The prevalence of vancomycin-resistant enterococci (VRE) colonization or infection in the hospital setting has increased globally. Many previous studies had analysed the risk factors for acquiring VRE, based on cross-sectional studies or prevalent cases. However, the actual incidence of and risk factors for VRE remain unclear. The present study was conducted in order to clarify the incidence of and risk factors for VRE in the intensive care unit (ICU). From 1st April 2008 to 31st March 2009, all patients admitted to a surgical ICU (SICU) were put on active surveillance for VRE. The surveillance cultures, obtained by rectal swab, were taken on admission, weekly while staying in the SICU, and on discharge from the SICU. A total of 871 patients were screened. Among them, 34 were found to carry VRE before their admission to the SICU, and 47 acquired VRE during their stay in the SICU, five of whom developed VRE infections. The incidence of newly acquired VRE during ICU stay was 21.9 per 1000 patient-days (95% confidence interval [CI], 16.4–29.1). Using multivariate analysis by logistic regression, we found that the length of ICU stay was an independent risk factor for new acquisition of VRE. In contrast, patients with prior exposure to first-generation cephalosporin were significantly less likely to acquire VRE. Strategies to reduce the duration of ICU stay and prudent usage of broad-spectrum antibiotics are the keys to controlling VRE transmission.
Small hepatocellular carcinoma (HCC) affects millions of individuals worldwide. Surveillance of high-risk patients increases the early detection of small HCC.
To identify prognostic factors affecting the overall survival (OS) and recurrence-free survival (RFS) of patients with small HCC.
The present prospective study enrolled 140 Taiwanese patients with stage I or stage II small HCC. Clinical parameters of interest included operation type, tumour size, tumour histology, Child-Pugh class, presence of hepatitis B surface antigen and liver cirrhosis, hepatitis C status, alpha-fetoprotein, total bilirubin and serum albumin levels, and administration of antiviral and salvage therapies.
Tumour size correlated significantly with poorer OS in patients with stage I small HCC (P=0.014); however, patients with stage II small HCC experienced a significantly poorer RFS (P=0.033). OS rates did not differ significantly between patients with stage I and stage II small HCC. Tumour margins, tumour histology and cirrhosis did not significantly affect OS or RFS (P>0.05).
Increasing tumour size has generally been associated with poorer prognoses in cases of HCC. The present study verified the relationship between small HCC tumour size and OS; however, a reduction in OS with increasing tumour size was demonstrated for patients with stage I – but not for stage II – small HCC.
Patients with stage II small HCC may benefit from aggressive surveillance for tumour recurrence and appropriate salvage treatment. Further studies are needed for additional stratification of stage I patients to identify those at increased risk of death.
Prognosis; Small hepatocellular carcinoma; Tumour size
Regulatory approaches for evaluating therapeutic equivalence of multisource (or generic) drug products vary among different countries and/or regions. Harmonization of these approaches may decrease the number of in vivo bioequivalence studies and avoid unnecessary drug exposure to humans. Global harmonization for regulatory requirements may be promoted by a better understanding of factors underlying product performance and expectations from different regulatory authorities. This workshop provided an opportunity for pharmaceutical scientists from academia, industry and regulatory agencies to have open discussions on current regulatory issues and industry practices, facilitating harmonization of regulatory approaches for establishing therapeutic equivalence and interchangeability of multisource drug products.
bioequivalence; harmonization; interchangeability; regulatory standards; therapeutic equivalence
Lactic acid bacteria that exist in the urinogenital system play an important role in maintaining the health of the host. Here, we report the finished and annotated genome of a Lactococcus strain that was isolated from the vaginas of healthy women and shows probiotic properties, including nisin A production and adhesion to vaginal epithelial cells.
