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Objective To establish the theoretical basis of the probiotic application among very low birth weight(VLBW) infants,the efficacy of probiotics on the gut microbiota of VLBW infants on the 14th postnatal day was studied.Method The VLBW infants admitted to BaoAn Maternal and Child Care Hospital from January 2015 to December 2015 were randomly assigned into probiotics group and placebo group.From the first feeding to corrected gestational age of 36 weeks,probiotics group was treated with a combination of Bifidobacterium and Lactobacillus while placebo group with placebo.Fecal samples were collected on the 1st and 14th postnatal day.Total bacterial DNA was extracted and sequenced using high-throughput 16S rRNA gene sequencing on MiSeq sequencing platform.Result A total of 21 VLBW infants were enrolled,9 in probiotics group and other 12 placebo group.No significant differences of clinical data existed between the two group (P ＞ 0.05),The abundance and diversity of microflora (P ＞ 0.05) on the first day between the two group were similar.Compared with placebo group,the relative abundance of Bifidobacterium and Lactobacillales in stool samples on the 14th day was significantly increased,while the Halomonas was significantly decreased.The relative abundance of the Shannon-index was increased,but without significant difference (P =0.16).Conclusion Enteral supplementation of probiotics in VLBW infants may increase probiotic bacterium and microflora diversity.
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Objective To investigate the carrying status and characteristics of Clostridium difficile isolated from infants.Methods Two hundred and thirty-eight stool specimens were collected from infant younger than 1 year old,that were hospitalized or outpatient from August to November 2015.Immunochromatography targeted GDH and toxin A&B of C.difficile was used for C.difficile screening,and those positive specimens were inoculated in CDIF and anaerobic culture.C.difficile isolates were genotyped by using slpA sequence typing (slpA ST),and tcdA,tcdB,cdtA and cdtB of C.difficile isolates were detected by PCR.Results Fifty C.difficile strains were isolated from 238 stool samples,and the isolated rates of C.difficile from <3 months,3 months to <6 months,and 6 months to 1 years old groups were 9.3%,17.6% and 27.3%(χ2=6.940,P=0.031<0.05),respectively.52.0%(26/50) of the C.difficile isolates were toxigenic,and 69.2% (18/26) toxigenic isolates harbored tcdA+tcdB+cdtA-cdtB-.Fifty C.difficile isolates were genotyped as 11 slpA STs,slpA ST fr-02 and kr-02 were the commonest genotypes in toxigenic C.difficile isolates;however,that was slpA ST xr-03 in non-toxigenic isolates.Conclusion High C.difficile carriage is found in infants younger than 1 year old,and more than half of C.difficile isolates are toxigenic.Most of toxigenic isolates harbored toxin A and B.The genotype of C.difficile isolates is different between toxigenic isolates and non-toxigenic isolates.
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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.
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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 .
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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 .
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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.
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Objective To compare real time PCR with PCR-reverse dot blot hybridization (PCR-RDB)for detecting human pap-illomavirus (HPV)infection in women.Methods A total of 109 genital specimens from women were collected in the study.All specimens were tested HPV by using real time PCR and PCR-RDB,discrepant samples were tested again by PCR-xMAP.Results The concordant rate was 83.5%(91/109)between real time PCR and PCR-RDB (kappa=0.671),the other 18 discrepant samples were retested by PCR-xMAP,7 of those were identical with real time PCR and 11 with PCR-RDB.No differences of PCR-RDB pos-itive rates were found between the high and low viral load groups (χ2 =1.476,P =0.224).Conclusion It demonstrated moderate consistency between real time PCR and PCR-RDB.The HPV positive rates of PCR-RDB were stable when the viral loads were 103-108 .
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Objective To investigate the female human papillomavirus(HPV)infection situation in Baoan district,the HPV pos-itive rates in different age groups and the subtypes distribution.Methods PCR followed with reverse dot blot was performed to ex-amine 23 kinds of HPV genotypes in 2 627 female patients in our hospital from the January 2011 to December 2012.Results In 2 627 samples,the positive rate of HPV was 23.94% (629 cases),in which the infection rate of single low-risk type was 15.1%(95 cases),the main HPV genotype was HPV43 (7.79%);the infection rate of high-risk type was 55.17% (347 cases),the 3 most prevalent HPV genotypes were HPV52 (12.56%),HPV16 (9.86%)and HPV58 (7.79%).The multiple HPV infection ac-counted for 29.73% (187 cases).The HPV infection rates in different age groups were 50.0% in age 15-20 years,24.7% in age 21-30 years,20.8% in age 31-40 years,25.8% in age 41 -50 years,42.1% in age >50 years respectively,the differences had statistical significance.Conclusion The HPV infection rate is 23.94% in Bao′an district.The most prevalent HPV genotypes are HPV 52,16,58,43.Women in age 15-20 years old have a higher infection rate.
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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,P0.05).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.
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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.