Objective To establish a multiplex polymerase chain reaction (PCR) method for rapid detection of three kinds of di‐arrheagenic Escherichia coli(EPEC ,EIEC ,EHEC)simultaneously .Methods The eae gene of EPEC ,ipaH gene of EIEC and stx1 gene of EHEC were selected to design primers ;the reaction system and condition were adjusted to optimize the multiplex PCR sys‐tem .Results The target gene fragments were amplified correctly with these primers .The three target bacteria could be detected at the same time by multiplex PCR .Conclusion A rapid multiplex PCR system were successfully established for detection of three di‐arrheagenic Escherichia coli ,and this system could be suitable for rapid screening in food safety .

Objective To understand the origin and variation of the hemagglutinin gene of isolates viruses from 3 novel influenza A( H1N1 ) deaths in Changsha ( A/Hunan Kaifu/SWL4142/2009 ( H1N1 ) , A/Hunan Changsha/SWL4346/2009 ( H1 N1 ) and A/Hunan Furong/SWL4224/2009( H1N1 )). Methods The nasopharyngeal swab specimens from the 3 novel influenza A( H1N1 ) deaths in Changsha were tested by RT-PCR and influenza viruses were isolated simultaneously. With the sequencing primers recommended by World Health Organization (WHO), the HA gene of sequences of 3 novel influenza A( H1N1 ) deaths were tested by CEQTM 8000 Genetic Analysis System, through dye terminator cycle sequencing. The sequencing results were submitted to GenBank, then the results were analyzed for amino acid alignment and phylogenetic tree analysis with ClustalX and Mega4.1 software. Results All the nucleotide homologies of HA gene sequences in A/Hunan Kaifu/SWL4142/2009 ( H1N1 ), A/Hunan Changsha/SWL4346/2009 ( H1N1 ) and A/Hunan Furong/SWL4224/2009( H1N1 ) are 99% as compared with the novel influenza A( H1N1 ) virus strains of A/NewYork/3502/2009 ( H1N1 ), A/Shanghai/71T/2009 ( H1N1 ) and A/Chita/01/2009 ( H1N1 )The nucleotide homology of the 3 HA gene sequences are more than 99. 5% the same compared with the novel influenza A( H1N1 ) virus strain ( A/Sichuan/1/2009( H1N1 ) ) in China. Phylogenetic tree analysis reveals that 2009 novel influenza A(H1N1 ) viruses including 3 HA gene sequences of A/Hunan Kaifu/SWL4142/2009 ( H1 N1 ), A/Hunan Changsha/SWL4346/2009 ( H1N1 ), A/Hunan Furong/SWL4224/2009( H1N1 ) had a close evolutionary relationship with the swine H1 virus isolates in North America ( A/Swine/Indiana/P12439/00), but a distant evolutionary relationship with those human seasonal A( H1 N1 ) influenza virus and avian. After comparing with genes of A/Swine/Indiana/P12439/00, we found that the HA gene sequences of the 3 viruses isolated had 28,30 and 27 amino acids with mutation respectively, but only one (R53K) amino acids mutation at 21 important antigenic sites in the 3 viruses isolated. Multiple alignment of 364 HA genes sequences of novel influenza A ( H1N1 ) viruses in the world showed they had 119 nonconserved amino acids, 5 non-conserved position at important antigenic sites. Conclusions The HA gene sequences from 3 viruses isolated in this study and other influenza A ( H1N1 ) viruses might originate from swine A( H1N1 ) in North America by variation. The 3 HA gene sequences of viruses isolated have high homology as compared with the novel influenza A ( H1N1 ) virus strains worldwide, and the 3 HA gene sequences of viruses isolated are in stable condition as the vast majority of novel influenza A( H1N1 ) virus strains in the world.

Objective To explore the relationship between the expression of CD44v6,P16 protein and the invasion and metastasis in gastric carcinoma and to investigate the correlation between the expression of CD44v6 and P16 protein.Methods Immunohistochemical methods were used to detect the expression of CD44v6 and P16 protein in 110 cases of gastric carcinoma.Results In 110 cases of gastric carcinoma,the expression of CD44v6 and P16 protein were 72.7%(80/110)and 47.3%(52/110)respectively. The high level expression of CD44v6 protein and low level expression of P16 protein were positively correlated with the TNM staging,serosa infiltration,lympho node and liver metastasis of gastric carcinoma(P

