[Purification of the heparin-binding haemagglutinin adhesin of Mycobacterium tuberculosis and its application in diagnosis of tuberculosis].

OBJECTIVE: To investigate the application of heparin-binding haemagglutinin adhesin (HBHA) in tuberculosis (TB) diagnosis. METHODS: We prepared native HBHA from cultivated Mycobacterium Bovis Calmetta Guerin (BCG) in Suton liquid medium. After BCG grew to the stationary status, native HBHA was acquired by specific CL-6B chromatography column binding heparin. At the same time, we cloned hbhA gene from Mycobacterium tuberculosis into PET-32alpha (+) expression vector. Recombinant HBHA from E. coli was obtained. Based on the native HBHA and recombinant HBHA, we chose 4 groups of pulmonary TB, extra-pulmonary TB, PPD (-) and PPD (+) healthy control with 47 in each group and conducted ELISA from serum for specific HBHA antibody level. At last we calculated the sensitivity and specificity in TB diagnosis by detection of anti-HBHA antibody level. RESULTS: The native HBHA could be diluted and purified with the PBS containing the 375 mmol/L NaCl by specific CL-6B chromatography column binding heparin; There was no significant difference in experimental result based on the natural and recombinant HBHA protein, also no difference between PPD (-) and PPD (+) healthy control groups. Serum antibody level by ELISA could distinguish pulmonary TB and ertra-pulmonary TB (t = 12.224, P< 0.05). The antibody level of the TB groups (pulmonary TB and ertra-pulmonary TB) was higher than the healthy control groups [PPD (+) and PPD (-) healthy control] (t =25.909, P<0.05). CONCLUSION: Both recombinant and native HBHA can be used as immunological diagnosis in TB. It can be used in TB and especially extra-pulmonary TB diagnosis.

[Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates by denaturing high-performance liquid chromatography and DNA sequencing].

OBJECTIVE: To determine the rpsL and rrs gene mutation in Mycobacterium tuberculosis (M. tuberculosis) and compare the consistency between the results of denaturing high-performance liquid chromatography (DHPLC) and those of DNA sequencing. METHODS: The values of streptomycin minimum inhibitory concentration (MIC) against 215 M. tuberculosis clinical isolates, 115 being streptomycin-resistant and 100 being susceptible by a routine proportional method, were tested by DHPLC. DNA sequencing was conducted to detect the rpsL and rrs mutation. RESULTS: 98 of the 115 streptomycin-resistant isolates (85.2%) harbored rpsL and/or rrs mutation, 76.5% of which being rpsL mutation (88/115). There was no significant correlation between the MIC values and mutation types. No mutation was found in all the susceptible isolates. There was a complete consistency between the DHPLC results and those of DNA sequencing. CONCLUSION: DHPLC can be regarded as a useful and powerful tool to detect the streptomycin resistance detection in M. tuberculosis.

[Optimization and evaluation of a new antischistosomal drug QH917 self-microemulsifying drug delivery system].

To screen a new poorly water-soluble antischistosomal drug QH917 self-microemulsifying drug delivery system which has steady release in vitro and absorption in situ separately. The formulation was optimized using central composite design-response surface methodology. Independent variables were oil content (%) and the weight ratio of surfactant and cosurfactant (Km), while response variables were self-microemulsifying time (t), mean particle size (PS) and polydispersity index (PI). The effects of ionic strength, food, pH, rotation speed and medium volume on drug release of the optimized formulation were evaluated under conditions simulating in vivo physiological situations. The absorption of the optimized formulation was studied using in situ intestinal permeability technique of rats. The optimized formulation was as follows: the content of media chain triglyceride (MCT) was 30%-34% (w/w); and the weight ratio of surfactant polyoxyl 40 hydrogenated castor oil (Cremophor RH40) and co-surfactant ethanol was 4.8-5.2. Release of QH917 from the optimized formulation was nearly unaffected by ionic strength, food, pH, rotation speed and medium volume. There was no marked difference of the absorption rate between rats with and without ligated bile duct in rat intestinal permeability technique. Inter-individual variability in absorption of the optimized formulation was negligible. Central composite design-response surface methodology is an efficient approach for optimizing formulations of self-microemulsifying drug delivery system; drug release in vitro and absorption behavior in situ of the optimized formulation is steady.

[Polymerase chain reaction-single strand conformation polymorphism analysis combined with nucleic acid hybridization to detect ofloxacin-resistant Mycobacterium tuberculosis mutation selected in vitro].

OBJECTIVE: To explore the feasibility of utilizing polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis combined with Southern blot to detect ofloxacin (OFLX)-resistant Mycobacterium tuberculosis, and figure out the boundary between the OFLX-susceptible and resistant Mycobacterium tuberculosis. METHODS: OFLX-resistant Mycobacterium tuberculosis bacilli were induced in vitro and their minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) were determined. The 320 bp segments of gyrA gene were amplified by PCR and their polymorphism was tested by SSCP from H(37)Rv, H(37)Ra and those induced OFLX-resistant strains. To prove the repeatability of PCR-SSCP combined with Southern blot, 36 clinical isolated OFLX-resistant strains were tested. RESULTS: All of the 85 OFLX-resistant mutations involved in this study were identified to have gyrA mutations. The results showed that there was an overlap if OFLX 10 microg/ml was used as a criteria for discriminating susceptible and resistant strains. Compared with the H(37)Ra, the similar single-stranded shift caused by the single-stranded conformation change was viewed in the 36 clinical isolated OFLX-resistant strains by PCR-SSCP combined with Southern hybridization. CONCLUSIONS: PCR-SSCP analysis combined with Southern blot can clearly distinguish OFLX-susceptible from OFLX-resistant strains. The results suggest that as a cut-off point between OFLX-susceptible and low-level OFLX-resistant to Mycobacterium tuberculosis, OFLX concentration of 10 microg/ml is considered to be a better one.