One of the major goals for the immunotherapy of autoimmune diseases is the induction of regulatory T cells that mediate immunologic tolerance. Parenteral administration of anti-CD3 monoclonal antibody is an approved therapy for transplantation in humans and is effective in autoimmune diabetes. We have found that oral administration of anti-CD3 monoclonal antibody is biologically active in the gut and suppresses experimental autoimmune encephalomyelitis both prior to disease induction and at the height of disease. Oral anti-CD3 antibody acts by inducing a unique type of regulatory T cell characterized by latency-associated peptide (LAP) on its cell surface that functions in vivo and in vitro via TGF-β dependent mechanism. Orally delivered antibody would not have side effects including cytokine release syndromes, thus oral anti-CD3 antibody is clinically applicable for chronic therapy. These findings identify a novel and powerful immunologic approach that is widely applicable for the treatment of human autoimmune conditions.
regulatory cell; TGF-β; antibody; multiple sclerosis; autoimmunity
A case of lipomatous tubular adenoma (LTA) with syringocystadenom papilliferum (SCAP) arising from the external auditory canal in a 25-year-old man is described and to the best of our knowledge through literature review, this kind of morphologic entity has not been reported before. Herein we reported the first case in the English literature in the world.
tubular adenoma; syringocystadenoma papilliferum; external auditory canal
Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.
Rhizopus oryzae lipase (ROL); Yeast surface display; Codon optimization; Whole-cell biocatalyst
Modified release products are complex dosage forms designed to release drug in a controlled manner to achieve desired efficacy and safety. Inappropriate control of drug release from such products may result in reduced efficacy or increased toxicity. This workshop provided an opportunity for pharmaceutical scientists from academia, industry, and regulatory agencies to discuss current industry practices and regulatory expectations for demonstrating pharmaceutical equivalence and bioequivalence of MR products, further facilitating the establishment of regulatory standards for ensuring therapeutic equivalence of these products.
bioequivalence; interchangeability; modified release; pharmaceutical equivalence; therapeutic equivalence
Akt-deficiency causes resistance to replicative senescence, oxidative stress- or oncogenic Ras-induced premature senescence, and to reactive oxygen species (ROS)-mediated apoptosis. Akt activation induces premature senescence and sensitizes cells to ROS-mediated apoptosis by increasing intracellular ROS through increased oxygen consumption and by inhibiting the expression of ROS-scavengers downstream of FoxO, particularly sestrin3 expression. This uncovers an Achilles’ heel of Akt, since in contrast to its ability to inhibit apoptosis induced by multiple apoptotic stimuli; Akt could not inhibit ROS-mediated apoptosis. Furthermore, treatment with rapamycin that led to further Akt activation and resistance to etoposide, hypersensitized cancer cells to ROS-mediated apoptosis. Given that rapamycin alone is mainly cytostatic, this constitutes a strategy for cancer therapy that selectively eradicates cancer cells via Akt activation.
Vancomycin-resistant Enterococcus faecium (VRE) has become an important health care-associated pathogen because of its rapid spread, limited therapeutic options, and possible transfer of vancomycin resistance to more-virulent pathogens. In this study, we compared the ability to detect clonal relationships among VRE isolates by an automated repetitive-sequence-based PCR (Rep-PCR) system (DiversiLab system) to pulsed-field gel electrophoresis (PFGE), the reference method for molecular typing of VRE. Two sets of VRE isolates evaluated in this study were collected by active microbial surveillance at a large teaching hospital in Taiwan during 2008. The first set included 90 isolates randomly selected from the surveillance cohort. The first set consisted of 34 pulsotypes and 10 Rep-PCR types. There was good correlation between the two methods (P < 0.001). The second set included 68 VRE isolates collected from eight clusters of colonization. A dominant clone was detected in five out of eight clusters by both methods. Two clusters were characterized by Rep-PCR as being caused by a dominant clone, whereas PFGE showed polyclonal origins. One cluster was shown to be polyclonal by both methods. A single Rep-PCR clone type was detected among 12 of 14 vancomycin-intermediate enterococci, whereas PFGE detected six pulsotypes. In conclusion, the Rep-PCR method correlated well with PFGE typing but was less discriminative than PFGE in defining clonal relationships. The ease of use and more rapid turnaround time of Rep-PCR compared to PFGE offers a rapid screening method to detect outbreaks of VRE and more rapidly implement control measures. PFGE remains the preferred method to confirm clonal spread.
