WO2010087110A1 - 微生物自動分析装置および微生物自動分析方法 - Google Patents
微生物自動分析装置および微生物自動分析方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
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- the present invention performs an automatic microorganism analysis apparatus and method, particularly blood culture tests and identification / drug susceptibility tests continuously without separate culture, greatly reducing the time (number of days) to report the results and reporting the next day.
- the present invention relates to an apparatus and method for automatically analyzing microorganisms, which have an improved effect.
- Blood culture test is an important test in microbial testing. Rapid detection of bacteria, fungi, and the like from blood that is inherently sterile is very important in sepsis and bacteremia, which are serious infections. On the other hand, finally identifying the species of microorganisms, quickly measuring the sensitivity to antibiotics, determining the type and concentration of an effective drug, and establishing a treatment strategy will lead to appropriate antimicrobial treatment.
- the collected blood is cultured in a blood culture test apparatus, and after a positive determination is made, the sample is removed from the culture bottle, applied to the culture medium, and cultured for several hours to overnight (separation) Cultivation), the formed colonies are fished to prepare a bacterial suspension and inoculated into the measuring device of the identification / drug sensitivity testing apparatus. That is, after a test with a blood culture test apparatus, after several hours to an overnight culture, a test is performed using another identification / drug sensitivity test apparatus.
- Patent Document 1 shows an example in which a certain amount of bacteria can be collected with a simple grooved rod.
- concentration adjustment is shown.
- these rods or bottles are excellent for human work, but the steps are complicated and not suitable for automation by the apparatus.
- the problem to be solved by the present invention is that, after blood culture, a positive sample is transferred and transferred to an identification / sensitivity test as it is without being separated and cultured overnight to produce colonies.
- the purpose is to reduce the time required to report the test results and enable the next day report.
- the in-bottle culture medium that is positive in blood culture is used and directly inoculated into the device of the identification / sensitivity test apparatus, the components in the blood culture bottle affect the drug contained in the identification / sensitivity test apparatus device.
- the present invention provides a bacterial test apparatus and method that solves the above-mentioned problems and allows the sample to be transferred to identification / sensitivity measurement as it is without going through separation culture by transplanting, greatly reducing the test time and reporting the next day.
- the purpose is to do.
- Another object of the present invention is to provide a method and apparatus for testing bacteria in consideration of biohazard countermeasures such as infection by automatically connecting blood culture tests and identification / drug sensitivity tests without human intervention. .
- the above problem is solved by providing an apparatus having a mechanism for pre-processing a sample collected from a positive bottle after blood culture. That is, a mechanism for filtering positive samples, a mechanism for adjusting the pH of a sample, a mechanism for concentrating the number of bacteria in a sample to a predetermined concentration, and inoculating bacteria in a sample into a liquid medium to obtain a predetermined amount of bacterial solution It is solved by providing a mechanism that can.
- the components in the culture solution of blood culture can be removed by a mechanism for filtering the sample after positive blood culture.
- the blood culture bottle may contain activated carbon, an ion-adsorbing resin, and other components for adsorbing the administered antibacterial agent contained in the blood of a patient specimen already undergoing chemotherapy.
- the influence on the drug used for identification / sensitivity test can be removed.
- the identification / sensitivity test it is essential that the amount of inoculated bacteria on the device is kept constant at a predetermined concentration with good accuracy and reproducibility.
- the culture solution is colored by hemocytes and other components in addition to activated carbon and ion-adsorbing resin, and turbidity occurs, and the bacterial solution cannot be adjusted well. It becomes possible to measure. Further, during blood culture, the pH of the culture solution is lowered by carbon dioxide metabolized and produced as microorganisms grow.
- the influence on the identification / sensitivity test measurement reagent can be eliminated by returning the pH of the culture solution to an appropriate range by the addition mechanism of the pH adjusting reagent. Furthermore, in order to obtain an accurate result in the identification / sensitivity test, it is necessary to inoculate the measuring device with a certain concentration of bacterial solution, but in many cases the bacterial count ( The concentration of bacteria) is insufficient, and the sample cannot be used in the identification / sensitivity testing device as it is. Therefore, in the present invention, in order to obtain a bacterial solution having a certain bacterial count concentration or more, the positive bottle is centrifuged, the sediment is suspended in physiological saline or a dedicated test solution, and is collected by further centrifugation as necessary.
