Pre-Analytical Modification of Serum miRNAs: Diagnostic Reliability of Serum miRNAs in Hemolytic Diseases.

Circulating microRNAs (miRNAs) are useful biomarkers of hemolysis. Since blood cells are the main origins of circulating miRNAs, we evaluated blood cell-related pre-analytical modification of the miRNA signatures during blood drawing and serum processing. The levels of miRNA before and after ex vivo blood drawing were analyzed with the reverse transcriptase-based polymerase chain reaction method. Furthermore, the changes of miRNA signatures caused by different time-lag between blood drawing and serum preparation by 24 h were evaluated. Finally, we compared the miRNA levels between leftover samples and samples of hemolytic diseases. Blood drawing procedure induced increments of red blood cell (RBC)-related miRNAs (miR-451a, miR-486) about 2-fold. One hour standing of blood samples before serum separation induced almost the same increases in RBC-related miRNAs. To test the clinical usefulness of miR-451a as a biomarker of hemolytic diseases, we analyzed miRNAs of samples from 10 normal subjects, 30 leftover samples in the clinical laboratory, and 20 samples from patients with hemolytic diseases. Serum miR-451a significantly increased in patients with hemolytic anemia more than the levels of pre-analytical modification. In conclusion, the pre-analytical modification of serum miRNAs did not disturb the usefulness of RBC-derived miRNAs as biomarkers of hemolytic diseases.

[On the identification accuracy of MALDI-TOF MS influence of pretreatment methods, types of medium, coating skills, and preservation methods].

In bacterial identification by MALDI-TOF MS, there are many reports of usefulness concerning direct identification from blood culture and identification of bacteria which cannot be identified with automatic analysis equipment. On the other hand, there are very few studies that investigate how various conditions influence on identification accuracy, such as the type of medium used for bacterial isolation and pure culture, the pretreatment methods, the difference in coating technique, and preservation methods. Therefore, we examined 10 strains of 2 drug-resistant bacteria species and 9 strains of 1 unnormal bacterium species. As a result, no significant differences were found in accuracy of identifying all strains of the target bacteria incubated for 24 hours and changing the types of medium, the pretreatment methods, and the coating techniques. In particular, methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum ß-lactamase (ESBL) producing Escherichia coli showed little change in the score value and the mass spectrum that assayed every 24 hours during the preservation period in all of the medium. In the case of Vibrio vulnificus, however, identification accuracy was decreased by the specific medium and storage conditions. It is suggested as this factor that the growth state of bacteria may have influenced the identification accuracy.

[On the performance evaluation of VersaTREK introduced in blood culture].

We evaluated performance of Versa TREK, blood culture system used in our hospital. Compared with BacT/ALERT 3D, the detection time of bacteria in the VersaTREK was shorter in most of strains. Compared with BacT/ALERT Virtuo, there was little difference in the detection time of bacteria. In addition, VersaTREK was able to detect Helicobacter cinaedi which could not be detected by other equipment, and H. cinaedi was detected in clinical specimens within 2 days. There were 147 bottles judged to be false positives at our facility, of which 7,290 were 2,0% of the total. Ninety one points eight percentage of the cause was due to the change in the temperature inside the device, 3.4% was due to incorrect procedure. So, it is considered that false positives are further decreased by appropriate management of the installation and sample collection.

[Evaluation of novel nucleic acid detection kit for Mycoplasma pneumoniae].

For diagnosis of Mycoplasma pneumoniae infection, highly sensitive and rapid diagnosis is important. Because antibiotics are limited for the treatment of M. pneumoniae infection. In this study, we evaluated new rapid nucleic acid detection kit for M. pneumoniae. This kit does not require excessive pretreatment of specimens and molecular diagnosis of M. pneumoniae is possible within 40 min. Using 120 nasopharyngeal specimens, we compared this kit with a commercially available molecular diagnostic reagent (LAMP). 51 of 120 cases were M. pneumoniae positive, and the results of both assays were all consistent. In addition, sequencing of 23S rRNA gene was performed on 51 cases positive for M. pneumoniae. As a result, macrolide resistance mutation (2063A>G) was observed in 19 cases (37.3%). The gene mutations estimated by this kit coincided completely with the sequencing. In conclusion, new rapid nucleic acid detection kit could detect M. pneumoniae with the same sensitivity as other molecular diagnostics, in a simple process.

[Development and Evaluation of a New Selective Culture Medium, KBM Anaero RS-GNR, for Detection of Anaerobic Gram Negative Rods].

The laboratory culture methods for isolating drug-resistant pathogens has been the gold standard in medical microbiology, and play pivotal roles in the overall management of infectious diseases. Recently, several reports have emphasized the development of antibiotics-resistance among anaerobic gram-negative rods, especially Genus Bacteroides and Prevotella. Therefore, a selective culture method to detect these pathogens is needed. We developed here the new selective culture medium, termed "KBM Anaero RS-GNR," for detecting anaerobic Gram-negative rods. Growth capability and selectivity of the agar medium were assessed by using the pure culture suspensions of more than 100 bacterial strains as well as the 13 samples experimentally contaminated with these bacterial strains. This new medium, "KBM Anaero RS-GNR," successfully showed the selective isolation of anaerobic Gram-negative rods. Compared with commercially available medium, "PV Brucella HK Agar, " which is also designed to detect anaerobic Gram-negative rods, there was no significant difference of the overall detection efficiency between two media. However, "KBM Anaero RS-GNR" showed superior to selectivity for anaerobic Gram-negative rods, especially from the samples contaminated with Candida species. Thus, the culture method using KBM Anaero RS-GNR is relevant for isolation of anaerobic Gram-negative rods especially from clinical specimens.