1、1臨床常見病原體檢測工作 Examination of clinical common pathogens2目的Objective確定感染的發生和性質，及早明確診斷；Determine the occuring and nature of infection, and make diagnosis early;盡早選擇適當的治療方案；Select the appropriate treatment as soon as possible;采取有效的預防措施，防止感染可能廣泛傳播所造成的危害。Take effective prevention measures to prevent infect

2、ion to cause widespread damage. 3基本程序Basic Procedures正確采集和運送標本；Correct collection and transportation of specimens初步診斷：直接鏡檢，免疫學和分子生物學檢測；Initial diagnosis: direct microscopy, immunology and molecular biology examination;確定診斷：病原體的分離、鑒定及藥敏實驗，報告結果；Determined diagnosis: pathogen isolation, identification

3、and sensitivity test, reporting results;合理用藥Appropriate drug use4第一節Section One標本的采集運送、實驗室評價和檢查方法Transportation and collection of specimen, laboratory evaluation and examination methods5一、標本采集和運送One. Collection and transportation of Specimen 6基本原則Basic principles根據病史與臨床表現確定標本采集的時間、部位、種類和數量，盡量采集病變明顯部

4、位標本；According to history and clinical manifestations to determine the collection time, location, type and quantity of specimens, collect specimens at significant parts of lesions; 無菌操作，防止污染；Use aseptic technique to prevent contamination; 采取適宜的方式進行儲存，盡快送檢；Appropriate way to store, as soon as possible

5、 for examination;要視所有標本為傳染品，高度危險性的標本要有明顯標識，急癥或危重患者標本要特別注明。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適應癥：疑為菌血癥、敗血癥或膿毒血癥的病人；Indications: suspected bacteremia, sepsis or sepsis p

6、atients; 一般在抗生素使用前，于發熱初期或高峰期采血；已用過抗菌藥物治療者，在下次用藥前采集；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;以無菌法由肘靜脈穿刺，一般成人采血量每次1020ml，嬰兒和兒童1-5ml ，在床邊接種；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;注明抗生素使用情況，選擇合適類型的培養瓶；Indicate the use of antibiotics, select the appropriate type of culture flask;24小時內在不同部位采血3次可提高陽性率。Collecting blood at different parts 3 times within 24 hours can

8、increase the positive rate.8血液標本的采集方法Collection method of blood samples9（二）尿液(Two) Urine無菌采集中段尿；Sterile collection of mid-portion urine;如考慮厭氧菌感染，采取膀胱穿刺法采集標本，無菌厭氧小瓶運送；If consideration of anaerobic infections, apply bladder puncture to collect specimens, transport them with sterile anaerobic vial;排尿困難

9、者考慮導尿采集標本。Apply catheterization to collect specimens for dysuria patients.10（三）糞便(Three) Stool挑取膿、血或粘液部分于清潔容器中送檢；Pick pus, blood or mucus from stoll into clean containers for examination;排便困難者或嬰兒采用直腸拭子采集，置于有保存液的試管內送檢；Apply rectal swab for infants or difficult defecation, place it with preservation s

10、olution in the test tube for examination; 懷疑霍亂弧菌感染引起的腹瀉，將標本置于堿性蛋白胨水或卡-布（Cary-Blair）運送培養液送檢；Suspected infections caused by Vibrio cholerae, the specimens were placed in alkaline peptone water or card - cloth (Cary-Blair) transport medium for examination;傳染性腹瀉應連續送檢3次。Continuous examination 3 times for

11、 infectious diarrhea.11（四）呼吸道標本(Four) Respiratory specimens類型：鼻咽拭子，痰和經氣管采集的標本；Types: nasopharyngeal swab, sputum, and specimens collected through tracheal上呼吸道存在正常菌群，在采集標本與結果分析時應予考慮。There is normal flora in upper respiratory tract, the specimens collection and results analysis should be considered.12

