[Susceptibility test of the Mycobacterium avium complex to sixteen anti-infective agents].

OBJECTIVE: To compare the in vitro activities of 13 anti-infective agents and 3 new quinolones (sitafloxacin, gatifloxacin and moxifloxacin) against Mycobacterium avium complex (MAC) isolates, and therefore to explore the possibility of using these quinolones to treat MAC diseases. METHODS: The minimal inhibitory concentration (MIC) of the above 16 anti-infective agents, including sitafloxacin, gatifloxacin and moxifloxacin against MAC isolates was determined by using agar dilution methods, and then the MIC90s of the different anti-infective agents were compared. RESULTS: The MICs of M. avium isolates showed a wider range and was less sensitive to most of the anti-infective agents as compared with M. intracellulare isolates. The MIC90s of clarithromycin against M. avium and M. intracellulare isolates were 32 mg/L and 16 mg/L, respectively, which were the lowest among 4 macrolide compounds. The MIC90 of rifalazil were 0.5 mg/L and 0.25 mg/L, respectively, which were the lowest among 4 rifamycin compounds. The MIC90 of sitafloxacin against M. avium and M. intracellulare isolates were both 4 mg/L, which were the lowest among 5 quinolones. For gatifloxacin and moxifloxacin, the MIC90 against M. avium and M. intracellulare isolates were both 8 mg/L. Two clarithromycin-sensitive strains (MIC=0.5 mg/L) showed a similar MIC of the lower limit for other compounds. Three clarithromycin-insensitive strains (MIC=64 mg/L) showed a similar MIC of the upper limit for other compounds except quinolones. CONCLUSION: Rifalazil, sitafloxacin, gatifloxacin and moxifloxacin showed acceptable in vitro activities against MAC isolates.

[Rapid testing on drug susceptibility of Mycobacterium tuberculosis with nitrate reducrase assay].

OBJECTIVE: To establish a rapid method for testing drug sussceptibility on Mycobacterium tuberculosis. METHODS: Taking absolute Concentration method for drug susceptibility testing of M. tuberculosis as the "gold standard", we examined the drug-resistant of M. tuberculosis strain with nitrate reducrase assay (NRA) and the drug-resistant of M. tuberculosis germ in sputum with NRA. RESULTS: NRA and absolute concentration method was basically comparable with NRA susceptibility as 96.5% and the specificity was 100%, When comparing with traditional absolute concentration method, NRA could shorten the time about 3 weeks. Using NRA to test the drug-resistant of M. tuberculosis germ in sputum, its susceptibility was more than 66.7% and specificity was 100%, within 10-20 days. CONCLUSION: NRA could be used as a rapid drug susceptibility testing on M. tuberculosis.

[Correlation between the mRNA levels of carcinoembryonic antigen and cytokeratin 19 in peripheral blood with staging, treatment response and prognosis in patients with lung cancer].

OBJECTIVE: To investigate the expression of carcinoembryonic antigen (CEA) and cytokeratin19 (CK19) mRNAs in peripheral blood of patients with lung cancer and their correlation with staging, treatment response and prognosis. METHODS: CEA and CK19 mRNAs in peripheral blood were detected by Taq Man reverse transcriptase-polymerase chain reaction (RT-PCR) in 78 patients with lung cancer before and after treatment, 30 patients with benign lung diseases and 30 healthy subjects. Serum CEA and CYFRA21-1 levels were also measured by enzyme linked immunosorbent assay (ELISA) in the 78 patients with lung cancer before treatment. The patients were followed for 2 years. RESULTS: The positive rates of CEA mRNA and CK19 mRNA in patients with lung cancer were 69.2% (54/78) and 62.8% (49/78), respectively, which were significantly higher than those in patients with benign lung diseases and the healthy controls (P < 0.01). The positive rate of CEA mRNA was the highest in adenocarcinoma, while the positive rate of CK19 mRNA was the highest in squamous cell carcinoma. There was no statistically significant difference among different stages (P > 0.05), The positive rates of CEA mRNA and CK19 mRNA were higher than those of serum CEA and CYFRA21-1. The positive rates of CEA mRNA and CK19 mRNA decreased significantly after surgical operation, but there was no significant change after chemotherapy. The median survival time (MST) for patients with a positive CEA mRNA before chemotherapy was shorter than those with a negative CEA mRNA (8.5 month and 11.7 month, respectively). The MST for patients with a positive CK19 mRNA before chemotherapy was shorter than those with a negative CK19 mRNA (8.9 month and 12.3 month, respectively). The rate of relapse and metastasis was higher in patients (29.4%) with a positive CEA mRNA preoperatively than those with a negative CEA mRNA (7.7%). The rate of relapse and metastasis was also higher in patients with a positive CK19 mRNA preoperatively (18.8%) as compared to those with a negative PCR result (7.1%). CONCLUSIONS: CEA and CK19 mRNAs can be used as markers in the detection of tumor micrometastases in lung cancer, and in evaluating surgical response and prognosis. The results suggest that the gene markers are better than the serum ones, and therefore may be useful for the early diagnosis of lung cancer.

