Wenying Xia

Double recognition of oligonucleotide and protein in the detection of DNA methylation with surface plasmon resonance biosensors

DNA methylation plays an essential role in maintenance of cellular function. A growing number of human diseases have been found to be associated with aberrant DNA methylation, especially cancer. However, current technologies used in DNA methylation detection are complicated and time consuming. A promotor of the Adenomatous polyposis coli (APC) gene, a well-studied tumor suppressor gene, was used as the detection target DNA sequence. The double recognition mechanism was realized with oligonucleotide probe hybridization and specific protein binding. First, complementary target DNA was captured by the probe immobilized onto a surface plasmon resonance (SPR) sensor chip. Then, the recombinant methyl-CpG binding domain (MBD) protein was passed over the surface to recognize and bind to methylated CpG sites. Binding resulted in an increase in the refractive index, and a detectable optical signal was generated. Five picomoles of methylated APC promotor DNA could be easily detected with this method. The entire detection could be completed within 1h. This work represents the first SPR based biosensor technology, which achieves simple and specific DNA methylation detection and avoids complicated bisulfite treatment and methylation-sensitive restriction digestion. It will improve our ability to detect DNA methylation specifically and rapidly, and promote our understanding of the role of DNA methylation in gene regulation and diseases.

Epidemiology of carbapenem resistant Enterobacteriaceae (CRE) during 2000-2012 in Asia

BACKGROUND Over the past decade, the worldwide emergence of carbapenem resistance in Enterobacteriaceae has become a severe public health issue. This meta-analysis aims to describe the epidemiology of carbapenem resistant Enterobacteriaceae (CRE) during the years of 2000-2012 in Asian area. METHODS PubMed and Embase databases were searched to identify the qualified papers. Random or fixed-effect model was used to deal with the data. RESULTS Over all the 49 Asian countries (or regions), only 37.5% [19] of them contributed epidemiology data of CRE, and the rest ones provided either only case reports or no information at all. In Asia, the prevalence of CRE was still low during the study period with average resistance rates of 0.6% (95% CI, 0.6-0.8%, imipenem) and 0.9% (95% CI, 0.7-1.2%, meropenem). Resistance rates to imipenem and meropenem in Enterobacteriaceae exhibited stably escalating trend. Similar trend can also be observed among each Enterobacteriaceae genus, such as E. coli, Klebsiella spp. and Enterobacer spp. Klebsiella spp. accounted for the largest proportion among the isolates resistant to imipenem, and then followed by E. coli and Serratia. The rank order of resistance rates to imipenem among Enterobacteriaceae genus during the period of 2000-2012 was as follows: Serratia spp. (1.8%) > Proteus spp. (1.6%) > Klebsiella spp. (0.8%) = Citrobacter spp. (0.8%) > Enterobacer spp. (0.7%) > E. coli (0.2%). CONCLUSIONS Given the fact that the prevalence of CRE was increasing during the past decade, it is urgent for us to establish regional surveillance worldwide, carry out more effective antibiotic stewardship and infection control measures to prevent further spread of carbapenem resistance in Enterobacteriaceae.

MALDI-TOF MS versus VITEK 2 ANC card for identification of anaerobic bacteria

BACKGROUND Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an accurate, rapid and inexpensive technique that has initiated a revolution in the clinical microbiology laboratory for identification of pathogens. The Vitek 2 anaerobe and Corynebacterium (ANC) identification card is a newly developed method for identification of corynebacteria and anaerobic species. The aim of this study was to evaluate the effectiveness of the ANC card and MALDI-TOF MS techniques for identification of clinical anaerobic isolates. METHODS Five reference strains and a total of 50 anaerobic bacteria clinical isolates comprising ten different genera and 14 species were identified and analyzed by the ANC card together with Vitek 2 identification system and Vitek MS together with version 2.0 database respectively. 16S rRNA gene sequencing was used as reference method for accuracy in the identification. RESULTS Vitek 2 ANC card and Vitek MS provided comparable results at species level for the five reference strains. Of 50 clinical strains, the Vitek MS provided identification for 46 strains (92%) to the species level, 47 (94%) to genus level, one (2%) low discrimination, two (4%) no identification and one (2%) misidentification. The Vitek 2 ANC card provided identification for 43 strains (86%) correct to the species level, 47 (94%) correct to the genus level, three (6%) low discrimination, three (6%) no identification and one (2%) misidentification. CONCLUSIONS Both Vitek MS and Vitek 2 ANC card can be used for accurate routine clinical anaerobe identification. Comparing to the Vitek 2 ANC card, Vitek MS is easier, faster and more economic for each test. The databases currently available for both systems should be updated and further developed to enhance performance.

Necrotizing pneumonia and empyema caused by Neisseria flavescens infection.

