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Objective:To explore the structure and diversity of intestinal flora in patients with community acquired pneumonia (CAP) before and after treatment with cefotaxime combined with levofloxacin.Methods:From October to December 2018, 6 patients with CAP in the Department of Infection, Zhejiang Provincial Hospital of Tongde, were treated with cefotaxime injection 2.0 g (once/8 h) combined with 0.5 g levofloxacin injection (once a day). A total of 12 fecal samples were collected before and after 7 days of treatment. The stool samples before and after treatment were analyzed by 16S rRNA sequencing.Results:⑴ The structure of intestinal flora before and after treatment : at the phylum level: Firmicutes 59.2% vs 40.8%, Proteobacteria 18.6% vs 35.5%, Bacteroidetes 14.8% vs 20.8%, Actinobacteria 5.6% vs 1.2%; At the family level: Ruminococcaceae 34.5% vs 13.0%, Lachnospiraceae 15.9% vs 9.7%, Veillonellaceae 1.8% vs 3.3%, Lactobacillaceae 0.3% vs 8.0%, Streptococcaceae 2.9% vs 1.1%, Enterococcaceae 0.02% vs 5.2%, Enterobacteriaceae 16.4% vs 34.6%, Bacteroidaceae 13.3% vs 16.8%, Porphyromonadaceae 0.3% vs 3.4%, Adlercreutzia 4.4% vs 0.5%. There was no significant difference in the composition and structure of intestinal flora before and after treatment ( P>0.05). ⑵ The diversity of intestinal flora before and after treatment: operational taxonomic units (OTU) mean (150.5±59.0) vs (93.2±34.1), t=2.72, P=0.04; Chao1 index (169.25±49.61) vs (117.92±35.06), t=3.22, P=0.02; shannon index (3.61±0.83) vs (2.31±0.73), t=4.54, P=0.01; simpson index (0.80±0.10) vs (0.61±0.20), t=2.76, P=0.04. There were significant differences in the diversity of intestinal flora before and after treatment ( P<0.05). ⑶ There was significant difference in desulfovibrio between the two groups before and after treatment (LDA=2.03, P=0.02). Conclusions:After intravenous infusion of cefotaxime combined with levofloxacin for one week , the diversity of intestinal flora was significantly reduced after treatment. Desulfovibrio was the flora with statistical differences between before and after treatment.
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Objective To evaluate the clinical value of combined detection of T cell receptor rear-rangement excision circles ( TRECs) and CD31+ regulatory T ( Treg) cells for accessing the recent thymic output in patients with chronic hepatitis B. Methods Four groups involving 135 subjects were set up in this study as follows: mild chronic hepatitis B ( Mild CHB, n=35 ) , moderate chronic hepatitis B ( Moderate CHB, n=35 ) , severe chronic hepatitis B ( Severe CHB, n=35 ) and healthy control ( HCs, n=30 ) groups. CD4+CD25+Treg cells in these subjects were sorted out using magnetic cell separation. The ratio of peripheral CD31+Treg cells to Treg cells in each group was analyzed by flow cytometry. Real-time PCR was performed to detect TRECs in CD4+CD25+Treg cells. The percentages of CD3+, CD4+ and CD8+T cell sub-sets were also measured. Results The ratios of CD31+Treg/Treg cells and the numbers of TRECs in pe-ripheral blood of the Moderate CHB and Severe CHB groups were significantly lower than those of the Mild CHB and HCs groups (P<0. 05), while no statistical difference was found between the mild CHB and HC groups (P>0. 05). No significant difference in the percentages of CD3+, CD4+ or CD8+ T cell subsets was observed between the four groups (P>0. 05). CD31+ Treg/Treg cell ratio had a positive correlation with the number of TRECs (r=0. 551, P=0. 014). Conclusions Both CD31+Treg/Treg cell ratio and the number of TRECs were reduced in the peripheral blood of patients with moderate or severe CHB. CD31+Treg/Treg cell ratio and the number of TRECs were positively correlated and could be used as new indices to evaluate recent thymus output.
