臨床微生物學(xué)實(shí)驗(yàn)室建設(shè)基本要求專家共識(shí)二
發(fā)布時(shí)間：
2022-12-27
作者：
限度檢查成品培養(yǎng)基
臨床微生物學(xué)實(shí)驗(yàn)室建設(shè)基本要求專家共識(shí)二
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非培養(yǎng)診斷技術(shù)：
根據(jù)臨床需求可使用PCR、基因測(cè)序、芯片、質(zhì)譜等技術(shù)進(jìn)行病原菌鑒定/分型/耐藥性檢測(cè)和同源性分析?？砷_(kāi)展病原體血清免疫學(xué)檢測(cè)、降鈣素原、真菌－(1，3)－β－D－葡聚糖檢測(cè)、真菌半乳甘露聚糖檢測(cè)、結(jié)核T細(xì)胞斑點(diǎn)試驗(yàn)等項(xiàng)目。
質(zhì)量和服務(wù)
1
檢驗(yàn)前程序
1
檢驗(yàn)申請(qǐng)單的設(shè)計(jì)和應(yīng)用：
由于微生物學(xué)檢驗(yàn)的特殊性，所以檢驗(yàn)申請(qǐng)單除常規(guī)信息外還應(yīng)包括：標(biāo)本采集方法(如中段尿、導(dǎo)尿、膀胱穿刺尿等)、采集部位、采集時(shí)間、采集前是否已使用抗菌藥物、臨床擬診感染類型和(或)可疑目標(biāo)菌等信息。
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標(biāo)本采集的指導(dǎo)和培訓(xùn)：
通過(guò)編寫(xiě)《微生物標(biāo)本采集手冊(cè)》(供醫(yī)護(hù)人員使用)，《痰、尿、便標(biāo)本采集和送檢須知》(供患者使用)，配合多媒體及視頻宣傳等多種形式進(jìn)行標(biāo)本采集方法指導(dǎo)和培訓(xùn)，提高有價(jià)值的標(biāo)本(如血液和無(wú)菌體液)以及合格標(biāo)本的送檢率，對(duì)不合格標(biāo)本應(yīng)及時(shí)反饋，并根據(jù)需要進(jìn)行溝通和再培訓(xùn)。
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標(biāo)本運(yùn)送：
(1)所有標(biāo)本均應(yīng)視為有潛在生物危險(xiǎn)，運(yùn)送過(guò)程中應(yīng)防止溢出；(2)應(yīng)根據(jù)標(biāo)本中的疑似病原菌選擇適宜的運(yùn)送培養(yǎng)基和保溫措施；(3)所有標(biāo)本均應(yīng)盡快運(yùn)送(在2 h內(nèi))，特殊標(biāo)本(如厭氧培養(yǎng)標(biāo)本、腦脊液標(biāo)本)應(yīng)立即運(yùn)送。
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標(biāo)本驗(yàn)收：
除常規(guī)的驗(yàn)收程序以外，應(yīng)重點(diǎn)關(guān)注：(1)送檢標(biāo)本的容器是否為無(wú)菌、封閉、無(wú)泄漏；(2)標(biāo)本類型是否與申請(qǐng)項(xiàng)目相符合(如痰液標(biāo)本申請(qǐng)項(xiàng)目為厭氧培養(yǎng)，則為不合格)；(3)肉眼觀察標(biāo)本性質(zhì)是否合格(如送檢的拭子已干燥，送檢的痰標(biāo)本為唾液等為不合格)；(4)標(biāo)本送檢時(shí)間是否已超過(guò)允許范圍。對(duì)不合格的標(biāo)本應(yīng)及時(shí)進(jìn)行登記和反饋。
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檢驗(yàn)程序
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染色鏡檢：
(1)能夠根據(jù)檢驗(yàn)申請(qǐng)、標(biāo)本類型及可疑的病原菌種類選擇正確的染色方法(如懷疑隱球菌性腦膜炎，選擇墨汁染色)。(2)對(duì)痰標(biāo)本應(yīng)進(jìn)行涂片革蘭染色，判斷標(biāo)本質(zhì)量，觀察細(xì)菌分布。(3)至少每周用已知的陽(yáng)性和陰性質(zhì)控菌株進(jìn)行1次質(zhì)控(若檢測(cè)頻率小于每周1次，則在實(shí)驗(yàn)當(dāng)日進(jìn)行質(zhì)控)。