Neisseria flavescens is an uncommon pathogen of human infection, pneumonia and empyema caused by N. flavescens is rarely reported. Herein, we report a 56-year-old diabetic patient presenting necrotising pneumonia and empyema due to N. flavescens infection. The main clinical manifestation of this patient was high fever, sticky pus and gradually aggravating dyspnea. The chest computed tomography (CT) scan showed there are mass of high density areas around hilus of the left lung, hollow sign with inflammation also appeared. A biopsy specimen was taken from the left principal bronchus by lung puncture biopsy and showed necrosis and inflammation. Microscopic examination of direct smear and culture of sticky pus, much more gram-negative diplococcus was present, pathogen was further identified by Vitek NH card, Vitek MS and confirmed as N. flavescens by 16S rRNA gene sequencing finally. Anti-infection therapy following the antimicrobial susceptibility test results was effectively. To our knowledge, this is the first report of pulmonary infection caused by N. flavescens.

Increasing resistance rate to carbapenem among blood culture isolates of Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa in a university-affiliated hospital in China, 2004-2011.

The objective of this study is to investigate the profile of antimicrobial resistance of Gram-negative bacteria in blood cultures from 2004–2011. Pathogens from positive blood cultures were subcultured, and identified in the First Affiliated Hospital of Nanjing Medical University from January 2004 to December 2011. The antibiotic resistance pattern was analyzed by WHONET 5.4. A total of 1224 cases of Gram-negative bacterial isolates were documented, accounting for 38.6% of the total pathogens isolated from positive blood cultures in the 8-year period. The isolation rates of Klebsiella pneumoniae and Acinetobacter baumannii increased nearly three times over the same time span. Most Gram-negative bacteria isolates, except the isolates of Pseudomonas aeruginosa, showed a significantly increased resistance rate to cephalosporins (in particular third/fourth generation cephalosporins). Noteworthy, the antimicrobial resistance of K. pneumoniae, A. baumannii and P. aeruginosa isolates to carbapenem (imipenem and meropenem) was significantly increased and the resistant rate to carbapenem was >80.0% in A. baumannii in 2011. The results from PCR detection for carbapenemases were as follows: 82.8% (24/29) isolates of K. pneumoniae carried the kpc-2 gene; only three metallo-beta-lactamase-positive P. aeruginosa isolates were detected; and 93.1% (67/72) A. baumannii isolates were blaOXA-23 positive. The antimicrobial resistance rate of Gram-negative bacteria isolated from blood cultures significantly increased from 2004 to 2011, with significant resistance to the third/fourth generation cephalosporins and carbapenem.

Changing trend of antimicrobial resistance among pathogens isolated from lower respiratory tract at a university-affiliated hospital of China, 2006-2010

OBJECTIVE To investigate the distribution and the antimicrobial resistance of pathogens in lower respiratory tract infection from 2006 to 2010. METHODS The sputum specimens from inpatients with lower respiratory tract infection in the First Affiliated Hospital of Nanjing Medical University during the past five years were cultured and identified; the antimicrobial resistance was analyzed by the software WHONET 5.4. RESULTS A total of 12,191 isolates were characterized in sputum samples: 73.5% were Gram-negative bacteria, 13.7% were Gram-positive bacteria, and 12.8% were fungi. The isolation rate of Acinetobacter was significantly increasing from 12.8% in 2006 to 26.4% in 2010. The Gram-negative bacterial resistance rate to the second and third generation cephalosporin increased year by year. Decreasing trend, 78.7% in 2006 decreased to 63.5% in 2010 (R(2)=0.93 and P<0.01), in resistance to clindamycin against Staphylococcus aureus was observed. Worth noting is the drug resistance of Acinetobacter and Klebsiella pneumoniae to carbapenem significantly increased (R(2)>0.3 and P≤0.05). CONCLUSIONS The antimicrobial resistance of pathogens in lower respiratory tract infection increased in recent years. The hospitals and government departments should strengthen management of the use of some antibiotics, such as the second/third generation cephalosporin and carbapenem, in order to enhance the effectiveness of medication.

