Rapid identification and susceptibility testing using the VITEK(R) 2 system using culture fluids from positive BacT/ALERT(R) blood cultures

BACKGROUND AND PURPOSE: In order to reduce the turnaround time for laboratory diagnosis of bacteremia, the efficacy of identification and antimicrobial susceptibility testing using samples taken directly from positive BacT/ALERT(R) standard aerobic and standard anaerobic blood culture bottles was evaluated. METHODS: 160 positive blood culture bottles were examined and incubated at 35 degrees C in 5% carbon dioxide for 4-24 h, and an aliquot of the culture fluid was Gram stained. Samples containing Gram-negative bacilli were inoculated on VITEK(R) 2 ID-GNB (identification-Gram-negative bacilli) and AST (antimicrobial susceptibility testing)-GN04 cards, and those containing Gram-positive cocci were inoculated on ID-GPC (identification-Gram-positive cocci) and AST-P526 cards. The same samples were also examined by the standard method, involving subculture from positive BacT/ALERT standard blood culture bottles. RESULTS: Eighty seven of 97 Gram-negative bacilli (89.7%) and 21 of 63 Gram-positive cocci (33.3%) were correctly identified to the species level. For antimicrobial susceptibility testing, the direct method had an overall error rate of 5.4% for Gram-negative bacilli, with 0.9% very major, 0.9% major, and 3.6% minor discrepancies compared to the standard method. The overall error rate in antimicrobial susceptibility testing for the 13 Staphylococcus spp. was 10.3%, with 6.0% very major, 2.6% major, and 1.7% minor discrepancies. CONCLUSION: These data suggest that VITEK 2 cards inoculated with samples taken directly from positive Bact/ALERT blood culture bottles would provide acceptable identification and antimicrobial susceptibility testing results for Gram-negative bacilli, but not for Gram-positive cocci. Compared to the standard method, the direct method would reduce turnaround time by at least 24 h.     

Characterization of an SAV organism and proposal of Mycobacterium triplex sp. nov.

Polyphasic taxonomic methods were employed to characterize a new species of slowly growing, nonpigmented mycobacteria. We propose the name Mycobacterium triplex sp. nov. for this new taxon. Conventional identification testing demonstrated a group of similar organisms that were geographically widespread in the United States. Commercially available nucleic-acid probes specific for the Mycobacterium avium complex were unreactive for these strains. High-performance liquid chromatography analysis of the mycolic acids revealed mycolate profiles that closely resembled Mycobacterium simiae. Comparative 16S rRNA sequence data confirmed the phylogenetic relationship of the strains with the slowly growing mycobacteria. Representative-type strains have been deposited in the American Type Culture Collection as strain ATCC 700071 [corrected].     

High selectivity of the i f channel to Na+ and K+ in rabbit isolated sinoatrial node cells

The ionic selectivity of the hyperpolarizationactivated inward current (i _f) channel to monovalent cations was investigated in single isolated sinoatrial node cells of the rabbit using the whole-cell patch-clamp technique. With a 140 mM K⁺ pipette, replacement of 90% external Na⁺ by Li⁺ caused a –24.5 mV shift of the fully activated current/voltage I/V curve without a significant decrease of the slope conductance. With a 140 mM Cs⁺ pipette, the i _f current decreased almost proportionally to the decrease in external [Na⁺]_o as Li⁺ was substituted. These responses are practically the same as those observed with N-methyl glucamine (NMG⁺) substitution, suggesting that the relative permeability of Li⁺ compared with Na⁺ for the i _f channel is as low as that of NMG⁺. When Cs⁺ or Rb⁺ was substituted for internal K⁺, the fully activated I/V relationship for i _f showed strong inward rectification with a positive reversal potential, indicating low permeability of the i _f channel for Cs⁺ and Rb⁺. These results show that the i _f channel is highly selective for Na⁺ and K⁺ and will not pass the similar ions Li⁺ and Rb⁺. Such a high degree of selectivity is unique and may imply that the structure of the i _f channel differs greatly from that of other Na⁺ and K⁺ conducting channels.     