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細(xì)菌分離培養(yǎng)：
(1)能夠根據(jù)檢驗(yàn)申請(qǐng)信息、標(biāo)本類型及可疑的病原菌選擇正確的培養(yǎng)基和培養(yǎng)條件(如培養(yǎng)溫度、氣體環(huán)境和培養(yǎng)時(shí)間)。(2)每一批培養(yǎng)基(包括自制或購(gòu)買)均應(yīng)進(jìn)行質(zhì)量和性能驗(yàn)證(無(wú)菌試驗(yàn)、生長(zhǎng)試驗(yàn)、必要時(shí)做生長(zhǎng)抑制試驗(yàn)和生化反應(yīng))。記錄培養(yǎng)基制備過(guò)程(僅對(duì)自制培養(yǎng)基)、生產(chǎn)日期、有效期、外觀和性能驗(yàn)證結(jié)果。應(yīng)在有效期內(nèi)使用。(3)能正確判別各類標(biāo)本在培養(yǎng)基上生長(zhǎng)的有意義的結(jié)果。(4)需要定量培養(yǎng)的標(biāo)本(如尿液、肺泡灌洗液)應(yīng)按要求接種，并進(jìn)行菌落計(jì)數(shù)。
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細(xì)菌鑒定：
(1)能根據(jù)細(xì)菌菌落形態(tài)和涂片染色結(jié)果選擇適當(dāng)?shù)蔫b定試驗(yàn)項(xiàng)目，并采用適宜的方法將細(xì)菌鑒定到種。(2)對(duì)細(xì)菌鑒定所需要使用的血漿凝固酶、觸酶、氧化酶試驗(yàn)及診斷性抗血清等至少應(yīng)在每個(gè)實(shí)驗(yàn)當(dāng)日做陽(yáng)性和陰性質(zhì)控。(3)對(duì)細(xì)菌鑒定所需的其他生化反應(yīng)試劑至少應(yīng)在每一批次產(chǎn)品使用前做陰性和陽(yáng)性質(zhì)控，并在有效期內(nèi)使用。
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藥敏試驗(yàn)：
(1)能根據(jù)本單位條件及所檢測(cè)的病原菌種類選擇紙片擴(kuò)散法、稀釋法、濃度梯度擴(kuò)散法(E試驗(yàn))或自動(dòng)化儀器法進(jìn)行抗菌藥物敏感性試驗(yàn)。(2)試驗(yàn)過(guò)程應(yīng)遵循標(biāo)準(zhǔn)化操作方法或制造商建議進(jìn)行操作。(3)藥物敏感性試驗(yàn)結(jié)果解釋至少應(yīng)遵循上一年度CLSI藥敏試驗(yàn)的判斷標(biāo)準(zhǔn)。(4)使用自動(dòng)化儀器進(jìn)行藥敏試驗(yàn)的實(shí)驗(yàn)室應(yīng)按制造商的要求進(jìn)行質(zhì)控。(5)應(yīng)定期(至少每1～2年1次)使用最新的CLSI文件標(biāo)準(zhǔn)對(duì)儀器的藥敏試驗(yàn)判斷折點(diǎn)進(jìn)行評(píng)估，僅報(bào)告符合文件標(biāo)準(zhǔn)的抗菌藥物藥敏試驗(yàn)結(jié)果，對(duì)評(píng)估不符合要求(如藥物稀釋度不包括藥敏判斷折點(diǎn)、折點(diǎn)設(shè)置錯(cuò)誤、檢測(cè)結(jié)果偏離)的儀器應(yīng)暫停使用，并與儀器供應(yīng)商溝通。(6)使用紙片擴(kuò)散法進(jìn)行藥敏試驗(yàn)的實(shí)驗(yàn)室應(yīng)按要求對(duì)每一批號(hào)的藥敏試驗(yàn)紙片進(jìn)行質(zhì)控，如果質(zhì)控在控，則改為每周質(zhì)控1次(若檢測(cè)頻率小于每周1次，則應(yīng)在每個(gè)檢測(cè)日進(jìn)行質(zhì)控)。
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檢驗(yàn)后程序
能夠?qū)Ω黝悩?biāo)本染色鏡檢和細(xì)菌培養(yǎng)鑒定結(jié)果進(jìn)行及時(shí)準(zhǔn)確的報(bào)告，并能進(jìn)行結(jié)果解釋，以及與臨床進(jìn)行良好的溝通。有檢驗(yàn)醫(yī)師的微生物室，可發(fā)出"臨床微生物檢驗(yàn)診斷報(bào)告" 。
