Objective To investigate the variability of katG gene in Mycobacterium tuberculosis strains isolated in China,and screen the new isoniazid resistance related mutation sites meanwhile. Methods 429 clinical Mycobacterium tuberculosis isolates were included in our research.PCR-RFLP method was applied to screen for the S315T mutation firstly.Whole katG gene sequencing was done in those resistant strains without S315T mutation to explore the unknown mutated sites associated with isoniazid resistance.Results S315 mutation were found in 76.9% (166/216)resistant stains. Complete or part deletion of katG gene was detected in 2 highly-resistant isolates.Sequencing in 48 resistant strains showed that 315,463 and 234 sites were the most frequent mutation sites,other sites were also found but distributed dispersedly with low prevalence rate as less than 5%.Besides S315T, S315N were also common in China (8.7%).The most common variation is still site mutation.Con- clusions The results from the study of genotypes associated with most common clinical resistant phe notypes can be helpful to develop new methods to detect the resistant M.tuberculosis.

Objective To develop a new Multiplex Allele-Specific polymerase chain reaction(MAS-PCR) assay to detect the main point mutations in the katG and rpoB genes, which has been reported to account for the majority of clinical Mycobaterium tuberculosis resistant to isoniazid and rifampin. Methods Based on the sequences of katG and rpoB genes, specific primers were designed to carry out the MAS-PCR to detect the most common mutations in codon315 of katG and codons 531,526,516 of rpoB gene. Results The purified DNA preparation of 96 clinical Mycobacterium tuberculosis strains were used to optimize PCR. No mutation was detected in 19 isoniazid-sensitive strains. The 315Ser point mutation was detected in 79.2%(61/77)of isoniazid-resistant strains, the type of mutation includes the most common S315T and the less common S315N, which could’t be detected by PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism). However, S315G couldn’t be detected by MAS-PCR and that will make a false negative. The mutations in codons 531,526,516 were detected by the MAS-PCR. Compared with the results of direct sequencing of rpoB gene, no mutation was detected in sensitive strains. For rifampin-resistant strains, the total sensitivity was 81.5%(66/81). Conclusions MAS-PCR is a new molecular method with high sensitivity and specificity, which can be used to detect the point mutation in katG and rpoB gene rapidly and economically. It can be used in clinical laboratories to detect drug-resistant tuberculosis strains. Simultaneous detection for katG and rpoB gene mutations in one MAS-PCR system will help to improve the efficiency of this method.

Objective To investigate the genotypic characteristics of hepatitis B virus(HBV) in Tibet.Methods Serum samples from 60 cases of hepatitis B in Tibet Autonomous Region were collected from January 2000 to March 2004.HBV genotypes were analyzed by sequencing S region and precore/core region.Results HBV isolated from 60 cases were all found to be D genotype by S region sequencing.Dc mixture was found in 59 cases,showing the recombination between their precore/core gene and genotype B virus at 1804-2299 nucleotide.The other one case showed Dbc mixture genotype,showing recombination between its precore/core gene and genotype B and C genes.Conclusions All Tibet cases in this study show mixture genotype D with recombination with genotvpe C or both genotype B and C at precore/core region.No case of pure genotype D is found.

Objective To investigate the risk factors associated with cranial nerve impairment in patients with tuberculous meningitis.Methods A total of 121 patients with tuberculous meningitis who were admitted to Huashan Hospital from 2000 to 2011 were reviewed retrospectively.Demographic data (gender,age),course of disease,initial results of cerebral spinal fluid (CSF) tests,occurrence of cranial nerve impairment and prognosis of these patients were collected.All the patients were followed up for at least 3 months,and for those with cranial nerve impairment,the minimum follow-up period was 1 year in order to judge the recovery of cranial nerve impairment.Multivariate analysis was performed to study the associated risk factors.Results Out of 121 patients,22 (18.2 ％)developed cranial nerve impairment.Nerves involved were abducens nerve,oculomotor nerve,optic nerve and auditory nerve,and impairment of single nerve occurred in 9 (40.9 ％),8 (36.4 ％),7(31.8％) and 1(4.5％) patient,respectively.Three cases had more than one group of cranial nerves involved,accounting for 13.6％ of the 22 patients with cranial nerve impairment.The incidence of conscious disturbance was significantly higher in patients with cranial nerve impairment than those without impairment (77 ％ vs 45 ％,P=0.020).Delay in diagnosis (OR =1.017,95 ％ CI:1.001-1.033,P=0.040) and occurrence of conscious disturbance (OR =3.242,95 ％ CI:1.142-9.205,P=0.027) were independent predictive factors of cranial nerve injury.During one-year follow-up,90.9％ of patients were fully recovered from cranial nerve impairment,with a median duration of 1 month (range 0.5-6.0 months).Conclusions Cranial nerve impairment is a common complication in patients with tuberculous meningitis.Delay in diagnosis and occurrence of conscious disturbance were independent predictive factors.Most cranial nerve impairment were reversible,and timely diagnosis and treatment are important ways to reduce complications.