12、（五）腦脊液與其他無菌體液(Five) Cerebrospinal fluid and other sterile body fluids腦脊液應立即保溫送檢或床邊接種；CSF should be transported immediately with insulation or cultured bedside；胸腔積液、腹腔積液和心包積液等應采集較大量標本送檢，離心后再接種培養。Pleural effusion, ascites and pericardial effusion with a large volume should be collected, centrifugated

13、and then cultured.13（六）眼、耳部標本(Six) Eye, ear specimens拭子采樣Swab samples14（七）泌尿生殖道標本(Seven) Urogenital specimens男性：無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：無菌采集陰道或宮頸分泌物。Female: vaginal or cervical secretions collected aseptically.15（八）創傷、組織和膿腫標本(Eight) Tra

14、uma, organization, and abscess specimens對損傷范圍較大的創傷，從不同部位采集多份標本；On a wider range of trauma injuries, specimens collected from many different parts; 采集部位應首先清除污物、消毒皮膚；Firstly remove dirt, disinfect the skin around collection site;標本較少則需加入無菌生理鹽水以防干燥；Fewer samples must be added to sterile saline to preve

15、nt drying;開放性膿腫及膿性分泌物：用無菌棉纖采取膿液或病灶深部分泌物；Open abscess and purulent secretions: collect deep discharge pus or lesions with sterile cotton fibers; 封閉性膿腫用注射器抽取；Collect specimen in closed abscess with a syringe;懷疑厭氧菌感染，應隔絕空氣采集。Suspected anaerobic infections, collection should be isolated from air.16（九）血清

16、(Nine) Serum用于檢測特異性抗體；For the detection of specific antibodies;血液自然凝固后吸取血清，滅活補體。Draw serum after natural coagulation of blood, then inactivate complement.17二、標本的實驗室質量評估標準Two. Standard of laboratory quality assessment of specimens18檢驗申請單的基本內容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標本接收和拒收準則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、檢查方法Three. Examination methods 22（一）直接顯微鏡檢查(One) Direct microscopic examination不染色標本檢查法：用于觀察病原體的生長、形態與運動等特性；Not stained specimen test method: to obs

20、erve the growth, shape and motion characteristics of pathogens;評價：部分病原體可借此初步診斷。Evaluation: for some pathogens, the initial diagnosis can be taken.23染色標本檢查法：觀察細菌的形態、染色性或觀察宿主細胞內包涵體的特征；Stained specimen test method: to observe bacterial morphology, staining or observe the characteristics of the inclusio

21、n bodies in host cell; 評價：為臨床初步診斷提供依據。Evaluation: available basis for the preliminary clinical diagnosis.24革蘭氏（染色）陽性球菌Gram (staining)-positive cocci革蘭氏（染色）陰性桿菌Gram (staining)-negative bacilli25抗酸染色陽性桿菌（抗酸桿菌）Acid-fast stain-positive bacilli (Acid-fast bacilli)26（二）病原體特異性抗原檢查(Two) Pathogen-specific an

22、tigen test免疫熒光技術Immunofluorescence technique酶聯免疫技術ELISA化學發光技術Chemiluminescence technology乳膠凝集試驗Latex agglutination test對流免疫電泳Counter immuno electrophoresis蛋白質芯片Protein chips27病原體特異性抗原檢測結果評價Evaluation of pathogen-specific antigen test results如果能排除交叉抗原的影響，病原體抗原檢測可明確感染的病原體。If the impact of cross-antige

23、n can be excluded, pathogen antigen test could clear the infection of pathogens.28（三）病原體核酸檢查(Three) Pathogen nucleic acid test聚合酶鏈式反應：擴增病原體微生物特異的DNA或 RNA片段；Polymerase chain reaction (PCR): amplify the specific fragments of DNA or RNA of microbial pathogens;實時熒光定量PCR技術；Real-time PCR; 核酸探針雜交技術：通過已知序列的

