Diagnostic Utility of a Mycobacterium Multiplex PCR Detection Panel for Tuberculosis and Nontuberculous Mycobacterial Infections.

The prevalence of nontuberculous mycobacterial diseases is increasing worldwide, and tuberculosis remains highly prevalent. Rapid and accurate microbial diagnoses of both tuberculosis and nontuberculous mycobacterial infections are required. A novel PCR-reverse sequence-specific oligonucleotide probe (PCR-rSSO) method-based mycobacterial detection panel (Myco-Panel) test was developed for the rapid identification of 30 mycobacterial species and subspecies. Clinical respiratory samples were collected from patients with suspected or confirmed tuberculosis and nontuberculous mycobacterial lung disease and those with other respiratory diseases. Myco-Panel tests were performed on the samples, and liquid mycobacterial culture and identification were performed for reference according to housekeeping gene sequences of mycobacteria in positive culture tubes. Furthermore, to assess the detection performance for several mycobacterial species rarely recovered in Japan, the accuracy of the Myco-Panel test was investigated using stock mycobacterial type strains and clinical isolates. A total of 178 clinical respiratory samples were analyzed. The Myco-Panel and sequence-based identification results for mycobacterial cultures were 83.1% concordant (kappa coefficient, 0.785 [95% confidence interval, 0.716 to 0.854]). The Myco-Panel correctly identified 281 of the 283 type strains and clinical isolates tested. The Myco-Panel test could accurately detect several mycobacterial species from clinical respiratory samples and mycobacterial suspensions. Rapid and accurate identification of pathogens using respiratory samples is possible using the Myco-Panel. IMPORTANCE Species identification is important for the diagnosis of mycobacterial infections and decisions on treatment regimens. The Myco-Panel test accurately detects clinically common mycobacterial species that cause respiratory infections from clinical respiratory samples and mycobacterial suspensions. The rapid identification of multiple mycobacterial species will provide clinically useful information for the management of patients. Although we understand that the current diagnostic criteria require mycobacterial culture results in general for the diagnosis of nontuberculous mycobacterial infection, mycobacterial culture examination is a time-consuming process. The detection of potentially causative agents directly from clinical samples will aid in practical diagnosis and decision-making for rapid treatment initiation. This is a new laboratory method for species identification, and evaluating its performance is important.

Structural basis for the high thermal stability and optimum pH of sphingomyelinase C from Streptomyces griseocarneus.

Sphingomyelinase C (SMC) hydrolyzes sphingomyelin to ceramide and phosphocholine. Prokaryotic SMCs share sequence homology with mammalian SMCs that have enzymatic pH optima at neutral pH. SMC from the nonpathogenic prokaryote Streptomyces griseocarneus shows notable enzymatic features such as higher optimum pH and thermostability than other prokaryotic SMCs. Determination of the three-dimensional structure of S. griseocarneus-SMC (Sg-SMC) and comparison with other SMC structures represents a promising strategy to elucidate the unique enzymatic features of Sg-SMC on a structural basis. Therefore, we determined the crystal structure of Sg-SMC at 2.0 Å resolution by X-ray crystallography. Comparison of the Sg-SMC structure with three other structurally known SMCs from Listeria ivanovii, Bacillus cereus, and Staphylococcus aureus indicated that Sg-SMC is more diverse in sequence and that structural differences in the main chain between these SMCs are primarily located on the molecular surface distant from the active site. Comparison of the surface area of the four SMCs revealed that Sg-SMC has the most compact structure, which may contribute to the enhanced thermostability of Sg-SMC. Regarding the hydrogen bond network in the active site of Sg-SMC, a basic amino acid, Arg278, is involved, whereas the corresponding residue in other SMCs (Ser or Asn) does not form hydrogen bonds with metal-coordinating water molecules. Hydrogen bond formation between Arg278 and a Mg2+ ion-coordinating water molecule may be responsible for the higher optimal pH of Sg-SMC compared to that of other SMCs.

Method to calculate frequency characteristics of reconstruction filter kernel in X-ray computed tomography.

