荧光原位杂交法快速鉴定血培养阳性葡萄球菌的临床应用

目的 快速鉴定血培养中的金黄色葡萄球菌和凝固酶阴性葡萄球菌(CoNS),结合临床快速判定是否为污染菌。方法 采用荧光原位杂交法鉴定血培养中的金黄色葡萄球菌和CoNS,杂交结果若为CoNS,根据临床资料进行判断,并与文献推荐的污染判断法进行结果比较。结果 探针的特异性经由标准菌株和临床分离菌株证实。金黄色葡萄球菌探针的特异性和敏感性均为100％,CoNS探针的特异性和敏感性分别为100％和95．5％。179株CoNS中117株判断为污染菌,污染率为68％,与文献推荐的污染判断方法一致。结论 荧光原位杂交法适用于血培养中的金黄色葡萄球菌和CoNS的快速鉴定,以排除CoNS污染。     

荧光原位杂交法快速鉴定金黄色葡萄球菌和凝固酶阴性葡萄球菌

目的 建立一种新的荧光原位杂交法,快速鉴定血培养中的金黄色葡萄球菌(金葡菌)和凝固酶阴性葡萄球菌。方法 将荧光同位素标记的针对金葡菌和凝固酶阴性葡萄球菌的核糖体16SrRNA序列的互补的DNA寡核苷酸探针,在同一张玻片上50℃杂交1 h,用杂交缓冲液50℃洗涤10min,干燥后置荧光显微镜下观察,全过程大约2 h。结果 探针的特异性经标准菌株和临床分离菌株证实。将259份临床标本测试与培养法比较,金葡菌的特异性和敏感性均为100％,CoNs的特异性和敏感性分别为100％和95.5％,荧光原位杂交法的最低检出限为103CFU/ml。结论 本方法适用于血培养中的金葡菌和凝固酶阴性葡萄球菌的快速鉴定。     

荧光原位杂交法快速鉴定血培养中白念珠菌的方法学评价

目的：评价荧光原位杂交法(FISH)快速鉴定血培养中阳性的白念珠菌的应用价值。方法：当BacT／ALERT报警后。涂片革兰染色，若发现念珠菌，与针对白念珠菌的特异性探针杂交，同时将分离菌转种沙保罗培养基，比较2种方法的鉴定结果。结果：探针的特异性经15株标准菌株证实，分离到的36株念珠菌中，杂交法在3h内可全部得到鉴定结果，与培养法比较，杂交法的特异性和敏感性均为100％．结论：使用FISH可直接快速鉴定报警阳性血培养中的白念珠菌，结果可靠。     

伤口感染常见病原菌的快速检测

目的建立一种新的荧光原位杂交法,快速检测鉴定伤口中的常见病原菌,指导临床早期合理使用抗生素。方法采用荧光同位素标记的,设计针对引起伤口感染的金黄色葡萄球菌、表皮葡萄球菌、铜绿假单胞菌、肠球菌和大肠埃希氏菌常见五种病原菌的核糖体16SrRNA序列的互补的DNA寡核苷酸探针,将探针置于玻片上于50℃杂交1h,用杂交缓冲液于50℃洗涤10min,干燥后置于荧光显微镜下观察,整个过程大约需要2h的时间。结果经438例临床标本测试,与培养法比较,荧光原位杂交法的敏感性和特异性分别为94．1％和81．1％。结论该方法适用于伤口感染常见病原菌的快速检测鉴定。     

量子点标记荧光原位杂交法在快速检测金黄色葡萄球菌中的应用

目的:应用量子点标记的荧光原位杂交方法以快速检测金黄色葡萄球菌。方法:采用两种量子点-亲和素-生物素-DNA探针,即A型肠毒素(SEA)基因与FemB基因探针与金黄色葡萄球菌临床分离株或感染金黄色葡萄球菌的粪便标本进行荧光原位杂交,同时与SEA基因聚合酶链反应(PCR)的检测结果比较。结果:10株临床金黄色葡萄球菌分离株中,2株SEA基因探针杂交和SEA基因PCR检测结果均为阳性,其余8株SEA基因探针杂交和SEA基因PCR检测结果均为阴性,10株FemB基因探针杂交结果均为阳性。3例分离培养为金黄色葡萄球菌的临床粪便标本直接进行荧光原位杂交,FemB及SEA基因探针杂交结果均为阳性,SEA基因PCR检测结果均为阳性。SEA、FemB基因探针与其他相关菌种未见非特异性反应。结论:量子点标记荧光原位杂交法检测金黄色葡萄球菌具有快速、简便、特异的特点,且可用于临床粪便标本的直接检测。     