24、探針與標本中的病原體的核苷酸雜交，用以了解病原體的有無；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;基因芯片技術。Gene chip or DNA microarray.29病原體核酸檢查結果評價Evaluation of pathogen DNA test results是檢測病原體微生物最靈敏的方

25、法，但具有一定的假陽性與假陰性；is the most sensitive detection method for microbial pathogens, but has some false positive and false negative;陽性只表明存在某種病原體的核酸，是否正被感染應結合臨床具體分析。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）病原體的分離培養與鑒定(Four) Isolation, culture and identification of pathogens1 細菌感染性疾病病原體的分離培養1 Isolation and culture the pathogens of bacterial infections 明確感染病原體；Clear the pathogens;為臨床提供體外抗微生物藥物敏感試驗結果。Provide vitro test results of anti-microbial drug sensitivity for clinical.312 不能人工培養的病原體感染性疾病

27、2 Not cultivated pathogens of infectious disease將標本接種易感動物、雞胚或行細胞培養。Specimens were inoculated into susceptible animals, eggs or cell lines.32（五）血清學實驗(Five) Serological test特異性 IgM可作為感染性疾病的早期診斷指標，且可區分原發與復發感染；Specific IgM can be indicator of early diagnosis for infectious diseases and can distinguish b

28、etween primary and recurrent infections;特異性 IgG，尤其雙份血清的滴度呈4倍或4倍以上升高，考慮現癥感染。For IgG, the titer of double serum higher 4 times or more than 4 times, current pathogen infection should be considered.33第二節 病原體耐藥性檢測Section two Detection of pathogen drug resistance 34抗生素壓力Antibiotic pressure35一、耐藥性及其發生機制On

29、e. Drug resistance and its mechanism 36（一）耐藥病原體(One) Drug resistant pathogens革蘭氏陰性桿菌： -內酰胺酶、超廣譜 -內酰胺酶(ESBL)、類 -內酰胺酶、多重耐藥等；Gram-negative bacillus: - lactamase, extended spectrum - lactamase (ESBL), type - lactamases, multidrug resistance, etc.;革蘭氏陽性球菌：耐甲氧西林葡萄球菌(MRS)、耐青霉素肺炎鏈球菌(PRSP)、耐萬古霉素腸球菌(VRE)、高耐氨基

30、糖苷類抗生素腸球菌。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）耐藥機制(Two) Drug resistance mechanism1 細菌水平和垂直傳播耐藥基因的整合子系統；1 H

31、orizontal and vertical transmission of bacterial drug resistance genes through integrons;2 產生滅活抗生素的水解酶和鈍化酶, 如ESBLs， AmpC -內酰胺酶，碳青霉烯酶，氨基糖苷類鈍化酶；2 proteolytic enzyme and inactive enzyme produced for inactivation of antibiotics, such as ESBLs, AmpC lactamase, carbapenemase, aminoglycoside inactive enzym

32、e; 383 細菌抗生素作用靶位的改變；3 Changes in the bacterial antibiotics target；4 細菌膜外排泵出系統；4 Bacterial membrane efflux pump system；5 細菌生物膜的形成。5 Bacterial biofilm (BF) formation.39二、檢查項目、結果和臨床應用examination projects, results and clinical application40（一）藥物敏感試驗Drug sensitivity test 抗微生物藥物敏感試驗(antimicrobial suscepti

33、bility test, AST)對敏感性不能預測的臨床分離菌株進行藥敏試驗，以指導臨床選擇治療藥物。 AST can not be predicted on the sensitivity of clinical isolates for susceptibility testing to guide clinical treatment of drug41目的Objective臨床分離菌株，如不能對抗生素敏感性進行預測，必須常規進行藥敏試驗。Clinical isolates, if they can not predict the sensitivity to antibiotics s

34、hould be routine susceptibility testing臨床治療效果差而考慮調整抗菌藥物時。Poor clinical treatment to consider adjusting the antimicrobial drugs.了解細菌耐藥的流行病學情況。Understand the epidemiology of bacterial resistance.評價新抗菌藥物的抗菌譜和抗菌活性等。Evaluation of new antibiotics and antibacterial activity of antibacterial spectrum.42方法Me