Objective To understand the clinical epidemiology of nosocomial candidemia in Huashan Hospital during a 10-year period. Methods One hundred and nine cases of nosocomial candidemia in Huashan Hospital affiliated Fudan University during the period of 1998- 2007 were retrospectively reviewed. The underlying conditions, risk factors, clinical manifestations, treatment and outcome were described. The prognostic factors were analyzed by chi square test or Fisher exact probability test. Multivariate analysis was done by multiple Logistic regression. Results The average annual incidence of nosocomial candidemia during the study period was 0.28/10 000 patients per day.The most common pathogen was C. albicans (59/109,54.1%), followed by C. tropicalis (20/109,18.3%), then C. parapsilosis (11/109, 10. 1%), C. glabrata (11/109, 10.1%), and other Candida spp. (8/109, 7.3% ). Underlying diseases frequently identified included diabetes (50,45.9%), solid malignancy (32, 29.4%), head trauma (13, 11. 9%) and stroke (12, 11.0%).There were 37 cases who died or deteriorated. The overall mortality was 34.0% and the attributable mortality was 22. 0% (24/109). In multivariate prognostic analysis, retention of central venous catheters (OR: 5.42, 95% CI: 1.68-17.41, P＝0.005), corticosteroid medication (OR: 3.69,95% CI: 1.10-12.34, P=0. 034), and severe sepsis on the day of candidemia (OR: 2.94, 95% CI:1.72-15. 21, P = 0. 003) were factors independently correlated to increased mortality. Furthermore,adequate antifungal therapy was the only independent predictor of decreased overall mortality (OR: 0. 27,95% CI: 0. 09-0. 78,P=0.015). Conclusions The incidence of nosocomial candidemia in our hospital has been increasing during the past decade. Timely diagnosis and treatment plays a key role in the management of nosocomial candidemia,

Objective To investigate the polymorphism profile of cytochrome P_(450)2C19 (CYP2C19) in Chinese patients with invasive fungal infections. Methods Two major single nucleotide polymorphism loci of the CYP2C19 gene (CYP2C19 * 2 and CYP2C19 * 3) were genotyped with PCR and restriction fragment length polymorphism (PCR-RFLP) in 134 patients with invasive fungal infections and 134 healthy volunteers. Allele frequencies and the proportions of metabolizer phenotypes were compared. Results In patients with invasive fungal infections, CYP2C19 * 1, CYP2C19 * 2 and CYP2C19 * 3 alleles showed frequencies of 58.2%, 36.6% and 5.2%. In healthy volunteers, the frequencies of CYP2C19 * 1, CYP2C19 * 2 and CYP2C19 * 3 were 63.4% , 34. 3% and 2. 2%. There was no significant difference in allele frequencies between the two groups. Of the patients with invasive fungal infections, 33. 6% were homozygous extensive metabolizers, 50.0% heterozygous extensive metabolizers and 16.4% poor metabolizers. Of the healthy volunteers, 40.3% were homozygous extensive metabolizers, 48.5% heterozygous extensive metabolizers and 11. 2% poor metabolizers. The proportions of metabolizer phenotypes were similar between the two groups. Conclusions Significant CYP2C19 polymorphism was detected in both groups. Approximately two thirds of the Chinese patients were either heterozygous extensive metabolizers or poor metabolizers. The genetic polymorphism may have important effect on drug metabolism in these patients

To develop a low-cost, reliable, easy-to-maintain and practical instrument for protein purification. Some ultraviolet luminescent diode was used to provide 280 nm light source, and high-sensitivity S1336 photo-electric detector was employed for real-time monitoring of purified protein solution flowing through quartz cell to supervise the concentration of the protein. The instrument gave voice alarm and stopped working in case the protein concentration was less than the standard one. The lower SCM monitored the liquid level of the protein collecting cup and the position of loading arm through laser infrared distance sensor, so that a cup full of protein might be replaced by another empty cup. The instrument involved in Samsung S5PV210 embedded master computer, Wince6.0 operating system, Keil4.0 and VS2005. Trials proved that the instrument could perform real-time monitoring and curve display of dual-channel ultraviolet absorption, and could realize auto collection of 735 ml protein solution and up to 5-hour standby. The instrument developed has simple structure, high reliability and easy maintenance, and meets the desired require-ments.

Objective To analyze the expression of interleukin (IL)-31 in tuberculous pleural effusion,and to evaluate its diagnostic value of tuberculous effusion.Methods Seventy-one patients with pleural effusion were enrolled,including 40 cases of tuberculous pleural effusion and 31 cases of malignant pleural effusion.Luminex method was applied to detect the IL-31 expression in pleural effusion.IL-31 levels were compared using non-parametric Mann-WhitneyU test,and the receiver operator characteristic (ROC)curve was used to elvaluate the diagnostic value of IL-31 .Results IL-31 expression in tuberculous pleural effusion was significantly higher than that in malignant pleural effusion with statistical significance (529.4 ng/L vs 13.8 ng/L,U =62,P <0.01 ).Based on the level of IL-31 expression,area under the ROC curve was 0.95 with the optimum cut-off value of 67.5 ng/L.Thus,the sensitivity and specificity of IL-31 ≥67.5 ng/L for diagnosis of tuberculous pleurisy were 82.5 % (95 %CI :73.3% - 94.2%)and 100.0% (95 %CI :91 .4%-100.0%),respectively.Conclusion IL-31 is highly sensitive and specific for the diagnosis of tuberculous pleural effusion, which favors the differentiation of tuberculosis from malignance.

