Prognostic significance of serum sP-selectin,sICAM-1 and immunoglobulin in patients with hepatitis B cirrhosis / 中国免疫学杂志

Objective:To investigate the significance of serum sP-selectin,sICAM-1 and immunoglobulin in the prognosis of hepatitis B cirrhosis.Methods:162 cases of hepatitis B patients with cirrhosis were treated as the research object in clinical laboratory of Chizhou People′s Hospital from January 2014 to December 2016,and detected in the serum of soluble P-selectin(sP-selectin), soluble intercellular adhesion molecule-1(sICAM-1),immunoglobulin and serum related index.Comparison of liver cirrhosis and liver cancer patients mean difference,Pearson analysis of serum soluble P-selectin(sP-selectin),soluble intercellular adhesion molecule-1 (sICAM-1),the correlation of immunoglobulin and serum indexes,logistic regression analysis of risk factors of liver cirrhosis to hepatocellular carcinoma,the receiver operating characteristic(ROC)OR>1 parametric curve evaluation of predictive value of hepatocellular carcinoma to liver cirrhosis.Results:The levels of sP-selectin,sICAM-1,IgM,IgG and IgA in patients with primary liver cancer were significantly higher than those in compensated cirrhosis and decompensated liver cirrhosis(P<0.05).The levels of sP-selectin,sICAM-1,IgM,IgG and IgA in patients with decompensated cirrhosis were significantly higher than those in decompensated liver cirrhosis(P<0.05).sP-selectin,sICAM-1,IgM,IgG,IgA were significantly higher in patients with grade C and grade B(P<0.05)in patients with different grades of cirrhosis.SP-selectin,sICAM-1,IgM,IgG,IgA were significantly higher in patients with grade B(P<0.05).There was no significant difference in the expressions of sP-selectin,sICAM-1 and immunoglobulin in patients with multiple foci of carcinoma and primary hepatocellular carcinoma(P>0.05).SP-selectin,sICAM-1 and immunoglobulin in patients with stage Ⅱprimary liver cancer were significantly higher than those in stage Ⅰ(P<0.05).There was a positive correlation between sP-selectin, sICAM-1 and immunoglobulin in patients with liver cirrhosis(P<0.05).In liver cirrhosis was the transfer of primary liver cancer as the dependent variable for Logistic regression analysis,the results showed that ALT,AST,sP-selectin,sICAM-1,IgM,IgG,IgA were risk factors of liver cirrhosis to hepatocellular carcinoma.ROC curve analysis showed that the sensitivity and specificity of sICAM-1 and sP-selectin in diagnosis of liver cirrhosis were higher than other factors.Conclusion:Serum sP-selectin,sICAM-1 has a higher accuracy of clinical diagnosis of hepatitis B cirrhosis prognosis evaluation,diagnostic specificity and sensitivity of liver cirrhosis to hepatocellular carcinoma was higher than that of simple detection of immunoglobulin,the combined detection in the diagnosis of liver cirrhosis is worthy of clinical prognosis.

Investigation of Ureaplasma urealyticum biovars and their relationship with antimicrobial resistance.

Purpose: To develop Taqman fluorescence quantitative polymerase chain reaction (PCR) method for investigating the characteristics of the distributions of Ureaplasma urealyticum (UU) biovars and to explore the relationship between UU biovars and antimicrobial resistance. Materials and Methods: By the method of culture, Ureaplasma species were detected. Taqman fluorescence quantitative PCR for detecting UU biovars were developed and UU clinical isolates were detected to distinguish biovars. The broth micro-dilution susceptibility testing methods were used to determine UU susceptibility. Results: By Taqman PCR method, UU biovars was successfully detected. Of 126 samples, biovar 1 was found in 73 (57.94%). There was a statistical difference between genital-urinary tract infection group and asymptomatic group (P<0.05). In the region, UU biovar 1 to 9 kinds of agents kept higher susceptibility rates, but biovar 2 maintained higher susceptibility rates only to tetracyclines. Compared with biovar 1, UU biovar 2 resistance rates to 7 kinds of agents were higher (P<0.05). Conclusions: (1) Our new established Taqman PCR method is a useful tool for screening UU biovars. (2) UU biovar 1 predominated in asymptomatic population; whereas in genital-urinary tract infection population UU biovar 2 had a higher proportion. (3) The characteristics of drug resistance were different between UU biovars. Overall, both two biovars remained higher susceptibility rates to tetracyclines. A majority of biovor 1 strains were sensitive to macrolides and quinolones; while only a small number of biovar 2 strains kept sensitive to roxithromycin and quinolones, a large proportion of biovar 2 strains were found in intermediate ranges.