35、thods1 K-B紙片瓊脂擴散法（Kirby-Bauer disc agar diffusion method）43參照NCCLS標準NCCLS standard reference敏感（susceptible，S）：表示測試菌能被測定藥物常規劑量給藥后在體內達到的血藥濃度所抑制或殺滅。Bacteria can be determined that the test drugs in the body after administration of conventional doses to achieve plasma concentrations inhibit or kill.耐藥（r

36、esistant，R）：表示測試菌不能被測定藥物常規劑量給藥后在體內達到的血藥濃度所抑制或殺滅，治療無效。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) : 該范圍作為敏感與耐藥之間的緩沖區，避免由于微小技術誤差影響實驗結果。The range as a buf

37、fer between sensitive and resistant to avoid the impact of the small technical error results.442 稀釋法（Dilution test） 最低抑菌濃度（MIC）：能夠抑制檢測菌肉眼可見生長的最低藥物濃度稱為測定藥物對檢測菌的最低抑菌濃度。 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試驗法（E test）4 耐藥篩選試驗Drug screening test5 折點敏感試驗Sensitive turning point test45（二）耐藥菌監測試驗Resistant to monitor test由于細菌存在一種或幾種耐藥機制，造成了細菌的多重耐藥性。單一的藥敏試驗已不能完全表示細菌的耐藥性，必須進行一些特殊的耐藥性監測試驗。 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)紙片的抑菌圈直徑10

40、mm，或MIC4g/ml的金黃色葡萄球菌, 1g苯唑青霉素紙片的抑菌圈直徑17mm，或的凝固酶陰性葡萄球菌稱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臨床意義 Clinical significance對所有-內酰胺類藥物均無效,并對氨基糖苷類、大環內酯類、克林霉素和四環素等抗生素多重耐藥。治療首選藥物為萬古霉素。 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、類抗生素高耐藥腸球菌High-resistant enterococci aminoglycosides對氨基糖苷類和青霉素等作用于細胞壁的等抗生素聯用無效。 The role of aminoglycosides and penicillin and other antibiotics in the cell wall combined with null and void.493. 耐青霉素的肺炎鏈球菌Penicillin-resistant Streptococcus pneumoniae定義：1g苯唑青霉素紙片的抑菌圈直徑2g/ml應視為耐青霉素肺炎鏈球菌（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臨床意義Clinical significancePRSP對氨芐西林、 氨芐西林/舒巴坦、 頭孢唑啉的臨床治療療效很差，治療時參考藥敏結果選擇藥物，經驗治療重癥感染時，可用頭孢曲松或頭孢噻肟聯合萬古霉素用藥。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. -內酰胺酶的檢測 -lactamase detection-內酰胺酶：可水解-內酰胺類抗生素。-lactamase

45、s: hydrolyzed -lactam antibiotics.臨床意義：（1）流感嗜血桿菌、淋病奈瑟菌以及卡拉莫拉菌等陽性，表示對青霉素、氨芐西林以及阿莫西林耐藥。（2）葡萄球菌屬以及腸球菌屬等，陽性表示對青霉素、氨基組青霉素、羧基組青霉素以及脲基組青霉素耐藥。 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. 產超廣譜-內酰胺酶(extend spectrum -lactamase, ESBL)的腸桿菌科細菌Producing ESBL Enterobacteriaceae超廣譜-內酰胺酶是由質粒介導的- 內酰胺酶，可水解青霉素

47、類、頭孢菌素和氨曲南，主要在大腸埃希菌和克雷伯菌屬等腸桿菌科細菌中產生。 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臨床意義Clinical significance產ESBL細菌，不論體外藥物敏感試驗結果如何，對

