ABL Genomic Editing Sufficiently Abolishes Oncogenesis of Human Chronic Myeloid Leukemia Cells In Vitro and In Vivo.

Chronic myelogenous leukemia (CML) is the most common type of leukemia in adults, and more than 90% of CML patients harbor the abnormal Philadelphia chromosome (Ph) that encodes the BCR-ABL oncoprotein. Although the ABL kinase inhibitor (imatinib) has proven to be very effective in achieving high remission rates and improving prognosis, up to 33% of CML patients still cannot achieve an optimal response. Here, we used CRISPR/Cas9 to specifically target the BCR-ABL junction region in K562 cells, resulting in the inhibition of cancer cell growth and oncogenesis. Due to the variety of BCR-ABL junctions in CML patients, we utilized gene editing of the human ABL gene for clinical applications. Using the ABL gene-edited virus in K562 cells, we detected 41.2% indels in ABL sgRNA_2-infected cells. The ABL-edited cells reveled significant suppression of BCR-ABL protein expression and downstream signals, inhibiting cell growth and increasing cell apoptosis. Next, we introduced the ABL gene-edited virus into a systemic K562 leukemia xenograft mouse model, and bioluminescence imaging of the mice showed a significant reduction in the leukemia cell population in ABL-targeted mice, compared to the scramble sgRNA virus-injected mice. In CML cells from clinical samples, infection with the ABL gene-edited virus resulted in more than 30.9% indels and significant cancer cell death. Notably, no off-target effects or bone marrow cell suppression was found using the ABL gene-edited virus, ensuring both user safety and treatment efficacy. This study demonstrated the critical role of the ABL gene in maintaining CML cell survival and tumorigenicity in vitro and in vivo. ABL gene editing-based therapy might provide a potential strategy for imatinib-insensitive or resistant CML patients.

Clinical Characteristics and Neonatal Outcomes of Pregnant Patients With COVID-19: A Systematic Review.

Background and Objective: Coronavirus disease 2019 (COVID-19) characterized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created serious concerns about its potential adverse effects. There are limited data on clinical, radiological, and neonatal outcomes of pregnant women with COVID-19 pneumonia. This study aimed to assess clinical manifestations and neonatal outcomes of pregnant women with COVID-19. Methods: We conducted a systematic article search of PubMed, EMBASE, Scopus, Google Scholar, and Web of Science for studies that discussed pregnant patients with confirmed COVID-19 between January 1, 2020, and April 20, 2020, with no restriction on language. Articles were independently evaluated by two expert authors. We included all retrospective studies that reported the clinical features and outcomes of pregnant patients with COVID-19. Results: Forty-seven articles were assessed for eligibility; 13 articles met the inclusion criteria for the systematic review. Data is reported for 235 pregnant women with COVID-19. The age range of patients was 25-40 years, and the gestational age ranged from 8 to 40 weeks plus 6 days. Clinical characteristics were fever [138/235 (58.72%)], cough [111/235 (47.23%)], and sore throat [21/235 (8.93%)]. One hundred fifty six out of 235 (66.38%) pregnant women had cesarean section, and 79 (33.62%) had a vaginal delivery. All the patients showed lung abnormalities in CT scan images, and none of the patients died. Neutrophil cell count, C-reactive protein (CRP) concentration, ALT, and AST were increased but lymphocyte count and albumin levels were decreased. Amniotic fluid, neonatal throat swab, and breastmilk samples were taken to test for SARS-CoV-2 but all found negativ results. Recent published evidence showed the possibility of vertical transmission up to 30%, and neonatal death up to 2.5%. Pre-eclampsia, fetal distress, PROM, pre-mature delivery were the major complications of pregnant women with COVID-19. Conclusions: Our study findings show that the clinical, laboratory and radiological characteristics of pregnant women with COVID-19 were similar to those of the general populations. The possibility of vertical transmission cannot be ignored but C-section should not be routinely recommended anymore according to latest evidences and, in any case, decisions should be taken after proper discussion with the family. Future studies are needed to confirm or refute these findings with a larger number of sample sizes and a long-term follow-up period.