Screening of tuberculosis specific antibody binding peptides / 中华预防医学杂志

<p><b>OBJECTIVE</b>To screen the specific antibody binding peptides of tuberculosis from phage-displayed random phage display (Ph.D.)-7 peptide libraries with purified IgG from tuberculosis serum, and to provide the basis for the development of serological detection reagent of tuberculosis.</p><p><b>METHODS</b>Purified IgG of tuberculosis serum was used as solid ligand to screen the binding peptide from the Ph.D.-7 peptide library, according to the biopanning process of absorption, elution and amplification. Purified IgG of health people serum was used as the molecule of counter selection during the second and third selection. Phages were enriched after 3 rounds of screening, then 20 single phages separately eluted by IgG of tuberculosis and health people were randomly selected on each direction of the determination plates and amplified. The single chains DNA were extracted as template for sequencing. The combination abilities of selected clones to IgG of tuberculosis and health people were tested by indirect enzyme linked immunosorbent assay (ELISA), and the positive clone was identified. Serum samples, from 47 patients with tuberculosis and 37 healthy people vaccinated with BCG, were verified by positive phage clones using phage-ELISA. The verifying results of 24 serum samples used for panning were separately analyzed statistically.</p><p><b>RESULTS</b>After 3 rounds of panning, remarkable enrichment of phages that could specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 12 sequences were obtained. 12 phage clones with different sequences were amplified and detected with indirect ELISA and single phage H12 showed higher affinity with IgG of tuberculosis (S/N ≥ 2.1) and was identified as the positive clone. It was found that, in indirect ELISA with single Phage H12, the A(450) value of tuberculosis patients (0.105 ± 0.010) was significantly higher than that of healthy individuals (0.070 ± 0.005), and the t value was 2.912 (P < 0.0001). The A(450) value of 12 serum samples of tuberculosis patients and 12 samples of health individuals used for panning were 0.144 ± 0.016 and 0.052 ± 0.004, and the t value was 5.69 (P < 0.0001).</p><p><b>CONCLUSION</b>By using phage-displayed random peptide libraries, we obtained the specific antibody binding peptides of tuberculosis, which showed specific binding activity with IgG of tuberculosis. It can provide a basis for the establishment of a new serological detection method of tuberculosis with peptide.</p>

Fast identification of mycobacteria in microtiter liquid culture / 中华预防医学杂志

<p><b>OBJECTIVE</b>This research was to establish a method for fast identification of mycobacteria in microtiter liquid culture and to evaluate its clinical value.</p><p><b>METHODS</b>2-thiophenecarboxylic acid hydrazide (TCH) and paranitrobenzoic acid (PNB) at different concentrations were added into liquid culture in 96-well plate. Different mycobacterium standard strains were incubated in liquid culture with PNB and TCH for 7 to 10 days. According to the growth assay for 15 mycobacterium strains and 30 mycobacterium tuberculosis strains, the best PNB and TCH concentration were determined. A total of 424 clinical mycobacterium isolates were identified by microtiter liquid culture at the best PNB and TCH concentration. The results of microtiter liquid culture were compared with those of PCR and DNA sequencing.</p><p><b>RESULTS</b>The best concentration of PNB was 200 µg/ml in microtiter liquid culture. Compared with the results of PCR, the sensitivity and specificity for identification of mycobacterium tuberculosis complex in microtiter liquid culture were 97.8% (306/313) and 100.0% (107/107) respectively and those for non-tuberculosis mycobacteria in microtiter liquid culture were 100.0% (107/107) and 96.5% (306/317) respectively. The best concentration of TCH was 0.5 µg/ml. Compared with the results of PCR, the sensitivity of mycobacterium tuberculosis in microtiter liquid culture was 100.0% (305/305). The specificity remained under and more studies were needed.</p><p><b>CONCLUSION</b>In microtiter liquid culture with PNB and TCH, mycobacteria can be identified in 7 to 10 days. The results were accurate and the process was simple without expensive equipments. This method meets clinical needs and can be used in all level hospitals in China.</p>

