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Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial components and selectively collect microorganisms are a prerequisite for successful identification, and a variety of home-brew and commercial protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We recently developed a method to collect bacteria from positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing. This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). The operation time required for processing using this novel kit is approximately 10 min, and the entire procedure can be completed within a biosafety cabinet. Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit as compared with the Sepsityper® kit. Performance of the rapid BACpro® II kit was evaluated using a total of 193 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the rapid BACpro® II kit or the Sepsityper® Kit, and isolated cells were subjected to direct identification by MS fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with >1.7 score and >2.0 score obtained using the rapid BACpro® II kit were 99.5% and 80.8%, respectively, whereas those obtained using the Sepsityper® Kit were 89.1% and 68.4%, respectively (P < 0.05 for >1.7 and P < 0.05 for >2.0 by Pearsons's chi-square). In Gram-positive cases, the rapid BACpro® II kit gave identification rate of 100% with >1.7 score and 69.4% with >2.0 score, whereas there were 84.7% and 56.8%, respectively by the Sepsityper® Kit (P < 0.05 for >1.7). These results are preliminary, but considering that this new kit is easy to perform and the identification rates are promising, the rapid BACpro® II kit deserves assessment in a larger-scale study with a variety of platforms for MS-based bacterial identification., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: A concentration of specimen is recommended for the effective recovery of Mycobacterium tuberculosis (MTB), but the bacteriological efficiency is not well evaluated. The present study evaluated the factors contributing to concentration efficiency of centrifugation and bead-based technique (TB-Beads; Microsens, UK) to recover MTB by using simple in vitro specimens., Methods: Four specimens were prepared (6.5×10(3); 8.1×10(4); 7.9×10(5); and 6.4×10(6)cfu/mL) of different concentrations with or without 5×10(4) of THP-1 cells (RIKEN BRC, Japan). Specimens were subjected to centrifugation at 2000, 3000, and 4000g for 15min, and to TB-Beads. The concentration and recovery rate were calculated to evaluate the efficiency of each method., Results: The specimens containing a higher number of bacteria and THP-1 cells had a tendency to yield a higher concentration and recovery rate (p=0.001-0.083). MTB was recovered more efficiently with THP-1 cells from the 6.5×10(3)cfu/mL specimen by centrifugation (p⩽0.001) than without them; 24.7-54.4% of MTB were recovered with THP-1 cells by centrifugation at 3000g for 15min, while the recovery using TB-Beads was a maximum of 12.7%., Conclusions: The efficiency of centrifugation depends on the bacterial density and the co-existence of THP-1 cells. The efficiency of TB-Beads was not as high as centrifugation., (Copyright © 2015 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.)

Background: Hospital cleanliness in hospitals with a tendency toward long-term care in Japan remains unevaluated. We therefore visualized hospital cleanliness in Japan over a 2-month period by two distinct popular methods: ATP bioluminescence (ATP method) and the standard stamp agar method (stamp method)., Methods: The surfaces of 752 sites within nurse and patient areas in three hospitals located in a central area of Sapporo, Japan were evaluated by the ATP and stamp methods, and each surface was sampled 8 times in 2 months. These areas were located in different ward units (Internal Medicine, Surgery, and Obstetrics and Gynecology). Detection limits for the ATP and stamp methods were determined by spike experiments with a diluted bacterial solution and a wipe test on student tables not in use during winter vacation, respectively. Values were expressed as the fold change over the detection limit, and a sample with a value higher than the detection limit by either method was defined as positive., Results: The detection limits were determined to be 127 relative light units (RLU) per 100 cm2 for the ATP method and 5.3 colony-forming units (CFU) per 10 cm2 for the stamp method. The positive frequency of the ATP and stamp methods was 59.8% (450/752) and 47.7% (359/752), respectively, although no significant difference in the positive frequency among the hospitals was seen. Both methods revealed the presence of a wide range of organic contamination spread via hand touching, including microbial contamination, with a preponderance on the entrance floor and in patient rooms. Interestingly, the data of both methods indicated considerable variability regardless of daily visual assessment with usual wiping, and positive surfaces were irregularly seen. Nurse areas were relatively cleaner than patient areas. Finally, there was no significant correlation between the number of patients or medical personnel in the hospital and organic or microbiological contamination., Conclusions: Ongoing daily hospital cleanliness is not sufficient in Japanese hospitals with a tendency toward long-term care.