荧光原位杂交法(FISH)快速检测尿液中金黄色葡萄球菌

目的 利用荧光原位杂交法快速检测尿液中的金黄色葡萄球菌,筛查金黄色葡萄球菌所致的尿路感染.方法 针对金黄色葡萄球菌16sRNA设计荧光标记的核苷酸探针,利用荧光原位杂交技术(FISH)对132例疑似尿路感染患者中段尿标本进行检测;同时进行中段尿培养.结果 荧光原位杂交法检测阳性的有9例,与中段尿培养比较,其敏感度为100.0%,特异度为99.2%,阳性预期值为90.0%,阴性预期值为100.0%.结论 荧光原位杂交能快速检测尿液中的金黄色葡萄球菌,对金黄色葡萄球菌所致的尿路感染进行快速诊断.     

荧光原位杂交法快速鉴定临床标本中的粪肠球菌和屎肠球菌

目的建立荧光原位杂交法,快速鉴定临床标本中的粪肠球菌和屎肠球菌。方法将荧光同位素标记的粪肠球菌或屎肠球菌的核糖体16S rRNA及23S rRNA序列互补的寡核苷酸探针,在同一张玻片上杂交30min至1h,用杂交缓冲液洗涤10min,干燥后置显微镜下观察。结果探针的特异性经标准菌株证实,对163例临床标本测试结果与培养法结果比较,2种探针的特异性和敏感性均为100％。结论荧光原位杂交法适宜于临床标本中粪肠球菌和屎肠球菌的快速鉴定。     

志贺菌TaqMan-MGB探针实时荧光聚合酶链反应快速检测方法的应用研究

目的建立志贺菌TaqMan-MGB探针实时荧光PCR快速检测技术,为从患者、食品和环境中快速分离和鉴定志贺菌提供技术支撑。方法根据志贺菌ipaH基因序列设计一对特异性引物和TaqMan-MGB探针;通过对PCR扩增体系和反应条件的优化,建立志贺菌TaqMan-MGB探针实时荧光PCR快速检测方法;用添加已知志贺菌浓度的样本验证方法敏感性;用志贺菌标准菌株、分离株以及大肠埃希菌、沙门菌、副溶血性弧菌、金黄色葡萄球菌等致病菌验证方法特异性。结果用本研究建立的志贺菌TaqMan-MGB探针实时荧光PCR检测方法检测志贺菌,其Ct值与模板浓度的对数值具有很好的对应关系（Y=-3.93×log（X）＋37.34,R=0.999）,最低检测浓度为30cfu/ml,3株志贺菌标准株,30株志贺菌分离株检测结果均为阳性;而沙门菌、副溶血性弧菌、大肠埃希菌、金黄色葡萄球菌等91株其他细菌的Ct值均〉35或扩增曲线成一平滑直线。与常规分离鉴定方法比较差异无统计学意义（P〉0.05,χ^2=0.27）。对于纯菌和食品样品整个检测过程仅需2h和10h。结论志贺菌TaqMan-MGB探针实时荧光PCR检测技术具有特异性强,敏感性高,易操作等优点,有很好的应用前景和研究价值。     

实时荧光PCR快速检测耐甲氧西林金黄色葡萄球菌的临床评估

目的:采用实时荧光PCR检测耐甲氧西林金黄色葡萄球菌(MRSA),并评价该方法在临床上的应用价值。方法:将临床分离的62株金黄色葡萄球菌(金葡菌)和35株非金黄色葡萄球菌同时采用分离培养及药物敏感试验(药敏试验)和实时荧光PCR法进行检测并比较,对2种方法检测结果不符的菌株辅以测序法进行最后鉴定,并确定实时荧光PCR法的检测灵敏度。结果:分离培养法与实时荧光PCR法对金葡菌检测的一致率为100%,都检测出了62株金葡菌阳性株。实时荧光PCR法与分离培养药敏试验法MRSA检测的符合率为96.77%,而分离培养药敏试验法检测为阴性而实时荧光PCR法检测为阳性的2株待检菌的测序结果显示为MRSA阳性株。最后确定实时荧光PCR法检测MRSA的临床灵敏度为1×103 cfu/mL。结论:实时荧光PCR法可用于临床对MRSA的快速检测,具有较高的临床价值。     

