细菌微量快速生化鉴定方法学研究

目的建立细菌微量快速生化鉴定系统,用于临床实验室细菌学的快速诊断。方法应用所研制的27种微量快速生化反应试剂与传统试管方法检测208株临床分离菌及8种质控菌株生化反应符合率,依据杭州24 h的JY Z-15E及JY Z-11E肠杆菌生化编码鉴定管的项目要求组合了该法的肠杆菌15种(RE 15n)及肠杆菌11种(RE 11n)生化鉴定试验条,用208株临床分离菌及8种质控菌菌株进行鉴定试验。结果27种细菌微量快速生化反应试剂与传统试管法生化反应总符合率为93.5%~100%。对208株临床分离菌及8株质控菌的菌种鉴定,其完全符合率15种(RE 15n)为99.9%,11种(RE 11n)为100%。8种质控菌株鉴定完全正确。结论用细菌微量快速生化反应系统鉴定细菌,具有简单、快速的特点,无需特殊设备,成本低廉,所得结果准确、可靠,能够满足临床需要。     

革兰阴性细菌鉴定编码系统（GN—30）对肠杆菌科细菌的鉴定评价

目的 用常规细菌鉴定方法和API20系统两种方法对我们自行设计的革兰阴性细菌鉴定编码系统（GN－30）进行评价。方法 用三种方法对16株质控菌株（14株全国质控菌株,2株省质控菌株）,42株临床菌株进行重新鉴定,分析鉴定结果。结果 GN－30细菌鉴定板与常规鉴定方法符合率为100％,API 20E系统与常规鉴定方法、CN－30系统完全符合者37株,符合率为85．7％,三种方法在统计学上无显著性差异（P＞0．05）。结论 GN－30系统在肠杆菌科细菌鉴定方面使用较为满意,可作为一种适用于各级医院临床常规细菌鉴定的较为可靠的方法。     

弧菌科30V12D微量生化菌种鉴定系统的应用效果评价

在致病性弧菌检验中，用30V12D微量生化菌种鉴定系统，对弧菌科细菌鉴定，多年来共鉴定出弧菌科细菌五种计126株，并与常规的鉴定方法相比较，该系统简便、迅速且准确，鉴定符合率达98．39％。除气单胞菌属符合率为94．89％外，所检鉴定菌株符合率均达100％，并在鉴定中发现一株海栖弧菌。本文包括以下内容：①30V12D微量生化菌种鉴定系统应用中所采用的材料和方法；②30V12D微量生化菌种鉴定系统应用所获取的相关结果；③30V12D微量生化菌种鉴定系统应用过程中得到的相关结论。     

Microflex~(TM) MALDI-TOF MS和Vitek 2 Compact全自动微生物分析系统对肠杆菌科细菌鉴定能力的比较

目的比较MicroflexTM基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)和Vitek 2 Compact全自动微生物分析系统(简称Vitek 2 Compact)对肠杆菌科细菌的鉴定能力。方法采用MicroflexTMMALDI-TOF MS和Vitek 2 Compact同时对545株肠杆菌科质控菌株和临床分离菌株进行鉴定,鉴定结果不一致者采用沙门菌血清凝集试验或细菌16S r DNA基因测序予以确证。结果 MicroflexTMMALDI-TOF MS对545株细菌的种和属的鉴定率分别为97.1%、2.9%。Vitek 2 Compact对545株细菌的种、群、属的鉴定率分别为83.3%、13.9%和2.2%,鉴定错误率为0.2%,未鉴定率为0.4%。结论 MicroflexTMMALDI-TOF MS对肠杆菌科细菌的鉴定符合率高于Vitek 2 Compact,且操作快速、简便,成本低,可用于临床肠杆菌科细菌的常规快速鉴定。     

