Comparative study of rapid ATP bioluminescence assay and conventional plate count method for development of rapid disinfecting activity test.

Antimicrobial activity is tested when developing disinfectants, pharmaceutical products, cosmetics, and many other consumer products. However, the plate count method, the conventional way to count the number of microorganisms, needs several days of culture. Consequently, a means of rapid microbial detection is strongly desired to replace this method. We have already developed a rapid and sensitive microbial adenosine triphosphate (ATP) detection system utilizing ATP bioluminescence, which can quantify microbial ATP within 1 h. To apply this technique to antibacterial activity tests, the ATP method should be proved equal or superior to the conventional method. In this study, we conducted disinfectant activity tests comparing the ATP method and the plate count method, using polyhexamethylene biguanide (PHMB) in different concentrations (0-10 ppm) as a model disinfectant against Staphylococcus aureus and Aspergillus brasiliensis. We found that the log reduction of intracellular ATP had a positive correlation with the log reduction of the plate count. Moreover, the ATP method was able to distinguish different conditions of injured microbial cells that were observed using scanning electron microscopy, whereas colony counting detects only culturable cells. The ATP method is thus a rapid and useful alternative to the conventional method in the field of antimicrobial activity testing.

A Rapid ATP Bioluminescence-based Test for Detecting Levofloxacin Resistance Starting from Positive Blood Culture Bottles.

Administering appropriate antimicrobial therapy as early as possible is important for rescuing bacteremic patients. Therefore, rapid antimicrobial susceptibility tests in positive blood culture specimens have been diligently sought. Adenosine triphosphate (ATP) bioluminescence-based methods have been used for rapid antimicrobial susceptibility tests. However, blood culture specimens have not been examined in many studies, possibly due to abundant intracellular ATP in blood corpuscles resulting in false-susceptible results. In this study, we developed a rapid ATP bioluminescence-based method for detecting antibiotic resistance starting from positive blood culture. To minimize background ATP originating from blood corpuscles, specimens were centrifuged and the supernatant diluted with broth, and an ATP-eliminating reagent was then added to the bacterial suspension at the beginning of incubation. This newly devised procedure reduced the background ATP by more than five orders of magnitude. In a pilot study using levofloxacin, no false-susceptible results were observed in 15 clinical specimens. Furthermore, the results indicated that the rapid method provided additional information about bacterial activities with high resolution, in contrast to the less-thorough findings with the conventional turbidity method. Therefore, our approach will contribute to the treatment of infectious diseases as a rapid antimicrobial susceptibility test.

Attomol-level ATP bioluminometer for detecting single bacterium.

We have developed an automated high-sensitive ATP bioluminometer for detecting single bacterium. The apparatus consists of a tube rack for setting reagents and samples, two washing baths for preventing sample carry-over from dispenser nozzle, and x-, y-, z- actuators for moving the dispenser, and an high-sensitive optical system. The reaction tube was selected to reduce the background signal intensities for the ATP bioluminescence measurement. The background signal intensity of the reaction tube was 18 RLU, which is almost the same as the dark counts of the photomultiplier (16 RLU). The ATP calibration curve was linear from 0 to 5 amol (its slope = 22.4 RLU/amol and 3.3 SD of the blank sample signal = 17.9 RLU), and the detection limit of 0.8 amol was obtained. The relationship between intracellular ATP and CFU in Escherichia coli (ATCC25922) was kept linearity from 0 to 20 CFU, and the intracellular ATP (amol) per CFU was calculated to be 3.3 amol/CFU (R2 = 0.9713). Moreover, the relationship between intracellular ATP and CFU in Staphylococcus aureus (ATCC25923) was also kept linearity from 0 to 30 CFU, and the amol/CFU was calculated to be 1.6 amol/CFU (R2 = 0.9847). The automated ATP bioluminometer has ultra-high sensitivity and will be a powerful tool for measuring ATP luminescence derived from small number of bacteria.

A 1-year, prospective, observational study of Japanese outpatients with type 1 and type 2 diabetes switching from insulin glargine or detemir to insulin degludec in basal-bolus insulin therapy (Kumamoto Insulin Degludec Observational study).

