临床常见病原体检测工作

1、1临床常见病原体检测工作 Examination of clinical common pathogens2目的Objective确定感染的发生和性质，及早明确诊断；Determine the occuring and nature of infection, and make diagnosis early;尽早选择适当的治疗方案；Select the appropriate treatment as soon as possible;采取有效的预防措施，防止感染可能广泛传播所造成的危害。Take effective prevention measures to prevent infect

2、ion to cause widespread damage. 3基本程序Basic Procedures正确采集和运送标本；Correct collection and transportation of specimens初步诊断：直接镜检，免疫学和分子生物学检测；Initial diagnosis: direct microscopy, immunology and molecular biology examination;确定诊断：病原体的分离、鉴定及药敏实验，报告结果；Determined diagnosis: pathogen isolation, identification

3、and sensitivity test, reporting results;合理用药Appropriate drug use4第一节Section One标本的采集运送、实验室评价和检查方法Transportation and collection of specimen, laboratory evaluation and examination methods5一、标本采集和运送One. Collection and transportation of Specimen 6基本原则Basic principles根据病史与临床表现确定标本采集的时间、部位、种类和数量，尽量采集病变明显部

4、位标本；According to history and clinical manifestations to determine the collection time, location, type and quantity of specimens, collect specimens at significant parts of lesions; 无菌操作，防止污染；Use aseptic technique to prevent contamination; 采取适宜的方式进行储存，尽快送检；Appropriate way to store, as soon as possible

5、 for examination;要视所有标本为传染品，高度危险性的标本要有明显标识，急症或危重患者标本要特别注明。To view all the specimens for infectious, high risk specimens should be clearly marked, acute or critically ill patient specimens should be specified.7（一）血液(One) Blood适应症：疑为菌血症、败血症或脓毒血症的病人；Indications: suspected bacteremia, sepsis or sepsis p

6、atients; 一般在抗生素使用前，于发热初期或高峰期采血；已用过抗菌药物治疗者，在下次用药前采集；In general, prior to use of antibiotics, in the early or peak heat, to collect blood; if antibiotic treatment has been used, collect blood before the next administration;以无菌法由肘静脉穿刺，一般成人采血量每次1020ml，婴儿和儿童1-5ml ，在床边接种；Puncture cubital vein by a sterile

7、 method, in general, 10 20ml blood per time for adult, 1-5ml for infants and children, and inoculate at the bedside;注明抗生素使用情况，选择合适类型的培养瓶；Indicate the use of antibiotics, select the appropriate type of culture flask;24小时内在不同部位采血3次可提高阳性率。Collecting blood at different parts 3 times within 24 hours can

8、increase the positive rate.8血液标本的采集方法Collection method of blood samples9（二）尿液(Two) Urine无菌采集中段尿；Sterile collection of mid-portion urine;如考虑厌氧菌感染，采取膀胱穿刺法采集标本，无菌厌氧小瓶运送；If consideration of anaerobic infections, apply bladder puncture to collect specimens, transport them with sterile anaerobic vial;排尿困难

9、者考虑导尿采集标本。Apply catheterization to collect specimens for dysuria patients.10（三）粪便(Three) Stool挑取脓、血或粘液部分于清洁容器中送检；Pick pus, blood or mucus from stoll into clean containers for examination;排便困难者或婴儿采用直肠拭子采集，置于有保存液的试管内送检；Apply rectal swab for infants or difficult defecation, place it with preservation s

10、olution in the test tube for examination; 怀疑霍乱弧菌感染引起的腹泻，将标本置于碱性蛋白胨水或卡-布（Cary-Blair）运送培养液送检；Suspected infections caused by Vibrio cholerae, the specimens were placed in alkaline peptone water or card - cloth (Cary-Blair) transport medium for examination;传染性腹泻应连续送检3次。Continuous examination 3 times for

11、 infectious diarrhea.11（四）呼吸道标本(Four) Respiratory specimens类型：鼻咽拭子，痰和经气管采集的标本；Types: nasopharyngeal swab, sputum, and specimens collected through tracheal上呼吸道存在正常菌群，在采集标本与结果分析时应予考虑。There is normal flora in upper respiratory tract, the specimens collection and results analysis should be considered.12