- the bacterial solution can be concentrated by using an ultrafiltration membrane or the like.
- a small amount of concentrated bacterial solution collected by centrifuging the bottle is inoculated into a new liquid medium and cultured for a short time so that a bacterial solution having a predetermined bacterial concentration can be obtained.
- a microorganism testing apparatus and method capable of significantly reducing the time required until the time has been realized.
- FIG. 1 shows a workflow of using the apparatus of the present invention and a conventional inspection.
- the patient sample, blood arrives at 9:00 on the first day.
- the laboratory technician immediately sets the blood culture apparatus and starts culturing. Usually, culture is monitored continuously for 5 to 7 days, but 90% of the cultures are positive in 1 to 2 days. Here, the cells were cultured for 18 hours, and a positive determination result was obtained at 3 o'clock in the night.
- the laboratory is unattended at midnight, and positive specimens are placed directly in the blood culture device.
- the laboratory work starts at 8:00 am on the second day, the blood sample is applied to the plate medium from the positive bottle at 9:00 and is cultured in a thermostatic chamber from 10:00. This is a separate culture for transplantation. Colonies are observed on the medium as early as 8 hours or more, that is, after 18:00 in the evening.
- colonies are generated at noon on the second day.
- the judgment after 9 hours comes at midnight on the second day, that is, at 0:00 on the third day.
- medication information is given to the patient at the shortest time from the midnight on the third day to the early morning.
- the flow in the case of the present invention is as follows.
- the sample arrives at 9 o'clock on the first day, and the positive determination is at 3 o'clock in the night.
- the sample is not applied to the flat plate from the positive bottle, and pretreatment such as filtration and pH adjustment is automatically performed in the apparatus.
- the time required for preparing the bacterial solution is at most one hour at the latest. If it is set in the identification / sensitivity apparatus at 4 o'clock in the early morning, the measurement result can be reported to the doctor after 9 hours, that is, at 13:00 on the second day, and the time can be shortened by 2 days or more.
- FIG. 2 shows the principle diagram of the automatic microorganism analyzer.
- reference numeral 1 denotes a blood culture unit.
- a large number of blood culture bottles 2 such as 60 can be installed.
- the blood culture unit 1 is maintained at a predetermined temperature.
- 3 is the whole pre-processing unit.
- the pretreatment unit 3 includes a filtering mechanism 5, a pH adjustment mechanism 6, a centrifuge unit 7, a bacterial solution adjustment unit 9, a liquid culture unit 11, and the like.
- Reference numeral 12 denotes an identification / drug sensitivity analysis unit.
- the bacterial solution adjusted through the pretreatment unit 3 is filled in the culture / measurement device 13 by the inoculation mechanism 14.
- the device is filled with a number of different concentrations and types of antibiotics or nutrient media for bacterial species identification.
- the device is maintained at a constant temperature in the culture unit 15, and the inoculated microorganism is cultured.
- the device 13 is pulled out of the culture unit at a constant time interval by the transport unit 16 and optically photometrically measured by the detection unit 17.
- 18 is a microcomputer, 19 is an interface, 20 is a Log converter and A / D converter, 21 is a printer, 22 is a CRT, 23 is a hard disk as a storage device, and 24 is an operation panel.
- the operator inputs analysis request information using the operation panel 24.
- the input analysis request information is stored in a memory in the microcomputer 18.
- the blood sample placed in the blood culture container 2 and set at a predetermined position of the blood culture unit 1 is cultured for a certain time.
- microorganisms contained in the blood grow, metabolites such as carbon dioxide are produced, and the pH of the medium changes. This change is detected by the pH sensor.
- Measurement is performed at regular time intervals, and the microcomputer 18 makes a positive / negative determination using a predetermined algorithm.
