Effect of Antigen Retrieval on Genomic DNA From Immunodissected Samples.

Immunohistochemical (IHC) staining is an established technique for visualizing proteins in tissue sections for research studies and clinical applications. IHC is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immunodissection, computer-aided laser dissection (CALD), expression microdissection (xMD), and other techniques. The initial antigen retrieval (AR) process increases epitope availability and improves staining characteristics; however, the procedure can damage DNA. To better understand the effects of AR on DNA quality and quantity in immunodissected samples, both clinical specimens (KRAS gene mutation positive cases) and model system samples (lung cancer patient-derived xenograft tissue) were subjected to commonly employed AR methods (heat induced epitope retrieval [HIER], protease digestion) and the effects on DNA were assessed by Qubit, fragment analysis, quantitative PCR, digital droplet PCR (ddPCR), library preparation, and targeted sequencing. The data showed that HIER resulted in optimal IHC staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased overall yields. However, neither of the AR methods combined with IHC prevented ddPCR amplification of small amplicons and gene mutations were successfully identified from immunodissected clinical samples. The results indicate for the first time that DNA recovered from immunostained slides after standard AR and IHC processing can be successfully employed for genomic mutation analysis via ddPCR and next-generation sequencing (NGS) short-read methods.

The association of molecular typing, vancomycin MIC, and clinical outcome for patients with methicillin-resistant Staphylococcus aureus infections.

BACKGROUND/PURPOSE: There are reports of an increase in vancomycin minimum inhibitory concentration (MIC) against methicillin-resistant Staphylococcus aureus (MRSA) over time, a phenomenon referred to as "MIC creep", but some studies have conflicting results. The aim of this study is to evaluate the association of molecular typing, vancomycin MIC, and clinical outcome for patients with MRSA infections. METHODS: Thirty-two MRSA isolates from Taichung Veterans General Hospital (TCVGH), Taichung, Taiwan during the period of 2003 to 2008 were analyzed for the association of sequence typing, vancomycin MIC, and the correlated clinical outcome for patients with MRSA infections. The vancomycin MICs of 28 additional isolates from 2014 were used for the detection of MIC creep. RESULTS: Among the genotypes of 32 isolates, there were 17 (53.1%) isolates with ST239-SCCmecIII, seven (21.9%) isolates with ST5-SCCmecII, six (18.8%) isolates with ST59-SCCmecIV, and two (6.2%) isolates with ST59-SCCmecVT. Two isolates had an MIC of 2 µg/mL and were identified as ST239-SCCmecIII. No statistically significant change in the distribution of MICs of all isolates was observed between 2003 and 2014 (p = 0.263). There was no significant difference in the mortality rates between two groups of patients with vancomycin MICs < 2 µg/mL and ≥ 2 µg/mL (p = > 0.99). CONCLUSION: There was no vancomycin MIC creep in the period from 2003 to 2014 in this study. Appropriate prognostic models for assessment of the association among sequence types, vancomycin MICs, and clinical outcome warrant further investigation.

Impact of revised susceptibility breakpoints on bacteremia of Klebsiella pneumoniae: Minimum inhibitory concentration of cefazolin and clinical outcomes.

