ES2577452T3 - Método de diagnóstico in vitro de una micosis invasiva por espectrometría de masas de tipo MALDI-TOF - Google Patents
Método de diagnóstico in vitro de una micosis invasiva por espectrometría de masas de tipo MALDI-TOF Download PDFInfo
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- ES2577452T3 ES2577452T3 ES13756524.8T ES13756524T ES2577452T3 ES 2577452 T3 ES2577452 T3 ES 2577452T3 ES 13756524 T ES13756524 T ES 13756524T ES 2577452 T3 ES2577452 T3 ES 2577452T3
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- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 title abstract description 5
- 206010052366 systemic mycosis Diseases 0.000 title abstract description 4
- 238000002405 diagnostic procedure Methods 0.000 title abstract 2
- 238000000338 in vitro Methods 0.000 title abstract 2
- 229920001542 oligosaccharide Polymers 0.000 abstract description 7
- 150000002482 oligosaccharides Chemical class 0.000 abstract description 7
- 150000001875 compounds Chemical class 0.000 abstract description 5
- 150000003839 salts Chemical class 0.000 abstract description 5
- 241000124008 Mammalia Species 0.000 abstract description 4
- 230000002538 fungal effect Effects 0.000 abstract description 4
- 238000004949 mass spectrometry Methods 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 150000002632 lipids Chemical class 0.000 abstract description 3
- 230000001717 pathogenic effect Effects 0.000 abstract description 3
- 150000004676 glycans Chemical class 0.000 abstract 4
- 150000002772 monosaccharides Chemical class 0.000 abstract 4
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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- 238000004458 analytical method Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
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- 239000011707 mineral Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000011734 sodium Chemical class 0.000 description 2
- 241000222122 Candida albicans Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
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- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical class C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
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- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
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- WISCONQAEAWCKO-UHFFFAOYSA-N cyclohexa-2,4-diene-1-carboxylic acid Chemical compound OC(=O)C1CC=CC=C1 WISCONQAEAWCKO-UHFFFAOYSA-N 0.000 description 1
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- LUEWUZLMQUOBSB-GFVSVBBRSA-N mannan Chemical class O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-GFVSVBBRSA-N 0.000 description 1
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- 239000011591 potassium Chemical class 0.000 description 1
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- 238000011002 quantification Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5002—Partitioning blood components
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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- G—PHYSICS
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
- G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
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- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
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- G—PHYSICS
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- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
- G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
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- G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
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Abstract
Método de diagnóstico in vitro de una micosis invasiva ocasionada por un microorganismo fúngico patógeno, caracterizado por: - proporcionar una muestra de un líquido biológico que procede de un mamífero, en donde dicho líquido biológico contiene en especial proteínas y/o lípidos y/o sales y/o polisacáridos y/o oligosacáridos y/o monosacáridos capaces de formar complejos con dichas proteínas y/o lípidos y/o sales; - tratar dicha muestra de líquido biológico para extraer dichos polisacáridos y/o oligosacáridos y/o monosacáridos; - determinar por espectrometría de masas de tipo MALDI-TOF la presencia o no, entre dichos polisacáridos, oligosacáridos y/o monosacáridos extraídos, de al menos un compuesto de interés dado que procede de dicho microorganismo fúngico y elegido entre los polisacáridos, los oligosacáridos y los monosacáridos; - deducir que si dicho compuesto de interés dado está presente en dicha muestra, dicho mamífero está afectado por una micosis invasiva.
Description
mamífero, y más en particular de un humano, y que contiene agua, al menos un tipo de proteína y/o al menos un tipo de lípido y/o al menos un tipo de sal mineral, presente en forma de ion, tal como, por ejemplo, sodio, potasio, en donde el ion puede ser metálico. En el sentido de la presente invención, se define una sal como una sal mineral en forma de ion.
Los experimentos descritos a continuación en referencia con una micosis ocasionada por C. albicans se pueden reproducir con mamíferos infectados de modo artificial por uno, dos o varios microorganismos fúngicos patógenos a estudiar. Los experimentos que vienen a continuación pueden entonces servir para determinar el compuesto o los compuestos de interés aptos para servir de marcadores de la micosis invasiva ocasionada por el o los microorganismos fúngicos patógenos a estudiar, para caracterizar dicho o dichos compuestos de interés, y para conocer su relación con la infección.
Primera etapa: tratamiento de las muestras biológicas para la detección de los glucanos. Procedimiento aplicado a los sueros.
- •
- Distribuir 300 µl de las muestras a analizar en microtubos estériles de 1,5 ml.
- •
- Añadir 100 µl de la solución del tratamiento (solución ácida EDTA) en cada tubo.
- •
- Homogeneización vorticial.