Although CD8+ Treg-mediated suppression has been described, CD8+ Treg remain poorly characterized. Here we identify a novel subset of CD8+ Treg that express latency-associated peptide (LAP) on their cell surface (CD8+LAP+ cells) and exhibit regulatory activity in vitro and in vivo. Only a small fraction of CD8+LAP+ cells express Foxp3 or CD25, although the expression levels of Foxp3 for these cells are higher than their LAP− counterparts. In addition to TGF-β, CD8+LAP+ cells produce IFN-γ, and these cells suppress EAE that is dependent on both TGF-β and IFN-γ. In an adoptive co-transfer model, CD8+LAP+ cells suppress myelin oligodendrocyte glycoprotein (MOG)-specific immune responses by inducing or expanding Foxp3+ cells and by inhibiting proliferation and IFN-γ production in vivo. Furthermore, in vivo neutralization of IFN-γ and studies with IFN-γ-deficient mice demonstrate an important role for IFN-γ production in the function of CD8+LAP+ cells. Our findings identify the underlying mechanisms that account for the immunoregulatory activity of CD8+ T cells and suggest that induction or amplification of CD8+LAP+ cells may be a therapeutic strategy to help control autoimmune processes.
Autoimmunity; Treg; Tolerance
This study was designed to determine the strength of the association between the isolation of nonfermentative gram-negative bacilli (NFGNB) from tap water faucet aerators and the prevalence of colonization or infection of patients in intensive care units (ICUs). Surveillance cultures were obtained during a 4-month period from 162 faucet aerators located in seven different ICUs. The prevalence of colonization or infection of ICU patients with NFGNB was determined by prospective surveillance during the same period. Fifty four (33%) of the faucet aerators contained NFGNB. Among the 66 NFGNB isolated from faucet aerators, the most frequently encountered ones were Sphingomonas paucimobili (26 isolates), Pseudomonas aeruginosa (14 isolates), Chryseobacterium meningosepticum (13 isolates), Achromobacter xylosoxidans (6 isolates), Burkholderia cepacia (4 isolates), and Stenotrophomonas maltophilia (3 isolates). Acinetobacter baumannii was not recovered. The most common NFGNB isolated from ICU patients were P. aeruginosa and A. baumannii. There was a significant correlation between the overall prevalence of NFGNB in faucet aerators and their prevalence in exposed ICU patients (Spearman r = 0.821, P = 0.02). There was also a significant correlation between the prevalence of C. meningosepticum in faucet aerators and its prevalence among ICU patients (Spearman r = 0.847, P = 0.016). The electrokaryotypes of four clinical isolates of C. meningosepticum were similar to those of faucet isolates. Measures directed at making the water supply safe may prevent infection by C. meningosepticum and other waterborne pathogens.
Phenotypic analyses of mice null for the individual Akt isoforms suggested that they are functionally distinct and that only Akt2 plays a role in diabetes. We show here that Akt isoforms play compensatory and complementary roles in glucose homeostasis and diabetes. Insulin resistance in Akt2−/− mice was inhibited by haplodeficiency of Pten, suggesting that other Akt isoforms can compensate for Akt2 function. Haplodeficiency of Akt1 in Akt2−/− mice, however, converts prediabetes to overt type 2 diabetes, which is also reversed by haplodeficiency of Pten. Akt3 does not appear to contribute significantly to diabetes. Overt type 2 diabetes in Akt1+/− Akt2−/− mice is manifested by hyperglycemia due to beta-cell dysfunction combined with impaired glucose homeostasis due to markedly decreased leptin levels. Restoring leptin levels was sufficient to restore normal blood glucose and insulin levels in Akt1+/− Akt2−/− and Akt2−/− mice, suggesting that leptin-deficiency is the predominant cause of diabetes in these mice. These results uncover a new mechanism linking Akt to diabetes, provide a therapeutic strategy, and show that diabetes induced as a consequence of cancer therapy, via Akt inhibition, could be reversed by leptin therapy.