[The in-vitro interferon-gamma release assay for the diagnosis of tuberculosis and Mycobacterium tuberculosis infections].

OBJECTIVE: To investigate the in-vitro interferon-gamma (IFN-gamma) release assay based on three different Mycobacterium tuberculosis antigens in the diagnosis of tuberculosis and Mycobacterium tuberculosis infections. METHODS: The peripheral blood mononuclear cells (PBMC) were collected from the patients with tuberculosis (tuberculosis group, n = 57), patients with lung cancer (lung cancer group, n = 29), and healthy controls (healthy control group 2, n = 27). The PBMCs were co-cultured for 5 days with different antigens: purified protein derivatives (PPD) of tuberculin, early secretary antigenic target 6,000 protein (ESAT6) and 38,000 antigen. The protein levels of IFN-gamma were detected by ELISA, and the results were compared to those with the tuberculin skin test (TST). RESULTS: (1) For healthy controls, the TST was positively related to the history of BCG vaccination and the closeness of contact with sputum-positive tuberculosis patients (P = 0.047, P = 0.041 respectively). The ESAT6 based IFN-gamma release assay was only significantly related to the closeness of contact with sputum-positive tuberculosis patients (P = 0.005), but not to the history of BCG vaccination. (2) There was no significant difference of the TST results among the three groups (P > 0.05). (3) The receiver operating characteristic (ROC) curve analysis indicated that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of the IFN-gamma release assay based on 38,000 antigen were 64.9%, 89.3%, 77.0%, 86.0%, and 71.0% respectively for the diagnosis of tuberculosis. CONCLUSIONS: IFN-gamma release assay based on ESAT6 appears to be better than TST in the diagnosis of infection of Mycobacterium tuberculosis, while IFN-gamma release assay based on 38,000 may be helpful for the diagnosis of tuberculosis.

[Study on gene knock-out in Mycobacterium BCG].

OBJECTIVE: To establish the methodology of plasmid for gene knock-out in Mycobacterium BCG. METHODS: We designed two pairs of primers for amplification of MDP1 gene and inserted two fragments into pKO plasmid, and then the recombinant plasmid for MDP1 gene knock-out was obtained, and named pKO-MDP1. Gene exchange took place within the genome of BCG after pKO-MDP1 plasmid was transformed into Mycobacterium BCG. The strain of Mycobacterium BCG with MDP1 gene knock-out was selected, and the curve of growth rate was studied. RESULTS: The target strain was that of the positive strain by two step PCR and one step sucrose counter selection, without growth in culture media with hygomycin. The value of A(600) per 12 hours was detected for sixteen days. A "S" shaped growth curve was detected. However, there was no significant difference in the growth rates between the wild type Mycobacterium BCG strain and the gene knock-out Mycobacterium BCG strain. CONCLUSIONS: The plasmid of pKO was a useful tool for gene knock-out in Mycobacteria. MDP1 maybe one of the factors influencing the growth rate, but it was not the only one.