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<p><b>OBJECTIVE</b>To compare clinicopathological features and prognosis between patients with carcinoma in the remnant stomach (CRS) and with gastric cancer, and to investigate the prognostic factors in CRS patients.</p><p><b>METHODS</b>A retrospective cohort study was performed on clinicopathological data of 96 CRS patients (CRS group) and selected 440 patients with gastric cancer (GC group) treated at Harbin Medical University Cancer Hospital from January 1977 to December 2017.</p><p><b>INCLUSION CRITERIA</b>(1) undergoing gastrectomy; (2) diagnosed with CRS or gastric cancer through electronic gastroscopies and pathology; (3) without preoperative neoadjuvant radiotherapy or chemotherapy; (4) complete clinicopathological and follow-up data. The patients who died of other reasons or were lost during follow-up were excluded. Chi-square test and independent samples t-test were used to determine differences in clinicopathological factors between two groups. Survival analysis was conducted using the Kaplan-Meier method, and Log-rank test was used to compare survival difference between two groups. The prognosis of CRS patients was analyzed using Cox proportional hazards regression model.</p><p><b>RESULTS</b>As compared to GC group, CRS group had a higher proportion of female [30.2%(29/96) vs. 13.2%(58/ 440), χ=14.095, P=0.000], younger age [(56.4±10.1) years vs. (60.0±9.9) years, t=2.838, P=0.005], more distant metastasis and local organ infiltration [25.0%(24/96) vs. 16.1%(71/440), χ=4.246, P=0.039; 64.6% (62/96) vs. 24.5% (108/440), χ=58.331, P=0.000], lower prognostic nutritional index [(48.0±6.7) vs. (50.4±6.9), t=3.093, P=0.002], lower serum hemoglobin level [(115.0±24.7) g/L vs. (127.9±24.6) g/L, t=4.634, P=0.000], lower serum albumin level [(40.0±4.9) g/L vs. (41.2±5.0) g/L, t=2.038, P=0.042], and earlier occurrence of symptoms [(1.9±1.4) months vs. (3.7±3.2) months, t=5.431, P=0.000]. However, there were no statistically significant differences in TNM staging, postoperative hospital stay, and total hospitalization days between the two groups (all P>0.05). During follow-up, 24(25.0%) patients developed recurrence or distant metastasis and 68 (70.8%) patients died of tumor progression in CRS group, while 71(16.1%) patients developed recurrence or distant metastasis and 378(85.9%) patients died of tumor progression in GC group. The 5-year survival rate of CRS patients was 23.4%, which was higher than 15.0% of gastric cancer patients (P=0.032). Univariate analysis showed that the CRS patients with radical operation (P=0.000), earlier TNM stage (P=0.000), non-distant metastasis (P=0.022), serum hemoglobin level >130 g/L(P=0.013), and serum album level >40 g/L (P=0.042) had better prognosis. Multivariate analysis, enrolling above 5 factors, showed that TNM staging (HR=2.363, 95%CI: 1.478-3.776, P=0.000) and serum hemoglobin level >130 g/L(HR=0.449, 95%CI: 0.244-0.827, P=0.010) were independent factors influencing prognosis of CRS patients.</p><p><b>CONCLUSIONS</b>Although CRS patients have better prognosis than gastric cancer patients, but local organ invasion and distant metastasis occurs more readily. TNM staging and serum hemoglobin level are independent prognostic factors for CRS patients.</p>
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Aged , Female , Humans , Male , Middle Aged , Gastrectomy , Gastric Stump , Pathology , General Surgery , Lymphatic Metastasis , Multivariate Analysis , Neoplasm Recurrence, Local , Neoplasm Staging , Prognosis , Proportional Hazards Models , Retrospective Studies , Stomach Neoplasms , Pathology , General SurgeryABSTRACT