CD4+CD25+ regulatory T cells (Tregs) are essential for maintaining self-tolerance and immune homeostasis. Here we characterize a novel subset of CD4+CD25+ Tregs that express latency-associated peptide (LAP) on their cell surface (CD4+CD25+LAP+ cells). CD4+CD25+LAP+ cells express elevated levels of Foxp3 and Treg-associated molecules (CTLA4, glucocorticoid-induced TNFR-related gene), secrete TGFβ, and express both cell surface TGFβ and surface receptors for TGFβ. In vitro, the suppressive function of CD4+CD25+LAP+ cells is both cell contact and soluble factor dependent; this contrasts with CD4+CD25+LAP− cells, which are mainly cell contact dependent. In a model of experimental autoimmune encephalomyelitis, CD4+CD25+LAP+ cells exhibit more potent suppressive activity than CD4+CD25+LAP− cells, and the suppression is TGFβ dependent. We further show that CD4+CD25+LAP+ cells suppress myelin oligodendrocyte glycoprotein-specific immune responses by inducing Foxp3 and by inhibiting IL-17 production. Our findings demonstrate that CD4+CD25+ Tregs are a heterogeneous population and that the CD4+CD25+ subset that expresses LAP functions in a TGFβ-dependent manner and has greater in vivo suppressive properties. Our work helps elucidate the ambiguity concerning the role of TGFβ in CD4+CD25+ Treg-mediated suppression and indicates that LAP is an authentic marker able to identify a TGFβ-expressing CD4+CD25+ Treg subset.
A simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method using an atmospheric pressure chemical ionization source (APCI) for the quantification of fenretinide (4-HPR) in mouse plasma was developed and validated. After a simple protein precipitation of plasma sample by acetonitrile, 4-HPR was analyzed by LC-APCI-MS/MS. High performance liquid chromatography (HPLC) separation was conducted on a Hypurity C18 column (50 × 2.1 mm, 5μ) with a flow rate 0.60 mL/min using a gradient mobile phase comprised of 0.05% formic acid in water (A) and methanol (B), and a run time of 4.5 min. The elimination of a tedious sample preparation process and a shorter run time substantially reduced total analysis time. The method was linear over the range 0.5−100 ng/mL, with r>0.998. The intra- and inter-assay precisions were 1.4−9.2% and 5.1−8.2%, respectively, and the intra- and inter-assay accuracies were 93.9−98.6% and 92.7−95.3%, respectively. The absolute recoveries were 90.3% (1.5 ng/mL), 97.0% (7.5 ng/mL) and 92.1% (75.0 ng/mL) for 4-HPR, and 99.1% for the internal standard (150 ng/mL). The analytical method had excellent sensitivity using a small sample volume (30 μL) with the lower limit of quantification (LLOQ) 0.5 ng/mL. This method is robust and has been successfully employed in a pharmacokinetic study of 4-HPR in a mouse xenograft model of neuroblastoma.
Fenretinide (4-HPR); N-(4-methoxyphenyl) retinamide (4-MPR); LC-APCI-MS/MS; Plasma
To determine the predominant staphylococcal cassette chromosome (SCC) mec element in methicillin-resistant Staphylococcus aureus, we typed 190 isolates from a hospital in Taiwan. We found a shift from type IV to type III SCCmec element during 1992–2003, perhaps caused by selective pressure from indiscriminate use of antimicrobial drugs.
Methicillin-resistant Staphylococcus aureus; multilocus sequence typing; SCCmec; dispatch
We examined severe acute respiratory syndrome-associated coronavirus (SARS-CoV) RNA in plasma of 32 patients (probable SARS cases) by a quantitative real-time reverse transcription-PCR assay and reported that the highest detection rate, 75%, was found between day 5 and day 7 of illness, followed by rates of 64, 50, and 38% found between day 8 and day 11, day 2 and day 4, and day 12 and day 16, respectively. Analysis of sequential SARS-CoV load in plasma from six cases revealed different patterns of viremia, with the peak between day 4 and day 8. Our findings of the high detection rate of SARS-CoV RNA in plasma before day 11, together with the relative convenience of collecting and handling plasma, suggest that plasma can be used for early diagnosis of SARS.
