Objective To establish a convenient, fast, economic and hypersensitive low-density DNA array method to detect the genotypes of human papillomavirus (HPV) and evaluate its application in clinic services.Methods HPV in cervical swab samples from 355 suspected female patients collected in gynaecology and obstetrics clinic were genotyped by hybrid capture (HC) II method and low-density DNA array simultaneously. HPV in 730 clinic samples from the area of Pearl River delta were genotyped by low-density DNA array.Results Among 355 suspected samples positive HPV-DNA were detectable in 211 (59.4%) samples by DNA array and 222 (62.5%) samples by HCII method. The concordance rate between the two assays was 94.1%. In the HPV genotypes 15 high-risk type and 5 low-risk type were detected by low-density DNA array. The most common high-risk types were HPV-16, 52, 58 and 56. The peak age of HPV infection was 26-30 years. The distribution of genotypes was different from various degree of cervical changes.Conclusion Either single type or multiple type of HPV infection can be detected by low-density DNA array. The combination of HPV detection with cytology detection will provide instruction for cervical cancer screening.

Objective To develop a multiplex polymerase chain reaction (PCR )method for detecting and genotyping moxifloxacin-resistant Clostridium difficile (C.difficile)isolates.Methods Specific PCR primers of slpA genotypes gr,hr,fr,gc08 and 078 were designed according to the differences of slpA nucleotide sequences in different C.difficile genotypes,and the house-keeping gene tpi specific PCR primers were also added for the construction of multiplex PCR method.Nine common intestinal normal and pathogenic strains were used to verify the specificity of slpA multiplex PCR for the detection of C.difficile.Forty-six C.difficile reference strains,belonging to 11 slpA genotypes,were used to verify the ability of the multiplex PCR method for dectecting and genotyping.Thirty-nine moxifloxacin-resistant clinical isolates were genotyped by the multiplex PCR,and its clinical value was evaluated by comparing with slpA sequence typing (slpA ST)method.Results All the 9 intestinal normal and pathogenic strains were negative when detected by the multiplex PCR.And tpi of 46 C. difficile reference strains were positive,and 36 strains belonging to slpA genotypes gr,hr,fr,gc08 and 078 were genotyped correctly.Other 10 strains which belonged to other 6 genotypes were non-typeable. Among 39 moxifloxacin-resistant clinical isolates,all were positive of tpi,and 32 isolates were typed correctly by the multiplex PCR method,including 22 slpA genotypes gc08,6 genotypes hr,2 genotypes fr,and 2 genotypes 078,which were consistent with slpA ST.However,7 isolates could not be typed by multiplex PCR,which were identified as other genotypes not included in the multiplex PCR by slpA ST. Conclusions A convenient and rapid multiplex PCR method for the detection of C.difficile is established successfully,which can distinguish among five slpA genotypes.slpA genotype gc08 is the common genotype of moxifloxacin-resistant clinical isolates.

Objective To investigate the genotype and variance of toxin associated genes of moxifloxacin‐resistant Clostridium difficile clinical isolates in Sydney .Methods Twenty‐two moxifloxacin‐resistant Clostridium difficile clinical isolates were collected from Sydney ,which were genotyped by using sequencer capillary gel electrophoresis based PCR‐ribotyping ,and toxin A and B cod‐ing gene tcdA and tcdB ,and binary toxin coding gene cdtA and cdtB were detected by using PCR method .Toxin regulator gene tc‐dC was analyzed by using PCR‐sequencing ,and was aligned with reference sequence of VPI 10463 (Genbank accession number :X92982) ,and the tcdC sequence types of all 22 isolates were identified by using blast tool in NCBI .Results Twenty‐one isolates were genotyped as hypervirulent PCR‐ribotypes 027 (RT027) ,and one isolate as RT078 ;all 22 isolates contained tcdA and tcdB for toxin A and B and cdtA and cdtB for binary toxin (tcdA+ tcdB+ cdtA+ cdtB+ ) .The tcdC sequence types of the 21 RT027 i‐solates belong to sc1 ,and that of the one RT078 isolate belongs to WA39 .Compared with tcdC reference sequence of VPI 10463 ,a consecutive 18 bp deletion (nt341 to 379) and one nucleotide deletion at position 117 were found in the 21 RT027 isolates ,and a consecutive 39 bp deletion (nt330 to 368) and one nucleotide mutation at position 184(C> T) were found in the one RT078 isolate . Conclusion Clostridium difficile hypervirulent RT027 was the common moxifloxacin resistant genotype ;Clostridium difficile hy‐pervirulent RT027 and RT078 clinical isolates contained genes for toxin A and B and binary toxin ,and contained gene sequence mu‐tation in toxin regulator gene tcdC .