Objective To establish a rapid and high sensitive assay of real-time fluorescence loop-mediated isothermal amplification assay for clinical detection of HIV-1 DNA.Methods Four loop primers were constructed for loop-mediated isothermal amplification ( LAMP) assay based on six conserved regions selected from HIV gene sequence .SYBR Green Ⅰdye was added into the established LAMP assay and its specificity and sensitivity were evaluated .Results The real-time fluorescence LAMP assay for the detection of HIV-1 DNA was successfully established .It was used for the detection of HIV-1 DNA among 200 patients with HIV infection, of which 195 cases were positive.Moreover, 50 healthy subjects were found HIV-1 DNA negative tested by the real-time fluorescence LAMP assay .Quantitative testing for HIV-1 DNA showed that the lowest and the highest detectable amount were 51 copies/ml and 8.21×106 copies/ml respectively, and the average amount was 5.78×105 copies/ml.HIV viral loads ranging from 1×105 to 10×105 were detected in 162 of 200 patients (83.08%).The ten times dilution method showed that the lowest detection limit of the assay was 50 copies/ml.The crossover experiment indicated that the specificity of the assay was 100%as none of HBV-DNA, HSV-DNA and HCV-RNA was determined by the assay .Conclusion The present study shows that 97.5%of the patients with HIV infection are confirmed HIV-1 DNA positive by the real-time fluorescence LAMP assay , suggesting that the real-time fluorescence LAMP assay is a rapid and sensi-tive assay with high specificity and could be applied for clinical detection of HIV-1 DNA.
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Objective To establish a rapid, sensitive, and specific quantitative method to detect hepatitis C virus. Methods A primer set targeting HCV 5'UTR was designed. The isothermal amplification was performed by the Bst DNA polymerase and AMV reverse transcriptase, under the temperature of 60℃ for 60 min. The signal was monitored by SYBR Green Ⅰ. Results One hundred and twenty positive serum samples, confirmed by the real-time PCR. All were detected by the isothermal amplification, while 110 healthy subjects' samples were negative by the both methods. The lower detect limit was determined to 10 IU/ml HCV-RNA, by the assay on serial dilutions of the quality control standards obtained from clinical investigation center of MOH. Conclusion A real time reverse loop-mediated isothermal amplification method was developed to detect HCV, with the characteristic of rapidity, high sensitivity and specificity.
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Objective To investigate the correlation of T-lymphocyte expressing HLA-DR with serum HBV DNA and HBeAg contents in chronic hepatitis B. Methods Totally 134 chronic hepatitis B patients and 36 healthy blood donors were enrolled in the study. The T-lymphocytes (CD3 + HLA-DR + ,CD4 + HLA-DR+ and CD8 + HLA-DR+ T) expressing HLA-DR were detected by flow cytometry, the serum HBV viral loads were detected by the real-time quantitative PCR and HBeAg was detected by chemiluminescence method. According to serum HBV DNA viral loads patients were defined as HBV DNA negative (≤ 103 copies/mL), low (＞ 103 - 105 copies/mL), medium (＞ 105 - 107 copies/mL) and high groups (＞ 107 - 109 copies/mL) ; according to serum HBeAg levels, patients were defined as HBeAg negative (≤1 PEIU/mL), low (＞ 1 - 100 PEIU/mL), medium (＞ 100-1 000 PEIU/mL) and high groups (＞ 1 000-10 000 PEIU/mL). T test and one-way ANOVA were performed. Results With HBV DNA loads, HBeAg levels increased, the percentage of CD3 + HLA-DR + , CD4 + HLA-DR + and CD8 + HLA-DR +decreased, especially CD8 + HLA-DR +. Compared with HBV DNA negative group, the percentages of CD3 +HLA-DR + , CD4 + HLA-DR + and CD8 + HLA-DR + were significantly reduced in high group (t = 3. 686,4. 592 and 3. 216, P ＜ 0. 0l); the percentages of CD4 + HLA-DR + and CD8 + HLA-DR + were also reduced in medium group (t = 3. 761 and 2.862, P ＜ 0.01); while in low group, only the percentage of CD8 + HLA-DR + was reduced (t = 2.215, P ＜ 0.05). Compared with HBeAg negative group, the percentages of CD3 +HLA-DR+, CD4 + HLA-DR+ and CD8 + HLA-DR+ were significantly reduced in medium and high groups (thigher =3. 144, 2.222 and 4.035; tmiddle =3.311, 2.362 and 3.374, P ＜0.05), while in the low group,only the percentage of CD8+HLA-DR+ was reduced (t=2.029, P＜0. 05). Conclusion The combined measurement of HBV DNA, HBeAg and T-lymphocytes expressing HLA-DR in chronic hepatitis B patients may not only help to evaluate the immune status of patients, but also can predict the disease progression and clinical outcomes.