Evaluation of microscopic observation drug susceptibility for drug susceptibility testing of mycobacterium tuberculosis in smear-positive sputum / 中华预防医学杂志

<p><b>OBJECTIVE</b>To evaluate microscopic observation drug susceptibility (MODS) for mycobacterium tuberculosis drug susceptibility in smear-positive sputum.</p><p><b>METHODS</b>Drug susceptibility of mycobacterium tuberculosis in 275 smear-positive sputum samples collected from TB patients were detected directly by MODS. The susceptibility of seven antimicrobials including streptomycin, isoniazid, rifampicin, ethambutol, levofloxacin, amikacin and capromycin were detected MODS. At the same time the sputum sample were cultured in MGIT 960 tube and the positive isolates were tested for drug susceptibility by MGIT 960 system. The results of MODS were analyzed and compared with that of MGIT 960.</p><p><b>RESULTS</b>Of 275 smear-positive sputum, MODS detected 235 (85.45%). Results of MODS were obtained in a median time of 18 days (5 - 39 d). For the 235 MODS-positive samples, the compliance rates of MODS to MGIT of 7 drugs were 90.21% (212/235), 88.09% (207/235), 93.62% (220/235), 87.23% (205/235), 92.34% (217/235), 88.51% (208/235) and 86.81% (204/235) respectively. The sensitivity of MODS method were 83.33% (90/108), 85.11% (120/141), 90.74% (98/108), 85.71% (78/91), 86.73% (85/98), 76.92% (40/52) and 77.08% (37/48). The specificities of MODS method were 96.06% (122/127), 92.55% (87/94), 96.06% (122/127), 88.19% (127/144), 96.35% (132/137), 91.80% (168/183) and 89.30% (167/187) respectively.</p><p><b>CONCLUSION</b>MODS is an optimal alternative method for direct and rapid drug susceptibility of sputum with high accuracy in a timely and affordable way in resource-limited settings.</p>

Detection on drug resistance of Mycobacterium tuberculosis by microscopic observation drug susceptibility / 中华预防医学杂志

<p><b>OBJECTIVE</b>To evaluate the effects of microscopic observation drug susceptibility (MODS) in detecting susceptibility of Mycobacterium tuberculosis (MTB) onto four first line anti-tuberculosis drugs.</p><p><b>METHOD</b>The 24-hole cell culture plates were used to test drug susceptibility of MTB on liquid medium, and the best detecting condition of MODS assay was probed; 66 clinical isolates susceptibility to streptomycin (S), isoniazid (H), rifampin (R) and ethambutal (E) were evaluated by using MODS assay and Lowenstein-Jensen (L-J), thereafter, all the inconcordance of isolates between MODS and L-J were tested for the minimal inhibitory concentrations (MIC).</p><p><b>RESULTS</b>Concordance rate of the susceptibility to S, H, R and E in 66 clinical isolates detected by MODS and L-J was 97.0%, 90.9%, 95.5% and 86.4% respectively. If the results obtained by L-J were taken as a golden standard, the sensitivity, specificity, positive and negative predictive value (PPV and NPV) as well as accuracy of susceptibility test to S detected by MODS was 96.0%, 97.6%, 96.0%, 97.6% and 97.0%; 100%, 85.4%, 81.0%, 100% and 90.9% to H; 96.2%, 95%, 92.6%, 97.4% and 95.5% to R; 73.7%, 91.5%, 77.8%, 89.6% and 86.4% to E. There were 20 inconsistent results of 16 isolates by comparing MODS with L-J, and MIC yielded 16 results of those 14 isolates showing identical results with those of the MODS, while 4 results of other 4 isolates identical with L-J.</p><p><b>CONCLUSION</b>MODS method simultaneously provides drug susceptibility to S, H, R and E. MODS might be one of the rapid tools to diagnosing multidrug-resistant tuberculosis as it is rapid, simple, inexpensive and has high concordance with L-J drug susceptibility test.</p>