Objective To investigate the genotype and production of toxin A and B of C .difficile clinical isolates collected from Sydney ,Australia .Methods Sixty‐eight C .difficile clinical isolates were collected from Westmead Hospital ,the University of Sydney ,which were genotyped by using PCR‐ribotyping ,and toxin A ,B coding gene tcdA ,tcdB were detected by using PCR meth‐od .Results Thirty‐one PCR‐ribotypes (RTs) were confirmed in the 68 C .difficile clinical isolates ,RT014 (19 .1% ) and RT002 (11 .8% ) were the common genotypes .Sixty‐four of 68 (94 .1% ) isolates contained tcdA and tcdB for toxin A and B .Conclusion The common prevalent PCR‐ribotypes of C .difficile were RT014 and RT002 in Sydney ,most of the C .difficile clinical isolates contained toxin A and B .

Objective To develop a digital polymerase chain reaction (PCR) ribotyping method and database for Clostridium difficile genotyping.Methods Sequencer based fluorescence capillary gel electrophoresis was used,instead of agarose gel electrophoresis,to establish the digital PCR-ribotyping of Clostridium difficile.Forty Clostridium difficile reference strains,consisting of 10 PCR-ribotypes (RT),were genotyped by the new digital PCR-ribotyping method to set-up the database.Results The sequencer based fluorescence capillary gel electrophoresis correctly detected PCR-ribotyping products of the 40 reference strains,and showed as digital figure;significant differences of these digital figures were found between the 10 RT.High similar digital figures were shown in twenty-one RT027 strains,three RT002 strains and two RT014 strains.However,seven RT001 strains were typed as four subtypes,and two RT014 strains as two subtypes,respectively.Conclusion A digital PCR-ribotyping,and a reference database consisting of 10 RT are successfully established.

Objective To investigate 13 high-risk types of HPV (HR HPV) infection rates in women with different grades of cervical lesions.Methods A total of 350 women, who were hospitalized in the department of gynecology in Bao′an Maternity & Child health hospital, were enrolled for the study.TCT technology was used to evaluate the cervical epithelium.The group were divided according to the cytology results.Multiplex real time PCR (mRT PCR) was used to detect the viral loads.HR HPV infection rate of different groups were analyzed using χ2 test or Fisher exact test.HR HPV viral loads of patients in different grades of cervical lesion groups were compared using Kruskal-Wallis or Wilcoxon test, and the age distribution of HR HPV positive group and negative group was analyzed by using Wilcoxon test.Results The HR HPV infection rates of NILM, ASCUS, LSIL, HSIL were 3.4% (10/295), 20.0% (7/35), 78.6% (11/14) and 100.0% (6/6), respectively.HR HPV positivity in NILM was lower than ASCUS (χ2=14.43,P<0.01) and LSIL (χ2=107.69,P<0.01), HR HPV positivity in ASCUS was lower than LSIL (χ2=14.76,P<0.01). The median of HR HPV viral loads in NILM, ASCUS, LSIL and HSIL were 4.10 (3.38-6.27), 5.33 (3.63-6.66), 5.77 (4.01-7.01) and 5.58 (4.19-5.85) respectively (copies/ml,lg).Combined ASCUS, LSIL and HSIL groups into cervical lesion group, HR HPV viral load of which was higher than that of NILM (U=43.0, P<0.05).The median Ages of HR HPV positive group and negative group were 36 and 33, respectively.No statistical significance was found between them (U=4 544, P>0.05).Conclusions The present study revealed that HR HPV infection was related to cervical lesion, but there was no correlation between viral load and cervical lesion grade. In additional, no difference in age distribution was found between HR HPV positive group and negative group.

Objective To evaluate the incidence and to investigate risk factors of supraventricular arrhythmia (SVAs) in postoperative cancer patients in intensive care unit ( ICU ). Methods Data of 570 patients consecutively admitted to oncologic surgical ICU of Cancer Hospital of Chinese Academy of Medical Sciences from Nov. 2008 to Oct. 2009 were retrospectively collected. Univariate and multivariate logistic analysis were conducted for potential factors that influenced SAVs. Results Thirteen patients with a history of atrial fibrillation (AF) were excluded and 557 patients were eligible for the study. SVAs occurred in 72 patients ( 12. 93% ). Multivariate analysis showed four independent predictors of SVAs including age ( OR = 1. 066,95%CI: 1. 034 - 1. 099,P ＜0. 001 ) ,a history of coronary heart diseases ( OR = 2. 644,95% CI: 1. 459 - 4. 790,P ＜ 0. 05), sepsis ( OR = 2. 374,95% CI: 1. 098 - 5. 135, P ＜ 0. 05 ) and intra-thoracic procedure ( OR =2. 322,95 % CI: 1.061 - 5.084, P ＜ 0. 05 ) . ICU length of stay, severity ( APACHE Ⅱ scores in SVAs patients) were significantly greater in patients who were not affected by SVAs ( ICU stay: [2 ( 1 ～ 77 )]vs [3 ( 1 ～ 40 )]days,P ＜ 0. 001; APACHE Ⅱ score: [9 (0 ～ 37 )] vs [11 (3 ～ 38 )], P = 0. 001 ). Nine cases died in SVAs patients ( 12. 5% ) and 19 died in the non-SVAs patients (3.9%), with significant difference between the two groups( x2 = 9. 673, P = 0. 002). Conclusion In oncologic surgical ICU, the incidence of SVAs is high. Age,history of coronary heart diseases, sepsis and intra-thoracic procedure were independent rsik factors of SVAs. SVAs prolong ICU length of stay. SVAs is a marker of critical illness severity.