12、（五）脑脊液与其他无菌体液(Five) Cerebrospinal fluid and other sterile body fluids脑脊液应立即保温送检或床边接种；CSF should be transported immediately with insulation or cultured bedside；胸腔积液、腹腔积液和心包积液等应采集较大量标本送检，离心后再接种培养。Pleural effusion, ascites and pericardial effusion with a large volume should be collected, centrifugated

13、and then cultured.13（六）眼、耳部标本(Six) Eye, ear specimens拭子采样Swab samples14（七）泌尿生殖道标本(Seven) Urogenital specimens男性：无菌采集尿道口分泌物或前列腺液；Male: urethra or prostatic fluid secretions with sterile collection; 女性：无菌采集阴道或宫颈分泌物。Female: vaginal or cervical secretions collected aseptically.15（八）创伤、组织和脓肿标本(Eight) Tra

14、uma, organization, and abscess specimens对损伤范围较大的创伤，从不同部位采集多份标本；On a wider range of trauma injuries, specimens collected from many different parts; 采集部位应首先清除污物、消毒皮肤；Firstly remove dirt, disinfect the skin around collection site;标本较少则需加入无菌生理盐水以防干燥；Fewer samples must be added to sterile saline to preve

15、nt drying;开放性脓肿及脓性分泌物：用无菌棉纤采取脓液或病灶深部分泌物；Open abscess and purulent secretions: collect deep discharge pus or lesions with sterile cotton fibers; 封闭性脓肿用注射器抽取；Collect specimen in closed abscess with a syringe;怀疑厌氧菌感染，应隔绝空气采集。Suspected anaerobic infections, collection should be isolated from air.16（九）血清

16、(Nine) Serum用于检测特异性抗体；For the detection of specific antibodies;血液自然凝固后吸取血清，灭活补体。Draw serum after natural coagulation of blood, then inactivate complement.17二、标本的实验室质量评估标准Two. Standard of laboratory quality assessment of specimens18检验申请单的基本内容The basic content of an application for examinationThe pati

17、ent,s name and hospital number.Age and sex.Collection date and time.Suspected diagnosis.Exact nature and source of the specimen.Immunization history and antimicrobial therapy.Objective.Signed by physician.19标本接收和拒收准则Reception and rejection criteria for specimens1 The information on the label does no

18、t exist or not match the information on the requisition;2 Delay in delivery of the specimens;3 The specimen is leaking; 4 The specimen has been transported at the improper temperature or in improper medium.205 Contaminated samples;6 The quantity of specimen is insufficient for testing;7 Duplicate su

19、bmission of specimens (except blood cultures);8 The transportation of specimens of severe infectious diseases should comply with the relevant rules.21三、检查方法Three. Examination methods 22（一）直接显微镜检查(One) Direct microscopic examination不染色标本检查法：用于观察病原体的生长、形态与运动等特性；Not stained specimen test method: to obs

20、erve the growth, shape and motion characteristics of pathogens;评价：部分病原体可借此初步诊断。Evaluation: for some pathogens, the initial diagnosis can be taken.23染色标本检查法：观察细菌的形态、染色性或观察宿主细胞内包涵体的特征；Stained specimen test method: to observe bacterial morphology, staining or observe the characteristics of the inclusio

21、n bodies in host cell; 评价：为临床初步诊断提供依据。Evaluation: available basis for the preliminary clinical diagnosis.24革兰氏（染色）阳性球菌Gram (staining)-positive cocci革兰氏（染色）阴性杆菌Gram (staining)-negative bacilli25抗酸染色阳性杆菌（抗酸杆菌）Acid-fast stain-positive bacilli (Acid-fast bacilli)26（二）病原体特异性抗原检查(Two) Pathogen-specific an