HDAC1,2 Knock-Out and HDACi Induced Cell Apoptosis in Imatinib-Resistant K562 Cells.

Since imatinib (Glivec or Gleevec) has been used to target the BCR-ABL fusion protein, chronic myeloid leukemia (CML) has become a manageable chronic disease with long-term survival. However, 15%-20% of CML patients ultimately develop resistance to imatinib and then progress to an accelerated phase and eventually to a blast crisis, limiting treatment options and resulting in a poor survival rate. Thus, we investigated whether histone deacetylase inhibitors (HDACis) could be used as a potential anticancer therapy for imatinib-resistant CML (IR-CML) patients. By applying a noninvasive apoptosis detection sensor (NIADS), we found that panobinostat significantly enhanced cell apoptosis in K562 cells. A further investigation showed that panobinostat induced apoptosis in both K562 and imatinib-resistant K562 (IR-K562) cells mainly via H3 and H4 histone acetylation, whereas panobinostat targeted cancer stem cells (CSCs) in IR-K562 cells. Using CRISPR/Cas9 genomic editing, we found that HDAC1 and HDAC2 knockout cells significantly induced cell apoptosis, indicating that the regulation of HDAC1 and HDAC2 is extremely important in maintaining K562 cell survival. All information in this study indicates that regulating HDAC activity provides therapeutic benefits against CML and IR-CML in the clinic.

HDAC1 and HDAC2 Double Knockout Triggers Cell Apoptosis in Advanced Thyroid Cancer.

Anaplastic thyroid carcinoma (ATC) and squamous thyroid carcinoma (STC) are both rare and advanced thyroid malignancies with a very poor prognosis and an average median survival time of 5 months and less than 20% of affected patients are alive 1 year after diagnosis. The clinical management of both ATC and STC is very similar because they are not particularly responsive to radiotherapy and chemotherapy. This inspired us to explore a novel and effective clinically approved therapy for ATC treatment. Histone deacetylase inhibitor (HDACi) drugs are recently FDA-approved drug for malignancies, especially for blood cell cancers. Therefore, we investigated whether an HDACi drug acts as an effective anticancer drug for advanced thyroid cancers. Cell viability analysis of panobinostat treatment demonstrated a significant IC50 of 0.075 µM on SW579 STC cells. In addition, panobinostat exposure activated histone acetylation and triggered cell death mainly through cell cycle arrest and apoptosis-related protein activation. Using CRISPR/Cas9 to knock out HDAC1 and HDAC2 genes in SW579 cells, we observed that the histone acetylation level and cell cycle arrest were enhanced without any impact on cell growth. Furthermore, HDAC1 and HDAC2 double knockout (KO) cells showed dramatic cell apoptosis activation compared to HDAC1 and HDAC2 individual KO cells. This suggests expressional and biofunctional compensation between HDAC1 and HDAC2 on SW579 cells. This study provides strong evidence that panobinostat can potentially be used in the clinic of advanced thyroid cancer patients.

CRISPR/Cas9 Genome Editing of Epidermal Growth Factor Receptor Sufficiently Abolished Oncogenicity in Anaplastic Thyroid Cancer.

Anaplastic carcinoma of the thyroid (ATC), also called undifferentiated thyroid cancer, is the least common but most aggressive and deadly thyroid gland malignancy of all thyroid cancers. The aim of this study is to explore essential biomarker and use CRISPR/Cas9 with lentivirus delivery to establish a gene-target therapeutic platform in ATC cells. At the beginning, the gene expression datasets from 1036 cancers from CCLE and 8215 tumors from TCGA were collected and analyzed, showing EGFR is predominantly overexpressed in thyroid cancers than other type of cancers (P = 0.017 in CCLE and P = 0.001 in TCGA). Using CRISPR/Cas9 genomic edit system, ATC cells with EGFR sgRNA lentivirus transfection obtained great disruptions on gene and protein expression, resulting in cell cycle arrest, cell growth inhibition, and most importantly metastasis turn-off ability. In addition, the FDA-approved TKI of afatinib for EGFR targeting also illustrates great anticancer activity on cancer cell death occurrence, cell growth inhibition, and cell cycle arrest in SW579 cells, an EGFR expressing human ATC cell line. Furthermore, off-target effect of using EGFR sgRNAs was measured and found no genomic editing can be detected in off-target candidate gene. To conclude, this study provides potential ATC therapeutic strategies for current and future clinical needs, which may be possible in increasing the survival rate of ATC patients by translational medicine.

