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Objective:To investigate the pathogen spectrum of acquired immunodeficiency syndrome (AIDS) patients with pulmonary opportunistic infections in the local area, and to evaluate the clinical application of metagenomic next-generation sequencing (mNGS) in these patients.Methods:From January to December 2021, AIDS patients with pulmonary infections admitted to Zhongnan Hospital of Wuhan University were enrolled. Their bronchoalveolar lavage fluid (BALF) was subjected to mNGS and coventional pathogen detection.Routine pathogen detection methods included smear, culture, polymerase chain reaction (PCR), and immunochromatographic colloidal gold. Fisher′s exact probability method was used for statistical analysis.Results:A total of 69 patients were included, and all of them were tested positive for mNGS. Among them, 53 cases (76.8%) were positive for fungi and viruses, 40 cases (58.0%) were positive for bacteria (excluding Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM)), six cases were positive for MTB, 11 cases were positive for NTM, and seven cases were positive for other pathogens. Mixed infections with two or more pathogens were found in 89.9%(62/69) of the patients. Among the conventional pathogen detections of BALF, 79.7%(55/69) of the patients were positive for pathogens, including 42 cases positive for Pneumocystis jirovecii PCR, 16 cases positive for BALF culture, nine cases positive for MTB PCR, and five cases positive for Cryptococcus antigen. The total detection rate of mNGS was 100.0%(69/69), which was higher than that of the conventional pathogen detection rate of 79.7%(55/69), and the difference was statistically significant (Fisher′s exact probability method, P<0.001). The specificity of mNGS detection was 88.4%. Combining clinical and two detection methods, the top five pathogens were Pneumocystis jirovecii (62.3%(43/69)), Candida (29.0%(20/69)), MTB (20.3%(14/69)), NTM and Talaromyces marneffei (15.9%(11/69), each). Fifty-three patients (76.8%) had co-infection with virus. Conclusions:The main cause of pulmonary infection in AIDS patients in this area is mixed infection, and Pneumocystis jirovecii is the most common pathogen. mNGS could significantly improve the pathogen detection rate in AIDS patients with pulmonary infections.
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Objective:The matria-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS) and sequencing methods were performed to assess the methodology (biochemistry methods) for identifying the Pandoraea sputorum and provide the more preferred approach to identify Pandoraea species. Methods:This paper is a study on performance evaluation of identification method. Ten lines of Pandoraea sputorum were isolated from blood cultures of inpatients were collected at Zhongnan Hospital of Wuhan University from July to October 2018 and were confirmed by the cultural characteristics, colonial morphology and Gram′s stain. Further identification was carried out by using the manual biochemical method (API 20NE), automatic biochemistry systems(BioMerieuxVITEK 2 Compact, BD Phoenix-100andThemo ARIS 2X), MALDI-TOF MS (BioMerieuxVITEK MS and Bruker MALDI Biotyper) and the sequencing methods of the 16S rRNA to identify the Pandoraea sputorum. Results:Pandoraea sputorum was non-fermented gram-negative bacteria that are non-motile, oxidase positive, and catalase positive. Ten lines of Pandoraea sputorum were identified as Achromobacter denitrificans, Alcaligenes faecalis or Cupriavidus pauculus and the accuracy rate of genus and species identification was 0 by API 20NE. Among all the samples, six lines were identified as the Pandoraea spp. with the accuracy rate of genus identification was 6/10 by VITEK 2 Compact; whereas the other four lines were identified as the Burkholderia cepacia, Sphingomonas paucimobilis, Ralstonia pickettii, or "Low Discrim" . All of these were identified as "No Identification" by Phoenix-100, which the accuracy rate of genus and species identification was 0. Seven isolates were identified by ARIS 2X as Stenotrophomonas maltophilia, Acinetobacter lwoffii, Sphingomonas paucimobilis, Pseudomonas luteola, Acinetobacter baumannii/Acinetobacter haemolyticus; whereas the other three lines were identified as rare species, thus, the accuracy rate of genus and species identification was 0. Both VITEK MS and MALDI Biotyper indicated all the isolates were Pandoraea sputorum with the accuracy rate of genus and species identification was 10/10. 16S rRNA sequencing for the 10 isolates showed they have 100% of similarity to Pandoraea sputorum by blasting with Genebank. Conclusions:Methods based on biochemical reactions often failed to accurately identify the Pandoraea sputorum to species level. MALDL-TOF MS and 16S rRNA sequencing technology identify Pandoraea sputorum efficiently and precisely enough.