22、tigen test免疫荧光技术Immunofluorescence technique酶联免疫技术ELISA化学发光技术Chemiluminescence technology乳胶凝集试验Latex agglutination test对流免疫电泳Counter immuno electrophoresis蛋白质芯片Protein chips27病原体特异性抗原检测结果评价Evaluation of pathogen-specific antigen test results如果能排除交叉抗原的影响，病原体抗原检测可明确感染的病原体。If the impact of cross-antige

23、n can be excluded, pathogen antigen test could clear the infection of pathogens.28（三）病原体核酸检查(Three) Pathogen nucleic acid test聚合酶链式反应：扩增病原体微生物特异的DNA或 RNA片段；Polymerase chain reaction (PCR): amplify the specific fragments of DNA or RNA of microbial pathogens;实时荧光定量PCR技术；Real-time PCR; 核酸探针杂交技术：通过已知序列的

24、探针与标本中的病原体的核苷酸杂交，用以了解病原体的有无；Nucleic acid probe hybridization: hybridization between the known sequence of the probe and the nucleotide of pathogens in the samples, to explore whether pathogens exist;基因芯片技术。Gene chip or DNA microarray.29病原体核酸检查结果评价Evaluation of pathogen DNA test results是检测病原体微生物最灵敏的方

25、法，但具有一定的假阳性与假阴性；is the most sensitive detection method for microbial pathogens, but has some false positive and false negative;阳性只表明存在某种病原体的核酸，是否正被感染应结合临床具体分析。is the only show that the existence of a positive pathogen nucleic acid, whether being infected should be combined with clinical specific ana

26、lysis.30（四）病原体的分离培养与鉴定(Four) Isolation, culture and identification of pathogens1 细菌感染性疾病病原体的分离培养1 Isolation and culture the pathogens of bacterial infections 明确感染病原体；Clear the pathogens;为临床提供体外抗微生物药物敏感试验结果。Provide vitro test results of anti-microbial drug sensitivity for clinical.312 不能人工培养的病原体感染性疾病

27、2 Not cultivated pathogens of infectious disease将标本接种易感动物、鸡胚或行细胞培养。Specimens were inoculated into susceptible animals, eggs or cell lines.32（五）血清学实验(Five) Serological test特异性 IgM可作为感染性疾病的早期诊断指标，且可区分原发与复发感染；Specific IgM can be indicator of early diagnosis for infectious diseases and can distinguish b

28、etween primary and recurrent infections;特异性 IgG，尤其双份血清的滴度呈4倍或4倍以上升高，考虑现症感染。For IgG, the titer of double serum higher 4 times or more than 4 times, current pathogen infection should be considered.33第二节 病原体耐药性检测Section two Detection of pathogen drug resistance 34抗生素压力Antibiotic pressure35一、耐药性及其发生机制On

29、e. Drug resistance and its mechanism 36（一）耐药病原体(One) Drug resistant pathogens革兰氏阴性杆菌： -内酰胺酶、超广谱 -内酰胺酶(ESBL)、类 -内酰胺酶、多重耐药等；Gram-negative bacillus: - lactamase, extended spectrum - lactamase (ESBL), type - lactamases, multidrug resistance, etc.;革兰氏阳性球菌：耐甲氧西林葡萄球菌(MRS)、耐青霉素肺炎链球菌(PRSP)、耐万古霉素肠球菌(VRE)、高耐氨基

30、糖苷类抗生素肠球菌。Gram-positive coccus: methicillin-resistant Staphylococcus (MRS), penicillin-resistant Streptococcus pneumoniae (PRSP), vancomycin-resistant enterococcus (VRE), enterococcus with high resistance to aminoglycoside antibiotics.37（二）耐药机制(Two) Drug resistance mechanism1 细菌水平和垂直传播耐药基因的整合子系统；1 H

31、orizontal and vertical transmission of bacterial drug resistance genes through integrons;2 产生灭活抗生素的水解酶和钝化酶, 如ESBLs， AmpC -内酰胺酶，碳青霉烯酶，氨基糖苷类钝化酶；2 proteolytic enzyme and inactive enzyme produced for inactivation of antibiotics, such as ESBLs, AmpC lactamase, carbapenemase, aminoglycoside inactive enzym