A computed tomography (CT) image is generally reconstructed by a filtered back projection (FBP) algorithm. In an FBP algorithm, the image quality primarily depends on a reconstruction filter kernel. Although the details of the filter kernel are not disclosed to users, the frequency response of the filter kernel can theoretically be calculated using the relational formula of the filter kernel and the modulation transfer function (MTF) of the reconstruction algorithm (MTFA). In this study, we proposed a method to determine the frequency response of a filter kernel and verify its validity. Two clinical CT scanners were used to derive the filter kernel. The MTF was obtained and subsequently separated to the MTF of the scanner system and MTFA. Using the relational formula of the filter kernel and MTFA, we calculated the frequency response of the filter kernel. To verify the calculated result, we measured the noise power spectrum (NPS). Additionally, the filter kernel was calculated using the relational formula of the filter kernel and NPS. In both CT scanners, the filter kernels calculated by the two methods showed good agreement, and we confirmed the validity of the results and the effectiveness of the proposed method. Furthermore, the inherent image quality performance of the CT scanner could be clarified by the reconstruction filter kernel.

Estimation and validation of the frequency responses of a scanner system and an image reconstruction system in X-ray computed tomography.

In computed tomography, factors that theoretically affect the modulation transfer function (MTF) in the region near the isocenter are the frequency responses of the scanner system (MTFS) and reconstruction processing (MTFA). Although MTFS and MTFA are performance indices that are not disclosed to the users, both can be estimated by the measured MTF with the use of theoretical formulas. In this study, we proposed two methods to obtain the MTFS and MTFA, and confirm their validity. The first method to obtain the MTFS and MTFA uses a theoretical formula and the measured MTF. Another method uses the measured MTF and the noise power spectrum. In both the methods, the MTFS and MTFA were obtained separately. By our proposed methods, performance indices that are not usually disclosed to the users can be known.

Kinetic characterization and Mg2+ enhancement of Streptomyces griseocarneus sphingomyelinase C produced by recombinant Streptomyces lividans.

Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550-950 U/mg) and was obtained in high yields (approximately 120 mg/L of culture). SMC activity was enhanced by MgCl(2), and the maximum activity (542±25 U/mg) was observed in the presence of 1.5 mol/L (M) MgCl(2). Dynamic light scattering analysis proved that the highest specific SMC activity was obtained with the smallest mixed micelles of sphingomyelin (SM) and Triton X-100. The turnover rate (k(cat)), K(m) and k(cat)/K(m) values for SM were 346 s(-1), 0.458 mM, and 756 mM(-1)s(-1), respectively, in the presence of 1M MgCl(2). The k(cat) was strongly influenced by the MgCl(2) concentration. By contrast, the K(m) value was independent of the MgCl(2) concentration and was almost constant. Circular dichroism spectroscopy indicated that MgCl(2) did not cause local structural changes in SMC. From these results, we concluded that the SMC activity enhancement by MgCl(2) was caused by the increased specific surface area of the mixed micelles composed of substrate, SM, and Triton X-100.

Extracellular production of a sphingomyelinase from Streptomyces griseocarneus using Streptomyces lividans.

The structural gene for sphingomyelinase (SMase) from Streptomyces griseocarneus, was introduced into Streptomyces lividans using a shuttle vector, pUC702, for Escherichia coli/S. lividans. High-level secretory production of SMase was achieved using the promoter, signal sequence and terminator regions of phospholipase D from Streptoverticillium cinnamoneum. The transformant constitutively expressed a high specific activity of SMase extracellularly during batch culture. Maximum SMase activity (555 ± 114 U/mg protein) was with 1.75 M MgCl(2) which was about 50-fold more than that with 10 mM MgCl(2).

Preparation of cyanoacrylate nanoparticles using monosaccharides or disaccharides.

By using biomaterials, including monosaccharides and disaccharides, as polymeric stabilizing agents, the cyanoacrylate nanoparticles was prepared respectively, with particles diameter of approximately 200 nm or 300 nm. The new method was applied to load/encapsulate ampicillin (ABPC) and pDNA into nanoparticles. Loading efficiency of ABPC was increased compared to the existing method in which dextran is used as a stabilizer. The pDNA encapusulation rate was 68.7%, by using glucose.