對(duì)各類標(biāo)本培養(yǎng)陰性結(jié)果應(yīng)進(jìn)行規(guī)范報(bào)告：如血液增菌培養(yǎng)報(bào)告"培養(yǎng)5 d無(wú)菌生長(zhǎng)"；膿液、引流液、腦脊液、穿刺液等培養(yǎng)報(bào)告"培養(yǎng)××小時(shí)無(wú)菌生長(zhǎng)"；咽拭子、痰液等報(bào)告"正常菌群生長(zhǎng)"或"未分離出致病菌"；糞便或肛拭子等報(bào)告"未培養(yǎng)出志賀菌、沙門(mén)菌"或"未培養(yǎng)出霍亂弧菌"等。
血液、腦脊液、骨髓等無(wú)菌體液標(biāo)本檢出細(xì)菌(鏡檢或培養(yǎng))應(yīng)分級(jí)報(bào)告，并按危急值報(bào)告和登記。
當(dāng)鑒定出高致病性病原微生物(如布魯菌、弗朗西斯菌)時(shí)，應(yīng)按相關(guān)法規(guī)要求進(jìn)行上報(bào)和處理。
短時(shí)間(5～7 d)內(nèi)在同一科室分離出3株或以上同種病原菌，或某種耐藥菌分離率異常增高時(shí)應(yīng)報(bào)告醫(yī)院感染管理部門(mén)。
對(duì)藥敏試驗(yàn)結(jié)果應(yīng)按規(guī)定進(jìn)行審核和報(bào)告，應(yīng)特別關(guān)注天然耐藥、罕見(jiàn)耐藥菌株和特殊部位分離的病原菌藥敏試驗(yàn)結(jié)果的審核和報(bào)告。
對(duì)醫(yī)院感染管理規(guī)定監(jiān)測(cè)的多重耐藥菌要進(jìn)行報(bào)告和預(yù)警。
應(yīng)定期(至少每年1～2次)對(duì)抗菌藥物敏感性試驗(yàn)結(jié)果進(jìn)行統(tǒng)計(jì)分析，并向醫(yī)院感染部門(mén)和臨床醫(yī)師通報(bào)。
應(yīng)參加全國(guó)或地區(qū)性的耐藥監(jiān)測(cè)。
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儀器設(shè)備質(zhì)量控制和性能驗(yàn)證
對(duì)冰箱、培養(yǎng)箱和水浴箱至少每日觀察并記錄1次溫度；CO2培養(yǎng)箱應(yīng)每日記錄CO2濃度；紫外燈應(yīng)每日記錄消毒時(shí)間，應(yīng)定期(每6個(gè)月)監(jiān)測(cè)紫外線強(qiáng)度并監(jiān)測(cè)紫外燈消毒的效果。
壓力滅菌器應(yīng)每次使用化學(xué)指示劑，每周使用生物指示劑監(jiān)測(cè)滅菌效果。
細(xì)菌濁度儀至少每6個(gè)月應(yīng)進(jìn)行檢定或校準(zhǔn)1次。
生物安全柜的高效過(guò)濾器、氣流和負(fù)壓等參數(shù)，壓力滅菌器的壓力表以及卡尺、溫度計(jì)、濕度計(jì)、移液器的準(zhǔn)確性應(yīng)定期驗(yàn)證、檢定或校準(zhǔn)。
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能力驗(yàn)證
按要求參加相應(yīng)的能力驗(yàn)證/室間質(zhì)評(píng)。
應(yīng)對(duì)"不滿意"和"不合格"的能力驗(yàn)證/室間質(zhì)評(píng)結(jié)果進(jìn)行分析并采取糾正和預(yù)防措施。
對(duì)沒(méi)有開(kāi)展能力驗(yàn)證/室間質(zhì)評(píng)的項(xiàng)目，應(yīng)至少每6個(gè)月與其他實(shí)驗(yàn)室(或其他試驗(yàn)方法)進(jìn)行1次性能比對(duì)評(píng)估試驗(yàn)。
生物安全
臨床微生物室屬于二級(jí)生物安全實(shí)驗(yàn)室，應(yīng)按照相關(guān)法規(guī)要求進(jìn)行實(shí)驗(yàn)室設(shè)計(jì)和管理。
臨床微生物室應(yīng)定期(至少每年一次)進(jìn)行生物安全風(fēng)險(xiǎn)評(píng)估。