The Application of Non-Invasive Apoptosis Detection Sensor (NIADS) on Histone Deacetylation Inhibitor (HDACi)-Induced Breast Cancer Cell Death.

Breast cancer is the most common malignancy in women and the second leading cause of cancer death in women. Triple negative breast cancer (TNBC) subtype is a breast cancer subset without ER (estrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor 2) expression, limiting treatment options and presenting a poorer survival rate. Thus, we investigated whether histone deacetylation inhibitor (HDACi) could be used as potential anti-cancer therapy on breast cancer cells. In this study, we found TNBC and HER2-enriched breast cancers are extremely sensitive to Panobinostat, Belinostat of HDACi via experiments of cell viability assay, apoptotic marker identification and flow cytometry measurement. On the other hand, we developed a bioluminescence-based live cell non-invasive apoptosis detection sensor (NIADS) detection system to evaluate the quantitative and kinetic analyses of apoptotic cell death by HDAC treatment on breast cancer cells. In addition, the use of HDACi may also contribute a synergic anti-cancer effect with co-treatment of chemotherapeutic agent such as doxorubicin on TNBC cells (MDA-MB-231), but not in breast normal epithelia cells (MCF-10A), providing therapeutic benefits against breast tumor in the clinic.

A rapid and quantitative method to detect human circulating tumor cells in a preclinical animal model.

BACKGROUND: As cancer metastasis is the deadliest aspect of cancer, causing 90% of human deaths, evaluating the molecular mechanisms underlying this process is the major interest to those in the drug development field. Both therapeutic target identification and proof-of-concept experimentation in anti-cancer drug development require appropriate animal models, such as xenograft tumor transplantation in transgenic and knockout mice. In the progression of cancer metastasis, circulating tumor cells (CTCs) are the most critical factor in determining the prognosis of cancer patients. Several studies have demonstrated that measuring CTC-specific markers in a clinical setting (e.g., flow cytometry) can provide a current status of cancer development in patients. However, this useful technique has rarely been applied in the real-time monitoring of CTCs in preclinical animal models. METHODS: In this study, we designed a rapid and reliable detection method by combining a bioluminescent in vivo imaging system (IVIS) and quantitative polymerase chain reaction (QPCR)-based analysis to measure CTCs in animal blood. Using the IVIS Spectrum CT System with 3D-imaging on orthotropic-developed breast-tumor-bearing mice. RESULTS: In this manuscript, we established a quick and reliable method for measuring CTCs in a preclinical animal mode. The key to this technique is the use of specific human and mouse GUS primers on DNA/RNA of mouse peripheral blood under an absolute qPCR system. First, the high sensitivity of cancer cell detection on IVIS was presented by measuring the luciferase carried MDA-MB-231 cells from 5 to 5x1011 cell numbers with great correlation (R2 = 0.999). Next, the MDA-MB-231 cell numbers injected by tail vein and their IVIS radiance signals were strongly corrected with qPCR-calculated copy numbers (R2 > 0.99). Furthermore, by applying an orthotropic implantation animal model, we successfully distinguished xenograft tumor-bearing mice and control mice with a significant difference (p < 0.001), whereas IVIS Spectrum-CT 3D-visualization showed that blood of mice with lung metastasis contained more than twice the CTC numbers than ordinary tumor-bearing mice. We demonstrated a positive correlation between lung metastasis status and CTC numbers in peripheral mouse blood. CONCLUSION: Collectively, the techniques developed for this study resulted in the integration of CTC assessments into preclinical models both in vivo and ex vivo, which will facilitate translational targeted therapy in clinical practice.

Antiseptic Effect of Conventional Povidone-Iodine Scrub, Chlorhexidine Scrub, and Waterless Hand Rub in a Surgical Room: A Randomized Controlled Trial.

