MODS的方法和诊断结核病耐药结核病
1The Warren Alpert Medical School of Brown University, 2Laboratorio de Investigacion de Enfermedades Infecciosas,Universidad Peruana Cayetano Heredia, 3Department of International Health,Johns Hopkins Bloomberg School of Public Health, 4Wellcome Trust Centre for Clinical Tropical Medicine,Imperial College London
Summary
显微镜观察药物敏感性（MODS）的检测方法是一种低成本，低技术含量的高性能的检测结核病（TB）和耐多药结核病（MDRTB）工具。本视频介绍MODS的液体培养基中培养方法。
Abstract
活动性肺结核患者（TB）的感染每年10-15其他人，使诊断活动性结核病，既治愈病人，并防止新的感染至关重要。此外，耐药结核病（MDRTB）的出现意味着停止耐药菌株的传播耐药性的检测是必要的。显微镜观察药物敏感性（MODS）的检测方法是一种低成本，低技术含量的高性能的检测结核病和MDRTB工具。结核分枝杆菌（MTB）的增长速度比2固体培养基上，在液体介质）微观山地车的增长可以被检测到，早在液体介质等待宏观固体培养基上菌落外观，并MODS的检测是基于三个原则：1）增长是MTB的特点，使得它从非典型分枝杆菌或真菌或细菌污染3）杰出的药物异烟肼和利福平可分为MODS的检测纳入到允许MDRTB同时直接检测，这样就不必为亚文化执行间接的药敏试验。痰涂片的灵敏度低，长期拖延，直到诊断与固体培养基培养，与现有的液体培养基中培养的方法，成本过高，而且需要做间接药敏试验检测MDRTB亚文化阻碍了竞争的目前诊断。的非专有MODS的方法相反，一个结核病和MDRTB高灵敏度，是一个较快的文化方法，同时药物易感性测试为MDRTB，只是根据美元的结核病测试3资源有限的环境和MDRTB。
Protocol
准备库存解决方案磷酸盐缓冲库存 950毫升磷酸二氢钾解决方案（磷酸钾溶于千毫升蒸馏水二氢9.07克），搅拌混合950毫升钠磷酸氢二钠溶液（溶于千毫升蒸馏水磷酸钠二元9.47克）;保持每一个解决方案，回到50毫升调整如有必要的pH值调节pH值至6.8 ± 0.2：添加钠磷酸氢二钠溶液，以提高pH值;添加磷酸二氢钾溶液，以较低的pH值高压灭菌器在121-124 ° C下15分钟消毒</…
Discussion
MODS的实验是针对资源有限的环境。这是第一次，MODS的带来的快速液体培养法检测肺结核的能力和资源有限的环境测试3元多药耐药结核病。 MODS是一个非专有的，迭代的方法，和MODS的社会总是在其他实验室已设法使改善。
是一个经常性的关注，是因为液体可以溢出或雾化的液体介质中的结核病文化的生物安全。我们相信，MODS的检测是比任何实验，涉及的间接药敏试验BIOSAFE间接药敏试验，因为涉及操纵分?…
Acknowledgements
我们想确认为结核病的增长时间推移视频片段菲茨沃特肖恩和卡门Giannina月神科伦坡。马蒂罗珀我们感谢她彻底和极好的反馈，在编辑和共同创作的用户指南，从目前的协议大多是采取逐字。制作这个视频是由美国国立卫生研究院/ Fogarty国际中心的资助 http://www.fic.nih.gov/大卫AJ摩尔贡献威康信托在热带医学和读者临床研究员在英国伦敦帝国学院的传染病（奖学金获奖数量078067/Z/05）。马克F.布雷迪贡献作为美国国立卫生研究院/福格蒂国际中心研究员。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Refrigerator/ freezer | Equipment | to store pre-prepared broth and antibiotic stocks | ||
| Vortex | Equipment | to aid sputum decontamination | ||
| Centrifuge | Equipment | for sputum concentration; capable of reaching 3000 g; does not need to be refrigerated, but MUST be biosafe (buckets can be sealed) | ||
| Incubator (37 degree C) | Equipment | for culture; need not be CO2 enriched | ||
| Inverted light microscope | Microscope | to read MODS plates | ||
| Autoclave | Equipment | to sterilize media, PBS and used plates | ||
| Balance | Equipment | to weigh isoniazid, rifampicin and NALC | ||
| Middlebrook 7H9 broth (Difco) | Reagent | Fisher | DF0713-17-9 | 500gr/bottle; culture media base |
| Casitone (pancreatic digest casein) | Reagent | Fisher | DF0259‐17‐9 | 500gr/bottle; culture media base |
| Glycerol (glycerin) lyophilized | Reagent | Sigma | G‐33‐500 | 500ml/bottle; culture media base |
| PANTA (Antibiotic mixture lyophilized BD) | Reagent | Fisher | B4345114 | 6 bottles/pack; antibiotic media supplement |
| OADC (Middlebrook OADC enrichment BD) | Reagent | Fisher | B11886 | 10 x 20ml/pack; nutritional media supplement |