Thirty-one cases of severe acute respiratory syndrome (SARS) occurred after exposure in the emergency room at the National Taiwan University Hospital. The index patient was linked to an outbreak at a nearby municipal hospital. Three clusters were identified over a 3-week period. The first cluster (5 patients) and the second cluster (14 patients) occurred among patients, family members, and nursing aids. The third cluster (12 patients) occurred exclusively among healthcare workers. Six healthcare workers had close contact with SARS patients. Six others, with different working patterns, indicated that they did not have contact with a SARS patient. Environmental surveys found 9 of 119 samples of inanimate objects to be positive for SARS coronavirus RNA. These observations indicate that although transmission by direct contact with known SARS patients was responsible for most cases, environmental contamination with the SARS coronavirus may have lead to infection among healthcare workers without documented contact with known hospitalized SARS patients.
Severe acute respiratory syndrome; healthcare workers; environmental contamination; real-time reverse transcriptase–polymerase chain reaction
This is a summary report of the workshop, organized by the European Federation of Pharmaceutical Scientists in association with the American Association of Pharmaceutical Scientists, the European Agency for the Evaluation of Medicinal Products, the European Pharmacopoeia, the US Food and Drug Administration and the United States Pharmacopoeia, on “Assuring Quality and Performance of Sustained and Controlled Release Parenterals” held in Basel, Switzerland, February 2003. Experts from the pharmaceutical industry, regulatory authorities and academia participated in this workshop to review, discuss and debate formulation, processing and manufacture of sustained and controlled release parenterals, and identify critical process parameters and their control. This workshop was a follow-up workshop to a previous workshop on Assuring Quality and Performance of Sustained and Controlled Release Parenterals that was held in Washington, DC in April 2001. This report reflects the outcome of the Basel 2003 meeting and the advances in the field since the Washington, DC meeting in 2001. As necessary, the reader is referred to the report on the 2001 meeting. Areas were identified at the 2003 Basel meeting where research is needed in order to understand the performance of these drug delivery systems and to assist in the development of appropriate testing procedures. Recommendations were made for future workshops and meetings.
The gene encoding the 56-kDa protein of Orientia tsutsugamushi Shanxi was amplified by a nested PCR and cloned into the expression vector pQE30. The 56-kDa protein of O. tsutsugamushi Shanxi (Sxh56) was expressed as a fusion protein with the His6-binding protein of Escherichia coli by deleting the signal peptide-encoding sequence from the 5′ end of the open reading frame. The recombinant protein formed inclusion bodies when expressed in E. coli M15. The recombinant protein was examined for reactivity with mouse sera against three antigenic prototypes of O. tsutsugamushi by an immunoblot assay. The recombinant Sxh56 reacted only to polyclonal antiserum to O. tsutsugamushi Gilliam in an enzyme-linked immunosorbent assay (ELISA) and in an immunoblot assay. Recombinant Sxh56 was purified by Ni-nitrilotriacetic acid affinity chromatography and injected into mice to evaluate its ability to stimulate immune responses. High levels of immunoglobulin G and T-cell proliferation appeared in mice immunized with the recombinant protein. The recombinant Sxh56 was used in an ELISA to evaluate the ability of the method to detect antibodies to O. tsutsugamushi in human and animal sera. Thirty sera from mice infected with O. tsutsugamushi Gilliam or Shanxi and 55 sera from normal mice were detected in the ELISA with recombinant Sxh56, and the sensitivity and specificity were 96.67 and 100%, respectively. One hundred fifty-one positive sera and 412 negative sera to O. tsutsugamushi Gilliam were detected in an indirect immunofluorescence assay with the recombinant protein, and the sensitivity and specificity were 96.36 and 88.08%, respectively. These results strongly suggest that the recombinant Sxh56 is a suitable type-specific immunodiagnostic antigen and vaccine candidate.