OBJECTIVE Effective perioperative hand antisepsis is crucial for the safety of patients and medical staff in surgical rooms. The antimicrobial effectiveness of different antiseptic methods, including conventional hand scrubs and waterless hand rubs, has not been well evaluated. DESIGN, SETTING, AND PARTICIPANTS A randomized controlled trial was conducted to investigate the effectiveness of the 3 antiseptic methods among surgical staff of Taipei Medical University-Shuang Ho Hospital. For each method used, a group of 80 participants was enrolled. INTERVENTION Surgical hand cleansing with conventional 10% povidone-iodine scrub, conventional 4% chlorhexidine scrub, or waterless hand rub (1% chlorhexidine gluconate and 61% ethyl alcohol). RESULTS Colony-forming unit (CFU) counts were collected using the hand imprinting method before and after disinfection and after surgery. After surgical hand disinfection, the mean CFU counts of the conventional chlorhexidine (0.5±0.2, P<0.01) and waterless hand rub groups (1.4±0.7, P<0.05) were significantly lower than that of the conventional povidone group (4.3±1.3). No significant difference was observed in the mean CFU count among the groups after surgery. Similar results were obtained when preexisting differences before disinfection were considered in the analysis of covariance. Furthermore, multivariate regression indicated that the antiseptic method (P=.0036), but not other variables, predicted the mean CFU count. CONCLUSIONS Conventional chlorhexidine scrub and waterless hand rub were superior to a conventional povidone-iodine product in bacterial inhibition. We recommend using conventional chlorhexidine scrub as a standard method for perioperative hand antisepsis. Waterless hand rub may be used if the higher cost is affordable. Infect Control Hosp Epidemiol 2017;38:417-422.

Recombinant OmpA protein fragments mediate interleukin-17 regulation to prevent Escherichia coli meningitis.

BACKGROUND: Neonates are at a higher risk for bacterial meningitis than children of other age groups. Although the mortality rates have decreased over the past few decades, neonatal meningitis is still a severe disease with high morbidity. For bacterial meningitis, antibiotic therapy is the primary choice for management. However, neurologic complications often cannot be averted; ∼40% of survivors exhibit neurological sequelae. Escherichia coli infection is the common cause of neonatal meningitis. Previously, we have demonstrated that the recombinant loop 1-3, loop 2-3, and loop 2-4 fragments of OmpA protein can protect mice from death after intracerebral E. coli infection. In this study, the protective effects of the recombinant OmpA protein fragments in E. coli intracerebral infections were investigated. METHODS: The effects of E. coli intracerebral infection on cytokine and chemokine expression were determined. We also used various recombinant fragments of the OmpA protein to investigate the effects of these recombinant OmpA protein fragments on cytokine and chemokine expression. RESULTS: In this study, we demonstrated that the expression of interleukin-17 and other cytokines, chemokines, inducible nitric oxide synthase, and cyclooxygenase-2 are involved in the inflammatory processes of intracerebral E. coli infection. We also demonstrated that specific recombinant OmpA protein fragments (L1-3, L2-3, L2-4, and L3) can regulate cytokine, chemokine, nitric oxide synthase, and cyclooxygenase-2 expression and, subsequently, protect mice from death caused by intracerebral infection of E. coli. CONCLUSION: This finding indicates the potential for developing a new therapeutic approach to improve the prognosis of bacterial meningitis.

Recombinant outer membrane protein A fragments protect against Escherichia coli meningitis.