BACKGROUND: The Clinical and Laboratory Standards Institute (CLSI) revised the susceptibility breakpoints of cephalosporins for Enterobacteriaceae in 2010 and 2011. However, there is a lack of clinical data about the correlation of minimum inhibitory concentrations (MICs) and clinical outcome. Data for the distribution of MICs and clinical outcomes were analyzed in this study to evaluate the impact of changes in the CLSI breakpoints on the treatment of Klebsiella pneumoniae bacteremia. METHODS: Ninety-seven bacteremic K. pneumoniae isolates from Taichung Veterans General Hospital, Taichung, Taiwan were collected for study during the period 2009-2011. The cefazolin MIC was determined by the broth microdilution method according to the recommendations of the CLSI. The MIC distribution of cefazolin and the clinical responses to definitive cefazolin treatment were analyzed. RESULTS: The modal cefazolin MIC among the 97 isolates was 1 µg/mL and accounted for 73 (75.3%) isolates. There were 18 (18.6%) isolates with a cefazolin MIC of 2 µg/mL. The conventional dosage regimens of cefazolin (1 g every 6 hours or 8 hours) achieved a clinical cure in 70 (97.2%) of 72 patients in the group with a cefazolin MIC ≤1 µg/mL and in 14 (87.5%) of 16 patients in the group with a cefazolin MIC of 2 µg/mL. With the conventional dose, the cumulative clinical cure rate for K. pneumoniae bacteremia with cefazolin MIC ≤2 µg/mL was 95.5% (84/88 patients). CONCLUSION: The conventional cefazolin dose still can result in satisfactory clinical cure rates for bacteremic episodes due to K. pneumoniae with cefazolin MIC ≤2 µg/mL, the revised susceptible breakpoint of CLSI 2011.

The impact of revised CLSI cefazolin breakpoints on the clinical outcomes of Escherichia coli bacteremia.

BACKGROUND/PURPOSE: The susceptibility breakpoints of cephalosporins for Enterobacteriaceae were revised by the Clinical and Laboratory Standards Institute (CLSI) in 2010 and 2011. The clinical outcome and susceptibility data were analyzed to evaluate the impact of revised CLSI cefazolin breakpoints on the treatment of Escherichia coli bacteremia. METHODS: Forty-three bacteremic Escherichia coli isolates from Taichung Veterans General Hospital, Taichung, Taiwan, during the period from January 2013 to December 2013, were selected to analyze the minimum inhibitory concentration (MIC) distributions of cefazolin and the correlated clinical responses to cefazolin therapy. RESULTS: The modal cefazolin MIC among the 43 isolates was 1 µg/mL and accounted for 18 (42%) isolates. The cumulative percentage for MICs ≤ 2 µg/mL was 79%. The conventional dosing regimens achieved clinical cure in 33 (97%) of 34 patients with bacteremia due to E. coli with a cefazolin MIC ≤ 2 µg/mL, in all of the six patients with a cefazolin MIC of 4 µg/mL, and all of the three patients with a cefazolin MIC of 8 µg/mL. CONCLUSION: The microbiological data support the revised CLSI breakpoints of cefazolin. The conventional cefazolin dosing regimens can still achieve satisfactory clinical cure rates for bacteremia of E. coli with a cefazolin MIC ≤ 2 µg/mL in patients without severe septic shock. Before the approval of the efficacy of cefazolin for the treatment of E. coli isolates with a cefazolin MIC of 4 µg/mL, it is prudent to use cefazolin only when a high drug level can be achieved in the infection site, such as the urinary tract.

Molecular surveillance and clinical outcomes of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae infections.

BACKGROUND/PURPOSE: The emergence of carbapenem-resistant Enterobacteriaceae (CRE) is a cause for great concern. The aim of this study was to evaluate antimicrobial susceptibility, mechanisms of carbapenem-resistance in two members of the Enterobacteriaceae family (Escherichia coli and Klebsiella pneumoniae), and clinical outcomes of their infections. METHODS: The susceptibility tests of 16 E. coli and 60 K. pneumoniae isolates, collected from 2010 to 2011, were assessed. The minimal inhibitory concentrations of eight antimicrobial agents were assessed by the broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute. The detection of beta-lactamase genes was performed by polymerase chain reaction. The genetic relatedness of these isolates was determined by pulsed-field gel electrophoresis (PFGE) fingerprinting. RESULTS: The carbapenemase genes blaKPC-2 and blaOxA were detected in one and five K. pneumoniae isolates, respectively. The genetic combinations blaSHV-5-blaDHA and blaSHV-5-blaCTx-M-G9 were prevalent in 45% and 26.7% of 60 K. pneumoniae isolates, respectively. The susceptibility rates of 60 K. pneumoniae isolates to colistin and tigecycline were 58.3% and 50.0%, respectively. The 30-day mortality rates of the patients treated with carbapenem, colistin, or tigecycline were as high as 60.6%. Nine clusters of K. pneumoniae isolates were identified by PFGE fingerprinting. CONCLUSION: The findings of carbapenemase genes in a few isolates and small clusters of CRE indicated the emerging problems in the hospital. The high mortality rates were observed in the patients treated by colistin and tigecycline, although they were the only alternative treatment options for CRE infections. Active surveillance and an effective infection control strategy should be implemented to control the spread of CRE infections.