实时荧光定量PCR检测金黄色葡萄球菌方法的试验研究

目的建立金黄色葡萄球菌实时荧光定量PCR的快速检测方法,探讨该方法的可行性和应用价值。方法根据金黄色葡萄球菌femB基因序列设计引物和探针,采用基因重组技术构建用于金黄色葡萄球菌检测的定量标准品,建立实时荧光定量PCR检测金黄色葡萄球菌的方法。结果成功构建了金黄色葡萄球菌重组质粒标准品和金黄色葡萄球菌实时荧光定量PCR方法;通过特异性、敏感性、稳定性和重复性试验,结果表明具有较好的特异性、敏感性、稳定性和重复性;将模拟标本与分离培养对比,两者符合率为100%。结论金黄色葡萄球菌实时荧光定量PCR检测方法的建立,为金黄色葡萄球菌感染诊断及食源性金黄色葡萄球菌污染的快速检测提供依据,可用于临床感染诊断及食品卫生监管、商品检验检疫等。     

Identification of methicillin-resistant isolates of Staphylococcus aureus and coagulase-negative staphylococci responsible for bloodstream infections with the Phoenix system

We evaluated the reliability of the new Phoenix system (Becton Dickinson Microbiology Systems, Sparks, Md.) in species-level identification and detection of oxacillin (methicillin) resistance among 493 staphylococcal isolates (Staphylococcus aureus, n = 223; coagulase-negative staphylococci, CoNS, n = 270) recovered from patients with bacteremia. Identification results were concordant with those of the ID 32 STAPH system (bioMérieux, Marcy l'Etoile, France) for 100% of S. aureus (223/223) and 97.4% (263/270) of CoNS isolates. For S. aureus isolates, Phoenix oxacillin-susceptibility results fully concurred with those of mecA polymerase chain reaction (PCR) (reference method): 96 mecA-positive isolates identified as resistant, 127 mecA-negative strains as susceptible. Two of the 210 mecA-positive CoNS isolates were misclassified as susceptible by the Phoenix (sensitivity 99%, positive predictive value 97.6%). Five of 60 mecA-negative CoNS isolates were classified as resistant by the Phoenix (specificity 91.7%; negative predictive value 96.5%). The Phoenix system can provide accurate and reliable identification of methicillin-resistant staphylococci responsible for bloodstream infections.     

应用葡萄球菌A蛋白快速检测金黄色葡萄球菌的诊断价值

目的评价应用葡萄球菌A蛋白(SPA)快速检测金葡菌的临床诊断价值。方法 200株鉴定明确的菌株(金葡菌和非金葡菌各100株)采用SPA法检测,统计检测方法的灵敏度和特异度;118份ICU患者的痰标本,采用双盲法分别对其进行传统痰培养鉴定和SPA镜下快速检测,计算该诊断试验的评价指标,并对痰培养和SPA方法的相关性进行分析。结果 SPA检测的灵敏度和特异度均为100%;双盲法痰标本SPA检测敏感度为100%,特异度97.7%,SPA检测和痰培养的结果呈高度相关性(P>0.05)。结论 SPA检测法可早期诊断金葡菌感染,是一项快速的、灵敏度和特异度较高的诊断试验。     

Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit

The EVIGENE MRSA Detection Kit was evaluated on coagulase-negative staphylococci (CoNS) from agar plates and on staphylococci directly from positive spiked blood cultures. For the CoNS study, a total of 242 isolates were tested, and of these 237 gave valid test results. For the 237 valid tests, all gave correct mecA classification. For the blood culture procedure, a collection of 51 mecA-positive Staphylococcus aureus, 21 mecA-negative S. aureus, 31 mecA-positive CoNS and 28 mecA-negative CoNS were used for the simulated blood cultures. For the S. aureus strains, all gave valid test results and correct mecA classification. One of the MRSA isolates gave a very faint nuc signal, and another four isolates gave results close to the cut-off of the kit; however, these were still clearly positive when read by the naked eye. For the CoNS isolates, 51 of the 59 strains gave valid results. All of these 51 strains gave correct mecA status. Thus the EVIGENE MRSA Detection Kit can provide fast and accurate determination of methicillin resistance in CoNS. This preliminary study of the blood culture procedure indicates that it is possible to achieve determination of methicillin resistance in staphylococci 8 h after positivity of the blood culture, making same-day detection of methicillin resistance possible.     

