Objective To develop a high throughput sequencing method for pathogenic microbial metagenomics and verify its detection performance.Methods First,the library construction conditions with different input levels of nucleic acid were studied.Three differ-ent input levels of nucleic acid such as 5 ng,50 ng,and 100 ng,three fragmentation times such as 20 min,25 min,and 30 min,and three amplification cycles such as 7,5,and 3 cycles were tested.The optimal library construction conditions were determined by the li-brary concentration and fragment size.The representative bacteria covering fungi and gram-positive/negative bacteria,including Candi-da albicans,Staphylococcus aureus,Staphylococcus epidermidis,Escherichia coli and Cryptococcus Gattii,were selected.Three different concentrations of mixed bacterial suspensions were prepared at a 1∶1 ratio.Then,106/mL of T-cell suspensions were added to prepare reference samples.The performance of the self-established method(Method 1)was verified by the reference samples,and its consis-tency with Method 2(DNA sample library construction kit combined with probe anchored polymerase chain reaction sequencing method)was compared.Results The optimal library construction conditions of the sequencing method for pathogenic microbial metagenomics were determined as follows:a nucleic acid input of 50 ng,a nucleic acid fragmentation time of 20 min,and 5 amplification cycles for PCR.The positive coincidence rate(accuracy)and negative coincidence rate(specificity)of Method 1 were 100%,and its detection limit was 500 CFU/mL.The correlation coefficients(r)of the reads per million(RPM)in linear samples of various microorganisms were greater than 0.9.The CV of bacterial detection were less than 10%,while those of fungal detection were around 20%.The consis-tency between Method 1 and Method 2 was 100%.Conclusion A sequencing method for pathogenic microbial metagenomics is estab-lished successfully,which can acquire accurate,stable,and reliable results and is suitable for the pathogen detection of alveolar lavage fluid samples.

Objective To develop a high throughput sequencing method for pathogenic microbial metagenomics and verify its detection performance.Methods First,the library construction conditions with different input levels of nucleic acid were studied.Three differ-ent input levels of nucleic acid such as 5 ng,50 ng,and 100 ng,three fragmentation times such as 20 min,25 min,and 30 min,and three amplification cycles such as 7,5,and 3 cycles were tested.The optimal library construction conditions were determined by the li-brary concentration and fragment size.The representative bacteria covering fungi and gram-positive/negative bacteria,including Candi-da albicans,Staphylococcus aureus,Staphylococcus epidermidis,Escherichia coli and Cryptococcus Gattii,were selected.Three different concentrations of mixed bacterial suspensions were prepared at a 1∶1 ratio.Then,106/mL of T-cell suspensions were added to prepare reference samples.The performance of the self-established method(Method 1)was verified by the reference samples,and its consis-tency with Method 2(DNA sample library construction kit combined with probe anchored polymerase chain reaction sequencing method)was compared.Results The optimal library construction conditions of the sequencing method for pathogenic microbial metagenomics were determined as follows:a nucleic acid input of 50 ng,a nucleic acid fragmentation time of 20 min,and 5 amplification cycles for PCR.The positive coincidence rate(accuracy)and negative coincidence rate(specificity)of Method 1 were 100%,and its detection limit was 500 CFU/mL.The correlation coefficients(r)of the reads per million(RPM)in linear samples of various microorganisms were greater than 0.9.The CV of bacterial detection were less than 10%,while those of fungal detection were around 20%.The consis-tency between Method 1 and Method 2 was 100%.Conclusion A sequencing method for pathogenic microbial metagenomics is estab-lished successfully,which can acquire accurate,stable,and reliable results and is suitable for the pathogen detection of alveolar lavage fluid samples.

Objectives To identify and characterize 10 strains of Francisella philomiragia-like organisms isolated from blood samples and environmental water.Methods The 10 clinical and environmental isolates were identified by traditional morphological examination and biochemical characterization,matrix-assisted laser desorption/ionization time of flight(MALDI-TOF) mass spectrometry(MS) systems and sequencing based on 16S rRNA gene.The minimum inhibitory concentrations were tested by E-test methods.Results All the 10 isolates were gram-negative coccobacilli appearing tiny and faint counterstain of safranin,negative for urease,nitrate reduction and X and/or V factor requirement,but positive for oxidase and catalase.The isolates grew rapidly in sheep blood agar,chocolate agar and BCYE plate forming white opaque,colorless transparent or gray smooth colonies with about 2-mm diameters,but did not grow in M-H agar and MacConkey agar.The sequencing for 16S rRNA gene indicated that the 10 isolates shared more than 99.6％ similarity to Francisella philomiragia,and fell into the same clusters of Francisella philomiragia on phylogenetic tree.The MALDI-TOF MS analysis also showed the typical peaks with 6 153 m/z,5 180 m/z,7 757 m/z and 9 392 m/z which were similar to Francisella philomiragia ATCC 25015.However,they may be misidentified to be Sphingomonas paucimobilis by using Vitek 2 GN cards,Neisseria cinerea by using Vitek 2 NH cards,Myroides odoratimimus by using API 20NE strips and Haemophilus by using API NH cards.The results of antimicrobial susceptibility showed that they were all sensitive to chloramphenicol,doxycycline,tetracycline,gentamicin,ofloxacin and ciprofloxacin.Conclusion The 10 isolates could be identified as Francisella philomiragia,so we should pay more attention to the infrequent pathogen for its inactive biochemical reaction and the misidentification by commercial detection systems.

Objective To explore the risk factors of cognitive impairment in rural elderly patients from Changzhi urban. Methods Totally 118 elderly patients were examined with minimental state examination (MMSE). The common data and histories of coronary heart disease,hypertension,diabetes and chronic obstructive pulmonary disease were collected. Results The prevalence of cognitive impairment was 14.4％.Multivariate analysis showed that MMSE scores was associated with age,stroke history,homocysteine and low density lipoprotein(LDL-C) (x2 =21.166,19.323,13.672,3.698,P＜0.01 or P＜0.05),and cognitive impairment was not associated with gender,alive spouse,hypertension,coronary heart disease, diabetes and chronic obstructive pulmonary disease(P＞0.05).Age(OR=0.853,95％ CI:0.765-0.951),stroke history(OR=0.764,95％ CI:0.629-0.929),LDL-C (OR =0.208,95％ CI:0.062-0.701 ) and activities of daily living (ADL)(OR=0.839,95％ CI:0.725-0.972)were independent risk factors of cognitive impairment in rural elderly patients. Conclusions Age,stroke and LDL-C are independent risk factors for rural elderly patients in the community.

Objective To investigate the depression and its influential factors in the elderly with hypertension in communities,so as to provide the theoretical evidence for practicing intervention for community health workers.Methods According to uniform inclusion and exclusion criteria,350 elderly people aged 60 years and over in Changxing Community and Town God's Temple Community in Changzhi city included hypertensive patients and healthy persons who were extracted by stratified random sampling method.The face-to-face survey method was taken,and the questionnaire included general information and Geriatric Depression Scale (GDS).All data were through statistical analysis by SPSS 13.0.Results The incidence of depression was higher in elderly hypertensive patients (scores:11.70±6.67,56.4%) than in normal elderly persons [scores:(6.49+5.97,21.3)%,t=-7.314,x2=41.489,P<0.01].The results of multiple stepwise regression analysis showed the influencing factors of depression were subjective symptoms,complicating disease,knowledge of hypertension,co-living family and economic burden.Conclusions The incidence of depression is higher in elderly hypertensive patients than in normal elderly people.If community health workers do something to intervene the influencing factors of the depression in elderly hypertensive patients,the incidence may be reduced.