Changing trends in antimicrobial susceptibility of Streptococcus pneumoniae isolates in Taiwan, 2006-2007.

BACKGROUND: Multiple antibiotic-resistant clones of Streptococcus pneumoniae have spread throughout the world and continue to evolve under the selective pressure of antibiotics and vaccines. The aim of this study is to assess the susceptibility of S. pneumoniae isolates and to analyze the resistance trends in Taiwan. METHODS: Antimicrobial susceptibility tests were performed on 152 nonmeningeal isolates of S. pneumoniae that were collected from 13 different hospitals around Taiwan from 2006-2007. Tests were performed using the broth microdilution method according to recommendations of the Clinical and Laboratory Standards Institute. RESULTS: The minimal inhibitory concentrations (MIC(50)/MIC(90)) of penicillin, cefotaxime, vancomycin, and moxifloxacin were 0.5/1.0, 0.25/1.0, 0.25/0.5, and 0.06/0.12 µg/mL, respectively. The susceptibility rates of penicillin, cefotaxime, vancomycin, and moxifloxacin were 99.3%, 99.3%, 100%, and 98.7%, respectively. However, if the meningitis breakpoints were applied to these nonmeningeal isolates, the susceptibility rates of penicillin and cefotaxime were reduced to 18.4% and 76.3%, respectively. Compared with the findings from previous studies in Taiwan, our results show that the percentage of S. pneumoniae isolates with a penicillin MIC of 0.12-1.0 µg/mL increased from 43.3% in 1996-1997 to 73.7% in 2006-2007 (p < 0.001). The percentage of S. pneumoniae isolates with a cefotaxime MIC of 1.0 µg/mL increased from 11.3% in 1996-1997 to 23.0% in 2006-2007 (p < 0.001). Regarding the serial MIC intervals of the four antimicrobial agents, there was no significant difference between bacteremic and nonbacteremic isolates. CONCLUSION: Although nonmeningeal S. pneumoniae isolates remained susceptible to penicillin, the proportion of isolates with a penicillin MIC of 0.12-1.0 µg/mL or cefotaxime MIC of 1.0 µg/mL increased during the past decade in Taiwan. The ever-increasing resistance of S. pneumoniae has a great impact on the treatment of meningitis.

Phenotypic detection and polymerase chain reaction screening of extended-spectrum ß-lactamases produced by Pseudomonas aeruginosa isolates.

BACKGROUND/PURPOSE: A growing number of ß-lactamases have been reported in Pseudomonas aeruginosa isolates. The aims of this study were to survey the types of extended-spectrum ß-lactamases (ESBLs) by polymerase chain reaction (PCR), to evaluate the reliability of phenotypic tests for ESBLs, and to identify the clonal distribution by pulsed-field gel electrophoresis (PFGE) among P. aeruginosa isolates resistant to expanded-spectrum cephalosporins (ceftazidime, aztreonam, or cefepime). METHODS: The antimicrobial susceptibility of 57 P. aeruginosa isolates from blood specimens were examined according to the recommendations of the Clinical Laboratory Standards Institute. ESBL phenotypes were determined by using cloxacillin-containing double disc synergy test (DDST). The existence of 11 ß-lactamase genes was detected by PCR. RESULTS: Of the 57 P. aeruginosa isolates, 35 (61.4%) isolates were PCR-positive for ß-lactamase genes. Twelve of 35 isolates were PCR-positive for combination of ampC and ESBL genes, including TEM, GES, SHV, VEB and OXA-I genes. The sensitivity and specificity of cloxacillin-containing DDST (using the criteria of ceftazidime zone diameter increased ≧5 mm) were 84.1% and 54.5%, respectively. Nine clusters were classified among 35 PCR-positive isolates by PFGE. Isolates of clusters B and C were distributed in different wards of this hospital during a period of 3-4 years. CONCLUSION: ESBL genes are not uncommon in P. aeruginosa isolates. Cloxacillin-containing DDST can enhance the sensitivity and has a potential role for phenotypic detection of ESBL-producing P. aeruginosa, and PCR is also helpful for the identification of specific ß-lactamase genes. These P. aeruginosa isolates were classified into several diverse clones which could continue to spread in the hospital over a long period of time.