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Objective To develop an rapid, visuable method to detect the genotypes of HBV.Methods According to the published full-length sequence with known genotypes in GenBank, the specific primer and biotin-lablled probe were designed. We established a nucleic acid strip method for genotyping hepatitis B virus(HBV) isolates among 150 HBV infected patients and 20 healthy controls, and compared the results with those obtained by real-time PCR. Results There was 34.00% genotype B , 61.33% genotype C, 4.00% genotype B/C in 150 samples, respectively, while the remaining 0.67% unknown. The results for this assay were high comparable to the method of real-time PCR. Conclusion With the similar sensitivity and specifity when compared with real-time PCR, this rapid method is suitable to the clinical setting.
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Objective To investigate the association of primary liver cancer(PLC)with the mutations of HBV precore and basic core promoter(BCP)genes.Methods The serum markers of hepatitis B and the quantities of serum HBV DNA were detected in 144 HBsAg-positive PLC patients.The precore and BCP gene mutations in patients with HBeAg-negtive and HBV DNA-positive were detected by real-time PCR.One hundred and twenty chronic hepatitis B(CHB)patients were randomly selected to serve as the conol.Results There were 46(3 1.94%)patients with HBeAg-positive and 98(68.06%)patients with HBeAg-negative.In 98 HBeAg-negative patients,56(57.14%)were HBV DNA-positive,in which 43 (76.79%)were with precore 1896 gene mutations,50(89.29%)were with BCP1762/1764 gene mutations.and 38(67.86%)were with both gene mutations.Precore 1896 and BCP1762/1764 gene mutation rates in PLC patients were much higher than those in CHB patients(χ2=9.36 and 5.77,P＜0.05).Conclusion PLC may be associated with the mutations of HBV precore anti BCP genes.
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Objective To develop a rapid, accurate, specific method to detect causative agent of hand, foot and mouth disease (HFMD). Methods Specific primers and probe were designed based on highly conserved VP1 region of enterovirus 71, coxsackie virus A16 and enterovirus. The sensitivity and specificity of the real-time RT-PCR was evaluated with 35 stool samples collected from pediatric patients with suspected HFMD and 20 clinical samples from health pediatric patients. Results Out of 35 clinical samples from suspected HFMD, 35 samples were identified as positive for enterovirus, 25 clinical samples were identified as positive for enterovirus 71, 8 clinical samples were identified as positive for coxsackie virus A16, among which 3 clinical samples were identified as positive for enterovirus 71 and coxsackie virus A16. The clinical diagnostic accordance rate is 85.71%. Out of 20 clinical samples from normal pediatric patients, 5 clinical samples were identified as positive for enterovirus, 20 clinical samples were negative for enterovirns 71 and coxsackie virus AI6. Conclusion Our results indicate real-time RT-PCR offers a rapid, sensitive, specific and cheap method to detect pathogen of HFMD from clinical specimens.
