Detection of virulence genes of Clostridium difficile by multiplex PCR

Antikainen J, Pasanen T, Mero S, Tarkka E, Kirveskari J, Kotila S, Mentula S, Könönen E, Virolainen‐Julkunen A‐R, Vaara M, Tissari P. Detection of virulence genes of Clostridium difficile by multiplex PCR. APMIS 2009; 117: 607–13. Clostridium difficile strains belonging to the PCR ribotype 027, pulse‐field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027.     

Real‐time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis

A multiplex real‐time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage λ; the latter was used as an internal amplification control. The Y. pestis‐specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10–100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.     

左桡动脉在冠脉搭桥术中的应用

[目的]探讨左桡动脉作为移植血管在冠脉搭桥手术中的应用.[方法]93例冠脉搭桥术患者,手术测压试验决定是否切取桡动脉.桡动脉伴随静脉一起切取.术中采用不直接接触桡动脉的方法.[结果]平均桡动脉移植血管长(22±1)cm,术后流量(38±5)mL/min.与冠状动脉前降支、钝缘支、右冠脉血管单独或序贯吻合.无术中痉挛现象,围术期无出血栓塞合并症,术后左手无缺血合并症.[结论]桡动脉移植血管可常规应用于冠脉搭桥手术.本文介绍的切取方法可有效防止移植血管损伤及术后合并症.     

Changes in the probability of firing of motor units following electrical stimulation in human limb muscles

Changes in the probability of motor unit firing was studied in ten different muscles (six muscles in the upper extremity and four muscles in the lower extremity) of eleven healthy human subjects. The responses were elicited by the electrical stimulation of cutaneous or mixed nerves during weak voluntary contraction of the muscle studied, and were recorded by averaging the rectified surface electromyogram. In eight of the ten muscles, well-detectable, short and long latency excitatory phases were observed. The most constant and well-identified excitatory responses were observed in the first interosseus dorsalis muscle in the hand, and in the extensor digitorum brevis muscle in the foot. These two distal muscles seem to be the most useful muscles for routine determination of the short and long latency responses to cutaneous stimulation.     

Single right coronary artery assessed by contrast angiography and transoesophageal echocardiography

Contrast angiography is the gold standard for the assessmentof the site and severity of coronary artery narrowing. Transoesophagealechocardiography is recognized as a feasible technique in assessingcoronary artery anomalies. Our patient had arterioscleroticnarrowing in a single right coronary artery with poor distalopacification during contrast injection. Combining transoesophagealechocardiography with contrast angiography helped us in visualizinga surgically accessible vessel on the anterior wall of the leftventricle.     

A selective broth enrichment combined with real‐time nuc‐mecA‐PCR in the exclusion of MRSA

Pasanen T, Korkeila M, Mero S, Tarkka E, Piiparinen H, Vuopio‐Varkila J, Vaara M, Tissari P. A selective broth enrichment combined with real‐time nuc‐mecA‐PCR in the exclusion of MRSA. APMIS 2010; 118: 74–80. We analyzed the performance of a selective enrichment broth combined with Taqman‐based real‐time duplex nuc‐mecA‐PCR to expedite the screening of methicillin‐resistant Staphylococcus aureus (MRSA). We found the broth to be able to select MRSA strains (oxacillin MIC range 4–256 μg/ml) from MSSA strains. A total of 31 MRSA strains were found from 1250 clinical samples screened. The nuc‐mecA‐PCR was positive from all enrichment broths containing MRSA. From the remaining 1219 samples negative for MRSA on culture/subculture, 138 samples were nuc+/mecA+ in PCR. The sensitivity of the test was 93.5%, specificity 88.6%, positive predictive value 17.3%, and negative predictive value 99.8% as compared to culture. Thus, with this method, the negative MRSA results can be reliably reported within 24–48 h from sampling. The method is a practical additional alternative to those already described for the same purpose.