Clinical outcome of Mycobacterium abscessus infection and antimicrobial susceptibility testing.

BACKGROUND/PURPOSE: Mycobacterium abscessus is the most resistant and rapidly growing mycobacterium and causes a wide range of clinical infectious diseases. The relationship between antimicrobial susceptibility and clinical outcome needs to be further evaluated. METHODS: Forty M. abscessus isolates were obtained from clinical specimens of 40 patients at the Taichung Veterans General Hospital from January 2006 to December 2008. Antimicrobial susceptibility testing was performed using the broth microdilution method according to the recommendations of the National Committee for Clinical Laboratory Standards. The clinical manifestations and outcomes were reviewed from medical records. RESULTS: Twenty-two patients were diagnosed with M. abscessus infection. Cough (86.3%), hemoptysis (31.8%) and fever (18.1%) were the most common symptoms. The radiographic findings included reticulonodular opacities (50.0%), consolidation (31.8%) and cavitary lesions (18.1%). The 40 isolates were susceptible to amikacin (95.0%), cefoxitin (32.5%), ciprofloxacin (10.0%), clarithromycin (92.5%), doxycycline (7.5%), imipenem (12.5%), moxifloxacin (22.5%), sulfamethoxazole (7.5%) and tigecycline (100%). The rate of treatment failure was 27.3% at the end of the 12(th) month after the start of treatment, although these patients were treated with a combination of clarithromycin and other antimicrobial agents. CONCLUSION: M. abscessus is naturally susceptible to clarithromycin and amikacin, variably susceptible to cefoxitin and imipenem, and resistant to most other antimicrobial drugs. Combination therapy with clarithromycin, amikacin and other active antimicrobial agents may lead to clinical improvement; however, the rate of treatment failure is still high.

Antimicrobial susceptibility and multiplex PCR screening of AmpC genes from isolates of Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens.

BACKGROUND/PURPOSE: The emergence of multiple drug resistance in Enterobacteriaceae is of particular concern. The aim of this study was to evaluate the antimicrobial susceptibility and screen for the ampC gene in three members of the Enterobacteriaceae family (Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens) found at Taichung Veterans General Hospital during the past 5 years using multiplex polymerase chain reaction (PCR). METHODS: The susceptibility of thirty isolates from each of the three Enterobacteriaceae family members to five antimicrobial agents (ceftazidime, flomoxef, imipenem, moxifloxacin, and colistin) was assessed. The susceptibility was analyzed by disk diffusion, screening and confirmatory tests for extended-spectrum ß-lactamases (ESBL) and minimum inhibitory concentration tests according to the recommendations of the Clinical and Laboratory Standards Institute. The detection of ampC genes (3 families, including DHA, EBC and CIT) was performed by multiplex PCR. To detect the coexistence of ESBL genes, PCR was performed using five primer pairs: TEM, SHV, SHV-5, CTX-M-3, and CTX-M-14. RESULTS: Of the 90 isolates, 53 (58.9%) were positive in the screening test for ESBL. Resistance genes were detected in 12 (22.6%) of these isolates: ampC gene of DHA type in one E. cloacae isolate and EBC type in three E. cloacae isolates; ampC gene of CIT type in four C. freundii isolates; CTX-M-3-like in one C. freundii isolate and one S. marcescens isolate; TEM in three E. cloacae isolates, three C. freundii isolates and two S. marcescens isolates; SHV in one C. freundii isolate. CONCLUSION: Antibiotic phenotypes cannot accurately distinguish the resistance mechanisms caused by ampC or ESBL, and especially in ESBL-ampC combinations. However, PCR is a useful technique for the identification of the different types of resistance genes.