應(yīng)根據(jù)風(fēng)險(xiǎn)評(píng)估結(jié)果配備必要的生物安全防護(hù)設(shè)備，如生物安全柜、高壓消毒滅菌器、紫外燈、應(yīng)急燈、洗眼和噴淋設(shè)施。根據(jù)所配備的生物防護(hù)設(shè)備設(shè)施和個(gè)體防護(hù)能力明確規(guī)定本實(shí)驗(yàn)室所能開(kāi)展的檢驗(yàn)項(xiàng)目。
應(yīng)根據(jù)風(fēng)險(xiǎn)評(píng)估結(jié)果對(duì)檢驗(yàn)人員進(jìn)行生物安全防護(hù)教育，并配備個(gè)人防護(hù)裝備和用品(如手套、口罩、帽子、實(shí)驗(yàn)用鞋、防護(hù)服、防護(hù)眼罩等)。
二級(jí)醫(yī)院不建議保存菌種(質(zhì)控菌株和需要進(jìn)一步確認(rèn)的臨床菌株除外)，三級(jí)醫(yī)院可以保存非高致病性病原微生物菌種，但應(yīng)指定專人負(fù)責(zé)，應(yīng)有存儲(chǔ)、使用、轉(zhuǎn)運(yùn)、銷毀記錄，謹(jǐn)防濫用、誤用。實(shí)驗(yàn)中如遇到疑似高致病性菌(按照《人間傳染的病原微生物名錄》)，應(yīng)立即向上級(jí)匯報(bào)，不要再進(jìn)行后續(xù)的試驗(yàn)和標(biāo)本處理。
對(duì)超過(guò)規(guī)定保存時(shí)間的標(biāo)本和培養(yǎng)物應(yīng)高壓滅菌后再交保潔人員進(jìn)行處理，并進(jìn)行交接記錄。
'針對(duì)在試驗(yàn)過(guò)程中可能產(chǎn)生的生物安全意外事故(如針刺傷、皮膚、黏膜或環(huán)境污染)應(yīng)有相應(yīng)的應(yīng)急預(yù)案和事故記錄。
本文來(lái)源：節(jié)選自《中華檢驗(yàn)醫(yī)學(xué)雜志》
The establishment of clinical microbiology laboratory requires the consensus of experts
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Non-culture diagnosis technology:
According to the clinical requirements, PCR, gene sequencing, microarray, mass spectrometry and other techniques can be used for pathogen identification/typing/drug resistance detection and homology analysis. Projects such as pathogen serum immunology detection, calcitonin progenitor, fungal -(1,3)- vacuum-d-glucan detection, fungal galactomannan detection, and tuberculosis t-cell spot test can be carried out.
Quality and service
1
Pre-test procedure
1
Design and application of inspection application form:
Due to the particularity of microbiology test, so the inspection application form in addition to the regular information should include: the specimen collection methods (such as urine, urethral catheterization, bladder puncture urine, etc.) in middle and gathering place, have before sampling time, use of antibacterial drugs, infection of clinical examination type and (or) suspicious information such as target bacteria.
