Developing and expanding hospital antimicrobial stewardship: The Ontario experience.

Mount Sinai Hospital and University Health Network, two academic health science centres in Toronto, Ontario, jointly established a robust, well-resourced antimicrobial stewardship program (ASP). Over the course of four years, we spread our program to five intensive care units (ICUs), learned which change management practices worked and which did not, and leveraged our ICU successes to other areas of our hospitals. We identified the following two factors as critical to establishing ASPs in hospitals: strong leadership with clear accountability; and valid, reliable data to monitor progress. Subsequently we have led the spread of our program to 14 academic hospital ICUs, and more recently we leveraged to help community hospitals implement ASPs without in-house infectious diseases specialists. We introduced three new data fields into the provincial critical care information system: days of antibacterial therapy, days of antifungal therapy, and ICU-onset C. difficile, which will help standardize data collection moving forward. This model-starting with academic health sciences centres, and antimicrobial stewardship experts and leaders who are then supported to mentor and develop new experts and leaders-could be copied in other jurisdictions both within and outside of Canada.

Troglitazone inhibits cell growth and induces apoptosis of B-cell acute lymphoblastic leukemia cells with t(14;18).

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear receptor superfamily, has been detected in several human leukemia cells. Recent studies reported that PPARgamma ligands inhibit cell proliferation and induce apoptosis in both normal and malignant B-lineage cells. We investigated the expression of PPARgamma and the effects of PPARgamma ligands on UTree-O2, Bay91 and 380, three B-cell acute lymphoblastic leukemia (B-ALL) cell lines with t(14;18), which show a poor prognosis, accompanying c-myc abnormality. Western blot analysis identified expression of PPARgamma protein and real-time PCR that of PPARgamma mRNA on the three cell lines. Troglitazone (TGZ), a synthetic PPARgamma ligand, inhibited cell growth in these cell lines in a dose-dependent manner, which was associated with G(1) cell cycle arrest and apoptosis. We also found this effect PPARgamma independent since PPARgamma antagonists failed to reverse this effect. We assessed the expression of c-myc, an apoptosis-regulatory gene, since c-myc abnormality was detected in most B-ALL cells with t(14;18). TGZ was found to dose-dependently downregulate the expression of c-myc mRNA and c-myc protein in the three cell lines. These results suggest that TGZ inhibits cell growth via induction of G(1) cell cycle arrest and apoptosis in these cell lines and that TGZ-induced apoptosis, at least in part, may be related to the downregulation of c-myc expression. Moreover, the downregulation of c-myc expression by TGZ may depend on a PPARgamma-independent mechanism. Further studies indicate that PPARgamma ligands may serve as a therapeutic agent in B-ALL with t(14;18).

Modification of cytokine milieu by A2A adenosine receptor signaling--possible application for inflammatory diseases.

Pro-inflammatory cytokine TNF-alpha (TNF) production from in vitro lipopolysaccharide (LPS)-stimulated human peripheral blood CD14+ cells (PB-CD14) was inhibited by A2A adenosine receptor (AdoR) (A2AR) or beta2 adrenergic receptor (ADR) (beta2R) signaling in a concentration-dependent manner. These inhibitory effects were presumably mediated by the increase in intracellular cAMP. Furthermore A2AR agonist and beta2R agonist synergistically inhibited the TNF production of LPS-stimulated PB-CD14 cells. These results suggest that the anti-inflammatory effect of extracellular adenosine is, at least in part, due to the modification of the cytokine milieu via A2A signaling, and that the targeting of both A2AR and beta2R may have strong therapeutic potential for the inflammatory diseases.

Quantitative nested reverse transcriptase PCR vs. real-time PCR for measuring AML1/ETO (MTG8) transcripts.