Organic anion transporting polypeptide 1B1 (OATP1B1) is an important liver-specific uptake transporter, which mediates transport of numerous endogenous substances and drugs from blood into hepatocytes. To identify and investigate potential modulators of OATP1B1 from natural products, the effect of 21 frequently used natural compounds and extracts on OATP1B1-mediated fluorescein methotrexate transport was studied by using Chinese hamster ovary cells stably expressing OATP1B1 (CHO-OATP1B1) in 96-well plates. This method could be used for the screening of large compound libraries. Our studies showed that some flavonoids (e.g., quercetin, quercitrin, rutin, chrysanthemum flavonoids and mulberrin) and triterpenoids (e.g., glycyrrhetinic acid and glycyrrhizic acid) were inhibitors of OATP1B1 with IC50 values less than 16 µmol · L(-1). The IC50 value of glycyrrhetinic acid on OATP1B1 was comparable to its blood concentration in clinics, indicating an OATPlB1-mediated drug-drug interaction could occur. Structure-activity relationship analysis showed that flavonoids had much higher inhibitory activity than their glycosides. Furthermore, the type and length of saccharides had a significant effect on their activity. In addition, we used OATP1B1 substrates fluvastatin and rosuvastatin as probe drugs to investigate the substrate-dependent effect of several natural compounds on the function of OATP1B1 in vitro. Our results demonstrated that the effect of these natural products on the function of OATPlB1 was substrate-dependent. In summary, this study would be conducive to predicting and avoiding potential OATP1B1-mediated drug-drug and drug-food interactions and thus provide the experimental basis and guidance for rational drug use.

Objective To develop a new multiplex allele-specific PCR(MAS-PCR) assay to detect mutation in codon 315 of katG gene of Mycobacterium tuberculosis , mutation in this codon has been reported to be able to account for the Mycobacterium tuberculosis clinical isolates resistant to isoniazid(INH).Method Based on the sequence of katG gene of Mycobacterium tuberculosis , three specific primers are designed to carry out the MAS-PCR, 84 purified DNA preparation with known katG 315 variation detected by PCR-restriction fragment length polymorphism (PCR-RFLP) are used to optimize PCR.Results 84 Mycobacterium tuberculosis clinical stains are detected by the MAS-PCR and PCR-RFLP, respectively.The sensitivity of detection by MAS-PCR is 77.8%,and the specificity is 95.2%.katG mutation S315N(AGC→AAC), neglected in RFLP, can be detected by MAS-PCR.Conclusion MAS-PCR assay is sensitive, specific, economic and easy to carry out , can be used in clinical laboratories to detect the INH-resistant Mycobacterium tuberculosis strains.

70%. The cluster analysis and factorial analysis showed that the fierceness, cruelty, and vital qi damaging were often clustered with each other and constituted the factors of main characteristics of Du-pathogen. In the Logistic regression analysis, it was discovered that fierceness, cruelty, and multi-damaging were closely related with the recognition of "the excessive pathogen damaging the health" (P