32、e; 383 细菌抗生素作用靶位的改变；3 Changes in the bacterial antibiotics target；4 细菌膜外排泵出系统；4 Bacterial membrane efflux pump system；5 细菌生物膜的形成。5 Bacterial biofilm (BF) formation.39二、检查项目、结果和临床应用examination projects, results and clinical application40（一）药物敏感试验Drug sensitivity test 抗微生物药物敏感试验(antimicrobial suscepti

33、bility test, AST)对敏感性不能预测的临床分离菌株进行药敏试验，以指导临床选择治疗药物。 AST can not be predicted on the sensitivity of clinical isolates for susceptibility testing to guide clinical treatment of drug41目的Objective临床分离菌株，如不能对抗生素敏感性进行预测，必须常规进行药敏试验。Clinical isolates, if they can not predict the sensitivity to antibiotics s

34、hould be routine susceptibility testing临床治疗效果差而考虑调整抗菌药物时。Poor clinical treatment to consider adjusting the antimicrobial drugs.了解细菌耐药的流行病学情况。Understand the epidemiology of bacterial resistance.评价新抗菌药物的抗菌谱和抗菌活性等。Evaluation of new antibiotics and antibacterial activity of antibacterial spectrum.42方法Me

35、thods1 K-B纸片琼脂扩散法（Kirby-Bauer disc agar diffusion method）43参照NCCLS标准NCCLS standard reference敏感（susceptible，S）：表示测试菌能被测定药物常规剂量给药后在体内达到的血药浓度所抑制或杀灭。Bacteria can be determined that the test drugs in the body after administration of conventional doses to achieve plasma concentrations inhibit or kill.耐药（r

36、esistant，R）：表示测试菌不能被测定药物常规剂量给药后在体内达到的血药浓度所抑制或杀灭，治疗无效。Test bacteria that can not be measured after administration of conventional dose of drug in the body to the plasma concentrations inhibit or kill, the treatment ineffective.中介 (intermediate，I) : 该范围作为敏感与耐药之间的缓冲区，避免由于微小技术误差影响实验结果。The range as a buf

37、fer between sensitive and resistant to avoid the impact of the small technical error results.442 稀释法（Dilution test） 最低抑菌浓度（MIC）：能够抑制检测菌肉眼可见生长的最低药物浓度称为测定药物对检测菌的最低抑菌浓度。 Minimum inhibitory concentration (MIC): Detection of bacteria able to inhibit visible growth of the minimum drug concentration determ

38、ination of drugs on the detection of bacteria known as the minimum inhibitory concentration.3 E试验法（E test）4 耐药筛选试验Drug screening test5 折点敏感试验Sensitive turning point test45（二）耐药菌监测试验Resistant to monitor test由于细菌存在一种或几种耐药机制，造成了细菌的多重耐药性。单一的药敏试验已不能完全表示细菌的耐药性，必须进行一些特殊的耐药性监测试验。 Because there is one or sev

39、eral bacterial resistance mechanisms, resulting in multiple drug resistance of bacteria. Single susceptibility test can not fully express the drug resistance of bacteria to be resistant to some special monitoring tests.461. 耐甲氧西林的葡萄球菌（methecillin resistance staphylococcus，MRS）1g甲氧西林(苯唑青霉素)纸片的抑菌圈直径10

40、mm，或MIC4g/ml的金黄色葡萄球菌, 1g苯唑青霉素纸片的抑菌圈直径17mm，或的凝固酶阴性葡萄球菌称耐甲氧西林葡萄球菌。1g of methicillin (oxacillin) paper of the inhibition zone diameter 10mm, or MIC 4g/ml Staphylococcus aureus, 1g oxacillin disk inhibition zone diameter 17mm, or MIC 0.5g / ml of coagulase-negative methicillin-resistant Staphylococcus a

