[Selection of Laboratory Procedures to Detect Toxigenic by the 2-Step Method].

The 2-step method is an algorithm to detect toxigenic Clostridium difficile. We herein compared the sensitivities and specificities of an enzyme immunoassay (toxin A/B-EIA), toxigenic culture (TC-EIA), Loop-Mediated Isothermal Amplification assay (LAMP), and Xpert C. difficile (Xpert) with the detection of the toxin B gene by a polymerase chain reaction (PCR). The results obtained showed that the sensitivities and specificities of toxin A/B-EIA, Xpert, TC-EIA, and LAMP were 30 and 100%, 87.2 and 100%, 97.5 and 89.7%, and 95 and 100%, respectively. We also evaluated the turnaround time (TAT) and cost of toxigenic C. difficile detection. Our hospital TAT for toxin A/B-EIA and TC-EIA are 37 min and 5 days, respectively. We estimated the TAT of Xpert, LAMP, and PCR to be 105 min, 5 days, and 6 days, respectively. On the other hand, the cost to detect toxigenic C. difficile increased in the order of TC-EIA, LAMP, Xpert, and PCR. We have never experienced outbreak of Clostridium difficile infection (CDI) in our hospital, and there is less the number of CDI than other place. So we selected TC-EIA that is good sensitivity and low cost per specimen. Hereafter it'll be necessary to solve a problem it takes time, because we have to respond to outbreak of CDI quickly if it happens.

[Evaluation of MRSA rapid detection by real-time PCR directly from positive blood cultures].

Methicillin-resistant Staphylococcus aureus (MRSA), a well-known causative multidrug-resistant pathogen responsible for nosocomial and community-acquired infections, particularly in blood stream infection, often proves difficult and expensive to treat. Despite the need for rapid, accurate MRSA detection for treatment and infection control, conventional testing including culture, have sensitivity and turn-around time (TAT) problems. We evaluated BD GeneOhm MRSA Detection Kit rapid detection performance directly from positive blood culture using real-time PCR. The kit recognizes, a specific part of the staphylococcal cassette chromosome mec (SCCmec) gene, not a mecA gene. Compared to conventional culture in 138 samples with gram stains showing gram-positive cocci (GPC) clusters, the kit's sensitivity was 100%, specificity 97.3%, positive predictive value 90% and negative predictive value 100%. Three of the 27 MSSA isolates found was false-positive, indicating that the kit detected SCCmec/orfX region sequences lacking mecA. Coupled with direct tube coagulase testing to rapidly differentiate MRSA from methicillin-susceptible S. aureus (MSSA) could provide optimum treatment through appropriate antibiotic use. The kit thus appears to be useful in rapidly diagnosing MRSA from blood culture, improving the prognosis and reducing medical cost.

[Evaluation of the MRSA rapid detection assay (BD GeneOhm MRSA detection kit) by a real-time PCR].

Methicillin-resistant Staphylococcus aureus (MRSA) is well known to be a causative pathogen of skin and soft tissue or blood stream infections, and also to be a nosocomial drug-resistant bacteria in healthcare settings. Although a rapid and accurate detection of MRSA is indispensable for infection control, the conventional tests including culture method have some problems of sensitivity, procedure time, and so on. We evaluated the performance of the rapid detection assay of MRSA (BD GeneOhm MRSA Detection Kit) directly from specimens by a real-time PCR. The principle of this kit is characterized by recognizing not a mecA gene, but a specific part of SCCmec gene. Limits of detection of this method was 810 CFU/mL. Compared to the results of mecA PCR assay in 105 clinically isolated samples, the sensitivity, specificity, positive predictive value and negative predictive value were 100%, 97.4%, 98.5% and 100%, respectively. One of the 38 mecA negative isolates was found to be a positive result, this finding suggested that this method detect sequences of SCCmec/orfX region lacking of mecA. Because of the rapid turn-around time and the excellent negative predictive value, this method appears to be a useful tool for rapid diagnosis of MRSA.

[Establishment of a microbiology laboratory open 365 days a year and its impact].

The microbiology laboratory of our university hospital aims to provide accurate and rapid microbiological results and useful information for healthcare workers involved in both the treatment of infectious diseases and infection control. For this purpose, we have been running a microbiology laboratory open 365 days a year since 2005. Before starting this laboratory, we formulated both a precise procedural manual and educational program to increase the number of microbiological technologists from 4 to 8 persons and improve their skills. Moreover, we reviewed the reporting system. As a result, we could report positive blood cultures up to 1.4 days earlier than previously possible, and significantly improved the prognosis of MRSA bacteremia patients by the early treatment of anti-MRSA antimicrobials within 48 hours after positive blood culture. In addition, the rate of MRSA/Staphylococcus aureus decreased to 35.8%. It is essential for the treatment of infectious diseases and infection control to accept only appropriate specimens and report the results rapidly and accurately.

[Microbiology laboratory as a base of information sending].

The goal of our microbiology laboratory is to provide an accurate microbiological result and a useful information for every healthcare workers (HCWs). For this purpose, we were trying to do several activities, such as improving the work-flow of microbiology testings, starting 365-day-open microbiology tests, providing some training courses of microbiology and sending many useful informations about infectious diseases and infection control. Before these activities, we needed another 5 microbiology technicians beside 3 technicians and had started the program to educate them. We have successfully finished it and enabled all plans begin in April, 2005. Since then we are open for 365 days and also sending HCWs many newsletters for performing effective microbiological testings via the intra-network system and having lectures for both doctors and nurses, especially for new resident doctors at the orientation. We had also the training course for certified infection control nurses and accepted two technicians from Africa, who came to study a basic microbiology via JICA. These activities have enabled every technician not only to report and analyze microbiological test result effectively but also to improve writing and presentation skills. Through these activities all technicians have realized that accurate and rapid information from a microbiology laboratory is a key to treat patients with infectious diseases and improve their prognosis. It is suggested that skill-up of technicians lead to report an accurate result in microbiology and at the same time improve the attitude for their job.