Impact of rapid in situ hybridization testing on coagulase-negative staphylococci positive blood cultures

OBJECTIVES: To evaluate the impact of the rapid differentiation of Staphylococcus aureus from coagulase-negative staphylococci (CoNS) in blood cultures using peptide nucleic acid fluorescence in situ hybridization (PNA FISH) on vancomycin usage, length of patient hospital stay and hospital costs. Design: This was a retrospective, cost-effective analysis of PNA FISH in its initial 3 month implementation period in 2004 in a 650 bed academic medical centre. Blood cultures with Gram-positive cocci in clusters (GPCC) that were negative for S. aureus using the PNA FISH assay were compared with an untested control group in the same period that had similar illness severity and location. We evaluated the effectiveness of the early identification of CoNS by ruling out S. aureus in conjunction with an antimicrobial team (AMT) on antimicrobial therapy, patient length of stay and hospital costs. RESULTS: A total of 139 blood cultures positive with GPCC had PNA FISH results while 84 in the control group did not. Evaluable criteria were met in 53 patients in the PNA FISH group and 34 in the control group. When comparing the results obtained from using the PNA FISH assay with those for the control group, there was a significant reduction in median length of hospital stay from 6 to 4 days (P < 0.05, CI 0.95-1.87) and a trend towards less vancomycin usage with a decrease in associated hospital costs of approximately Dollars 4000 per patient. CONCLUSIONS: The PNA FISH assay is rapid, accurate and reliable and in association with an AMT could decrease hospital length of stay in patients with CoNS bacteraemia in non-intensive care unit settings and prevent excessive vancomycin usage.     

Rapid, direct identification of Staphylococcus aureus and Streptococcus pneumoniae from blood cultures using commercial immunologic kits and modified conventional tests.

To develop safe and rapid methods for identification of Staphylococcus aureus and Streptococcus pneumoniae directly from positive blood culture bottles (BCB) (BACTEC, Johnston Laboratories), several commercial biochemical and immunological tests as well as modified conventional tests were evaluated. Preliminary studies demonstrated that both S. aureus and St. pneumoniae could be identified directly using only a small aliquot (100 microliters) of the blood culture broth obtained via vent without need for centrifugation or other separation steps. A simple tube coagulase exhibited 98% sensitivity and 100% specificity for 32 S. aureus isolates and 157 blood cultures positive for coagulase-negative staphylococci when read at 2 hr. All systems employed for direct identification of St. pneumoniae exhibited excellent sensitivity and specificity using aliquots from blood culture broths, but Pneumoslide (BBL Microbiology Systems, Cockeysville, MD) was easiest to perform and interpret. The results of this study show that S. aureus and St. pneumoniae can be identified directly from blood culture broth aliquots using rapid methods that eliminate the need for centrifugation or use of needles and syringes.     

Assessment of biofilm-forming ability of coagulase-negative staphylococci isolated from contaminated platelet preparations in Canada

BACKGROUND: Coagulase-negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet (PLT) preparations and have been implicated in adverse transfusion reactions worldwide. The most frequently identified contaminant is Staphylococcus epidermidis, which is noted for its ability to maintain chronic hospital-acquired infections by forming biofilms as a chief virulence mechanism. STUDY DESIGN AND METHODS: Strains of S. epidermidis isolated from contaminated PLT preparations in Canada were distinguished via gene-specific polymerase chain reaction (PCR) with divIVA as a marker. Biofilm-forming ability was assessed by the presence of the gene icaD, slime production on Congo red agar, and biofilm formation on polystyrene surfaces. Production of polysaccharide intercellular adhesin (PIA) was resolved by immunofluorescence. RESULTS: Eight of the 13 (62%) CoNS isolates under study were identified as S. epidermidis. Of these, four strains (50%) were classified as strong biofilm producers. Three of the four biofilm-positive strains (75%) produced slime, harbored the icaD gene, and had positive expression of PIA. CONCLUSIONS: Despite the presumable commensal origin of the CoNS isolates, a large proportion of S. epidermidis strains demonstrated a potential for enhanced virulence. Identification of contaminant staphylococci as biofilm producers is thus relevant and informative with regard to treatment approach in the circumstance of inadvertent infection of a PLT recipient.     