Objective To identify drug resistance status of Mycobacterium tuberculosis (MTB) strains by the GenoType MTBDRplus line-probe assay (LPA),compare its performance with traditional drug susceptibility testing (DST),and to assess its predictive value for the prognosis of patients with drug resistance tuberculosis.Methods Pulmonary tuberculosis patients who visited Zhuji People's Hospital,Zhejiang Province during February 2011 and January 2012 with a positive result of sputum smear at baseline were all recruited.A total of 275 culture positive specimens were collected,then isolated and cultured for Mycobacterium tuberculosis in the laboratory.DST were performed,meanwhile,GenoType MTBDRplus were also applied to detect resistance to isoniazid (INH) and rifampin (RMP).All the tuberculosis patients who were recruited were followed,including sputum culture and chest radiography.Results There were 192 strains showing drug resistance both by DST and MTBDRplus LPA.Fourteen multidrug resistant (MDR),21 INH mono-resistant and 2 RMP mono-resistant strains were detected by DST.As for GenoType MTBDRplus LPA,MDR,INH mono-resistant and RMP mono-resistant strains were 14,18 and 2,respectively.Taken DST as the gold standard,LPA was more accurate in the detection of resistance to RMP,while it failed to detect 23.8％ (5/21) of the INH-resistant strains.We analyzed the prognosis of patients with drug resistance by GenoType MTBDRplus LPA,the rates of treatment success were 84 ％ (110/131),9/15,3/11 in patients infected with susceptible,INH mono-resistant and MDR strains,respectively.For the 2 cases of RMP mono-resistanee,one was cured and the other failed.The predictive value of molecular drug resistance test for treatment failure in INH mono-resistant patients was 40.0 ％,while that was 83.5 ％ for treatment success in INH susceptible patients.The predictive value for treatment failure in RMP mono-resistant patients was 50.0％,while that was 81.5％ for treatment success in RMP susceptible patients.The predictive value for treatment failure in MDR patients was 72.7％,while that was 81.1％ for treatment success in patients without MDR.Conclusion The GenoType MTBDRplus LPA assay is a rapid and reliable diagnostic test for resistance of MTB,which can be used to predict the prognosis of drug resistant tuberculosis in the clinical practice.

Objective To evaluate a new enzyme-linked immunospot assay (TSPOT-TB) for the diagnosis of latent tuberculosis infection in patients with rheumatic diseases.Methods The rapid TSPOT-TB assay was applied to detect ESAT-6 and CFP-10 specific T cells in blood samples from 126 rheumatic disease patients.A PPD skin test was performed on all patients simultaneously.Results The positive rate of TSPOT assay was 23.8% and that of PPD skin test was 34.9%.The overall agreement between the 2 tests was 71.4%.Among PPD (-) patients (n=82),11 were TSPOT (+) ( 13.4% ).Among PPD (+) patients (n=44),25 were TSPOT(-) ( 56.8% ).The patients who got BCG vaccination showed a significantly higher rate of positive results of PPD skin test than those who did not(41% vs 19%,P＜0.05).While in TSPOT assay,the BCG vaccination did not show any influence on TSPOT results (22% vs 27%,P＞0.05).Conclusion BCG vaccina-tion affects the results of PPD test in patients with rheumatic diseases,but has no influence on TSPOT results.The infection rate of latent tuberculosis in patients with rheumatic diseases in our study is 23.8% detected by TSPOT.

Objective To evaluate the number and function of peripheral Vγ2Vδ2+T lymphocytes during Mycobacterium tuberculosis(MTB)infection in human immunodeficiency virus (HIV)infected individuals.Methods Seventy-six HIV/acquired immune deficiency syndrome(AIDS) patients co-infected with MTB were divided into active tuberculosis(TB)group(HIV+TB)and latent TB group(HIV+LTB).T cell subsets of peripheral blood lymphocytes were analysed by flow cytometry.Stimulated by protein purified derivative(PPD)and hydroxymethylbutenyl diphosphate (HMBPP),specific interferon(IFN)-γ producing T cells were detected using enzyme-linked immunospot(ELISPOT)and intracellular cytokine staining(ICS).Data were analyzed by t test.Results The absolute number of CD3'T cells and DroDortion of V72VB2'T cells in CD3+T cells in HIV+TB group were both significantly lower than those in HIV+LTB group(t=-3.67,P<0.01;t=-2.06,P<0.05).PPD-specific IFN-y-producing T cells and percentage of PPD-specific CD4+Tcells in CD3+T cells in HIV+LTB group were both similar with those in HIV+TB group.While HMBPP-specific IFN-γ-producing T cells and percentage of HMBPP-specific Vγ2Vδ2+T cells in CD3+Tcells in HIV+LTB group were both higher than those in HIV+TB group(t=2.71 and t=3.003,respectively;both P<0.0 1).Conclusion The number and function of Vγ2Vδ2+T cells were impaired in HIV/AIDS patients coinfected with active MTB infection,which indicates that Vγ2Vδ2+T cells may be the key immune cells against MTB in individuals with impaired CD4+T cells.