41、ureus, said47临床意义 Clinical significance对所有-内酰胺类药物均无效,并对氨基糖苷类、大环内酯类、克林霉素和四环素等抗生素多重耐药。治疗首选药物为万古霉素。 For all -lactam drugs are ineffective, and aminoglycosides, macrolides, clindamycin and tetracycline antibiotics such as multi-drug resistant. The preferred drug for the treatment of vancomycin.482. 氨基糖苷

42、类抗生素高耐药肠球菌High-resistant enterococci aminoglycosides对氨基糖苷类和青霉素等作用于细胞壁的等抗生素联用无效。 The role of aminoglycosides and penicillin and other antibiotics in the cell wall combined with null and void.493. 耐青霉素的肺炎链球菌Penicillin-resistant Streptococcus pneumoniae定义：1g苯唑青霉素纸片的抑菌圈直径2g/ml应视为耐青霉素肺炎链球菌（penicillin res

43、istant streptococcus pneumonia，PRSP）。Definition:1g oxacillin disk inhibition zone diameter 2g/ml should be regarded as penicillin-resistant Streptococcus pneumoniae 50临床意义Clinical significancePRSP对氨苄西林、 氨苄西林/舒巴坦、 头孢唑啉的临床治疗疗效很差，治疗时参考药敏结果选择药物，经验治疗重症感染时，可用头孢曲松或头孢噻肟联合万古霉素用药。PRSP to ampicillin, ampicilli

44、n / sulbactam, cefazolin poor clinical efficacy of the treatment drug of choice when drug susceptibility results for reference, experience the treatment of severe infection, can be combined ceftriaxone or cefotaxime to vancomycin therapy.514. -内酰胺酶的检测 -lactamase detection-内酰胺酶：可水解-内酰胺类抗生素。-lactamase

45、s: hydrolyzed -lactam antibiotics.临床意义：（1）流感嗜血杆菌、淋病奈瑟菌以及卡拉莫拉菌等阳性，表示对青霉素、氨苄西林以及阿莫西林耐药。（2）葡萄球菌属以及肠球菌属等，阳性表示对青霉素、氨基组青霉素、羧基组青霉素以及脲基组青霉素耐药。 Clinical significance: (1), Haemophilus influenzae, Neisseria gonorrhoeae, and karaoke-positive microorganisms in Mora, said to penicillin, ampicillin and amoxicilli

46、n resistance. (2) such as Staphylococcus and Enterococcus, the positive that penicillin, penicillin amino group, carboxyl group of penicillin and penicillin-resistant urea-based group.525. 产超广谱-内酰胺酶(extend spectrum -lactamase, ESBL)的肠杆菌科细菌Producing ESBL Enterobacteriaceae超广谱-内酰胺酶是由质粒介导的- 内酰胺酶，可水解青霉素

47、类、头孢菌素和氨曲南，主要在大肠埃希菌和克雷伯菌属等肠杆菌科细菌中产生。 Extended spectrum -lactamase mediated by plasmid -lactamases, can be hydrolyzed penicillins, cephalosporins and aztreonam, mainly in Escherichia coli and Klebsiella species and other Enterobacteriaceae Generated.53临床意义Clinical significance产ESBL细菌，不论体外药物敏感试验结果如何，对

48、青霉素类、头孢菌素类和氨曲南治疗均无效。ESBL producing bacteria, regardless of the outcome of in vitro drug sensitivity tests, to penicillin, cephalosporins and aztreonam treatment were ineffective.546. 耐万古霉素的肠球菌Vancomycin resistant enterococci定义: 对30g万古霉素纸片抑菌圈直径19mm， 或MIC32g/ml应视为耐万古霉素肠球菌（vancomycin resistant enteroco

49、ccus，VRE）。 Definition: paper 30g vancomycin inhibition zone diameter 19mm, or MIC 32g/ml should be considered as VRE.55临床意义Clinical significance耐万古霉素肠球菌目前尚无有效的治疗方法，但是对青霉素敏感的VRE可用青霉素和庆大霉素联合治疗，若对青霉素耐药而不是高水平耐氨基糖苷类可用白霉素和庆大霉素。另外氯霉素、红霉素、四环素（或多西环素、或米诺环素）及利福平可用于VRE株。Vancomycin resistant enterococci is curre