Performance of MRSA ID chromogenic medium for detection of methicillin-resistant Staphylococcus aureus directly from blood cultures and clinical specimens

MRSA ID was evaluated to see its performance in identifying methicillin-resistant Staphylococcus aureus (MRSA) directly from blood culture bottles (n = 837), wound swabs (n = 112), and abscesses (n = 18). Each positive blood culture and clinical specimen was directly inoculated on MRSA ID and the culture media routinely used. The sensitivity of MRSA ID was 97.8% after 24 h and 100% after 48 h for blood cultures, and 88.9% after 24 h and 100% after 48 h for wound samples. The specificity was 99.7% after 24 h and 99.6% after 48 h for blood cultures, and 100% after 24 and 48 h for wound samples. Four strains with green colonies indicating MRSA on MRSA ID were identified as methicillin-susceptible S. aureus (MSSA) by conventional methods. Three of these MSSA strains showed negative results with the mecA polymerase chain reaction, and 1 strain harbored the mecA gene. Using MRSA ID with primary culture media should decrease the time (18-24 h) to report a positive result compared with conventional methods.     

Rapid identification of Propionibacterium acnes from blood cultures by fluorescence in situ hybridization

A newly designed probe for rapid identification of Propionibacterium acnes by fluorescence in situ hybridization was evaluated using 111 isolates from subculture and showed 100% sensitivity and specificity. A sensitivity of 95% and a specificity of 100% were achieved with direct application on 55 blood cultures containing Gram-positive rods.     

高通量荧光PCR组合方法鉴定16种金黄色葡萄球菌肠毒素的方法建立及评估

目的 建立一种高效、特异的荧光探针熔解曲线方法，用于鉴定16种金黄色葡萄球菌肠毒素。 方法 根据金黄色葡萄球菌肠毒素SEA ~ SEQ特异基因序列设计不同杂交连接探针，建立金黄色葡萄球菌肠毒素检测体系，评估其最低检出限、特异度、可重复性等指标。 与普通PCR方法比较，采用荧光探针熔解曲线方法对本实验室的158株食物中毒金黄色葡萄球菌进行检测，评估新方法的灵敏度和特异度。 结果 荧光探针熔解曲线方法最低检出限为0.80 ~ 2.15 ng/μl，特异度为100.0%，无交叉荧光信号产生，变异系数＜1%。 与普通PCR方法比较，新方法的灵敏度为95.4%，特异度为100.0%，Kappa值为0.88。 结论 荧光探针熔解曲线技术检测金黄色葡萄球菌肠毒素的方法可覆盖金黄色葡萄球菌16种肠毒素，具有快速、准确、特异性高的特点。      

151株血培养分离菌的分布及耐药性分析

目的了解我院血培养分离病原菌的分布及耐药情况。方法收集2012年6月-2013年12月我院门诊及住院患者的1178份血液标本,采用Bact／Alert-3D全自动血培养仪进行培养,阳性标本采用VITEK—COMPACT2全自动细菌鉴定仪进行菌种鉴定,采用K—B法及VITEK—COMPACT2定量MIC药敏分析系统进行药敏试验,并采用WHONET5．4软件对菌种检出情况及耐药情况进行统计分析。结果1178份血液标本中共分离出病原菌151株,分离率为12．8％。151株病原菌中,革兰阴性菌97株（64．2％）,革兰阳性菌49株（32．5％）,真菌5株（3.3％）。97株革兰阴性菌中,分离率在前三位的为大肠埃希菌（39株,25．8％）、肺炎克雷伯菌（25株,16．6％）和马耳他布鲁菌（15株,9．9％）;49株革兰阳性菌中以金黄色葡萄球菌（24株,15．9％）和表皮葡萄球菌（8株,5-3％）为主,真菌多为白色念珠菌（4株,2．6％）。39株大肠埃希菌和25株肺炎克雷伯菌中,产超广谱β-内酰胺酶（extended spectrum betalactamases,ESBLs）的菌株分别为18株（46．2％,18／39）和8株（32．0％,8／25）;24株金黄色葡萄球菌中,耐甲氧西林金黄色葡萄球菌的检出率为50．0％（12／24）。大肠埃希菌和肺炎克雷伯菌对庆大霉素、左氧氟沙星和环丙沙星的耐药率在40．0％以上,金黄色葡萄球菌对庆大霉素、哌拉西林／他唑巴坦、头孢西丁和红霉素的耐药率在41．7％以上。结论我院血培养分离菌以革兰阴性杆菌居多,其中马耳他布鲁菌偏多是因为本地区牧民较多所致。分离病原菌的耐药情况严重,临床医师应根据细菌鉴定及药敏试验选择敏感药物治疗。