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OBJECTIVE To discuss the antibiotic resistance situation of Pseudomonas aeruginosa and provide basis for therapy and control of the nosocomical infection. METHODS Fourteen strains of P.aeruginosa which derived from 60 and more years aged patients in intensive care unit(ICU) were analyzed by conventional chemistry test,PCR inspection,antimicrobial susceptibility test,plasmid pattern analysis and enterobacterial repetitive intergenic consensus(ERIC)-PCR polymorphism analysis. RESULTS Fourteen strains had the same chemistry traits,many strains appeared multi-antibiotic resistance,10 strains had a plasmid of 23kb or so without obvious association with(multi-antibiotic) resistance,14 strains were divided into three clusters by ERIC-PCR technique and their banding pattern cluster analysis,their similarity coefficients were from 0.62 to 0.86. CONCLUSIONS Nosocomial infection of P.aeruginosa which screened from aged patients in ICU has taken on sporadic state;ERIC-PCR fingerprinting technique genotyping method has high discrimination,and good reproducibility and give faster genotyping result with less hands-on time,which serves as nosocomial infection epidemiologic surveillances of P.aeruginosa.
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Objective To study the drug susceptibility,potential toxin factors,and genotypes of Candida albicans.Methods Forty-six strains of C.albicans were isolated from clinical samples of lower respiratory tract infection.The genomic DNA was amplified by PCR to determine the 25S rDNA genotypes.The drug sensitivity was investigated by disk diffusion and broth dilution respectively.Adhesiveness test and protease activity assay were performed.Results Forty-six strains were divided into A(18),B(6),and C(22) three genotypes.Most trains displayed high sensitivity to natamycin,amphotericin B,nystatin,fluorocytosine,and itraconazole.MIC results showed that there was significant difference against fluconazole and 5-fluorocytosine between A and B as well as C genotypes(P0.01).The cell adhesiveness test demonstrated that the strain adhesiveness was proportional to the protease activity(r=0.977,P
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Objectives To study the characteristics of clinical distribution of enterococci, to test its sensitivity to antibiotics recommended by NCCLS, and to promote rational use of antibiotics. Methods A total of 381 strains of enterococci isolated from Hubei during January to December 2001 were tested for their sensitivity to antibiotics and production of beta-lactamases with paper disk diffusion method. Results Among 381 strains of enterococci, there were 78.0% of E. faecalis, 19.4% E. faecium, 1.6% E. durans, 0.53% E. avium, 0.3% E. hirae and 0.3% E. solitarium , and beta-lactamases-producing enterorocci accounted for 3.2% of the total. And, 46.5% of the strains were isolated from uroreproductive tracts samples, ranking the first of the total, possibly because of some samples from a hospital specialized in urology, 35.4 % from respiratory tract, ranking the next, and 11.0% form skin pustule and infected wound, ranking the third. Proportions of the strains resistant to varied antibiotics were 5.8%, 7.9%, 11.0%, 24.7%, 26.8%, 38.6%, 54.6%, 54.6%, 52.5% and 79.3% to vancomycin, teicoplanin, nitrofurantoin, ampicillin, penicillin, ciprofloxacin, chloromycetin, high concentration gentamycin, high concentration streptomycin, and tetracycline, respectively. Drug-resistant rate of E. faecium was higher than that of E. faecalis. Conclusions Enterococci, most of them identified as E. faecalis and E. faecium, were isolated from samples of uroreproductive and respiratory system, and the most susceptible to vancomycin. E. faecium was more resistant to antibiotics than E. faecalis.
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AIM: To confirm the expression level of the gene which corresponding to JT8 tag decreased in coronary artery disease (CAD). METHODS: The validity and reliability of the results gotten by serial analysis of gene expression method was verified by RT-PCR and Northern blot. The expression level of the gene which corresponding to JT8 tag was compared with the expression level of GAPDH and ?-actin in JT and WY, while according to SAGE results that the gene expression level of JT8 gene was 8 times higher in JT than in WY. RESULTS: It was found that the results of RT-PCR and Northern bolt were identified with the results of SAGE. The expression level of JT8 gene decreased in CAD. CONCLUSION: These results verified the validity of SAGE method and made a good foundation for further discovery of new candidate genes. [