Evaluation of pyrosequencing for the detection of rpoB gene mutation in Mycobacterium tuberculosis / 中华预防医学杂志

<p><b>OBJECTIVE</b>To detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates.</p><p><b>METHODS</b>Using the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC).</p><p><b>RESULTS</b>Among the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively.</p><p><b>CONCLUSION</b>Not only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.</p>

Expression and purification of CFP32 of Mycobacterium tuberculosis and its serodiagnostic analysis / 中华预防医学杂志

<p><b>OBJECTIVE</b>To establish a recombinant plasmid of CFP32 of Mycobacterium tuberculosis in E. coli, and to analyze its antigenicity.</p><p><b>METHODS</b>Rv0577 gene was amplified by polymerase chain reaction from genome of Mycobacterium tuberculosis, and then cloned into vector pMD18-T followed by the subclone into the expression vector pET21a. Recombinant CFP32 was expressed and purified. The antigenicity of the recombinant protein was analyzed by using Western-blot. The purified recombinant CFP32 protein was used as an antigen to screen the sera of 7 pulmonary TB patients (n = 97), as well as the other pulmonary disease patients (n = 25), and the clinically healthy controls (n = 38) by ELISA.</p><p><b>RESULTS</b>Recombinant plasmid of CFP32 was established, and be expressed efficiently in E. coli BL21 (DE3). The relative molecular mass of the protein was about 300,000 by SDS-PAGE analysis. The protein purified by Ni-NTA was in a purity over 90%, which was confirmed by Western-blot analysis. ELISA analysis showed its sensitivity and specificity were 63.9% (62/97) and 96.8% (2/63) respectively.</p><p><b>CONCLUSION</b>The recombinant expression plasmid pET21a CFP32 has been constructed and CFP32 proteins has been successfully expressed and be purified in E. coli and, ELISA analysis has identified the recombinant CFP32 as a candidate antigen for TB serodiagnosis.</p>

Identification of novel genetic markers in Mycobacterium tuberculosis Beijing genotype strains / 中华传染病杂志

Objective To identify a unique protein as a novel genetic marker for rapid molecular typing of Mycobacteriutn tuberculosis Beijing genotype strains by comparing the proteome of Beijing genotype strains with non-Beijing strains.Methods Fifty-six clinical isolates of Mycobacterium tuber- culosis were analyzed by spoligotyping to determine genotypes.The two-dimensional electrophoresis (2 DE)was used to compare the global protein patterns between Beijing genotype strains and non Bei jing strains.Differential expressed proteins were measured by matrix assisted laser desorption ioniza tion lime of flight mass spectrometry(MALDI-TOF-MS).The data obtained from peptide mass fingerprinting were compared in protein database.The genes encoding differential expressed proteins and their upstream were sequenced.Results Forty nine of the 56 isolates were Beijing genotype strains and 7 isolates were non-Beijing strains.A unique protein Rv0927c was identified,which is absent in Beijing genotype strains compared with 7 non Beijing strains and H37Rv.There were two characteristic mutations in Beijing genotype strains,a deletion of AGC at nucleotide position 421 of Rv0927c and a 127 G→A muta- tion in the upstream of Rv0927c.but not in non Beijing strains and H37Rv.Conclusion Characteris tic mutations of Rv0927c in Beijing genotype strains can be used as a novel genetic marker for rapid molecular typing of Mycobacteriuln tuberculosis Beijing genotype strains and non Beijing strains.