Chryseobacterium meningosepticum infection: antibiotic susceptibility and risk factors for mortality.

BACKGROUND AND PURPOSE: A limited range of antibiotic classes are available for treatment of Chryseobacterium meningosepticum infections. Although the role of C. meningosepticum in newborn infections and immunocompromised hosts has been recognized, clinical data detailing these infections remain limited. This retrospective study investigated the risk factors for mortality in patients with C. meningosepticum infections and the antibiotic susceptibility of clinical isolates. METHODS: Information on demographic characteristics, clinical parameters, antibiotic treatment, and outcomes was collected. Statistical significance of potential prognostic parameters was analyzed by Fisher's exact test. The antimicrobial susceptibility of 19 isolates to seven antibiotics was determined, and susceptibility results were presented as minimal inhibitory concentration (MIC) range, MIC at which 50% of isolates were inhibited (MIC(50)), and MIC at which 90% of isolates were inhibited (MIC(90)). RESULTS: Hypoalbuminemia (<2.5 g/dL) [p=0.02] and increased pulse rate (p=0.008) at the onset of infection, and presence of an indwelling central venous line (p=0.04) were associated with poor outcomes. Use of appropriate antibiotics was not significantly associated with the clinical outcome (p=0.21). MIC values of levofloxacin (MIC(50)/(90), 0.12/2 microg/mL) were lower than those of ciprofloxacin (0.5/4 microg/mL). CONCLUSION: Hypoalbuminemia, increased pulse rate at the onset of infection and presence of central venous line infection were associated with a poor outcome in patients with C. meningosepticum.

Both cGMP and peroxynitrite mediate chronic interleukin-6-induced negative inotropy in adult rat ventricular myocytes.

We previously showed that chronic exposure to interleukin (IL)-6 decreases contractile and sarcoplasmic reticular (SR) function assessed by postrest potentiation (PRP) via a nitric oxide (NO)-dependent mechanism in adult rat ventricular myocytes (ARVM). Cyclic GMP (cGMP) has been associated with NO-associated negative inotropic effects of IL-6 during acute exposure; however, its role in chronic cardiac effects of IL-6 remains unclear. The present study examined the roles of cGMP and peroxynitrite (ONOO-) in chronic IL-6-induced negative inotropy in ARVM. After ARVM were exposed to IL-6 for 2-24 h, intracellular cGMP contents were time dependently increased; this was mimicked by a NO donor and abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), or Rp-8-Br-cGMP, an inhibitor of cGMP-dependent protein kinase G (PKG). Meanwhile, the IL-6-induced decrease in PRP at 2 h was blocked by ODQ or Rp-8-Br-cGMP. By contrast, ODQ or Rp-8-Br-cGMP only attenuated the inhibition of PRP induced by IL-6 after 24 h exposure. Furthermore, IL-6 time dependently increased superoxide anion production and ONOO- formation; the latter was abolished by 5,10,15,20-tetrakis-(4-sulphonatophenyl)-porphyrinato iron (III) (FeTPPS), an ONOO- decomposition catalyst. Interestingly, FeTPPS had no effect on the IL-6-elicited decrease in PRP at 2 h, but attenuated it after 24 h exposure. Moreover, inhibition of sGC/cGMP/PKG, but not ONOO- formation, abolished the IL-6-induced inhibition of kinetics of myocyte contraction during 24 h exposure. We conclude that while the sGC/cGMP/PKG pathway was the primary mechanism for chronic IL-6-induced negative inotropy at 2 h, both sGC/cGMP/PKG and ONOO-, at least in part, mediate the IL-6-induced inhibition of SR function after 24 h exposure.