- a certain amount of the culture solution in the blood culture container 2 that has been determined to be positive is collected by the fractionation nozzle 4.
- the filter preferably has a pore size of about 0.2 to several micrometers that allows microorganisms to pass through and remove carbon particles, ion adsorption resin, and blood cell components.
- a pH adjusting mechanism which is equipped with a pH adjusting reagent.
- a pH adjusting reagent By adding to the culture solution after blood culture, it is possible to adjust the pH of the culture solution lowered by the carbon dioxide that is metabolized and produced as the microorganism grows, and to remove the influence on the reactivity of the identification test.
- a bacterial solution having a predetermined accurate bacterial amount (concentration) is required. This is because when the amount of bacteria is insufficient or excessive, accurate results cannot be obtained.
- Reference numeral 7 denotes a centrifugal unit. In many cases, the microorganism concentration in the culture medium positively determined in the blood culture is less than a certain number.
- the culture solution after blood culture is centrifuged to obtain a sediment containing microorganisms.
- the blood cell component and the carbon particles / ion adsorbing resin may be removed by this centrifugation.
- the sediment is collected by the microbial collection mechanism 8 and dissolved and suspended in physiological saline or a dedicated test solution in the microbial solution adjustment unit 9.
- the floating bacterial solution suspended in physiological saline is optically measured by the photometer 10.
- Various methods can be used for the optical system as long as the degree of turbidity of the microorganism suspension such as absorbance, turbidity, and scattering intensity can be measured. It is usually desirable to prepare a series of dilutions to result in 5 ⁇ 10 5 CFU / mL.
- a new liquid medium in the liquid culture unit 11 may be automatically inoculated to increase the number of bacteria.
- the liquid culture unit 11 is maintained at a constant temperature, and in many cases, a sufficient amount of bacterial concentration sample can be obtained by culturing within 12 hours. In order to shorten the culture time, shaking culture may be performed.
- the bacterial solution in the liquid culture is automatically collected by the bacterial collection mechanism 8, and remains in the liquid medium in the bacterial solution adjustment unit 9, or is physiological saline or It is dissolved and suspended in a dedicated test solution, optically measured by the photometer 10, and prepared to be 5 ⁇ 10 5 CFU / mL. Even when the enrichment process is in this way, it automatically shifts to a subculture by liquid culture as soon as the blood culture is positive, so the final result is at least 8-12 hours shorter than the conventional method of generating colonies. Is possible.
- the bacterial solution prepared to 5 ⁇ 10 5 CFU / mL is filled in the culture / measurement device 13 of 13 identification / drug susceptibility analysis units by the inoculation mechanism 14, maintained at a constant temperature in the culture unit 15, and cultured.
- the bacterial solution will not become cloudy. If there is no effect, the microorganisms will grow and become cloudy. This minimum inhibitory concentration is the MIC and is reported to the clinical side.
- the device 13 is withdrawn from the culture unit at regular time intervals by the transport unit 16 and optically photometrically measured at regular intervals. The measured optical signal is taken into the microcomputer 18 via the logarithmic converter, A / D converter 20 and interface 19. That is, the turbidity of bacterial growth can be detected with high sensitivity by the optical system of the detection unit 17, and the rise of the growth can be determined by a predetermined algorithm in the microcomputer 18 to obtain a quick result determination.
- the mechanism of the pre-processing unit in the invention may be used partially if necessary, and even if all are used, an effective result can be obtained.
- the order of unit use is not determined, and any pre-processing may be performed with priority.
- the liquid culture unit may be used before the filtering unit.
- Example 1 in order to collect microorganisms in a culture solution pretreated by filtering or pH adjustment, the concentration of bacteria can be obtained using an ultrafiltration membrane. This method leaves only necessary components like a dialysis tube and discharges unnecessary solvents and moisture. The bacterial suspension may be adjusted after sufficient concentration, and it is also effective to increase the efficiency in combination with centrifugation.