Motor outcome according to the integrity of the corticospinal tract determined by diffusion tensor tractography in the early stage of corona radiata infarct

Diffusion tensor tractography (DTT) is useful for exploring the state of the corticospinal tract (CST). An accurate estimation of the integrity of the CST in the early stage of a cerebral infarct would enable a determination of motor recovery. DTT was performed to classify CST integrity following a corona radiata infarct to evaluate if the procedure could characterize the motor outcome of the affected hand. Fifty-five patients with completely paralyzed hands due to a corona radiata infarct were recruited for the study, and DTT images were obtained within 7–30 days after a stroke. The DTI findings for the patients were classified into four groups. In type A, the CST was preserved around the infarct; in type B, the CST originated from a cortex other than the primary motor cortex; in type C, the CST was interrupted at the infarct; in type D, the CST failed to reach the infarct due to degeneration. Six months after a stroke, the motor function of the affected hand was evaluated with the motricity index (MI) for the hand, the Medical Research Council score (MRC) for finger extensors and the modified Brunnstrom classification (MBC). These indices were significantly influenced by the DTT type (p < 0.05). The highest MI, MRC and MBC were seen in the DTT type A patients; the lowest MI, MRC and MBC were seen in the DTT type D patients (p < 0.05). The integrity of the corticospinal tract determined by DTT obtained during the early stage of a corona radiata infarct seems to be helpful in predicting the motor outcome of the affected hand.     

Evaluation of PCR-based assay for diagnosis of spotted fever group rickettsiosis in human serum samples

A nested PCR assay was developed for the detection of spotted fever group (SFG) rickettsiae in serum samples. The assay was based on specific primers derived from the rickettsial outer membrane protein B gene (rompB) of Rickettsia conorii. An SFG rickettsia-specific signal is obtained from R. akari, R. japonica, R. sibirica, and R. conorii. Other bacterial species tested did not generate any signal, attesting to the specificity of the assay. As few as seven copies of the rompB gene of R. conorii could be detected in 200 microl of serum sample. The assay was evaluated with a panel of sera obtained from patients with acute-phase febrile disease tested by immunofluorescent antibody assay (IFA). The SFG rickettsia-specific DNA fragment was detected in 71 out of 100 sera, which were proven to have immunoglobulin M antibodies against SFG rickettsial antigen by IFA. The results were further confirmed by restriction fragment length polymorphism and sequencing analysis of the DNA fragments. The results indicated that this PCR assay is suitable for the diagnosis of spotted fever group rickettsiosis in Korea.     

Immunohistochemical analysis of Smac/DIABLO expression in human carcinomas and sarcomas

Second mitochondria-derived activator of caspases (Smac/DIABLO) is released from mitochondria into the cytosol during apoptosis, promoting caspase activation by neutralizing the inhibition of inhibitor of apoptosis proteins (IAPs) on caspases. Alteration of apoptosis is essential for cancer development, and cancer cell death by radiation and chemotherapy is largely dependent upon apoptosis. In this study, archival tissues of 100 carcinomas and 50 sarcomas from various origins were analyzed by immunohistochemistry for the expression of Smac/DIABLO. Smac/DIABLO immunoreactivity was seen in 62 of 100 (62%) carcinomas, including 42 of 60 stomach carcinomas, 7 of 10 colorectal carcinomas, 4 of 10 lung carcinomas, 7 of 10 ovarian carcinomas, and 2 of 10 prostate carcinomas. Smac/DIABLO is expressed in 11 of 50 (22%) sarcomas, including 2 of 8 malignant schwannomas, 5 of 11 rhabdomyosarcomas, 2 of 7 malignant fibrous histiocytomas, 1 of 6 leiomyosarcomas, 0 of 8 angiosarcomas, 0 of 8 liposarcomas, and 1 of 2 Ewing's sarcomas. These data demonstrated that Smac/DIABLO expression levels vary depending on the individual cancer types. Furthermore, the present study showed that many human cancers do not express Smac/DIABLO, and suggest that lack of Smac/DIABLO expression in the cancer cells may inhibit apoptosis, thereby promoting their survival.     