Vitek—AMS在细菌室间质评中的应用

目的：Ｖｉｔｅｋ－ＡＭＳ在细菌室间质评中的应用价值。方法：用Ｖｉｔｅｋ－ＡＭＳ（自动微生物鉴定系统）对１０８株不同质控菌株进行鉴定并作了８３例次药敏平行试验，结果；Ｖｉｔｅｋ－ＡＭＳ对质控菌株鉴定的总正确率达９２．６％，Ｖｉｔｅｋ－ＡＭＳ对肠杆菌科，非发酵菌，酵母菌以及葡萄球菌与链球菌属鉴定结果可靠；对弧菌科，棒状杆菌较差，Ｖｉｔｅｋ－ＡＭＳ测定的药敏结果与质控菌药敏标准值的完全符合率达９７．６％     

MicroscanAS—4自动微生物分析仪在细菌室间质控中的评价

应用 Microscan AS- 4(以下简称为 AS- 4)自动微生物鉴定 /药敏测试系统 ,对细菌室间质评中的 39株不同质控菌株进行鉴定 ,并作了 110对次平行药敏试验。AS- 4对质控菌株鉴定的总正确率为 89.7%。对肠杆菌科、非发酵菌、苛养菌、弧菌科以及葡萄球菌、链球菌属鉴定具有较稳定的良好效果 ;对真菌 (酵母菌 )、李斯特菌属鉴定较局限 ;对马红球菌、棒状杆菌属不能鉴定。对沙门菌属、志贺菌属仍须借助血清学分型鉴定。 AS- 4检测的 MIC与质控测定菌株药敏靶值的完全符合率达 98.2 % ,比药敏纸片的 K- B法测定符合率略高。     

Microscan AS-4自动微生物分析仪在细菌室间质控中的评价

应用Microscan AS-4(以下简称为AS-4)自动微生物鉴定/药敏测试系统,对细菌室间质评中的39株不同质控菌株进行鉴定,并作了110对次平行药敏试验.AS-4对质控菌株鉴定的总正确率为89.7%.对肠杆菌科、非发酵菌、苛养菌、弧菌科以及葡萄球菌、链球菌属鉴定具有较稳定的良好效果;对真菌(酵母菌)、李斯特菌属鉴定较局限;对马红球菌、棒状杆菌属不能鉴定.对沙门菌属、志贺菌属仍须借助血清学分型鉴定.AS-4检测的MIC与质控测定菌株药敏靶值的完全符合率达98.2%,比药敏纸片的K-B法测定符合率略高.     

革兰阴性杆菌超广谱β-内酰胺酶的检测与思考

[目的]了解肠杆菌科及非发酵菌超广谱β-内酰胺酶（ESBLs）的携带情况，为临床及时掌握和有效遏制细菌由于ESBLs而导致的耐药性提供实验依据。[方法]细菌的分离按全国临床检验操作规程第2版进行。细菌的鉴定主要采用仪器法（autoscan-4-DADE．U．S．A）。ESBLs的检测采用纸片扩散法初筛和确证，部分菌株辅以E．test法，以保证结果的准确性。[结果]2大类阴性杆菌1462株，产ESBLs的507株，阳性率35％；其中肠杆菌科细菌880株，产ESBLs309株，阳性率42％；此类菌中，大肠埃希菌和弗劳地枸椽酸杆菌产ESBLs最高，都在50％以上；肺炎克雷伯菌和阴沟肠杆菌居第2，在45％以上；产酸克雷伯菌位于第3，达30％。非发酵菌523株，产ESBLs的126株，阳性率24％；这类菌中，脑膜炎败血性黄杆菌和嗜麦芽窄食单胞菌产ESBLs最高，分别达56％和46％。[结论]2大类阴性杆菌产ESBLs都高，且有逐步增高的趋势。此外．2大类阴性杆菌比较，肠杆菌科细菌更易产ESBLs，P〈0．01；只有扩大ESBLs的检测面，与临床共同努力，才能减缓细菌耐药的进程。     

应用电脑鉴定编码系统微量快速生化法鉴定肠杆菌科

参照丹麦本特·尼森的《细菌的酶学鉴定方法》，研制了微量快速生化反应板鉴定肠杆菌。鉴定了23株标准菌株，结果全部正确。测试了临床标本130例，并与传统鉴定法比较，符合率92％．可认为：该鉴定板快速、准确。特异性和敏感性均佳，广泛适用于大、中、小型医院临床细菌学的鉴定工作．     