A quantitative nested reverse transcriptase polymerase chain reaction (QN-RT-PCR) method was developed using a plasmid cDNA containing the AML1/ETO (MTG8) fusion transcript from Kasumi-1 cells, an acute-myelogenous leukemia cell line with the t(8;21) translocation. In this method, the plasmid was detectable at a concentration of 10(-17) m. The fusion transcript in a mixture of 10(7) Rice94 (Burkitt lymphoma cell line) cells containing two Kasumi-1 cells was detectable at 10(-17) m. In a previously published real-time PCR method, the plasmid containing the fusion transcript was detectable at 10(-16) m or higher, and 20 or more Kasumi-1 cells were detectable in 10(7) Rice94 cells. Thus, this QN-RT-PCR method is more sensitive than the real-time PCR. When the same samples were examined by real-time PCR and our QN-RT-PCR method, in one patient in clinical remission after chemotherapy and allogeneic-bone marrow transplantation (BMT), the transcript was detected by QN-RT-PCR 60 days prior to hematological relapse, in contrast to 10 days before hematological relapse by real-time PCR. The transcript level was below 10(-17) m (undetectable) with this QN-RT-PCR in patients in clinical remission after chemotherapy and BMT, while it was 10(-15)-10(-16) m in patients in clinical remission after chemotherapy alone. The quantitative difference of the transcript level in minimal residual disease (MRD) between these two different types of clinical remission was estimated to be at least 10(2)-fold. This QN-RT-PCR method is useful for predicting hematological relapse and for quantitatively estimating MRD in different types of clinical remission.

Activation-induced cytidine deaminase expression in follicular lymphoma: association between AID expression and ongoing mutation in FL.

Activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) gene. AID has been reported to be specifically expressed in the germinal center (GC). Follicular lymphoma (FL) cells are known to be exposed to GC reaction, as characterized by a high degree of SHM with some heterogeneity in terms of intraclonal microheterogeneity and antigen selection. The heterogeneity of SHM pattern in FL intrigued us to investigate the AID expression. AID expression was investigated in 19 FL materials consisting of 15 cases of FL fresh cells and four cell lines. In all, 10 fresh cells and three cell lines expressed AID, but the others did not. SHM was investigated in 12 fresh cells and four cell lines. The ongoing mutation was significantly different between AID-positive and AID-negative FL fresh cells (unpaired Student's t-test, P=0.047). Ongoing mutation was not seen in any of the cell lines. AID expression was associated with the ongoing mutation in FL fresh cells (two-tailed Pearson's coefficient correlation, r=0.899, P=0.01). The switch off of AID expression may start in the B-lineage differentiation stage counterpart of FL after optimizing SHM, indicated by the cessation of the ongoing mutation in AID-negative FL fresh cells.

Hospital outbreak of MEN-1-derived extended spectrum beta-lactamase-producing Klebsiella pneumoniae.

An outbreak of Klebsiella pneumoniae resistant to broad spectrum cephalosporins occurred in a hospital in the Kinki area in Japan. During 18 months, from February 1998 to July 1999, 23 strains were isolated from 21 patients (10 with pneumonia, 4 with urinary tract infection, 1 with sepsis, 1 with vaginosis, 1 with a wound infection, and 1 with both pneumonia and sepsis; 3 patients showed noninfective colonization with K. pneumoniae) in seven wards, including the intensive care unit. MEN-1-derived gene was detected by polymerase chain reaction from the majority of the strains. Ninety-nine strains of K. pneumoniae were isolated during this period. The isolation rate of K. pneumoniae resistant to broad spectrum cephalosporins was 21%. We distinguished three clones by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis, and one of them was isolated from 18 patients. The presence of an R-plasmid of more than 160 kb was confirmed by plasmid analysis, but it was not possible to obtain transconjugants from all strains. This outbreak of K. pneumoniae was immediately confirmed by genetic analysis, and it was promptly ended by the infection control procedures. This is the first hospital outbreak of MEN-1-producing K. pneumoniae in Japan.

The hippurate ratio as an indicator of functional hepatic reserve for resection of hepatocellular carcinoma in cirrhotic patients.