- •
- Colocar los microtubos cerrados herméticamente con grapas de sellado durante 6 min a 120 ºC en un bloque calefactor.
- •
- Retirar los tubos y centrifugarlos 10 min a 10.000 g.
- •
- El sobrenadante (150 µl) se transfiere a un microtubo de 1,5 ml estéril.
Los tratamientos posteriores para la detección de los glucanos mediante espectrometría de masas se efectúan sobre este sobrenadante. Este tratamiento libera o disocia los mananos y los galactomananos de los complejos proteicos coagulados en el sedimento; no altera en absoluto la dosis colorimétrica del glucano (kit Fungitell®) para el cual da unos resultados que concuerdan con la misma muestra sin tratar. En cambio, este tratamiento permite eliminar las interferencias relacionadas con el exceso de proteínas, de triglicéridos, de bilirrubina y/o de hemoglobina.
Segunda etapa: tratamiento de los sobrenadantes para purificar y concentrar los oligosacáridos antes de la espectrometría de masas.
Se depositan 100 µl del sobrenadante en un cartucho de extracción de fase sólida (SPE) (referencia SPE-C18: C18 Sep-Pak cartridge (Waters)) (1 volumen) (columna con un relleno hidrófobo) que se ha acondicionado previamente con 5 volúmenes de una solución de acetonitrilo (ANC) y agua (75:25, vol/vol) y que se ha lavado con 10 volúmenes de agua. Después de que penetre en el cartucho el volumen del sobrenadante recogido, el cartucho se enjuaga con dos volúmenes de agua que se recogen. El eluido de la columna de C18 se deposita sobre un cartucho SPE que contiene una mezcla en peso con la misma cantidad de carbón activo comercial y de Célite® (volumen equivalente) acondicionada anteriormente con 5 volúmenes de una solución ANC/H2O (75:25, vol/vol), y luego se enjuaga con 10 volúmenes de agua. Después de que penetre en el cartucho el volumen del sobrenadante, el cartucho se enjuaga con 20 volúmenes de agua. Se desecha el eluido. La columna se enjuaga con dos volúmenes de una solución ANC/H2O (25:75, vol/vol). Se desechan los primeros 100 µl. Se recogen los 300 µl siguientes. Se seca el eluido (ACN/H2O).
Tercera etapa: Análisis de los oligosacáridos por espectrometría de masas.
El espectrómetro de masas utilizado es un espectrómetro de tipo MALDI-TOF Voyager Elite DE-STR (Perspective Biosystems Framingham, MA). El eluido seco se solubiliza en agua. A 1 µl de solución se le añade 1 µl de una solución de ácido dihidrobenzoico (10 mg/ml en ACN/H2O 50:50). Se deposita 1 µl de la solución en una placa de MALDI-TOF (placa de Applied Biosystems, acero inoxidable, 96 depósitos, cercada) y se cristaliza a 50 ºC. El análisis por MALDI-TOF se realiza en modo positivo-reflectrón según los parámetros de adquisición optimizados para la observación de los oligosacáridos neutros que presentan una relación m/z inferior a 1000. Después de la adquisición de los datos, se establece la presencia de los oligoglucanos en la mezcla mediante la observación de los aductos [M+Na]+ generados. Las masas (M) de interés para el diagnóstico se calculan según la fórmula M Hexn = 180 + [162](n–1) + 23. La cuantificación relativa de las especies moleculares se efectúa mediante el cálculo de la relación de las señales M Hexn, bien en función de una señal ubicua endógena, bien en función de una referencia externa introducida en la muestra durante la primera fase de la segunda etapa. Este día, la señal que presenta un interés para el diagnóstico es M Hex2 = 365. Los ejemplos de análisis presentados en el resto del texto se basan en el cálculo de la relación de las señales 365/361.