微量快速生化法鉴定肠杆菌科——应用电脑鉴定编码系统

参照丹麦专家本特·尼森的《细菌的酶学鉴定方法》,我们研制了微量快速生化反应扳、鉴定肠杆菌科。鉴定了23株标准菌株,结果全部正确。测试了临床标本130例、与传统鉴定法比较、符合率92％。该扳鉴定快速、准确、特异生和敏感性增多佳,可广泛适用于临床细菌学的鉴定工作。     

Use of and cost savings with morphologic criteria and the spot indole test as a routine means of identification of Escherichia coli

The use of primary isolation plate colonial morphologic criteria (CMC) of a flat, nonmucoid, lactose-fermenting, gram-negative rod on MacConkey agar and the spot indole (SI) test from the sheep blood agar plate was evaluated as a means for identification of Escherichia coli in comparison to kit (Micro-ID, API-20E) and conventional biochemical testing. In this preliminary phase of comparison of accuracy, 427 isolates of E. coli (69.8%) from a total of 612 isolates of lactose-fermenting gram-negative rods were evaluated. Of these E. coli isolates, 357 (83.6%) fit the CMC and were SI positive; 3 (less than 1% error rate) were not E. coli. In the second phase of the evaluation, using CMC and SI alone as a means for identification of E. coli, 472 (57.6%) E. coli isolates from a total of 820 Enterobacteriaceae isolates were assessed. Of these E. coli isolates, 326 could be identified using only CMC and SI (69.1% of the E. coli isolates and 39.8% of all Enterobacteriaceae isolates); 146 (30.9%) required additional biochemical testing because of atypical colonial morphology, because of the investigator's inability to differentiate colony types on both media or lack of isolated colonies on either of the two required media, or because as isolates from sterile body sites they were processed directly to Micro-ID kits. A minimum of 40% savings on Enterobacteriaceae identification schemes without compromising accuracy was calculated. As of November 1983, a direct (labor and materials) cost savings of approximately +200.80 per 100 Enterobacteriaceae identifications was projected.     

Identification of methicillin-resistant Staphylococcus aureus (MRSA): Comparison of a new molecular genetic test kit (GenoType MRSA) with standard diagnostic methods

Results obtained from 188 isolates of staphylococci using standard diagnostic methods for identifying MRSA were compared with those achieved with a newly available molecular genetic test kit, the GenoType, Version 1, MRSA (Hain Lifescience GmbH, Nehren, Germany). The GenoType MRSA detects the mecA gene and, in addition, a highly specific sequence for Staphylococcus aureus (S. aureus) by polymerase chain reaction (PCR) and reverse hybridization. There was a 100% overall correlation between the results of conventional and molecular genetic testing. 143 isolates were tested positive for MRSA, 10 isolates were identified as oxacillin-sensitive Staphylococcus aureus strains (MSSA), and 35 isolates were coagulase-negative staphylococci of various species. However, five of the 143 MRSA strains yielded ambiguous results with the first line standard tests and therefore required additional testing leading to delay of definitive diagnosis. As expected, mecA could not only be detected in MRSA strains, but also in coagulase-negative staphylococci. The reliable identification as S. aureus from the same isolate is therefore an essential prerequisite for MRSA diagnosis. The GenoType MRSA fulfills this requirement by parallel detection of a S. aureus-specific sequence and the mecA gene. Molecular genetic testing with the GenoType MRSA kit needs much less time than conventional microbiological methods. Therefore genetic testing provides not only a considerable advantage with respect to reliability but also to speed.     

Application of quadFERM+ for the identification of fastidious gram-positive and gram-negative bacilli

The 2-hr quadFERM+ kit (qF) (Analytab Products, Plainview, NY) was compared with conventional tube tests for the identification of the HACEK bacteria (Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), other Haemophilus and Kingella spp., Capnocytophaga, Corynbacterium, and Moraxella spp. Test results were identical for 296 comparisons with 74 isolates (74%). In the remaining 104 comparisons for 26 isolates, 50 reactions were identical, and qF produced a positive result in 46 of 54 discrepancies.     

Evaluation of Syngene DNA-DNA probe assays for the identification of the Mycobacterium tuberculosis complex and the Mycobacterium avium complex.

