Rinsho Biseibutsu Jinsoku Shindan Kenkyukai shi = JARMAM : Journal of the Association for Rapid Method and Automation in Microbiology最新文献

Pathogenic bacteria of Mycoplasma pneumonia, Mycoplasma pneumoniae, do not respond to β-lactam antimicrobial agents. Therefore, it is clinically important to promptly identify infection with this type of bacteria. However, conventional examination methods such as culture and antibody tests are not useful for the rapid diagnosis of this bacterial type. In this study, we examined a Loopamp Mycoplasma P-detecting Reagent Kit developed based on the LAMP (Loop-mediated isothermal amplification) method, which may overcome the limitations of the conventional methods. The consistency rate for the conventional polymerase chain reaction (PCR) method was 98.3%. That for a rapid antibody test, immunocard Mycoplasma antibody, was 54.0%, suggesting the limitation of the rapid antibody test. We compared a pretreatment method using a Loopamp PURE DNA Extraction Kit, as a simple DNA extraction method, with that using a QIAamp DNA Mini Kit. The consistency rate was 91.4%, suggesting the usefulness of the simple DNA extraction method. The use of this kit may facilitate a more rapid diagnosis. The Loopamp Mycoplasma P-detecting Reagent Kit is useful for the accurate and rapid diagnosis of Mycoplasma pneumoniae infection.

Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenged for the identification of Nocardia species. However, the standard ethanol-formic acid extraction alone is insufficient in allowing the membrane proteins of Nocardia species to be ionized by the matrix. We therefore aimed to establish our new extraction method for the MALDI-TOF MS-based identification of Nocardia species isolates. Our modified extraction procedure is through dissociation in 0.5% Tween-20 followed by bacterial heat-inactivation, mechanical breaking of the cell wall by acid-washed glass beads and protein extraction with formic acid and acetonitrile. As reference methods for species identification, full-length 16S rRNA gene sequencing and some phenotypical tests were used. In a first step, we made our own Nocardia database by analyzing 13 strains (13 different species including N. elegans, N. otitidiscaviarum, N. asiatica, N. abscessus, N. brasiliensis, N. thailandica, N. farcinica, N. nova, N. mikamii, N. cyriacigeorgica, N. asteroids, Nocardiopsis alba, and Micromonospora sp.) and registered to the MALDI BioTyper database. Then we established our database. The analysis of 12 challenge strains using the our database gave a 100% correct identification, including 8 strains identified to the species level and 4 strains to the genus level (N. elegans, N. nova, N. farcinica, Micromonospora sp.) according to the manufacture's log score specifications. In the estimation of reproducibility of our method intended for 4 strains, both within-run and between-run reproducibility were excellent. These data indicates that our method for rapid identification of Nocardia species is with reliability, reproducibility and cost effective.

Since we had experienced a case of misidentification for group B Streptococcus (GBS) (Streptococcus agalactiae) by automated identification system RAISUS, we have investigated the identification accuracy for GBS by automated identification system RAISUS system using our stock isolated of GBS. Among 31 strains including standard strain ATCC13813 of GBS, 30 strains were identified as GBS, while only 1 strain was misidentified as Streptococcus constellatus. We have needed 3 hours to identify 29 true GBS strains, while it has taken 6 hours for misidentified strain as S. constellatus. Since we have needed more than 4 hours for our misidentified strain of GBS, the tendency for identification time to become long has been recognized. Longer identification time might be associated with the misidentification. The improvement for galactose and glycerol tests of RAISUS by the manufacturer has resulted in better identification. As for the strains to be identified as S. constellatus by RAISUS, we might re-check it with other identification kits when there is higher clinical necessity.

On 11 March 2011, an earthquake measuring 9.0 on the Richter scale off the northeast coast of Honshu Island, Japan, produced a devastating tsunami that destroyed many towns and villages near the coast in Iwate, Miyagi, and Fukushima prefectures. Miyagi Prefecture was the area most severely devastated by the tsunami, with extensive loss of life and property; hundreds of thousands of people lost their houses and were forced to move to evacuation areas. In the days and weeks following devastating natural disasters, the threat of infectious disease outbreak is high. Rapid diagnostic tests can be performed at or near the site of patient care and the tests were very useful in this disaster, because they enabled us to manage patients appropriately in the settings where medical resources were limited. Here we report actual cases where the rapid diagnostic tests for infectious diseases were useful in the patient management.

The centrifuge method with the use of Semi-Alkalin Proteinase (SAP) and NALC-NaOH, recommended by the "2007 edition of the assay guideline for detection of Mycobacterium tuberculosis," has significantly contributed to improving the sensitivities and specificities of both smear and culture tests for detection of acid fast bacilli (AFB). However, this method poses some challenges in terms of its cumbersome and time-consuming assay protocol. "TB-beads (Kyokuto Pharmaceutical Industrial Co., Ltd.)" is a newly-developed method for detection of AFB utilizing magnetic beads. We evaluated the quality of this method in comparison with the centrifuge method, focusing on the results of smear and culture tests. This evaluation study was conducted using both 5 positive and 5 negative sputum samples. The sensitivity of TB-beads for fluorescent smear tests, conducted using "Acri-stain," was almost the same as that of the centrifuge method. One advantage of TB-beads, however, was that it was very convenient to practice microscopic observation due to the clear background of the smeared glass slides. The comparison of the contamination rates between the two methods showed that TB-beads suggested significantly lower contamination rates. The centrifuge method resulted in 50% and 60% of contamination rates for HK Semisolid Isolation Medium and BacT/ALERT MP, respectively. On the other hand, the contamination rates of TB-beads for both of the culture methods were only 10%. With regard to the 5 positive sputum samples, the comparison of the detection rates between the centrifuge and TB-Beads method was made utilizing Myco Acid, Ogawa K, and BacT/ALERT MP. The TB-Beads method suggested higher detection rates for Myco Acid and Ogawa K, while there were no significant differences between the two methods for BacT/ALERT MP (16-23 days). TB-beads is an easy method that allows to simplify the process of smear tests, and contributes to significantly reducing the contamination rate of culture tests. It also contributes to improving the sensitivity and detection rate of AFB testing. Furthermore, it does not require centrifugation. Ultimately, TB-beads is an innovative, safe, and convenient testing method for detection of AFB, which enables laboratory technicians to save time for routine work.