BACKGROUND: Although the mortality rates have decreased over the past few decades, neonatal meningitis is still a severe disease with high morbidity. Moreover, approximately 40% of survivors exhibit neurological sequelae. Escherichia coli is the major Gram-negative bacterial pathogen in neonatal meningitis. The N-terminal ß-barrel domain of the outer membrane protein A (OmpA) of E. coli is essential for effective protein conformation and function and contains four surface-exposed hydrophilic loops. In this study, we expressed different fragments of the four ring structures of the N-terminal domain, and investigated whether these recombinant OmpA fragments can protect mice from death after E. coli infection. METHODS: We expressed the recombinant proteins of the following OmpA fragments by using molecular cloning of Loop 1-2, Loop 1-3, Loop 1-4, Loop 2-3, Loop 2-4, and Loop 3-4. Animal experiments were subsequently performed to investigate the effects of these recombinant OmpA fragments on the survival of C57BL/6 mice after intracerebral E. coli RS218 administration. RESULTS: This study demonstrated that the recombinant Loop 1-3, Loop 2-3, and Loop 2-4 fragments of OmpA can protect mice from intracerebral E. coli infection. CONCLUSION: In bacterial meningitis, although antibiotic therapy is the first choice for management, neurological complications can seldom be averted. Based on the results of the present study, we intend to establish an effective therapeutic application for E. coli meningitis.

Identification of DHA-23, a novel plasmid-mediated and inducible AmpC beta-lactamase from Enterobacteriaceae in Northern Taiwan.

OBJECTIVES: AmpC ß-lactamases are classified as Amber Class C and Bush Group 1. AmpC ß-lactamases can hydrolyze broad and extended-spectrum cephalosporins, and are not inhibited by ß-lactamase inhibitors such as clavulanic acid. This study was conducted to identify DHA-23, a novel plasmid-mediated and inducible AmpC ß-lactamase obtained from Enterobacteriaceae. METHODS: A total of 210 carbapenem-resistant Enterobacteriaceae isolates were collected from a medical center (comprising two branches) in Northern Taiwan during 2009-2012. AmpC ß-lactamase genes were analyzed through a polymerase chain reaction using plasmid DNA templates and gene sequencing. The genetic relationships of the isolates were typed using pulsed-field gel electrophoresis following the digestion of intact genomic DNA by using XbaI. RESULTS: Three enterobacterial isolates (one Escherichia coli and two Klebsiella pneumoniae) were obtained from three hospitalized patients. All three isolates were resistant or intermediately susceptible to all ß-lactams, and exhibited reduced susceptibility to carbapenems. These three isolates expressed a novel AmpC ß-lactamase, designated DHA-23, approved by the curators of the Lahey website. DHA-23 differs from DHA-1 and DHA-6 by one amino acid substitution (Ser245Ala), exhibiting three amino acid changes compared with DHA-7 and DHA-Morganella morganii; three amino acid changes compared with DHA-3; four amino acid changes compared with DHA-5; and eight amino acid changes compared with DHA-2 (>97% identity). This AmpC ß-lactamase is inducible using a system involving ampR. CONCLUSION: This is the first report to address DHA-23, a novel AmpC ß-lactamase. DHA-type ß-lactamases are continuous threat in Taiwan.

Types and prevalence of carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii complex in Northern Taiwan.

The frequency of the carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii (CRACB) complex increases annually in our hospitals. However, the types and prevalence of carbapenemases among isolates still remain unclear. In this study, we identified and collected 672 carbapenem-resistant isolates from a medical center in Northern Taiwan between April and December of 2010. There were 577 genospecies 2 (Acinetobacter baumannii), 79 genospecies 13TU, and 16 genospecies 3 isolates. The isolates had an acquired blaOXA-24-like gene, which was confirmed by sequencing for the encoded OXA-72 carbapenemase, and were often associated with high-level carbapenem resistance. These CRACB complex isolates remained susceptible to colistin (100%). The genotyping of isolates was conducted using pulsed-field gel electrophoresis with ApaI digestion. In most clonally related groups, patients were from both branch hospitals. The results indicate that interhospital dissemination of clones occurred. This study provides updated data on the types and prevalence of the CRACB complex. In addition, it presents a warning on the emergence and spread of CRACB complex harboring blaOXA-24-like genes in northern Taiwan.

Molecular epidemiology and phylodynamics of the human respiratory syncytial virus fusion protein in northern Taiwan.