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Objective To study the difference in detection rate of Mycobacterium tuberculosis in diffenent types of sumples.MethodsThe sputum,bronchial fluid,blood of 52 patients with clinical diagnosis of patients with pulmonary tuberculosis were tested with fluorescence quantitative PCR.Results45 cases were identiified in sputum and 51 cases were identified in bronehial perfusate and 15case were identified in blood.?2 was 4.875(P
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Objective To investigate the distribution of hepatitis B virus(HBV)genotypes in Zhejiang.Methods HBV genotyps were detected by fluorimetric real-time PCR in 240 HBVDNA positive patients who were born in Zhejiang(Hangzhou,Huzhou,Jinhua,Shaoxing,Taizhou,Ningbo each 40 positive patients).Results of the 240 HNBDNA positive patients,82(34.17%)were genotype B,and 140(58.33%)were genotype C,15(6.25%)were genotype B and C,3(1.25%)were genotype D.No genotype A、E and F found in the studied subjects.Conclusions HBV genotype B,genotype C,genotype B and C,genotype D existed in Zhejiang and there is no difference in ferms of the distribution of genotypes in six areas metioned above.
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Objective To investigate the distribution and virologic characteristics of HBV genotypes,and possible association with the severity of liver disease.Methods HBV genotype was determined,using oligonuleotides(oligo) microarray method.Results 37patients were differentiated as genotype B,69 genotype C.The clinical manifestations demonstrated that the serum lever of HBVDNA and HBeAg positive rate in patients of genotype C was 7.02?1.26 and 60.87% higher than 5.62?1.02and 32.43% in those of genotype B,the serum HBeAb positive rate in patients of genotype B was 67.57% higher than 39.13% in those of genotype C.The occurrence rates of those developed to chronic hepatitis B liver cirrhosis and hepatoma in those of genotype C were 46.38%,30.44% and 11.59% highter than that of 40.54%,13.51%and 8.11% in those of genotype B.Patients with genotype B were much younger than those with genotype C.Conclusions Genotype B and C exsited in Hangzhou,genotype C is a predominated genotype.The serum leverl of HBVDNA in those of genotype C is highter than that of genotype B.The positive rate of HBeAg in those of genotype B is highter than that of genotype C.The damage to liver induced by genotype C is severe than that of genotype B.
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Objective To investigate the distribution of hepatitis B virus genotypes in Hangzhou area and preliminarily identify and evaluate the applied characteristics of oligonuleotides(oligo)microarray for genotyping hepatitis B virus(HBV).Methods HBV PCR products were hybridized with oligonucleotide probes,which were prestablized on the chip.The hybridized results were colorized.According to the hybridization signal and the corresponding probe sequence,HBV genotype was determined.Results Of the 106 HBV DNA positive patients 37(34.91%)were genotype B,and 69(65.09%)were genotype C,No genotype A,E and F were found in the studied subjects.Conclusions HBV genotype B and C exsited in Hangzhou No HBV genotype A,D,E and F were found in the studied subjects.We should take further investigation for HBV genotypes by enlarging study population.The advantages of this assay are sensitive accurate,fast and economical when using it for HBV genotype test comparing with other relative methods.
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OBJECTIVE To discuss the infection rate of Chlamydia trachomatis(Ct) and Ureaplasma urealyticum(Uu) in patients with non-gonococcal infection.METHODS Fluorescence quantitative PCR method was used on 1025 cases and 30 cases of NGU patients for Ct and Uu detection.RESULTS Of 1025 NGU patients,positive Ct alone accounted for 156 cases,the positive rate was 15.22%.505 cases were separate Uu,the positive rate was 49.27%.Ct,Uu mixed in 217 cases,the positive rate was 21.17%.The detection rate was 85.66%.Uu infection rate in women was more than that in men(?2 = 104.56 P0.05).of control group,the Ct Uu Results negative.CONCLUSIONS In NGH patients,Uu is most common pathgen in man and woman.To diagnosis of NGU,Uu and Ct should be followed by Ct infection rate but no gender tested at the same time to avoid missed diagnosis.