48、青霉素類、頭孢菌素類和氨曲南治療均無效。ESBL producing bacteria, regardless of the outcome of in vitro drug sensitivity tests, to penicillin, cephalosporins and aztreonam treatment were ineffective.546. 耐萬古霉素的腸球菌Vancomycin resistant enterococci定義: 對30g萬古霉素紙片抑菌圈直徑19mm， 或MIC32g/ml應視為耐萬古霉素腸球菌（vancomycin resistant enteroco

49、ccus，VRE）。 Definition: paper 30g vancomycin inhibition zone diameter 19mm, or MIC 32g/ml should be considered as VRE.55臨床意義Clinical significance耐萬古霉素腸球菌目前尚無有效的治療方法，但是對青霉素敏感的VRE可用青霉素和慶大霉素聯合治療，若對青霉素耐藥而不是高水平耐氨基糖苷類可用白霉素和慶大霉素。另外氯霉素、紅霉素、四環素（或多西環素、或米諾環素）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）病原菌耐藥基因的檢測Detection of pathogen resistance genes更早Earlier 確證Confirmed準確Accurate金標準Gold standard57第三節 臨床感染常見病原體檢查Check the common pathogens of clinical infection 58感染性疾病指各種生物性病原體（病原微生物、寄生蟲）寄生人體所引起的傳染性感染疾病和非傳染性

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、流行病學和臨床類型Epidemiology and clinical types60（一）流行病學Epidemiology新傳染病陸續被發現，老傳染病死灰復燃Been discovered new infectious diseases, the re

53、surgence of old diseases多重耐藥株Multi-drug resistant strains新醫療技術的開展The launching of new medical technologies61（二）臨床類型Clinical types臨床常見病原體為：細菌、真菌、病毒、支原體、衣原體、螺旋體、立克次體以及寄生蟲等。Common clinical pathogens: bacteria, fungi, viruses, mycoplasma, chlamydia, spirochetes, rickettsia, and parasites.62 二、檢查項目和臨床應用

54、inspection items and clinical applications 63（一）細菌感染檢查項目的選擇The selection of checking projects on bacterial infection普通細菌：常采用直接鏡檢、分離培養與鑒定、抗原檢測。Common bacteria: direct microscopic examination is often used, Isolation and identification of antigen.病原體抗體檢測。Antibody detection of pathogens 難培養或培養要求高的細菌：綜合

55、運用免疫學或分子生物學檢測，如結核分枝桿菌感染以及幽門螺桿菌感染等。Difficult for high culture or bacterial culture: the integrated use of immunology or molecular biology, such as Mycobacterium tuberculosis and Helicobacter pylori infection.細菌培養是最重要的確診方法。Bacterial culture is the most important diagnostic method.64（二）實驗結果分析和臨床應用Analy

56、sis and clinical application of experimental results顯微鏡檢查或分離培養的陰性結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應；Antigenic cross-reactivity caused by the common核酸檢測的假陽性；False-positive nucleic acid detection血清學的動態檢測。Dynamic detec

57、tion of serological65第六節 醫院感染常見病原體檢測Detection of common pathogens of nosocomial infection 66醫院感染（nosocomial infection）：又稱醫院獲得性感染（hospital acquired infection），指患者在入院時不存在，也不處于潛伏期，而在醫院內發生的感染，包括醫院內獲得的而在出院后發病的感染。廣義的醫院感染對象不僅指住院患者，還包括門診病人、醫院職工、探視人員與陪護人員等。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、流行病學和臨床類型Epidemiology and clinical types68 （一）流行病學Epidemiology69病原學Pathogen細菌是最常見的病原體。 Bacterial is the most common pathogens.變化趨勢：革蘭氏陰性桿菌比例在增加，革蘭氏陽性球菌比例在減少。Trend: increasing the proportion of Gram-negative bacteria, Gram-positive cocci ratio decreased.罕見細菌變成流行株：陰溝腸桿菌、洋蔥假單孢菌，軍團菌等Strains of bacteria become rare: Enterobacter cloacae, Pseu