The kenkiporter II (KP II) transport system is commonly used in many hospitals in Japan for transporting bacterial specimens to microbiology laboratories. Recently, the BBL Port-A-Cul (PAC) fluid vial became available. However, no reports thus far have compared the effectiveness of these two transport systems. We chose 4 aerobic and facultative anaerobic bacteria as well as 8 anaerobic organisms, and prepared three strains of each bacterium in culture media for placement into PAC and KP II containers. We compared the effectiveness of each transport system for preserving each organism at 6, 24, and 48 h after inoculation at room temperature. Thirty-six strains out of 12 bacteria were used in this study. The PAC system yielded better recovery in quantity of organisms than the KP II system at 6, 24 and 48 h. More strains were significantly recovered with the PAC system than with the KP II at 24 h (36/36 vs. 23/36, P < 0.001) and 48 h (30/36 vs. 12/36, P < 0.001). The PAC system was better in the recovery of viable organisms counted at 24 and 48 h after inoculation compared with the KP II system. The PAC system may be recommended for the transfer of bacterial specimens in clinical settings., (Copyright © 2013 Japanese Society of Chemotherapy and the Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: The urinary antigen detection kit for Streptococcus pneumoniae was tested., Objectives: It was our aim to evaluate the usefulness of the immunochromatographic membrane test by doing a large prospective study of community-acquired pneumonia (CAP) in Japan., Methods: We prospectively evaluated the use of the S. pneumoniae urinary antigen detection kit and analyzed the treatment and clinical effect seen in patients with positive test kit results. One hundred and fifty-six patients with CAP admitted to our hospital between October 2001 and September 2003 were evaluated., Results: In 49% of these CAP patients, the causative microorganisms were isolated. S. pneumoniae was suspected to be the causative microorganism in 15%, but positive results of the urinary antigen detection kit indicated S. pneumoniae to be a probable microorganism in 28%, even though antibiotics had previously been administered to half of the patients. The kit was particularly useful for diagnosing patients with poor quality sputum in whom antibiotics treatment nevertheless had to be selected. Antibiotics appropriate for S. pneumoniae (mainly penicillin) were given. The treatment was found to have excellent clinical results in 89% of the CAP patients., Conclusions: The S. pneumoniae urinary antigen detection kit was considered to be useful in selecting treatment since there was a high level of clinical effectiveness when the most suitable antibiotics were immediately administered to positive patients. The use of the S. pneumoniae urinary antigen kit is rapid and simple compared with conventional microbiological procedures., (Copyright (c) 2006 S. Karger AG, Basel.)

In the study presented here, the performance of the BDProbeTec ET system (Becton Dickinson, USA) was compared with the Roche Cobas Amplicor-PCR (Roche, Switzerland) to detect Mycobacterium tuberculosis complex (MTB) in clinical respiratory samples. The Bactec MGIT 960 liquid culture system (Becton Dickinson) was used as a reference method. A total of 411 samples were tested. Of the 93 culture-positive samples, both the BDProbeTec ET system and the Cobas Amplicor-PCR detected 87 (sensitivity, 93.5%). When only smear-negative samples were considered, the BDProbeTec ET exhibited a sensitivity of 50% and the Cobas Amplicor-PCR 60%. Specificity was 99.7% for the BDProbeTec ET system and 100% for the Cobas Amplicor-PCR. Percent agreement between the two nucleic amplification methods was 98.7%. Inhibition occurred in three (0.7%) samples in the BDProbeTec ET system. The high sensitivity and specificity of the BDProbeTec ET system suggest it is a useful method for the rapid and direct detection of MTB in smear-positive respiratory samples.

Interest in DNA sequencing has increased in recent years and research into new instrument systems to optimize and automate the process has been intensified. A number of commercial devices, from Europe, Japan, and the United States, which instrument aspects of (mainly) the Sanger method, are now available and several new DNA sequencing techniques have been reported. This review briefly describes current methods of DNA sequencing and provides an account of systems presently available or under development for instrumenting the technology. The problems of automation for large-scale DNA sequencing are discussed.