BACKGROUND AND AIMS: The glycoprotein (G protein) and fusion protein (F protein) of respiratory syncytial virus (RSV) both show genetic variability, but few studies have examined the F protein gene. This study aimed to characterize the molecular epidemiology and phylodynamics of the F protein gene in clinical RSV strains isolated in northern Taiwan from 2000-2011. METHODS: RSV isolates from children presenting with acute respiratory symptoms between July 2000 and June 2011 were typed based on F protein gene sequences. Phylogeny construction and evaluation were performed using the neighbor-joining (NJ) and maximum likelihood (ML) methods. Phylodynamic patterns in RSV F protein genes were analyzed using the Bayesian Markov Chain Monte Carlo framework. Selection pressure on the F protein gene was detected using the Datamonkey website interface. RESULTS: From a total of 325 clinical RSV strains studied, phylogenetic analysis showed that 83 subgroup A strains (RSV-A) could be further divided into three clusters, whereas 58 subgroup B strains (RSV-B) had no significant clustering. Three amino acids were observed to differ between RSV-A and -B (positions 111, 113, and 114) in CTL HLA-B*57- and HLA-A*01-restricted epitopes. One positive selection site was observed in RSV-B, while none was observed in RSV-A. The evolution rate of the virus had very little change before 2000, then slowed down between 2000 and 2005, and evolved significantly faster after 2005. The dominant subtypes of RSV-A in each epidemic were replaced by different subtypes in the subsequent epidemic. CONCLUSIONS: Before 2004, RSV-A infections were involved in several small epidemics and only very limited numbers of strains evolved and re-emerged in subsequent years. After 2005, the circulating RSV-A strains were different from those of the previous years and continued evolving through 2010. Phylodynamic pattern showed the evolutionary divergence of RSV increased significantly in the recent 5 years in northern Taiwan.

The characteristics of new semi-quantitative method for diagnosing proteinuria by using random urine samples.

We assessed the characteristics of the new semi-quantitative test paper (Clinitek ATLAS Pro(12)) using random urine samples. Three hundred urine samples were analyzed using either the new test paper, conventional dipsticks, quantitative (P/C ratio), or immunological quantitative methods (A/C ratio). Our study showed that the new test paper is highly sensitive and specific for the detection of urinary protein. The new test paper also detected the urine protein more accurately than the conventional test and has a lower false-positive rate. In addition, the new test paper detected 14 of the 300 patients (4.7%) as dilute urine samples needing reassessment. Seventeen of the 300 samples tested were negative with conventional dipsticks but positive with the new test paper. The new semi-quantitative test paper not only has higher sensitivity than the conventional dipstick method, but also has potential to detect dilute samples.

A method for lactate and pyruvate determination in filter-paper dried blood spots.

Lactic acidemia is commonly associated with severe diseases in pediatric patients. Quantitation of blood lactate and pyruvate is important for the diagnosis and clinical management. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using dried blood spots (DBS) was developed and could be used for simultaneous quantification of blood lactate and pyruvate. The applicability of the developed method was tested and confirmed by the regression analysis between LC-MS/MS method and enzymatic assay. Lactate and pyruvate were extracted from DBS obtained from 580 full-term, 120 pre-term infants (gestations ranging from 24 to 36 weeks), and 65 patients with suspected lactic acidemia, with methanolic internal standard (IS) solutions of sodium L-lactate-(13)C(3) and pyruvate-(13)C(3). An API-2000 LC-MS/MS system with multiple reaction monitoring (MRM) mode was applied. The within-run and between-run precisions (CV%) were determined and the results were 1.9% and 3.9% for lactate (n=20) and 5.7% and 7.3% for pyruvate (n=20). The linearity of lactate (r=0.9986) and pyruvate (r=0.9973) based on the IS was excellent. The parameter r squared (r(2)) of linear regression between LC-MS/MS method and enzymatic assay was 0.9405 for lactate and 0.9447 for pyruvate, respectively, and the agreement between these methods was consistent and acceptable. The stability of lactate and pyruvate on DBS was also confirmed. The LC-MS/MS method we developed is a specific, sensitive, and reproducible method for measuring blood lactate and pyruvate concentrations. The use of DBS in this method makes it particularly attractive for pediatric patients.

OmpA is the critical component for Escherichia coli invasion-induced astrocyte activation.