This is a summary report of the American Association of Pharmaceutical Scientists, the Food and Drug Administration and the United States Pharmacopoeia cosponsored workshop on “Assuring Quality and Performance of Sustained and Controlled Release Parenterals.” Experts from the pharmaceutical industry, the regulatory authorities and academia participated in this workshop to review, discuss and debate formulation, processing and manufacture of sustained and controlled release parenterals and identify critical process parameters and their control. Areas were identified where research is needed in order to understand the performance of these drug delivery systems and to assist in the development of appropriate testing procedures. Recommendations were made for future workshops, meetings and working groups in this area.
To determine the distribution and antimicrobial drug resistance in bacterial pathogens causing nosocomial infections, surveillance data on nosocomial infections documented from 1981 to 1999 at National Taiwan University Hospital were analyzed. During this period, 35,580 bacterial pathogens causing nosocomial infections were identified. Candida species increased considerably, ranking first by 1999 in the incidence of pathogens causing all nosocomial infections, followed by Staphylococcus aureus and Pseudomonas aeruginosa. Candida species also increased in importance as bloodstream infection isolates, from 1.0% in 1981-1986 to 16.2% in 1999. The most frequent isolates from urinary tract infections were Candida species (23.6%), followed by Escherichia coli (18.6%) and P. aeruginosa (11.0%). P. aeruginosa remained the most frequent isolates for respiratory tract and surgical site infections in the past 13 years. A remarkable increase in incidence was found in methicillin-resistant S. aureus (from 4.3% in 1981-1986 to 58.9% in 1993-1998), cefotaxime-resistant E. coli (from 0% in 1981-1986 to 6.1% in 1993-1998), and cefotaxime-resistant Klebsiella pneumoniae (from 4.0% in 1981-1986 to 25.8% in 1993-1998). Etiologic shifts in nosocomial infections and an upsurge of antimicrobial resistance among these pathogens, particularly those isolated from intensive care units, are impressive and alarming.
nosocomial infections; antimicrobial resistance; Taiwan; bacterial pathogens; hospital-based surveillance
We report the case of a 56-year-old man who developed bacterial mediastinitis with methicillin-resistant Staphylococcus aureus after undergoing heart transplantation. He had a history of insulin-dependent diabetes mellitus and prior cardiac surgery. To find the source of nosocomial infection, we cultured nasal swab specimens from all hospital personnel involved in this operation. We used antibiotic sensitivity profiling and pulsed-field gel electrophoresis to subtype the involved microorganism. The S. aureus isolates from the patient and the perfusionist were identical to each other and were different from the strains previously found in our hospital. It is almost certain that the S. aureus mediastinitis in this patient was transmitted from the perfusionist. We recommend obtaining cultures from hospital staff members when there is an outbreak of staphylococcal infection.
Cross infection/epidemiology; electrophoresis, gel, pulsed-field; heart transplantation/adverse effects; immunosuppression/adverse effects; mediastinitis/etiology/therapy; methicillin resistance/genetics; postoperative period; staphylococcal infections/etiology; Staphylococcus aureus/classification; surgical wound infection/therapy
Background Children with oppositional defiant symptoms (ODS) are highly related to parental stress, especially in mothers. This study is the first to investigate the quality of life (QOL) of mothers of children with ODS in a community sample.
Methods Randomly selected mothers of children attending an elementary school were contacted, and 387 who completed the questionnaire participated in this study. The children's ODS status was determined by the maternal rating of the Chinese Swanson, Nolan, and Pelham rating scale, version IV. The mothers' QOL was estimated by maternal reports from the World Health Organization Quality of Life – BREF (WHOQOL‐BREF) instrument. The relationship between the children's ODS status and maternal QOL was examined by analysis of covariance (ANCOVA) with the participants' sociodemographic factors as covariables.
Results Sixty‐three children, mostly boys, met the screening criteria for ODS. The positive screening rate for ODS was 16.49%. The children's ODS status was a significant predictor for the maternal physical capacity, psychological wellbeing and environment domains of QOL. Mothers of children with ODS who rented a house were younger and had lower education levels and worse QOL in all domains.
Conclusion A high positive screening rate for ODS children in the elementary school and a relationship between poor maternal QOL and children's ODS were found in this study. Routine screening for ODS in children and mental health services for these children and their mothers are warranted.
mother; oppositional defiant disorder; quality of life