drug is sensitive to the acid-fast bacterium based on a time-dependent change in the luminescence amount obtained in the step A and the step B.

. Here, it is preferable to heat-treat the acid-fast bacterium-containing sample before adding the reagent in step A and step B, and it is more preferable to add an ATP extraction reagent to the sample before the heat treatment. Further, it is preferable that the determination in the step C is made based on a change in the ratio of the luminescence amount obtained in the steps A and B with time.

Further, the present invention relates to an apparatus for performing the method for measuring the number of acid-fast bacteria and the method for testing drug sensitivity of acid-fast bacteria. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the RLU measurement procedure,

FIG. 2 is a diagram showing the relationship between the heating conditions of the sample and the amount of luminescence,

FIG. 3 is a diagram showing the relationship between the number of days of culture of the sample and the amount of luminescence, and

FIG. 4 is a diagram showing a culture procedure. BEST MODE FOR CARRYING OUT THE INVENTION

Mycobacteria count reagent

The reagent for determining the number of acid-fast bacteria according to the present invention contains a component that emits light when reacted with ATP. Such components are, for example, luciferin, luciferase and magnesium sulfate, and a reagent containing these components is commercially available from Kikkoman Co., Ltd. under the trade name Lucifer-LU Plus. Known luciferases are used. Any organism can be used as long as it can be used and emits light in response to ATP. In the present invention, luciferases derived from insects, for example, insects belonging to the family Echinacea (for example, Genji firefly, Heike firefly, Hime firefly, American firefly, etc.) and insects belonging to the family Beetle family (for example, Hikari click beetle, Jamaican beetle, etc.) Luciferase can be used. When this reagent is used, light is emitted by the following reaction mechanism.

Luciferase

Luciferin + AT P

Mg ^{2 +}

Luciferin 'Lucifera-Zee AMP + P-P

Luciferase

Luciferin - AM P + 0 ₂ → product + light magnesium ions are supplied from any magnesium salts. Examples of the magnesium salt include compounds such as magnesium acetate and magnesium sulfate. The reagent for measuring the number of acid-fast bacteria according to the present invention may contain components such as a buffer and a stabilizer. For example, HE PES buffer, EDTA and the like can be mentioned. The concentration of each component in the acid-fast bacterium measurement reagent is appropriately determined by those skilled in the art according to the type and concentration of the acid-fast bacterium. Method for Counting Mycobacteria The number of mycobacteria is determined by (1) adding the above reagent to a sample that may contain mycobacteria, (2) measuring the amount of luminescence, and ( 3) Includes a step of calculating the number of acid-fast bacteria by conventional means based on the measured luminescence. Regarding the reagent addition step (1) above, what kind of sample should be used (for example, whether or not a diluted stock solution should be used), how much sample should be used, and how much reagent should be used A person skilled in the art will appropriately determine whether or not to add the degree. In the luminescence measuring step (2), the luminescence is preferably measured at around 27 ° C. Usually, the luminescence is measured with a Lumi counter. Also, In the step (3) of calculating the number of acid-fast bacilli, the amount of luminescence is proportional to the amount of ATP, so the amount of ATP can be calculated by proportional calculation, and based on the amount of ATP per target acid-fast bacterium, The number of bacteria can be determined from the calculated amount of ATP.

From the viewpoint of improving the sensitivity, it is preferable to introduce (0) a step of heating the sample before the step (1) of adding the reagent. By this step, the cell wall of the acid-fast bacterium in the sample is broken, and ATP in the acid-fast bacterium can be efficiently extracted. In order to further improve the sensitivity, it is preferable to introduce a step of adding the ATP extraction reagent to the (0) ′ sample and heating the sample before the step (1) of adding the reagent. Heating in the presence of the ATP extraction reagent has the effect of further improving the ATP extraction efficiency. The ATP extraction reagent is not particularly limited, and a known reagent such as a mixed solution of ethanol and ammonia, methanol, ethanol, a surfactant (such as Triton XI 100), trichloroacetic acid, perchloric acid, and the like. ATP extraction reagents can be used. Further, an ATP extraction reagent attached to a commercially available kit, for example, Lucifer-1 LU LU Plus (manufactured by Kikkoman) may be used. Various conditions such as the heating temperature, the heating time, and the reagent concentration are appropriately selected according to the state of the sample, the capability of the measuring device, and the like. However, it is preferable to select a heating temperature of 35 ° C. or higher. For example, for reference, Table 1 shows the results obtained by measuring the luminescence amount of the same sample while changing the heating temperature and heating time according to the procedure shown in FIG.