ObjectiveTo evaluate the application of a whole blood interferon-γ (IFN-γ) release assay QuantiFERON-TB Gold In Tube (QFT-GIT) in the diagnosis of tuberculous pleural effusion.Methods IFN-γ released by specific T cells stimulated by early secreted antigenic target 6 × 103protein (ESAT-6),culture filtrate protein 10 (CFP -10) and TB7.7 were measured by QFT-GIT test in 44 tuberculous pleural effusion patients and 16 non-tuberculous pleurisy controls.The IFN-γ release level between groups was compared by Mann-Whitmey test.ResultsThe positive rates of QFT-GIT in patients with tuberculous pleural effusion and non tuberculous pleurisy were 95.5％ and 12.5％,respectively.The sensitivity,specificity,positive predictive value and negative predictive value of QFT-GIT were 95.6％,87.5％,95.6％ and 87.5％,respectively.The antigen-specific IFN-γ release level in the patients with tuberculous pleural effusion was significantly higher than that in non-tuberculous pleurisy controls (P＜0.01).Conclusions The whole blood INF-γ release assay QFT-GIT is a sensitive and specific assay for detecting pleural tuberculosis infection.It could be a useful diagnostic tool for the diagnosis of tuberculous pleural effusion in China.

Objective To analyze the clinical features of patients with uncommon fungal infections in central nervous system (CNS).Methods Thirty-five patients with uncommon CNS fungal infections who were admitted to Huashan Hospital from 1997 to 2010 were retrospectively reviewed.The pathogens,symptoms and signs.treatments of patients were evaluated.The data were analyzed by rank sum test and Fisher'S exact test.Results Twenty-nine of the 35 patients met the definition criteria of prover CNS fungal infections,while the other 6 had probable diagnosis.Predisposing factors were found in 86% of all patients.The most common pathogens were Aspergillus and Candida species.The symptoms and signs commonly occurred including fever(22 cases),headache(19 cases), cranial neuropathy(12 cases),and meningeal irritation sign(12 cases).High white blood cell count,high protein level,and low glucose level were the main findings of cerebrospinal fluid (CSF) analysis.Patients with cerebral aspergillosis were more frequently accompanied with immunocompromised conditions, and they often got CNS aspergillosis from hematogenous dissemination or direct extension of paranasal sinus infection.Cerebral granuloma and abscess were the common clinical characteristics of CNS aspergillosis.Cerebral candidiasis often arose from neurosurgical surgery or traumatic brain injury,and these patients were usually presented with meningitis.All patients were treated with antifungal drugs and (or) surgical intervention and 77%(27/35) of the patients achieved complete or partial responses. Antifungal agents combined with surgical resection might improve outcome of patients with CNS aspergillosis; while removal or replacement of drainage tubes in combination with antifungal treatment showed satisfactory efficacy in patients with cerebral candidiasis who usually had shunt manipulation. Conclusions The incidence of CNS fungal infection, such as cerebral aspergillosis and candidiasis, is increasing. Early diagnose and therapeutic intervention are crucial for improving outcome.

Objective To describe the distribution of mannose binding lectin (MBL) genetic polymorphisms in non-acquired immunodeficiency syndrome (AIDS) patients with cryptococcosis in China and to verify the association of MBL polymorphisms with susceptibility to cryptococcosis.Methods The case-controlled genetic association study was conducted and 167 non-AIDS patients with cryptococcosis and 208 healthy controls were recruited. Genome DNA was extracted from the peripheral blood and MBL gene was amplified by polymerase chain reaction (PCR). Six singlenucleotide polymorphisms ( SNP) of MBL gene were sequenced. The association of MBL polymorphisms with susceptibility to cryptococcosis were analyzed. The comparison between patients and controls was performed by chi square test or Fisher's exact test. The differences of MBL plasma concentrations between groups with different MBL genotypes were compared by single factor variance analysis. Results There were no differences between patients and controls in terms of MBL genotype frequencies, haplotypes and genotypes (all P>0. 05). Compared with healthy control, the deficient MBL-producing genotypes were strongly associated with cryptococcal meningitis (16. 5% vs 8. 7%,χ2=4.25, P=0.0392, OR = 2.09), particularly in patients without underlying immunocompromised conditions (21. 4% vs 8. 7%, χ2 =7. 15, P = 0. 0075, OR = 2. 88). Individuals with MBL deficiency genotypes showed significantly higher rates of central nervous system (CNS) cryptococcal infection rather than non-CNS cryptococcosis (16. 5% vs 3. 1%, Fisher's exact test, P = 0. 010, OR = 6. 13).The difference was even more significant in the immunocompetent patients (21. 4% vs 4. 0%, P =0.009, OR= 6. 55). Conclusion MBL deficiency is associated with cryptococcal meningitis and may play a role in CNS Cryptococcus infection.