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Sample collection instruction and training:
By writing "manual of microbiological specimen collection" (for the use of health care workers), the sputum, urine, stool specimen collection and inspection guidelines "(for the use of patients), with a variety of forms such as multimedia and video propaganda specimen collection method guidance and training, improve the valuable specimens (such as blood and aseptic humoral) and specimens of qualified sampling rate, the unqualified specimens should be timely feedback, and according to the need to communicate and retraining.
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Specimen transport:
(1) all specimens shall be deemed to have potential biological hazards and overflow shall be prevented during transportation; (2) appropriate transport media and heat preservation measures should be selected according to the suspected pathogenic bacteria in the specimen; (3) all specimens shall be sent as soon as possible (within 2 h), and special specimens (such as anaerobic culture specimens and cerebrospinal fluid specimens) shall be sent immediately.
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Specimen acceptance:
In addition to the routine acceptance procedures, attention should be paid to: (2) whether the specimen type is consistent with the application project (if the sputum specimen application project is anaerobic culture, it is unqualified); (3) visually observe whether the specimen is qualified (if the swab is dry and the sputum specimen is saliva, etc.); (4) whether the specimen delivery time has exceeded the allowed range. Unqualified specimens should be registered and feedback timely.
2
Inspection procedures
1
Stain microscopy:
(1) the correct staining method can be selected according to the test application, specimen type and suspect pathogen species (for example, if cryptococcal meningitis is suspected, ink staining is selected). (2) sputum samples should be stained with smear gram to determine the sample quality and observe the distribution of bacteria. (3) conduct quality control with known positive and negative quality control strains at least once a week (if the test frequency is less than once a week, conduct quality control on the test day).
2
Isolation and culture of bacteria:
(1) the right medium and conditions (such as incubation temperature, gas environment and incubation time) can be selected according to test application information, specimen types and suspected pathogenic bacteria. (2) quality and performance verification (aseptic test, growth test, growth inhibition test and biochemical reaction if necessary) shall be conducted for each batch of culture medium (including self-made or purchased). Record the results of the preparation process (for the homemade medium only), production date, expiry date, appearance and performance verification. It should be used within the validity period. (3) meaningful results that can accurately identify the growth of various specimens in the medium. (4) specimens requiring quantitative culture (such as urine and alveolar lavage fluid) shall be inoculated as required and colonies shall be counted.
3
Bacterial identification:
(1) appropriate identification test items can be selected according to the bacterial colony morphology and smear staining results, and appropriate methods can be adopted to identify bacteria into species. (2) the plasma coagulase, thixoenzyme, oxidase test and diagnostic antiserum test required for the identification of bacteria shall be subject to positive and negative quality control at least on each experimental day. (3) other biochemical reagents needed for the identification of bacteria shall be used for negative and positive quality control at least before the use of each batch of products and within the effective period.
4
Drug sensitivity test:
Can (1) according to its condition and detection of pathogenic bacteria species selection by disc diffusion method, dilution method, the concentration gradient diffusion method (E) or automation instrument method for antibacterial drug sensitivity test. (2) the test process shall follow the standardized operation method or the manufacturer's Suggestions. (3) the interpretation of drug sensitivity test results should follow the judgment criteria of CLSI drug sensitivity test at least last year. (4) laboratories using automatic instruments for drug sensitivity testing shall conduct quality control according to the requirements of the manufacturer. (5) should be regularly (at least once every 1 ~ 2 years) using the latest CLSI file standard of instrument to evaluate the drug sensitive test of judgment fold point, only report file standard antimicrobial susceptibility test results, the assessment does not conform to the requirements (such as drug dilution degrees, not including susceptibility to judge fold point, fold point setting error and deviation of test result) of the instruments used shall be suspended, and communicate with equipment suppliers. (6) using disc diffusion method for drug sensitive test laboratory shall according to the requirements for each batch number of drug sensitive test pieces for quality control, and if the quality control, or quality control 1 times a week (if the testing frequency is less than 1 times a week, should be in each detection, quality control).
3
Post-test procedure
It can timely and accurately report the results of staining microscopy and bacterial culture identification of various specimens, interpret the results, and communicate well with the clinic. A microbiological laboratory with an inspector may issue a "clinical microbiological diagnostic report".