[Analysis and evaluation of phage amplified biologically assay, DNA sequencing analysis and single-strand conformational polymorphism for the drug susceptibility testing of Mycobacterium tuberculosis].

OBJECTIVE: To rapidly identify drug-resistant Mycobacterium tuberculosis using phenotypic and genotypic methods and to evaluate the clinical significance of rapid phenotypic susceptibility test by phage amplified biologically assay (PhaB). METHODS: PhaB, DNA sequencing and polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) were performed on the 91 rifampicin (RFP)-resistant strains (including 82 multi-drug resistant Mycobacterium tuberculosis strains), 42 RFP-susceptible strains, 75 ofloxacin (OFLX)-resistant strains and 40 OFLX-susceptible strains at the same time. RESULTS: The results obtained using PhaB assay, DNA sequencing and PCR-SSCP were compared with the absolute concentration method. For RFP susceptibility, the accordance, sensitivity and specificity of PhaB were 93%, 92% and 95% respectively; the accordance, sensitivity and specificity of DNA sequencing were 93%, 90% and 100% respectively; those of PCR-SSCP were 90%, 86% and 100% respectively. For OFLX susceptibility, the accordance, sensitivity and specificity of PhaB assay were 95%, 95% and 95%; those of DNA sequencing were 80%, 71% and 98% respectively; those of PCR-SSCP were 75%, 63% and 98% respectively. CONCLUSIONS: PhaB assay is a low-cost, rapid, and sensitive method and shows high accordance with absolute concentration technology. It can give drug susceptibility test results within 48 - 96 h, and is a promising technology in clinical laboratory.

[A study on the association of 3'UTR polymorphisms of NRAMP1 gene with susceptibility to tuberculosis in Hans].

OBJECTIVE: To determine whether 3'UTR polymorphisms of the NRAMP1 gene are associated with tuberculosis in Hans. METHODS: 3'UTR polymorphisms of NRAMP1 gene were typed by PCR-RFLP among 147 patients with active tuberculosis and 145 healthy individuals. The relationship between 3'UTR polymorphisms and susceptibility to tuberculosis was studied, and cases were grouped according to genotypes. RESULTS: In the tuberculosis patients, genotype TGTG/TGTG, TGTG/TGTG deleted, and TGTG deleted/TGTG deleted were observed in 95, 50 and 2 cases respectively, while the genotypes of the healthy controls were TGTG/TGTG in 115, TGTG/TGTG deleted in 29 and TGTG deleted/TGTG deleted in 1 case. The frequency of the genotype TGTG/TGTG was found more often among controls than that in patients (chi(2) = 7.79, P < 0.01). The frequency of allele TGTG and the frequency of variant allele were 0.85 and 0.15 respectively. CONCLUSIONS: 3'UTR polymorphisms of NRAMP1 gene are associated with susceptibility to tuberculosis in Hans. The variant allele observed in Hans is more common than that in Caucasians. These observations might explain in part why Hans have greater susceptibility to tuberculosis than Caucasians.

[Nitric oxide production and expression of cytokines by macrophages infected by M. tuberculosis H(37)R(v)].

OBJECTIVE: To study nitric oxide (NO) production and cytokine expression by macrophages infected by M. tuberculosis H(37)R(v), and to compare the difference between dead and live M. tuberculosis in the induction of immune responses, and thus to show if dead bacteria could be a possible candidate for new vaccines. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and ELISA were used to measure the production of NO and cytokines in macrophages infected by H(37)R(v). RESULTS: Macrophages infected by viable M. tuberculosis produced more NO, IL-1, IL-12, IL-18, TNF-alpha and inducible nitric oxide synthases (iNOS), as compared with macrophages infected by dead bacteria. The number of bacteria was also an important factor determining the production of NO and cytokines. CONCLUSIONS: Viable M. tuberculosis H(37)R(v) can induce the activation of macrophages and the production of more NO and cytokines which play important roles in the host immune response. Heat-killed M. tuberculosis H(37)R(v) failed to induce activation of macrophages and the production of NO and cytokines, which makes it unlikely to be a candidate for vaccine development.