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OBJECTIVE To find out the imipenem-resistan metallo-?-lactamase in P.aeruginosa to provide the proof of treatment for clinic.METHODS A multi-disk was used to detect the metallo-?-lactamase of P.aeruginosa and the K-B disk method was used for monitoring of the antibiotic-resistance to 15 antibiotics.RESULTS Fifteen strains producing metallo-?-lactamase were isolated from 93 imipenem-resistant P.aeruginosa strains.The positive rate with metallo-?-lactamase was 16.1%(15/93).The susceptibility tests showed the lower resistance was to cefoperazone sulbactam(Sulperazone),amikacin and piperacillinl tazobactam(Tazocin).their resistance rate respectively was 16.8%,24.8% and 26.6%.The resistance rate to ciprofloxacin and ceftazidime was 43.7% and 33.6%.The resistance rate over 90.0% was to ampicillin,ampicilillin/sulbactam,cefazolin,cefpodoxime,cefuroxime and so on.CONCLUSIONS P.aeruginosa with imipenem-resistance shows seriou multidrug-resistance.The metallo-?-lactamase is the main reason for P.aeruginosa resistance to imipenem and cephalosporins.The doctors should choose antibiotics reasonably according to the susceptibility test when taking effective treatment to P.aeruginosa infection.

Objective To develop a novel padlock probe and rolling circle amplification(RCA) to detect 5 common cell culture contaminant Mollicute( Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycomplasma orale, and Acholeplasma laidlawii ). Methods "Universal" primers ( SPS1, SPA2 and SPS2, SPA1) were used to amplify the Mollicute 16S-23S rRNA intergenic spacer region.Amplicon was ligate Mollicute specific padlock probe. Probe amplified and monitored using a Corbett RotorGeneTM 6000 machine. Results Five reference Mollicute strains were correctly detected by RCA method.There was no cross-reaction. RCA method can sensitive detect 10 copies templates and show strong positive signal. Sixty-two cell culture specimens were detected. Thirty-seven samples were contained single specie Mollicute and 14 samples contained two Mollicutes. Eleven samples did not contained Mollicute. RCA detection results were concordant with previously species-specific PCR. Conclusion RCA can rapid, sensitive and specific detect contamination Mollicute.

Objective To detect point mutations of TAC1 gene in fluconazole-resistant Candida albi-cans isolates with rolling circle amplification (RCA), develop an accurate, rapid and specific assay to detect single nucleotide polymorphisms (SNPs), and to estimate the relationship between mutations of TAC1 gene and resistance to fluconazole. Methods A total of 33 fluconazole-resistant Candida albicana isolates, including 8 strains from America and 25 from Australia, were collected. Four TAC1-specific padlock probes were designed according to previously reported mutations. DNA was extracted from these tested strains, subjected to amplification of three targeted fragments of TAC1 gene with PCR. Then, RCA was performed to detect point mutations of TAC1 gene in resistant Candida albicans strains. At the same time, the target fragments underwent sequencing analysis, and the results of RCA were compared with those of sequencing. Results Two types of resistance-associated mutations were found in 5 out of the 33 fluconazole-resistant strains. Among the 5 strains, 4 were from America, 1 harbored T225A mutation and 4 carried A736V mutation. No related mutation was found in TAC1 gene of 4 fluconazole-sensitive isolates. Conclusions RCA assay could accurately and rapidly detect point mutations of genes. Further studies are required to clarify the relationship between TAC1 point mutations and fluconazole resistance.

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.

ObjectiveTo explore the effect of rehabilitation evaluation and treatment on Guillain-Barré syndrome.MethodsThe rehabilitation treatment was performed in 21 patients with Guillain-Barré syndrome, and the therapeutic effect was evaluated with Lindmark Assessment.ResultsThe total effective rate of all patients was 90.47% in the upper limps and wrists, 85.71% in the hands and 95.23% in the lower limps.ConclusionThe rehabilitation can improve the therapeutic effect of Guillain-Barré syndrome.

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 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 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 assess the implementation of National Leprosy Control Plan (2011-2020) in Zhejiang Province, so as to provide insights into leprosy control. Methods: Leprosy control data were collected from the National Leprosy Management Information System of China, and health administrative sectors, centers for disease control and prevention and professional leprosy control institutions at each level in Zhejiang Province. According to the scheme for the final evaluation of National Leprosy Control Plan (2011-2020), the implementation of 12 indicators in Zhejiang Province in 2020 was investigated, including the current number of leprosy patients, prevalence of leprosy, proportion of training on leprosy control skills, proportion of regular leprosy treatment, proportion of treatment of adverse reactions, annual examination rate of close contacts, proportion of early case identification and awareness of leprosy control knowledge. Results: There were 50 registered leprosy cases in Zhejiang Province by the end of 2020, with a decrease of 50.98% relative to in 2010. The prevalence of leprosy was less than 1/105 in 93 counties (districts) of Zhejiang Province, and the rate of training on leprosy control skills, rate of regular leprosy treatment, rate of treatment of adverse reactions, annual examination rate of close contacts, rate of early case identification were all 100% in Zhejiang Province. There were no new confirmed cases with diagnosis of leprosy having grade 2 deformity or disability, or no new cases with deformity or disability within 2 years following anti-leprosy therapy. In addition, the awareness of leprosy control knowledge was 91.67% among the public and 98.12% among the close contacts. All of the 12 indicators reached the requirements of the National Leprosy Control Plan (2011-2020). Conclusions The implementation of the National Leprosy Control Plan (2011-2020) in Zhejiang Province had achieved the targets defined in the final evaluation of the plan. Intensifying multi-sectoral joint leprosy prevention and control and improving early identification and standardized therapy of leprosy cases are recommended for future leprosy control in Zhejiang Province.