Can plasma DNA monitoring be employed in personalized chemotherapy for patients with advanced lung cancer

Personalized chemotherapy is the ideal treatment usually chosen to help improve the survival chances of patients with advanced lung cancer. However, there is no short-term evaluation protocol for predicting the efficacy of the therapy. The aim of this study was to determine the value of using plasma DNA to monitor chemotherapeutic efficacy and to select most appropriate chemotherapeutic regimen for patients with advanced lung cancer. Eighty-eight lung cancer patients and 200 healthy controls were included in this study. Plasma DNA was extracted from plasma samples with internal controls by using the BILATEST DNA Kit. The quantity of plasma DNA was determined by using duplex real-time quantitative PCR. After first-line chemotherapy, plasma DNA levels of partial response patients were significantly different from those of stable disease patients or progressive disease patients, but with no statistical difference from healthy controls (P=0.014, P<0.001 and P=0.418, respectively). Survival analysis showed a statistically better survival time in patients who had lower levels of plasma DNA after the third cycle chemotherapy (P=0.031). In this study, the correlation of the kinetics of DNA concentrations with chemotherapeutic efficacy during the whole therapy was also observed. The quantification of plasma DNA is a sensitive indicator of chemotherapeutic efficacy in advanced lung cancer patients, and it can be useful in predicting response to therapy and guiding medication.

Emergence of linezolid resistance in a clinical Staphylococcus capitis isolate from Jiangsu Province of China in 2012

Linezolid (LZD) is an important antimicrobial agent for the treatment of infections caused by Gram-positive organisms, including methicillin-resistant Staphylococci. And until now, LZD resistance in clinical is still rare. Here we reported the first case of LZD resistance Staphylococcus capitis in Jiangsu, China. This strain was isolated from a 92-year old female who received long-term and repeatedly antibiotics treatment because of recurrent pulmonary infections in August 2012. Isolated from blood, the Staphylococcus capitis showed a resistance to LZD with a minimal inhibitory concentration (MIC) of 64 µg/mL, and the followed gene detection showed that the isolates existed C2190T and C2561Y point mutations in the 23S rRNA. Moreover, the isolation was also found carrying the cfr gene.

The Study on Newly Developed McAb NJ001 Specific to Non-Small Cell Lung Cancer and Its Biological Characteristics

Monoclonal antibody (McAb) is the key tool for cancer immunodiagnosis and immunotherapy. McAb-based immunotherapy that targets tumor antigens has had great achivement. In this study, a cell clone which kept secreting high-titer IgG1-type McAb named NJ001 against human non-small cell lung cancer (NSCLC) cells was obtained. The titer of purified NJ001 was 2×106. The antigen named SP70 of NSCLC specifically identified by NJ001 was proved to be a protein with the relative molecular mass (Mr) of 70 kDa. The results of immunohistochemical staining indicated that NJ001 could positively react to NSCLC, but weak positively or negatively react to human small-cell lung cancer (SCLC), pulmonary pseudotumor and other epithelial tumors. In soft agar assay, the colony formation efficiency in NJ001 groups decreased in a dose-dependent manner. For the concentration of 100 µg/ml, 200 µg/ml and 400 µg/ml, the inhibition ratio of colony formation was 23.4%, 62.5% and 100% respectively. Meanwhile, NJ001 caused significant reduction in tumor volume and tumor weight compared to control mice in lung cancer xenograft model. The tumor growth inhibition ratio in 200 µg, 400 µg and 800 µg NJ001 groups was 10.44%, 37.29% and 44.04%, respectively. NJ001 also led to cytomorphological changes and induced the apoptosis of human lung adenocarcinoma cell line SPC-A1 significantly. The newly developed NJ001 selectively reacted to NSCLC and exhibited anti-tumor activity both in vitro and in vivo. NJ001 is of great value concerning immunodiagnostics and immunotherapy for NSCLC and holds promise for further research regarding the mechanism underlying tumor progression of NSCLC.

The Clinical Significance of Plasma DNA Quantification for Quake Trauma Patients

The aims of this study were to evaluate if the plasma DNA level, which can be an indicator of cell death in vivo, correlates with injury severity in trauma patients and whether its quantification could be used to monitor the efficacy of the treatment of trauma patients. A duplex real-time PCR assay with internal control was developed as a novel method for accurate quantification of plasma DNA and applied to determine the plasma DNA concentrations in a cohort of 1,187 Chinese healthy adults and 283 trauma patients in the deadly China Wenchuan earthquake. Plasma DNA concentrations determined by our newly developed real-time PCR method were not influenced by different DNA extraction protocols, PCR amplification efficiency and sample error. The median plasma DNA concentration of females (16.9 ng/ml) was significantly lower than that of males (22.6 ng/ml) (p < 0.0001). At the 95% confidence interval, the normal reference intervals of plasma DNA concentration were 0~50 ng/ml for males and 0~40 ng/ml for females. During the early stage of injury, the median plasma DNA level of patients was more than 100 ng/ml i.e., five times that of the healthy controls. There was a statistically significant positive correlation between ISS and plasma DNA concentration (R 2= 0.46, p < 0.0001). A statistically significant difference of plasma DNA concentration between patients with and without organ injury was observed (median, 168.9 vs. 96.7, p = 0.001). This newly developed plasma DNA quantification method allows accurate quantitative measurement and can be useful for determining injury severity stratification and disease processes.