Escherichia coli is the major Gram-negative bacterial pathogen in neonatal meningitis. Outer membrane protein A (OmpA) is a conserved major protein in the E. coli outer membrane and is involved in several host-cell interactions. To characterize the role of OmpA in the invasion of astrocytes by E. coli, we investigated OmpA-positive and OmpA-negative E. coli strains. Outer membrane protein A E44, E105, and E109 strains adhered to and invaded C6 glioma cells 10- to 15-fold more efficiently than OmpA-negative strains. Actin rearrangement, protein tyrosine kinase, and phosphoinositide 3-kinase activation were required for OmpA-mediated invasion by E. coli. In vitro infection of C6 cells and intracerebral injection into mice of the E44 strain induced expression of the astrocyte differentiation marker glial fibrillary acidic protein and the inflammatory mediators cyclooxygenase 2 and nitric oxide synthase 2. After intracerebral infection with E44, all C57BL/6 mice died within 36hours, whereas 80% of mice injected with E44 premixed with recombinant OmpA protein survived. Astrocyte activation and neutrophil infiltration were reduced in brain tissue sections in the mice given OmpA. Taken together, these data suggest that OmpA-mediated invasion plays an important role in the early stage of E.coli-induced brain damage, and that it may have therapeutic use in E. coli meningitis.

Evaluation of the VITEK 2 cards for identification and antimicrobial susceptibility testing of non-glucose-fermenting Gram-negative bacilli.

We evaluated VITEK 2 cards (NGNC and AST-GN10) for the accuracy of identification (ID) and antimicrobial susceptibility testing (AST) of non-glucose-fermenting Gram-negative bacilli (NGF-GNB). In a total of 201 strains, 190 strains (94.5%) were correctly identified, seven strains (3.5%)showed low discrimination, four strains (2.0%) had discrepancies, and no strain remained unidentified.Reference AST of amikacin, aztreonam, cefepime, cefotaxime, ceftazidime, ciprofloxacin, imipenem,levofloxacin, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole was performed by the agar dilution method. Approximately 82.5% of ID and 72.9% of AST were completed within 7 and 14 h,respectively. For NGF-GNB, other than Pseudomonas aeruginosa, Acinetobacter spp., Stenotrophomonas maltophilia, and the Burkholderia cepacia group, essential agreements (EAs) were 93.6-100.0%. Severe disagreements (resistant by the reference method to susceptible by AST-GN10) were observed for amikacin(0.9%), cefepime (1.8%), cefotaxime (1.8%), imipenem (0.9%), and piperacillin-tazobactam (0.9%).One major disagreement (susceptible to resistant) was observed for ceftazidime (0.1%). For P. aeruginosa,EAs were 85.7-100%, with severe disagreements observed for cefepime (4.8%) and piperacillin-tazobactam(4.8%). For Acinetobacter spp., EAs were 86.4-100% without disagreements. The VITEK 2 cards appear to be promising for rapid ID and reliable AST for most species of NGF-GNB.

Identification of individual DNA molecule of Mycobacterium tuberculosis by nested PCR-RFLP and capillary electrophoresis.

The improvement of sensitivity and differentiation in rapidly identifying a small amount of mycobacteria in sputum has significant implications for reducing tuberculosis transmission. We previously applied the conventional PCR and capillary electrophoresis (CE) to establish the restriction fragment length polymorphism (RFLP) pattern of mycobacterial 65-kDa heat shock protein (hsp65) gene from colony specimens. However, the previous analysis did not provide enough sensitivity for sputum specimens in which the limitation of analysis might be hindered by PCR inhibitors and primer-dimers formation during amplification. In the current study, nested PCR (nPCR) had been redesigned for PCR-RFLP analysis (PRA) of mycobacterial hsp65 gene using CE. The results show both Mycobacterium tuberculosis complex and mycobacteria other than tuberculosis could be identified in the presence of PCR inhibitors. The interference due to primer-dimers was also minimized. Based on the Poisson distribution, the repeatability of single DNA molecule detection was greatly affected by sampling probability and might be improved significantly by increasing the sample loading. The PRA using nPCR and CE is not only able to detect the individual mycobacterial DNA molecule but also potentially differentiate the species.