Two hundred mycobacterial cultures were used to evaluate two alkaline-phosphatase-labeled DNA probe (SNAP) kits developed by Syngene (San Diego, CA) for identification of Mycobacterium tuberculosis complex and M. avium complex. The M. tuberculosis complex SNAP probe, when compared with standard biochemical identification tests, gave results that were in agreement at 100% sensitivity and 98.7% specificity. Ninety-nine M. avium complex strains that were previously tested by the Gen-Probe M. avium complex probe assays and mycolic acid analysis were included to evaluate the M. avium complex SNAP assay which contained three probes, A (avium), I (intracellulare), and X. Eight strains identified as members of the M. avium complex by biochemical tests did not react with the three SNAP probes. These strains were also negative by the Gen-Probe assays. However, 23 strains identified as M. avium complex by biochemical tests and mycolic acid analysis and negative with the Gen-Probe assays gave positive results with the X probe and negative results with the A and I probes of the SNAP assay.     

产超广谱β-内酰胺酶细菌致医院感染的分析

目的 :了解我院产超广谱β-内酰胺酶 (ESBLs)细菌致医院感染的发生率、分布情况及耐药特点。方法 :ESBLs菌的检测采用双纸片协同试验和纸片确认试验 ,细菌鉴定及药敏试验分别用法国生物梅里埃API鉴定系统和K -B法进行。结果 :在 4 80株革兰氏阴性杆菌中 ,产ESBLs菌占 2 8 13% (135 / 4 80 ) ,其中大肠埃希菌和肺炎克雷伯菌的阳性率分别为 2 4 89%和 35 34% ,产ESBLs菌对抗生素的耐药率均显著高于非产ESBLs菌 (P <0 .0 1) ,本文尚未发现对亚胺培南耐药菌株。结论 :临床实验室细菌鉴定应常规进行ESBLs检测 ,将有助于控制产ESBLs细菌的传播和流行 ,亚胺培南是治疗产ESBLs菌感染的首选药物     

Identification of glycopeptide-resistant enterococci by VITEK 2 system and conventional and real-time polymerase chain reaction

Glycopeptide-resistant enterococci (GRE) are important causes of nosocomial infections in the United States and in Europe. Rapid detection of GRE is essential for the implementation of appropriate control measures to prevent the spread of GRE. In this study, we compared the reliability of 3 different methods, VITEK 2 automated system (bioMérieux), a conventional multiplex polymerase chain reaction (m-PCR) protocol, and a real-time PCR protocol performed on the LightCycler system (Roche) for identification of GRE in the routine microbiology laboratory. Species identification and glycopeptide resistance determination was tested with 80 enterococcal isolates with different glycopeptide resistance phenotypes. With the VITEK 2 system, 39% of the strains were correctly identified to species level. Resistance to vancomycin was detected in all isolates; however, discrepancies occurred in the correct detection of teicoplanin resistance. The PCR protocols proved to be suitable for detecting clinically relevant GRE; 90% of the isolates studied were correctly identified with the conventional m-PCR and 100% of vanA and vanB isolates with the real-time PCR protocol, respectively. High specificity and rapidity make the real-time PCR assays superior tools for identification of GRE in clinical samples; however, they do not have the ability to detect vanC.     

厦门地区血培养病原菌分布及耐药性分析

目的了解厦门地区血培养检出的病原菌分布及其对抗菌药物的耐药情况，为临床合理选择抗菌药物提供依据。方法2107例血标本经BacT／ALERT 120全自动血培养仪培养检测，检出的病原菌采用VITEK 2 compact全自动微生物鉴定药敏仪进行细菌鉴定及药敏分析。结果共检出230株病原菌，阳性率为10．9％，其中革兰阳性球菌占38．7％，革兰阴性杆菌占51．7％，真菌占9．6％，葡萄球菌属和肠杆菌科细菌是厦门地区菌血症或败血症的主要病原菌。有43、6％的大肠埃希菌和26．7％的肺炎克雷伯菌产超广谱β-内酰胺酶。结论血培养病原菌种类复杂，耐药率较高，应重视血培养及对血培养病原菌耐药性的监测，合理使用抗菌药物。     