Table 1 sword U j? CJ-'

Heating time (min) Non-heating 37 ° C 50 ° C 60 ° C 100 ° C

0 3,255 3,424 3,471 2,821 3,606

1 4,588 9,941 14,523 14,936 16,217

2 4,865 13,001 16,029 15,556 18,514

3 5,667 13,517 15,767 16,941 17,057

4 8,034 13,328 15,736 14,939 18,432

5 9,506 11, 961 15,546 16,763 15,097 Specimen strain: H 37 R v ATCC27294 From this table, it is possible to obtain 56 times the amount of luminescence when heated at 100 ° C for 2 minutes, for example, compared to non-heated (0 minutes). You can see that. At the time of measurement, it is preferable that the light emission amount is 100 000 RLU or more.Therefore, the temperature and heating time should be set so that the light emission amount is not less than the dotted line part in FIG. Is preferred. The heating is preferably performed at a temperature of 35 ° C. or higher, and particularly when a microplate is used, it is necessary to set the temperature at or below the heat resistance temperature of the microplate (about 75 ° C in a normal case). In this case, heating at 60 ° C. for 3 minutes is preferable. Method for Testing Drug Sensitivity of Mycobacteria The method for testing drug susceptibility of mycobacteria according to the present invention is an application of the above-described method for measuring the number of mycobacteria. This drug sensitivity test method comprises: (1) a first step of adding a drug to a sample containing the acid-fast bacterium, culturing it for a certain period of time, and then adding the above-mentioned reagent for measuring the number of acid-fast bacilli to measure the amount of luminescence. (2) a second step of culturing the same sample as in the first step without adding the drug thereto for the same time as the first step, and then adding the reagent to measure the amount of luminescence; and (3) the first step And a third step of determining whether or not the drug is sensitive to the acid-fast bacterium based on the luminescence amount obtained in the second step. Here, in the present specification, during the third step (and step C described later), the following R LU _{rat i} . The presence or absence of susceptibility to acid-fast bacilli is determined using a set of parameters, but this determination method is an example of the third step (and step C described below) and is not limited to this.

(Emission amount obtained in the first step)

R L U

(Luminescence obtained in the second step) (Luminescence of drug-containing medium) (Luminescence of control) Here, R LU _{rat i} . Determine the sensitivity (S) or resistance (R) according to the value of. For example, R LU _{rat i} below. If the value is 0.5 or less, set it to S. If it exceeds 0.5, set it to R. The test bacteria used in this drug susceptibility test method include fresh bacteria, precultured bacteria, and cryopreserved bacteria.As can be seen from Fig. 3, cryopreserved bacteria require time to grow. From the viewpoint of rapid diagnosis, fresh bacteria and pre-cultured bacteria are preferred. Here, a fresh bacterium refers to a bacterium cultured for 2 to 3 weeks in Ogawa medium, suspended in Myc0br0th (liquid medium), adjusted to a bacterial solution, and cultured. The pre-cultured bacteria (Myc 0 br 0th pre-cultured bacteria) refers to the microorganisms obtained by suspending the Ogawa medium culture in Mycobroth, adjusting the bacterial culture for 5 to 7 days, and then culturing the suspension. Cryopreserved bacteria (Ichi Ogawa 80 ° C storage bacteria) are bacteria that have been cultured in Ogawa medium culture at -80 ° C, suspended, adjusted, and cultured.