Distinct patterns of cleavage and translocation of cell cycle control proteins in CD95-induced and p53-induced apoptosis

Apoptotic cell death induced by p53 occurs at a late G1 cell cycle checkpoint termed the restriction (R) point, and it has been proposed that p53-induced apoptosis causes upregulation of CD95. However, as cells with defective in CD95 signaling pathway are still sensitive to p53-induced apoptosis, CD95 cannot be the sole factor resulting in apoptosis. In addition, unlike p53-induced apoptosis, the relationship between CD95-mediated apoptosis and the cell cycle is not clearly understood. It would therefore be worth investigating whether CD95-mediated cell death is pertinent with p53-induced apoptosis in view of cell cycle related molecules. In this report, biochemical analysis showed that etoposide-induced apoptosis caused the induction and the nuclear translocation of effector molecules involved in G1 cell cycle checkpoint. However, there was no such translocation in the case of CD95-mediated death. Thus, although both types of apoptosis involved caspase activation, the cell cycle related proteins responded differently. This argues against the idea that p53-induced apoptosis occurs through the induction of CD95/CD95L expression.     

A novel missense mutation (I344K) in the SPG4gene in a Korean family with autosomal-dominant hereditary spastic paraplegia

 Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by slowly progressive spasticity and weakness of the lower extremities. Among eight loci linked with autosomal-dominant (AD)-HSP, the SPG4 locus on chromosome 2p22 accounts for about 40% of all patients. Recently, mutations in a new member of the AAA protein family, called spastin, have been identified as responsible for SPG4-linked AD-HSP. Here, we describe a novel missense mutation (c.1031T>A; I344K) in exon 7 of the SPG4 gene identified in a Korean family with typical clinical features of pure AD-HSP. The mutation affects the third amino acid of the highly conserved AAA cassette domain, which is the most fore part of the domain altered by a missense mutation reported so far. Clinical presentations of affected individuals carrying the I344K mutation were not different from those of pure AD-HSP with SPG4 mutations reported previously. However, it is noteworthy that neither urinary dysfunction nor involvement of upper extremities was noticed in this family. To our knowledge, this is the first report of genetically confirmed AD-HSP in Korea. Received: February 20, 2002 / Accepted: May 21, 2002     

In vitro biocompatibility assessment of sulfonated polyrotaxane-immobilized polyurethane surfaces

Sulfonated polyrotaxanes (PRx-SO(3)'s), in which sulfonated alpha-cyclodextrins (alpha-CDs) were threaded onto the poly(ethylene glycol) (PEG) segments in a PEG-b-poly(propylene glycol) (PPG)-b-PEG triblock copolymer (Pluronic) capped with benzyloxycarbonyl (Z)-L-phenylalanine (Z-L-Phe), were prepared as a novel surface-modifying biomaterial. Surface modification of the polyurethane (PU) was carried out by blending the PRx-SO(3)'s with a PU solution, followed by solution casting. The incorporated PRx-SO(3)'s led to the enhanced hydrophilicity by changing the surface properties of the PU matrix. Modified PUs showed the stable entrapment of the PRx-SO(3)'s with little extraction into water and enhanced mechanical properties after exposure to water compared to the PU control. The incorporated PRx-SO(3)'s repelled the proteins and kept them from closely approaching the surface areas, prevented platelet activation by thrombin, and effectively repelled bacteria. These results suggest that both the supramolecular structure of the polyrotaxanes and exposure of the sulfonated groups onto the surfaces contribute to these phenomena. Thus, surface modification with PRx-SO(3)'s is suggested to be useful for the fabrication of biocompatible medical devices.