7
Claims (1)
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Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR1201796 | 2012-06-26 | ||
| FR1201796 | 2012-06-26 | ||
| PCT/FR2013/000158 WO2014001658A1 (fr) | 2012-06-26 | 2013-06-24 | Méthode de diagnostic in vitro d'une infection fongique invasive par spectrométrie de masse maldi-tof |
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| Publication Number | Publication Date |
|---|---|
| ES2577452T3 true ES2577452T3 (es) | 2016-07-15 |
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| Application Number | Title | Priority Date | Filing Date |
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| ES13756524.8T Active ES2577452T3 (es) | 2012-06-26 | 2013-06-24 | Método de diagnóstico in vitro de una micosis invasiva por espectrometría de masas de tipo MALDI-TOF |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US9360470B2 (es) |
| EP (1) | EP2877590B1 (es) |
| JP (1) | JP6254585B2 (es) |
| CN (1) | CN104411833B (es) |
| DK (1) | DK2877590T3 (es) |
| ES (1) | ES2577452T3 (es) |
| HU (1) | HUE028559T2 (es) |
| PL (1) | PL2877590T3 (es) |
| PT (1) | PT2877590E (es) |
| WO (1) | WO2014001658A1 (es) |
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| CN104977349B (zh) * | 2015-03-17 | 2019-04-23 | 北京毅新博创生物科技有限公司 | 全自动多肽提取飞行时间质谱检测仪 |
| CN105866407A (zh) * | 2016-04-22 | 2016-08-17 | 丹娜（天津）生物科技有限公司 | 一种曲霉菌半乳甘露聚糖抗原免疫检测试剂盒及其制备方法与应用 |
| JP2019207106A (ja) * | 2016-08-25 | 2019-12-05 | 日本たばこ産業株式会社 | 配糖体の分析方法 |
| CN114414341A (zh) * | 2022-01-25 | 2022-04-29 | 厦门元谱生物科技有限公司 | 一种血液培养报阳的检测方法 |
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| JPH04237496A (ja) * | 1991-01-17 | 1992-08-25 | Mitsubishi Kasei Corp | 環状イヌロオリゴ糖の製造方法 |
| JP2000041693A (ja) * | 1998-07-27 | 2000-02-15 | Morinaga Milk Ind Co Ltd | ガラクトオリゴ糖の製造方法 |
| FR2818647B1 (fr) * | 2000-12-21 | 2006-09-29 | Pasteur Institut | Glycopeptides immunogenes, criblage, preparation et applications |
| CN101104594A (zh) * | 2002-12-26 | 2008-01-16 | 盐野义制药株式会社 | 用捕捉糖链的分子纯化/浓缩糖链的方法和分析糖链结构的方法 |
| US20060057638A1 (en) * | 2004-04-15 | 2006-03-16 | Massachusetts Institute Of Technology | Methods and products related to the improved analysis of carbohydrates |
| JP4262289B2 (ja) | 2005-03-31 | 2009-05-13 | 財団法人野口研究所 | 生体試料の解析法及び疾患マーカーの検索法 |
| US8246832B2 (en) * | 2005-05-25 | 2012-08-21 | Bio-Rad Laboratories, Inc. | Fluidics device |
| JP2007145361A (ja) * | 2005-11-28 | 2007-06-14 | Toray Ind Inc | 納豆容器 |
| EP2010679A2 (en) | 2006-04-06 | 2009-01-07 | Ibis Biosciences, Inc. | Compositions for the use in identification of fungi |
| PL1920781T3 (pl) * | 2006-11-10 | 2015-06-30 | Glycotope Gmbh | Kompozycje zawierające core-1-dodatnie mikroorganizmy i ich zastosowanie w leczeniu lub profilaktyce nowotworów |
| US20100003699A1 (en) * | 2008-01-18 | 2010-01-07 | Glykos Finland Ltd. | Tissue carbohydrate compositions and analysis thereof |
-
2013
- 2013-06-24 WO PCT/FR2013/000158 patent/WO2014001658A1/fr active Application Filing
- 2013-06-24 ES ES13756524.8T patent/ES2577452T3/es active Active
- 2013-06-24 CN CN201380034007.9A patent/CN104411833B/zh active Active
- 2013-06-24 US US14/410,525 patent/US9360470B2/en active Active
- 2013-06-24 EP EP13756524.8A patent/EP2877590B1/fr active Active
- 2013-06-24 PT PT137565248T patent/PT2877590E/pt unknown
- 2013-06-24 PL PL13756524.8T patent/PL2877590T3/pl unknown
- 2013-06-24 HU HUE13756524A patent/HUE028559T2/en unknown
- 2013-06-24 DK DK13756524.8T patent/DK2877590T3/en active
- 2013-06-24 JP JP2015519273A patent/JP6254585B2/ja active Active
Also Published As
| Publication number | Publication date |
|---|---|
| WO2014001658A1 (fr) | 2014-01-03 |
| DK2877590T3 (en) | 2016-07-04 |
| JP6254585B2 (ja) | 2017-12-27 |
| PL2877590T3 (pl) | 2016-11-30 |
| US9360470B2 (en) | 2016-06-07 |
| PT2877590E (pt) | 2016-06-23 |
| US20150338391A1 (en) | 2015-11-26 |
| CN104411833A (zh) | 2015-03-11 |
| EP2877590B1 (fr) | 2016-05-18 |
| EP2877590A1 (fr) | 2015-06-03 |
| JP2015526711A (ja) | 2015-09-10 |
| WO2014001658A8 (fr) | 2014-06-05 |
| CN104411833B (zh) | 2017-11-14 |
| HUE028559T2 (en) | 2016-12-28 |
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