RLU _{rat i} . The value can vary depending on the culture time.In some cases, the culture may show sensitivity as time elapses even if it is initially resistant, so a certain amount of culture time is required to determine whether it is sensitive or resistant. Is done. The cultivation time varies depending on the type and concentration of the test bacteria, the type and concentration of the drug, etc., but the cultivation time is longer than the longest time among the R → S transition times (time to reach the judgment standard) required in various cases. The set Then, there is no need to consider the difference between these conditions. Therefore, according to the procedure shown in Fig. 1, the R → S transition time was examined for several cases in which the type and concentration of mycobacteria and the type and concentration of the drug were changed, and the results were shown in Tables 2 and 3. Show. Table 2

H 37 R v Ogawa bacteria 小. D. 0.22

R LU _{rat i} . Arrival time (days) Judgment Drug (μ.g / ml) ≤0.5 ≤0.3.5 5 7

R F P 0.01 R R

R F P 0.1 5 5 S S

R F P 1.0 3 5 S S

I N H 0.01 R R

I N H 0.1 5 7 S S

I N H 1.0 5 5 S S

E B 0.1 R R

E B 1.0 R R

E B 10.0 7 R S

S 0.01 R R

S M 0.1 R R

S M 1.0 5 5 S S

K 0.1 R R

K M 1.0 5 7 S S

K 10.0 3 5 S S

P Z A 1 R R

P Z A 10 R R

PZA 100 RR Sample strain: H 37 R v ATCC 27294 Table 3

Specimen strain: H 37 R v ATCC 27294 As can be seen from Tables 2 and 3, the R = S transition time (RLU _{rat i} , which is said to be less than 0.5) in these cases is as follows: 3-7 days. Therefore, based on these results, the culture time should be set to 7 days or more. However, the culture time of 7 days is based solely on the above results, and when performing a susceptibility test on new test bacteria / drugs, etc., perform the R → S transition time measurement test described above. The culture time may be set separately based on the result.

Or, RLU _{ra ti} . Do not wait for the value to drop below 0.5, and collect samples from the medium over time to obtain RLU _rat i. Was measured and RLU _rat i over time. You may compare the values. In this case, R LU _{rat i} . If the value decreases over time, it may be considered sensitive to the drug. For example, according to the procedure shown in FIG. 1, in order to determine the presence or absence of susceptibility of mycobacteria below against the following agents, over time RLU _rat i. The values were measured. Table 4 shows the results. Table 4

H37RvATCC35822 H37RvATCC35828 H37RvATCC35835

O.D.0.2 (INH-R) (PZA-K) ヽ O.D.0.2 (RFP-R) Drug (g / ml) ≤0.5 ≤0.3 5 7 ≤0.3 5 7 H 5 ≤0.3 5 7 ti

R F P 0.1 3 3 S S S S 33 3 S S s s R R R R

"

1 NH 1.0 RRRR 5 7 SR _D ss 7 9 S _D

E B 10.0 y 9 D C

RR _D f 7 RS _D cc 7 (7 RR ccoo

EB 20.0 9 9 RR _D RS _D 7 7 RR ss 7 7 RR ss

SM 1.0 5 7 SR, ss 5 5 SR _D ss 5 7 SR _D ss

K 5.0 3 5 S S s s 3 5 S S s s 5 5 S S s s

P Z A 100 5 9 S R I S R I R R R R 9 12 R R R S I

, P Z A 200 7 7 R S I S R I R R R R 9 9 R S I R S I

R R> 0.75 Resistance

RR _D resistance, decreased from the day before RLU

R S 0.75 ≥RS> 0.50 Resistance

R S I resistance, R L U increased from the day before

R S o tolerance, R L U decreased from the day before

S R 0.50≥S R> 0.30 Sensitivity

S R I sensitivity, R L U increased from the day before

SR _D susceptibility, decreased from the day before RLU

S S 0.3 Sensitivity

SSI sensitivity, increased from the day before RLU

In Table 4, RFP stands for Rifavicin, I NH stands for Isoniazid, EB stands for Ethambutol, SM stands for Streptomycin, and KM stands for Kanamycin. And PZA stand for Pyrazinamide, respectively.