50、ntly no effective treatment, but the VRE to penicillin-sensitive penicillin and gentamicin combination therapy can be used, if not the high-level resistance to penicillin-resistant available aminoglycosides neomycin and celebrate great white Adriamycin. Addition of chloramphenicol, erythromycin and

51、tetracycline (or doxycycline, or minocycline) and rifampin can be used for VRE strains.56（三）病原菌耐药基因的检测Detection of pathogen resistance genes更早Earlier 确证Confirmed准确Accurate金标准Gold standard57第三节 临床感染常见病原体检查Check the common pathogens of clinical infection 58感染性疾病指各种生物性病原体（病原微生物、寄生虫）寄生人体所引起的传染性感染疾病和非传染性

52、感染疾病。Refers to a variety of infectious diseases, biological pathogens (pathogens, parasites) caused by human infectious parasitic diseases and non-communicable diseases.59 一、流行病学和临床类型Epidemiology and clinical types60（一）流行病学Epidemiology新传染病陆续被发现，老传染病死灰复燃Been discovered new infectious diseases, the re

53、surgence of old diseases多重耐药株Multi-drug resistant strains新医疗技术的开展The launching of new medical technologies61（二）临床类型Clinical types临床常见病原体为：细菌、真菌、病毒、支原体、衣原体、螺旋体、立克次体以及寄生虫等。Common clinical pathogens: bacteria, fungi, viruses, mycoplasma, chlamydia, spirochetes, rickettsia, and parasites.62 二、检查项目和临床应用

54、inspection items and clinical applications 63（一）细菌感染检查项目的选择The selection of checking projects on bacterial infection普通细菌：常采用直接镜检、分离培养与鉴定、抗原检测。Common bacteria: direct microscopic examination is often used, Isolation and identification of antigen.病原体抗体检测。Antibody detection of pathogens 难培养或培养要求高的细菌：综合

55、运用免疫学或分子生物学检测，如结核分枝杆菌感染以及幽门螺杆菌感染等。Difficult for high culture or bacterial culture: the integrated use of immunology or molecular biology, such as Mycobacterium tuberculosis and Helicobacter pylori infection.细菌培养是最重要的确诊方法。Bacterial culture is the most important diagnostic method.64（二）实验结果分析和临床应用Analy

56、sis and clinical application of experimental results显微镜检查或分离培养的阴性结果不能完全排除感染；Microscopy or isolation and culture of negative results can not be completely ruled out infection共同抗原引起的交叉反应；Antigenic cross-reactivity caused by the common核酸检测的假阳性；False-positive nucleic acid detection血清学的动态检测。Dynamic detec

57、tion of serological65第六节 医院感染常见病原体检测Detection of common pathogens of nosocomial infection 66医院感染（nosocomial infection）：又称医院获得性感染（hospital acquired infection），指患者在入院时不存在，也不处于潜伏期，而在医院内发生的感染，包括医院内获得的而在出院后发病的感染。广义的医院感染对象不仅指住院患者，还包括门诊病人、医院职工、探视人员与陪护人员等。Nosocomial infection (nosocomial infection): also kn

58、own as hospital-acquired infection (hospital acquired infection), that does not exist on admission of patients, nor in the incubation period, while in the hospital infections occurred, including the hospital after discharge from hospital acquired disease in Infection. Generalized infection, not only

59、 by means of hospital inpatients, including outpatient, hospital staff, visiting officers and escort personnel.67 一、流行病学和临床类型Epidemiology and clinical types68 （一）流行病学Epidemiology69病原学Pathogen细菌是最常见的病原体。 Bacterial is the most common pathogens.变化趋势：革兰氏阴性杆菌比例在增加，革兰氏阳性球菌比例在减少。Trend: increasing the proportion of Gram-negative bacteria, Gram-positive cocci ratio decreased.罕见细菌变成流行株：阴沟肠杆菌、洋葱假单孢菌，军团菌等Strains of bacteria become rare: Enterobacter cloacae, Pseu