| Dimethyl sulphoxide (Hibri-Max) | Reagent | Sigma | D-2650 | 100ml/bottle; to prepare rifampicin stock |
| Antibiotic stocks: isoniazid | Reagent | Sigma | I-3377 | 50gr/bottle; direct susceptibility testing |
| Antibiotic stocks: rifampicin | Reagent | Sigma 99 | R-3501 | 1gr/bottle; direct susceptibility testing |
| Sodium hydroxide (pellets) | Reagent | Sigma | 221465 | 500gr/bottle; sputum decontamination |
| Sodium citrate (trisodium salt dihydrate) | Reagent | Sigma | S-4641 | 500gr/bottle; sputum decontamination |
| N-acetyl-L-cysteine | Reagent | Sigma | A-7250 | 50gr/bottle; sputum decontamination |
| Potassium Phosphate Monobasic crystal. KH2PO4 | Reagent | Sigma | P0662 | 500gr/bottle; sputum decontamination |
| Sodium Phosphate Dibasic, anhydrous. Na2HPO4 | Reagent | Sigma | S0876 | 500gr/bottle; sputum decontamination |
| Sodium hypochlorite | Reagent | household bleach | to discard contaminated waste | |
| 15ml centrifuge tubes (polypropylene 15ml Falcon 35‐2096) | Consumable | Fisher | 14‐959‐49B | 500ea/case; for sputum decontamination and concentration |
| 24 well plates (Plates Tissue 24 wells BD Falcon 35‐3047) | Consumable | Fisher | 08-772-1 | 50 plates/case; for culture and reading |
| Sealable polythene bags 6 X 6 “ (ziplock) | Consumable | for biosecurity to contain 24 well plate | ||
| Glass tubes with lid (16 x 100mm and 18 x 145mm) | Consumable | VWR | 47729-583 | 500 tubes/case; to store aliquots of prepared broth |
| Screw cap microcentrifuge tubes (1.5ml) | Consumable | Fisher | 05‐669‐22 | 1000ea/case; to store aliquots of antibiotic stocks |
| 0.22μm filters (aqueous solvents) Syringe filter Millex blue | Consumable | Fisher | SLGL 025 OS | 50 units/case; to filter antibiotic stocks |
| 0.22μm filters (organic solvents) Syringe filter Millex yellow | Consumable | Fisher | SLGV 033 RS | 50 units/case; to filter antibiotic stocks |
| Disposable Pasteur pipettes borosilicate glass 9″ | Consumable | Fisher | 13‐678‐20C | 720ea/case; to mix PANTA with media mix |
| Aerosol barrier tips 1000‐1300μl | Consumable | Fisher | 02‐707‐51 | 1000ea/pk; to dispense media into plate |
| USA Scientific Tips One 1‐200μl yellow tips | Consumable | Fisher | 1111‐0006 | 1000 tips/bag; to dilute antibiotic stocks |
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Tags
MODS MethodDiagnosisTuberculosisMultidrug Resistant TuberculosisActive Pulmonary TuberculosisDrug ResistanceMicroscopic-observation Drug-susceptibility AssayLiquid MediaSolid MediaMycobacterium TuberculosisAtypical MycobacteriaFungal ContaminationBacterial ContaminationIsoniazidRifampicinDirect DetectionSubcultureSensitivitySputum SmearCulture Methods
Citar este artigo
Brady, M. F., Coronel, J., Gilman, R. H., Moore, D. A. The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis. J. Vis. Exp. (18), e845, doi:10.3791/845 (2008).