From Table 4, R LU _{rat i} in all samples if sensitivity is indicated. A decrease in the value over time was observed. For example, H37RvATCC35822 showed an R for EB on both days 5 and 7, but was lower on day 7 than on day 5 and RLU _{rat i} on day 9. The value was below 0.5, indicating sensitivity. Thus, in the above case, R LU _{rat i on} days 5 and 7. From the values alone, it is possible to determine whether the test organism is susceptible to the test drug. Example

Clinical isolates with a known resistance pattern were cultured according to the procedure shown in Fig. 4 and susceptibility tests for various drugs were performed. Over time was sampled to measure the light emission amount of each sample in accordance with the procedure of FIG. 1 with the Lumitester K one 200 of Chicco ten thousand Inc. and R LU _{at i.} Value. Table 5 shows the results. As a result, according to the test method of the present invention, it was found that the same result as the conventional test method was obtained in a short time.

Table 5

SR-7 O.D.0.2 F11 O.D.0.2 K620 O.D.0.2

(RFP, INH, EB, SM-R) (RFP, INH, EB, SM-R) (RFP, INH, EB-R) Drug (ug / ml) ≤0.5 ≤0.3 5 days ≤0.5 ≤0.3 5 7 says ≤0.5 ≤0.3 5 says 7

R F P 1.0 R R R R R R R R R R R R

1 N H 1.0 R R R R R R R R R R R R

E B 10.0 R R R R R R R R R R R R

, EB 20.0 RRRS _D 7 7 RSSS 7 RSSR _D

S M 1.0 R R R R 5 5 S S S S R R R R R

K M 5.0 3 5 S S S S S R R R 5 5 S S S S

P Z A 100 R R R R R S R S I 5 5 S S S S I

P Z A 200 R R R R R R R R 5 5 S S S S I

* The definitions of the symbols in the table are the same as in Table 4,

Claims

The scope of the claims

1. An acid-fast bacterium count reagent containing a component that emits light in response to ATP.

2. The reagent according to claim 1, wherein said components are luciferin, luciferase and magnesium ion.

3. A method for measuring the number of acid-fast bacteria, comprising a step of adding the reagent according to claim 1 or 2 to a sample containing an acid-fast bacterium and measuring the amount of luminescence.

4. The method for counting the number of mycobacteria according to claim 3, wherein the sample containing mycobacteria is heated before adding the reagent.

5. The method for measuring the number of acid-fast bacteria according to claim 4, wherein an ATP extraction reagent is added to the sample prior to the heating.

6. In the method for testing the susceptibility of mycobacteria to drugs,

A first step of adding a drug to the sample containing the acid-fast bacterium and culturing it for a certain period of time, and then adding the reagent according to claim 1 and measuring the amount of luminescence;

A second step of culturing the same sample as in the first step without adding the drug for the same time as the first step, and then adding the reagent to measure the amount of luminescence; and

A third step of determining whether or not the drug is susceptible to the acid-fast bacterium based on the luminescence obtained in the first and second steps

A method that includes

7. The method according to claim 6, wherein in the first step and the second step, a heat treatment is performed before adding the reagent.

8. The method according to claim 7, wherein an ATP extraction reagent is added to the sample before the heat treatment.

9. The judgment according to any one of claims 6 to 8, wherein the judgment in the third step is made based on the magnitude of the ratio of the light emission amounts obtained in the first step and the second step. the method of.

10. In the method for testing the drug susceptibility of acid-fast bacilli,

Step A of adding a reagent to the sample containing the acid-fast bacterium and culturing the mixture, and then adding the reagent according to claim 1 and measuring the amount of luminescence over time;

A step B of culturing the same sample as in step A without adding the agent, and then adding the reagent over time to measure the amount of luminescence;

A step C of judging whether or not the drug is sensitive to the acid-fast bacterium based on the time-dependent change in the luminescence amount obtained in the steps A and B

A method that includes

11. The method according to claim 10, wherein in step A and step B, heat treatment is performed before adding the reagent.

12. The method according to claim 11, wherein an ATP extraction reagent is added to the sample before the heat treatment.

1 3. The judgment in step C was obtained in step A and step B

The method according to any one of claims 10 to 2, wherein the method is performed based on an increase / decrease of a temporal change in a ratio of light emission amounts.

14. An apparatus for performing the method of claim 3.