Evaluation of a new ultrafast real-time fluorescence polymerase chain reaction and common assays in the detection of novel Bunya virus / 中华传染病杂志

Objective:To investigate the specificity and sensitivity of four methods including ultrafastreal-time fluorescence polymerase chain reaction (PCR), real-time fluorescence (RT)-PCR, enzyme-linked immunosorbent assay (ELISA) and gold immunochromatography assay (GICA) for the detection of novel Bunya virus, so as to provide experimental basis for the early diagnosis of severe fever with thrombocytopenia syndrome (SFTS).Methods:Serum samples from 86 clinically diagnosis SFTS patients admitted to the Jinan Infectious Diseases Hospital Affiliated to Shandoug University were tested by ultrafast real-time fluorescence PCR, RT-PCR, ELISA and GICA during June 1 to September 30, 2017. Chi-square test was used for statistical analysis.Results:Among 86 serum samples, the positive rate of novel Bunya virus of ultrafast real-time fluorescence PCR, RT-PCR, IgM-ELISA, IgG-ELISA, IgM-GICA and IgG-GICA were 82(95.34%), 79(91.86%), 41(47.67%), 8(9.3%), 19(22.09%) and 3(3.49%), respectively. The specificity of ultrafast real-time fluorescence PCR was 100%, and the sensitivity was 1×10 3 copies/mL.Repeated amplification test showed that the variation coefficient of the computed tomography value was <2%.During phases one, two and three, the positive rates of ultrafast real-time fluorescence PCR were 41(97.62%), 34(94.44%) and 7(87.50%), and RT-PCR were 39(92.86%), 33(91.67%) and 7(87.50%), respectively. During phases one and two, the positive rate of ultrafast real-time fluorescence PCR was slightly higher. The positive rate of anti-novel Bunya virus antibody (IgM) tested by ELISA had a significant increase from phase one (28.57%)to phase three (87.50%). There were statistical differences between phase two and phase, as well as between phase three and phase one ( χ2=8.347 and 7.561, respectively, both P<0.01). IgM-GICA also had an increase from phase one (14.29%) to phase two (33.33%)( χ2=3.962, P<0.01), while it was still lower than the other tests.In phase one, the positive rate of RT-PCR was higher than those of ELISA(both IgM and IgG)and GICA(both IgM and IgG)( χ2=33.740, 55.080, 49.010 and 64.340, respectively, all P<0.01). In phase two, the positive rate of RT-PCR was higher than those of ELISA(both IgM and IgG)and GICA(both IgM and IgG) ( χ2=7.700, 46.720, 23.700 and 50.630, respectively, all P<0.01). In phase three, the positive rates of ultrafast real-time fluorescence PCR, RT-PCR and IgM-ELISA were equivalent, which were all higher than those of IgG-ELISA and GICA (both IgM and IgG). The positive rates of RT-PCR and IgG-ELISA, IgM-GICA and IgG-GICA were significantly different (all χ2=6.250, all P<0.05). Conclusion:In the early detection of novel Bunya virus, ultrafast real-time fluorescence PCR has higher sensitivity, specificity, good repeatability and high stability, which greatly reduces the amplification time compared with the traditional RT-PCR, and is of great value in the early and rapid diagnosis of SFTS.

Analysis of the drug resistance and the integron resistance gene cassette's characteristics of Shigella flexneri / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the correlation between Shigella flexneri multi-drug resistance and drug resistance gene cassette of integrons.</p><p><b>METHOD</b>All 79 strains of Shigella flexneri were isolated from the feces of children ranged in age from 6 months to 14 years in some hospitals of Jinan, between May 2009 and April 2012.The resistance was detected by Kirby Bauer agar diffusion method, 1, 2 and 3 integron gene was amplified by PCR, the variable region of positive strains treated with enzyme digestion and determined by Series Analysis.</p><p><b>RESULT</b>Among 79 Shigella flexneri strains, the resistance rate was 91% (72/79) to ampicillin, chloramphenicol, tetracycline, streptomycin, 70% (55/79) to sulfamethoxazole/trimethoprim, 30% (24/79), 23% (18/79), 33% (26/79) and 32% (25/79) to cefotaxime, ceftazidime, ciprofloxacin and levofloxacin.All 79 strains were susceptible to cefoxitin, imipenem, cefoperazone/sulbactam. The common drug resistance pattern is ampicillin tetracycline-chloramphenicol-streptomycin, accounted for 91% (72/79); 91% (72/79) strains carried integrons of class 1, 86% (68/79) strains carried integrons of class 2, No intI3 was detected. The resistance to ampicillin, streptomycin, tetracycline, chloramphenicol of atypical class 1 integron positive strains was significantly higher than the negative strains (χ² = 35.96, P<0.01). The sequencing results:dfrV was detected in class 1 integron variable regions of 9 strains, dfrA17-aadA5 in 2 strains, blaOXA-30-aadA1 in 70 strains, 2 strains were not detected resistance gene cassette, all resistance gene cassettes were dfrA1-sat1-aadA1 in class 2 integron variable regions.</p><p><b>CONCLUSION</b>The muti-drug resistance of Shigella flexneri in Jinan was closely associated with integrons.</p>