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Abstract
Description
2 血液培養ボトル
3 前処理ユニット
4 分手ノズル
5 フィルタリング機構
6 pH調整機構
7 遠心ユニット
8 集菌機構
9 菌液調整ユニット
10 光度計
11 液体培養ユニット
12 同定・薬剤感受性分析ユニット
13 培養・測定デバイス
14 接種機構
15 培養ユニット
16 搬送ユニット
17 検出ユニット
18 マイクロコンピュータ
19 インターフェイス
20 Log変換器およびA/D変換器
21 プリンタ
22 CRT
23 ハードディスク
24 操作パネル
Claims (18)
- 血液を培養する手段と、
前記血液中に含まれる微生物の増殖を検出する手段と、
前記培養後の培養液を分取する手段と、
前記分取した培養液を前処理する手段と、
前記前処理した試料を薬剤を含有する培養容器もしくは測定容器に充填する手段と、
前記培養容器もしくは測定容器に充填した微生物を含む試料を培養する手段と、
前記培養容器もしくは測定容器中の微生物の増殖を検出する手段と、を備えている微生物分析装置であって、
前記培養後の血液試料を含む培養液を前処理する手段は、血液培養時の影響を除去する機構および血液培養後の微生物濃度(菌数)を一定に調整し、所定濃度の菌液を調製する機構のいずれかもしくは両方を有し、
血液培養検査と同定・薬剤感受性検査を連続して自動的に実施するよう構成したことを特徴とする微生物自動分析装置。 - 請求項1に記載の装置において、血液培養後、分取した血液を含む培養液を前処理する手段は、血液培養時の培養時の培地中に添加された成分を除去するためのフィルターあるいはろ過装置であることを特徴とする微生物自動分析装置。
- 請求項1記載の装置において、血液培養後、血液培養の影響を除去する前処理手段として、さらに血液培養中に変化した培地のpHを調整する機能を有するよう構成したことを特徴とする微生物自動分析装置。
- 請求項1記載の装置において、血液培養後の微生物濃度(菌数)を一定に調整する手段は、培養後の血液培養容器を遠心分離し、微生物を含む沈渣を生理食塩水あるいは専用試液に浮遊させ、菌液調製するよう構成したことを特徴とする微生物自動分析装置。
- 請求項1記載の装置において、血液培養後の微生物濃度(菌数)を一定に調整する手段前は、一定菌数になるよう前記培養後の血液を含む培養液を濃縮する機能を有するよう構成したことを特徴とするた微生物自動分析装置。
- 請求項1記載の装置において、血液培養後の微生物濃度(菌数)を一定に調整する手段は、前記培養後の血液中の微生物を生理食塩水あるいは試液に懸濁させ、一定菌数もしくは一定濃度になるよう光学的手段にて測定し、調整するよう構成したことを特徴とする微生物自動分析装置。
- 請求項6記載の装置において、一定菌数もしくは一定濃度になるよう測定する光学的手段は吸光光度法,散乱光度法,濁度法,蛍光法などを用いた光学計であることを特徴とする微生物自動分析装置。
- 請求項1記載の装置において、さらに、血液培養後の培地中の微生物を新たな液体培地に植え継ぎ、培養する機能を有するよう構成したことを特徴とする微生物自動分析装置。
- 請求項8記載の装置において、血液培養後の培地中の微生物を新たな液体培地に植え継ぎ培養して得られた高濃縮菌液を、自動的に採取し生理食塩水もしくは試液に懸濁させ、所定濃度の菌液を調製する機能を有するよう構成したことを特徴とする微生物自動分析装置。
- 血液を培養するステップと、
前記血液中に含まれる微生物の増殖を検出するステップと、
前記培養後の培養液を分取するステップと、
前記分取した培養液を前処理するステップと、
前記前処理した試料を薬剤を含有する培養容器もしくは測定容器に充填するステップと、
前記培養容器もしくは測定容器に充填した微生物を含む試料を培養するステップと、
前記培養容器もしくは測定容器中の微生物の増殖を検出するステップと、を備えている微生物分析装置であって、
前記培養後の血液試料を前処理するステップは、血液培養時の影響を除去する第1前処理ステップおよび血液培養後の微生物濃度(菌数)を一定に調整し、所定濃度の菌液を調製する第2前処理ステップのいずれか、もしくは両方を有し、血液培養検査と同定・薬剤感受性検査を連続して自動的に実施するよう構成したことを特徴とする微生物自動分析方法。 - 請求項10記載の方法において、血液培養後、血液培養の影響を除去する第1前処理ステップは、血液培養時の培地中に添加された成分を除去するためにフィルタリングするよう構成したことを特徴とする微生物自動分析方法。