AIMS/INTRODUCTION: The aim of the present prospective observational study was to assess long-term efficacy and safety of insulin degludec as a part of a basal-bolus therapy for Japanese patients with type 1 or type 2 diabetes in routine clinical practice. MATERIALS AND METHODS: In the present study, 93 type 1 diabetes patients and 135 type 2 diabetes patients treated with insulin glargine or detemir were switched from their basal insulin to insulin degludec. The primary end-points were the changes in glycated hemoglobin (HbA1c) from baseline at 3, 6 and 12 months. The secondary end-points were changes in body mass index, insulin dose, frequency of hypoglycemia and adverse events. RESULTS: HbA1c levels from baseline were significantly reduced at 3, 6, and 12 months by 0.4, 0.4 and 0.3% in type 1 diabetes patients, respectively, and by 0.5, 0.5 and 0.3% in type 2 diabetes patients, respectively. Body mass index in type 1 diabetes patients increased significantly (P < 0.05), whereas that in type 2 diabetes patients did not change. Basal insulin dose decreased significantly at 3 months after switching (P < 0.05), and returned baseline dose at 12 months in type 1 diabetes and type 2 diabetes patients. The frequency of both total and nocturnal hypoglycemia decreased significantly in type 1 diabetes and type 2 diabetes patients (P < 0.05). The result of multiple regression analysis showed that baseline HbA1c was a significant independent variable of the percentage change in HbA1c with switching. CONCLUSION: In both type 1 diabetes and type 2 diabetes patients, switching from insulin glargine or insulin detemir to insulin degludec led to improvement of glycemic control with a significant reduction of hypoglycemia.

[The relationship between human papillomavirus (HPV) infection and penile cancer].

Human papillomavirus (HPV) may be carcinogenic effectors in a variety of human lower genital tract malignancies. We evaluated HPV status with respect to clinical and pathological features and prognosis of penile carcinoma. We searched for HPV infected cells (Koilocytosis) within the primary lesion of cancer tissue from 78 patients with penile squamous cell carcinoma. The following variables were recorded : age, tumor size, clinical stage, lymphatic and venous invasion, histologic and nuclear grade, Broders grade, infiltration status, and lymph node and distant metastasis. Koilocytosis were detected 55.1% (43 of 78) of patients. Tumors with Koilocytosis had better differentiation (p=0.0443) and lower grade (better keratinized) in Broders grading system (p=0.0116) than HPV negative tumors. No difference was found in the 5-year survival rate (p=0.5693). Our data suggest that the presence of Koilocytosis does not influence prognosis in penile cancer.

Automated bead alignment apparatus using a single bead capturing technique for fabrication of a miniaturized bead-based DNA probe array.

We have developed an automated bead alignment apparatus for fabricating a bead-based DNA probe array inside a capillary. The apparatus uses 16 micro vacuum tweezers to extract single beads from among a large amount of beads in bead stock wells. It then manipulates single beads into the probe array capillaries. Single 100-microm-diameter beads were successfully extracted from the water-contained bead-stock well by the vacuum tweezers, which have inner and outer diameters of 50 and 150 microm. An interesting aspect is that unexpected extra beads adsorbed on the outer wall of the vacuum tweezers can be removed using the surface tension force between the water and the atmosphere. In testing the total performance of this apparatus, the DNA probe arrays with 10 sets of probe-conjugated beads and 2 plain beads were produced in the intended order in the capillaries. The time needed to align the 12 beads was 10 min, and the 16 bead arrays were fabricated simultaneously. After hybridization experiments using these fabricated DNA probe arrays, fluorescence from each bead was clearly observed.

A bead-alignment device with a bead-sized microchamber on a rotating cylinder for fabrication of a miniaturized probe array.

We have developed a compact bead-alignment device with a bead-sized microchamber on a rotating cylinder. The cylinder fits inside a tube with bead-stock pipes containing different probe-conjugated beads and holes for bead-alignment capillaries. The cylinder rotates in the tube, and the microchamber transfers a single 100-microm-diameter bead from a pipe to one of the capillaries in 10 s. By using this process repeatedly, 'bead arrays', which are miniaturized DNA probe arrays in capillaries, were successfully fabricated.

DNA probes on beads arrayed in a capillary, 'Bead-array', exhibited high hybridization performance.

A DNA analysis platform called 'Bead-array' is presented and its features when used in hybridization detection are shown. In 'Bead-array', beads of 100- micro m diameter are lined in a determined order in a capillary. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1-300 micro l) into the capillary with reciprocal flow. For hybridization detection, as little as 1 amol of fluorescent-labeled oligo DNA was detected. The hybridization reaction was completed in 1 min irrespective of the amount of target DNA. When the number of target molecules was smaller than that of probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. 'Bead-array' enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform seems very promising for various genetic testing tasks.