Distribution of integrons and their gene cassettes in clinically isolated multi-drug resistant Shigella / 中华传染病杂志

Objective To analyze the distribution of class 1,2 and 3 integrons and their gene cassettes,and to explore its relationship with drug resistance in Shigella.Methods Antimicrobial susceptibility was detected by minimal inhibitory concentration (MIC) method.All the genes of integrons and gene cassettes were amplified by polymerase chain reaction (PCR).The amplicons were identified by amplified fragment length polymorphism (AFLP) and sequencing.Results Fifty seven multi-drug resistant strains were identified from a total of 62 Shigella strains (91.9％).Among multi drug resistant strains,52 strains carried integrons of class 1 (91.2 ％) and 55 strains carried integrons of class 2 (96.5％).Only 2 strains carried class 1 integrons alone,5 strains carried class 2 integrons alone and 50 strains had both class 1 and class 2 integrons.Class 3 integrons were not detected.The gene cassettes of typical class 1 integrons,dfrV and dfrA17-aadA5,were detected in 6 strains and 2 strains,respectively.Atypical class 1 integrons with gene cassettes blaOxA30 aadA1 were detected in 44 strains.The typical and atypical class 1 integrons coexisted in 6 Shigella flexneri strains.Gene cassettes for class 2 integrons were dfrA1 sat1-aadA1.Conclusions The multi-drug resistant Shigella strains are widely distributed in Ji'nan,and the atypical class 1 integrons and class 2 integrons are common in these strains.Coexistence of the two integrons is observed in some of the strains.

Detection of ESBLs produced by Escherichia coli and Klebsiella pheumoniae and drug resistance monitoring / 国际检验医学杂志

Objective To investigate the status of extended-spectrum β-lactamases (ESBLs) production by Escherichia coli and Klebsiella pheumoniae, analyze the characteristics of drug resistance of Escherichia coli and Klebsiella pheumoniae in our hospital from Jan 1998 to Dec 2006, in order to provide a guidance to clinical rational use of antibiotics.Methods Disc diffusion test (K-B method) was used to study the drug resistance of E. coli and K.pheumoniae isolates, and ESBLs-producing strains were identified with screening test and confirmatory test. WHONET5.4 software was apllied to performing data analysis.Results In 947 strains of E. coli, 311 strains (32.8%) were detected to produce ESBLs;in 293 strains of K.pheumoniae, 69 ESBLs-producing strains (23.5%) were detected. The resistance rate of ESBLs-producing strains was significantly higher than that of ESBLs non-producing ones (P<0.05), and but ESBLs-producing strains displayed multi-resistant.Conclusion The positive rate of ESBLs producing strains is higher in our hospital. Drug resistance monitoring should be strenghed to rationally use antibacterials and to prevent increase, prevalence and outbreak of ESBLs producing strains.

Study on plasmid-mediated extended spectrum β-lactamases and their resistance phenotypes in Shigella / 中华检验医学杂志

Objective To discuss the characteristics of extended-spectrum beta-lactamases(ESBLs)-producing Shigella and the relation between them and drug-resistance plasmid. Methods The suspicious ESBLs-producing isolates were screened by K-B disc diffusion method, and the ESBLs-producing strains were confirmed by confirmatory test recommended by the National Committee for Clinical Laboratory Standards. Furthermore, the partial blageneof these isolates were detected by PCR using universal primers for TEM, SHV, CTX-M-1 group, CTX-M-2 group and CTX-M-9 group, respectively. The entire blaCTX-M-9 and blaTEM were amplified by PCR using the primers outside the open reading frame (ORF) of these β-1actamases and products were directly sequenced. The conjugation experiment was performed to determine whether the resistance was transferable. Minimal inhibitory concentration (MIC) was detected with double agar dilution method. Results Of the 275 isolates, 12 strains were identified as ESBLs producers. Among them, 8 strains were CTX-M-14 carriers and 4 strains were CTX-M-3 carriers. All ESBLs-producing isolates are positive for plasmid conjugative transfer test. The transconjugants are only resistance to betalactams. Conclusions High resistance to beta-laetams in Shigella is caused by production of ESBLs in the local area. The ESBLs-produeing isolates can transfer the drug resistance through lateral transfer of plasmid.