- 請求項11記載の方法において、血液培養後、血液培養の影響を除去する第1前処理ステップは、さらに血液培養中に変化した培地のpHを調整する機能を有するよう構成したことを特徴とする微生物自動分析方法。
- 請求項10記載の方法において、血液培養後の微生物濃度(菌数)を一定に調整する第2前処理ステップは、培養後の血液培養容器を遠心分離し、微生物を含む沈渣を生理食塩水あるいは専用試液に浮遊させ、菌液調製するよう構成したことを特徴とする微生物自動分析方法。
- 請求項10記載の方法において、血液培養後の微生物濃度(菌数)を一定に調整する第2前処理ステップは、一定菌数になるよう前記培養後の血液を含む培養液を濃縮する機能を有するよう構成したことを特徴とする微生物自動分析方法。
- 請求項10記載の方法において、血液培養後の微生物濃度(菌数)を一定に調整する第2前処理ステップは、前記培養後の血液中の微生物を生理食塩水あるいは試液に懸濁させ、一定菌数もしくは一定濃度になるよう光学的手段にて測定し、調整するよう構成したことを特徴とする微生物自動分析方法。
- 請求項15記載の方法において、一定菌数もしくは一定濃度になるよう測定する光学的手段は吸光光度法,散乱光度法,濁度法,蛍光法などを用いた光学計であることを特徴とする微生物自動分析方法。
- 請求項10記載の方法において、さらに、血液培養後の培地中の微生物を新たな液体培地に植え継ぎ、培養する機能を有するよう構成したことを特徴とする微生物自動分析方法。
- 請求項17記載の方法において、血液培養後の培地中の微生物を新たな液体培地に植え継ぎ培養して得られた高濃度菌液を、自動的に採取し生理食塩水もしくは試液に懸濁させ、所定濃度の菌液を調製する機能を有するよう構成したことを特徴とする微生物自動分析方法。
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WO2017135524A1 (ko) * | 2016-02-03 | 2017-08-10 | 가톨릭대학교 산학협력단 | 혈액 배양 양성 검체에서 균종 동정 및 항균제 감수성 검사를 위한 전처리 방법 |
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CN114350495B (zh) * | 2022-01-17 | 2024-06-25 | 宁波天康生物科技有限公司 | 在线式非接触型血培养阳性检测装置及血培养仪 |
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JP2017502696A (ja) * | 2013-12-31 | 2017-01-26 | ニョ, ガンNIU, Gang | 全自動式微生物検出用集積システム及びその集積方法 |
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JPWO2016013394A1 (ja) * | 2014-07-22 | 2017-04-27 | 株式会社日立ハイテクノロジーズ | 細胞数濃度調整装置およびそれを用いた自動継代培養システム |
WO2017135524A1 (ko) * | 2016-02-03 | 2017-08-10 | 가톨릭대학교 산학협력단 | 혈액 배양 양성 검체에서 균종 동정 및 항균제 감수성 검사를 위한 전처리 방법 |
Also Published As
Publication number | Publication date |
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US20120021449A1 (en) | 2012-01-26 |
EP2392643B1 (en) | 2016-10-26 |
EP2392643A4 (en) | 2013-09-04 |
US9045726B2 (en) | 2015-06-02 |
JP5600603B2 (ja) | 2014-10-01 |
EP2392643A1 (en) | 2011-12-07 |
JPWO2010087110A1 (ja) | 2012-07-26 |
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