四川省细菌耐药监测网2011年川东地区细菌耐药性监测数据分析

目的了解川东地区2011年临床分离菌株的耐药情况,指导临床更加合理使用抗菌药物。方法应用法国生物默里埃半自动(API)、全自动(VITEK)微生物分析系统或手工方法进行菌株鉴定和药敏试验,以美国实验室与标准化研究所(CLSI)2010年折点标准判定药物敏感性,用WHO NET 5.5软件进行数据分析。结果 2011年1～12月川东地区6家医院临床共收集3810株非重复菌株,其中革兰阴性菌2771株(占72.7%),革兰阳性菌1039株(占27.3%)。耐甲氧西林金葡菌和耐甲氧西林凝固酶阴性葡萄球菌的检出率分别为31.2%和84.4%。未发现万古霉素、利奈唑胺中介株或耐药株。有3株万古霉素耐药屎肠球菌及2株万古霉素耐药粪肠球菌。产ESBI的大肠埃希菌、克雷伯菌属在各菌属中分别占53.5%、33.5%。铜绿假单胞菌对亚胺培南和美罗培南的耐药率分别为13.8%和15.6%。不动杆菌属对亚胺培南和美罗培南的耐药率分别为37.6%和42.7%。结论细菌耐药性不断增强,已出现多重耐药菌株。加强对细菌耐药性的定期监测,对正确选用抗菌药和控制耐药菌所到致感染十分重要。     

A miniaturised semiautomated system for the identification of Yersinia species within the genus Yersinia

Commercially available identification systems based on biochemical reactions of bacteria are not suited for typing the species of the genus Yersinia (Y.) or the biovars (BV) of the species Y. enterocolitica. This failure is caused by the limited number of biochemical reactions applied, resulting in the absence of important discriminatory key reactions. The MICRONAUT identification system (Merlin, Bornheim-Hersel) makes use of dried substrates/enzymes reactions in the wells of a 96-well microtitration plate, reading of the results by a scanner device and typing of the isolate by the calculation of probabilities according to a data base. For this study a special identification panel was designed on which 38 substrates and enzyme reactions were configurated including 20 reactions for the identification of the species of the genus and the Y. enterocolitica biovars. The database was calculated using the results obtained from a total of 250 Yersinia strains of the eleven species of the genus. Reevaluation of the results of these strains revealed an overall sensitivity of 98%, as only four strains were not identified satisfactorily. Considering also questionable results the sensitivity was still 85%. The system was also used to identify Y. pestis isolates, but in this case reading was done visually. The printouts usually cite species designation, identification quality and probabilities. The sealing of the plates in an aluminium bag guarantees long life and long lasting quality. However, an evaluation of the system with a considerable number of strains has to be done in a next step. The 'Yersinia identification set' can replace time-consuming tube testing in the future and is a big step forward towards a sensitive identification of Yersinia isolates in the routine laboratory.     

基于CLSI-M43国际标准改良的Mycoview-AST试剂盒检测性能评估

目的 基于美国临床标准化协会(clinical and laboratory standards institute,CLSI)M43国际标准改进的泌尿生殖道支原体Mycoview-AST试剂盒检测性能评估。方法 以法国MYCOFAST^○Rrevolution支原体试剂盒为比对标准,用CLSI-M43规定的质控株和有关指标及60例临床标本对新改进的试剂盒性能进行评估。结果 Mycoview-AST试剂盒以质控菌株检测的药敏结果与MYCOFAST11^○Rrevolution试剂盒一致。60例临床标本,阴性标本37例,解脲支原体(Uu)阳性16例,人型支原体(Mh)阳性5例,Uu与Mh均阳性的标本2例。两种试剂盒Uu和Mh的鉴定结果一致。23例阳性标本的药敏结果共161个,与进口比对试剂MYCOFAST^○R比对,154个结果符合,药敏结果符合率为95.6%(154/161)。结论 基于CLSI-M43国际标准改进后的Mycoview-AST试剂盒检测性能与国际知名品牌MYCOFAST^○Rrevolution试剂盒相当。