Predicting the ability of the cirrhotic liver to withstand resection remains a challenge for the surgeon. This study evaluates the use of the hippurate ratio, a novel assessment of glycine conjugation of para-aminobenzoic acid by the liver, as a preoperative indicator of functional hepatic reserve. Between 1998 and 2000, sixty-one cirrhotic patients were prospectively assessed for hepatic resection using the hippurate ratio, indocyanine green retention at 15 minutes (ICG R-15), and other standard measures of liver function. Twenty-six patients were excluded as candidates for resection on the basis of inadequate functional hepatic reserve. Patients excluded from resection had significantly higher ICG R-15 values (29% +/- 9% vs. 16% +/- 12%, P = 0.001), higher Child-Pugh scores (5.9 +/- 0.9 vs. 5.3 +/- 0.4, P = 0.01), and lower hippurate ratios (30% +/- 14% vs. 45% +/- 15%, P = 0.005). There was a significant correlation between the hippurate ratio and ICG R-15. Other indicators of liver function such as factor V, factor VII, albumin, bilirubin, prothrombin time, and transaminases were no different between patients who did and those who did not undergo resection. Of the 35 patients resected, there were seven (20%) who developed varying degrees of liver failure with three perioperative deaths (8.5%). Patients who had some degree of liver failure had significantly lower hippurate ratios than patients who had no liver failure (29% +/- 10% vs. 48% +/- 14%, P = 0.002). There was no difference in ICG R-15 values between patients who had liver failure and those who did not. The hippurate ratio offers information on hepatocellular reserve that is not provided by other measures of liver function and may allow better selection of cirrhotic patients for liver resection.

[Search for Staphylococcus aureus heterogeneously resistant to vancomycin (hetero-VRSA) in MRSA strains isolated from clinical samples during 1990s].

Hetero-VRSA was studied in 978 MRSA strains isolated from clinical samples during 1991 to 1998. Although no VRSA was detected, 23 strains (2.4%) were identified as hetero-VRSA by the vancomycin-resistance using MU3 agar plate. The frequency of hetero-VRSA was not increased in the course of time. MIC of the hetero-VRSA to vancomycin and teicoplanin was 1-2 micrograms/ml and 0.5-12 micrograms/ml, respectively. All of the hetero-VRSA strains were confirmed as a heterogeneous strain by a population analysis. Although 43% of the hetero-VRSA strains were coagulase type II, positive for TSST-1, and enterotoxin type C, others were various in the characteristics. In the gene analysis by pulse field gelelectrophoresis (PFGE), 4 sets of 2 strains were found to be identical among the 23 strains but the other 15 strains were genetically different. We speculate that hetero-VRSA strains were generated in 1991 secondary possibly by use of beta-lactam antibiotics.

[Detection methods for drug-resistant bacteria in routine examination--VISA].

MRSA strains which clinically showed low-level resistance to vancomycin(VISA, hetero-VRSA) were isolated. Vancomycin-intermediate Staphylococcus aureus(VISA) was found to show a vancomycin minimum inhibitory concentration(MIC) of 8 to 16 micrograms/ml. The subpopulation of hetero vancomycin resistant MRSA(hetero-VRSA) was resistant to vancomycin MIC of 1 to 4 micrograms/ml. VISA and hetero-VRSA did not have vanA, vanB, vanC genes from vancomycin-resistant enterococci(VRE). The disk diffusion test could not detect VISA and hetero-VRSA. The MIC tests can confirm VRSA well. To detect the hetero-VRSA, we need to use population analysis and growth on hetero-VRSA agar (MU3 agar plate).

Oguchi disease with sectoral retinitis pigmentosa harboring adenine deletion at position 1147 in the arrestin gene.

PURPOSE: To report a Japanese patient with Oguchi disease and sectoral retinitis pigmentosa harboring a homozygous adenine deletion at position 1147 (1147delA) in the arrestin gene. METHOD: Case report. RESULTS: In both eyes, golden discoloration with Mizuo-Nakamura phenomenon and tapetoretinal degeneration of the fundus were exhibited. Electroretinography showed abnormal a-wave and absent b-wave. The presence of 1147delA in the arrestin gene was demonstrated. CONCLUSIONS: Our case provides further evidence of the close association of 1147delA in the arrestin gene in Japanese patients with Oguchi disease. Coexpression of both phenotypes of Oguchi disease and retinitis pigmentosa may suggest the possible involvement of additional defects of genes encoding the phototransduction proteins.

Protein synthesis is required for the generation of oscillatory current responses in Xenopus oocyte.

In Xenopus oocyte, direct activation of G-proteins by AIF4- or injection of inositol 1, 4, 5-trisphosphate evoked Ca(2+)-gated Cl-current responses. The current responses were smooth and not oscillatory when tested immediately after the oocyte was isolated. After hours of incubation, the response became oscillatory. This change was prevented by cycloheximide, a protein synthesis inhibitor. Results indicate that the characteristics of Ca2+ signaling system have changed from not oscillatory to oscillatory with time and protein synthesis was required for this change.