The negative results of the culture of various specimens should be reported in a standardized way: for example, the report of blood enrichment culture "sterile growth of culture for 5 days"; Culture reports of abscess, drainage fluid, cerebrospinal fluid, puncture fluid, etc. Pharyngeal swab, sputum and other reports of "normal flora growth" or "unisolated pathogenic bacteria"; Reports of "no shigella, salmonella or vibrio cholerae" in feces or anal swabs.
Bacteria (microscopic examination or culture) detected in blood, cerebrospinal fluid, bone marrow and other sterile humoral specimens shall be classified and reported and registered according to the critical value.
When highly pathogenic pathogenic pathogenic microorganisms (such as brucella and Francis bacillus) are identified, they should be reported and treated in accordance with relevant regulations.
In a short period of time (5 ~ 7 d), 3 strains or more of the same pathogenic bacteria were isolated from the same department, or an abnormal increase in the separation rate of certain drug-resistant bacteria should be reported to the hospital infection management department.
The results of drug susceptibility test should be reviewed and reported in accordance with the regulations. Special attention should be paid to the review and report of the results of drug susceptibility test for pathogenic bacteria isolated from natural drug resistance, rare drug-resistant strains and special sites.
Multiple drug-resistant bacteria monitored in hospital infection management regulations should be reported and warned.
The results of antimicrobial susceptibility tests should be statistically analyzed regularly (at least once or twice a year) and reported to hospital infection departments and clinicians.
Participate in national or regional drug resistance monitoring.
4
Equipment quality control and performance verification
Observe and record the temperature of the refrigerator, incubator and water bath at least once a day. CO2 incubator should record CO2 concentration every day. The uv lamp shall record the disinfection time every day, and the uv intensity shall be monitored regularly (every 6 months) and the disinfection effect of the uv lamp shall be monitored.
The pressure sterilizer should use chemical indicator every time and use biological indicator every week to monitor the sterilization effect.
The bacterial turbidimeter should be calibrated or calibrated at least once every 6 months.
Hepa filter, flow and negative pressure of bio-safety cabinets, parameters such as pressure gauge pressure sterilizer and gauge, thermometer, hygrometer, the accuracy of the moving liquid should be regular verification, verification or calibration.
5
Ability to verify
Participate in the appropriate competency verification/inter-room quality assessment as required.
Analysis and corrective and preventive actions should be carried out on the results of the "dissatisfied" and "unqualified" capacity verification/interventricular quality assessment.
A performance comparison assessment test should be conducted at least once every 6 months with other laboratories (or other test methods) for projects that do not have capacity verification/interventricular quality assessment.
Biological safety
The clinical microbiome belongs to the second-level biosafety laboratory and should be designed and managed in accordance with relevant regulations.
The clinical microbiome should carry out biosafety risk assessment regularly (at least once a year).
The necessary biosafety protection equipment such as biosafety cabinet, high pressure sterilizer, uv lamp, emergency lamp, eye washing and spraying facilities should be equipped according to the risk assessment results. According to the equipped biological protection equipment facilities and the individual protection ability clearly define the laboratory can carry out the inspection project.
Should be according to the result of risk assessment on biosafety education inspection personnel, and equipped with personal protective equipment and supplies (such as gloves, mask, cap, experiment with shoes, protective clothing, protective goggles etc.).
Secondary hospitals do not recommend keep (quality control strains and the need to further confirm the clinical strains except), tertiary hospitals can preserve the highly pathogenic strains of pathogenic microorganisms, but should appoint someone who's in charge, storage, use, transfer, destruction of records, beware of abuse and misuse. If any suspected highly pathogenic bacteria are encountered in the experiment (according to the list of pathogenic microorganisms transmitted from human to human), they should report to the superior immediately, and no further test and sample treatment should be conducted.
Samples and culture materials over the specified preservation time shall be sterilized under high pressure and then handed over to the cleaning personnel for treatment and handover records.
'in the event of a potential biosafety accident (such as needle puncture, skin, mucous membrane or environmental pollution) in the course of the test, appropriate contingency plans and incident records shall be made.
Source: Chinese journal of laboratory medicine