Objective To explore the therapeutic efficacy of arthroscopy-assisted minimally invasive treatment for posterior tibial plateau fractures.Methods From July 2010 to June 2014,10 posterior tibial plateau fractures were treated at our department by arthroscopy-assisted minimally invasive treatment with percutaneous lag screws.They were 8 men and 2 women,with a median age of 31 years (from 18 to 52 years).All the fractures were closed and fresh,including 3 posteromedial tibial plateau ones,5 posterolateral tibial plateau ones,and 2 posteromedial and posterolateral tibial plateau ones.They were followed up periodically by radiological examinations.At the final follow-up,their knee functions were evaluated by Rasmussen scoring system,and their pain was evaluated by the visual analogue scale(VAS).Subjective factors included swelling,stairs climbing,joint stability,job participation and satisfaction with recovery.Results The follow-ups averaged 18 months (from 12 to 24 months).All fractures healed within 3 months postoperatively,with no infection or serious complications like implant failure.At 12 months postoperation,the mean Rasmussen score was 26 points (from 19 to 30 points).Eight cases were rated as excellent,one as good,and one as fair.Their mean VAS score was 1.2 points (from 0 to 4 points).Conclusion Arthroscopy-assisted minimally invasive management of posterior tibial plateau fractures with cannulated screw fixation is feasible,because it results in limited invasion,satisfactory reduction,reliable fixation,quick functional recovery and a low rate of complications.

Objective To analyze the therapeutic effect of Masquelet technique in the treatment of bone defects.Methods From January 2008 to December 2014,20 patients with bone defects were treated by Masquelet technique.There were 15 males and 5 females,from 18 to 69 years of age (average,38.4 years).Four cases had open bone defects and 16 infectious ones.At the first stage,radical debridement of the bone defects and soft tissue was conducted via conventional approaches.The bone defects ranged from 2 to 9 cm,averaging 6.1 cm.At the second stage,internal fixation was applied in 18 cases and external fixation in 2.The interval from the second stage to the first stage operation ranged from 6 to 23 weeks (average,11.5 weeks).The healing of bone defects and the functional recovery of adjacent joint were evaluated by Paley scoring at the last follow-up.Results The 20 patients were followed up for 12 to 50 months (average,19.7 months) after the second stage operation.All the patients obtained uneventful wound healing and control of infection after the first stage operation except the one with infectious defects who had to receive 2 operations to control the infection at the first stage operation.At the second stage operation,obvious injury and defect of the induced membrane occurred in 4 cases.All the patients achieved clinical healing of bone defects after 3 to 6 months (average,4.8 months).The bone defect healing was graded as excellent in all.After bone healing,all the patients resumed weight-bearing activities,with no breakage or infection of fixators,or recurrence of infection.By the Paley scoring at the last follow-up,the functional recovery of the adjacent joint was excellent in 8 cases,good in 10 and fair in 2,yielding an excellent and good rate of 90.0％.Conclusion As a kind of modified free bone grafting,Masquelet technique has advantages of simplicity,limited complications,a high rate of healing,and good control of bone infection.

Objective To construct a large animal model of ischemic heart disease induced by acute myocardial infarction (AMI) . Methods Male Diannan Mini-pigs were selected (n=8), weight 20 ± 4 Kg. After general anesthesia and endotracheal intubation, blood pressure and electrocardiograph were monitored. Parasternal incision was taken and thoracotomyv was performed at 4th-to-5th intercostalgap. Pericardium was opened and left anterior descending artery was ligated. Echocardiography was performed at baseline, postoperative 28 and 60 day. Euthanasia and heart explanted was conducted at 60 day of experimental pig. After gross examination of the heart, the histological examination was used to assess myocardial infarction by using H&E staining. Results All of the pigs were presented with ventricular arrhythmias in varying degrees after ligation of the left anterior descending artery, of whom 2 died of refractory ventricular fibrillation, the mortality was 25%. Echocardiography assessment showed the loss left ventricular function, the thinning of left ventricular apical and the enlargement of left ventricule. Histological examination revealed that myocardial fibrosis and fibrous scar were formed in myocardial infarction area. Conclusion The ligation of left anterior descending artery through thoracotomy under direct vision is a reliable strategy for construction of AMI porcine model and the experimental porcine can develop ischemic cardiac dysfunction progressively.