29 isoniazid (INH) resistant isolated strains and INH sensitive reference strain (H37Rv) of Mycobacterium tuberculosis were analysed by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and NciI restriction mapping for the detection of mutations in katG gene and inhA gene. The katG gene was divided into 3 parts (Akat, Bkat, Ckat; each part is about 800 bp) and amplified, inhA gene was amplified as a whole. Each of the amplified 800 bp DNA was digested into small fragments of less than 400 bp with restriction enzymes for the direct PCR-SSCP analysis. Firstly, 10 strains were analysed. All the 10 isolates showed clearly distinct SSCP patterns in Bkat from that of the reference strain, but only two isolates showed distinct SSCP patterns in Akat, and no isolated strain showed any distinct SSCP patterns in Ckat. 10 isolates also showed distinct SSCP patterns in inhA. NciI restriction mapping of Bkat showed mutation in codon 463 in 7 strains among 10 isolated strains. With these results an early detection strategy for the INH resistant M. tuberculosis was applied to the rest of 19 isolated INH resistant strains. Firstly, isolates were screened by Ncsl mapping in Bkat, and 13 strains showed mutations in codon 463. Secondly, the rest of 6 INH resistant isolates were analysed by PCR-SSCP with restriction enzyme digestion (PCR-SSCP-RE) in Bkat, and all the strains showed distinct SSCP patterns from that of the INH sensitive reference strain. This proved our strategy as effective and economic and time saving method in early detection of INH resistant M. tuberculosis.
Codon ; Diagnosis* ; Digestion ; DNA ; Isoniazid* ; Korea* ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymorphism, Single-Stranded Conformational ; Restriction Mapping ; Tuberculosis

One hundred and thirty-eight strains of Mycobacterium tuberculosis isolated from the 7th nationwide tuberculosis prevalence survey in 1995 were subjected to the restriction fragment length polymorphism (RFLP) analysis using IS6110 probe to define the representative fingerprinting patterns of Korean strains of M. tuberculosis and to evaluate the usefulness of DNA fingerprinting in tracing the transmission link of M. tuberculosis. Among 138 strains, 129 different IS6l10 RFLP types were identified. The number of bands in IS6110 RFLP types diversed from 1 to 20, and the majority (75%) was 9 to 14 bands. The RFLP patterns of 8 out of 15 strains isolated from the follow-up survey of one and half year later after the 7th national TB prevalence survey were unchanged when compared with previous RFLP patterns. Fifteen (11%) out of 138 strains were grouped in 6 IS6110 clusters; 2 with 10 copies, 2 with 12 copies, 1 with 14 copies, and 1 with 17 copies. These clusters were unable to be subclassified by IS1081 or (GTG) probes except one cluster by pTBN12 probe. The transmission links of 2 clusters were deducible; one from household and another from neighborhood, but those of remaining clusters were unclear because they had no contact one another. The results suggest that vigorous transmissions in tuberculosis are still progressing in Korea.
Dermatoglyphics ; DNA Fingerprinting* ; DNA* ; Family Characteristics ; Follow-Up Studies ; Korea ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymorphism, Restriction Fragment Length ; Prevalence* ; Residence Characteristics ; Tuberculosis*

HIV-1 p24 was cloned into multiple cloning site of pMV261, extrachromosomal expression vectors carrying BCG replication origin and BCG-specific heat-shock promoter, and then introduced into BCG and E. coli. Western blot experiments showed that the p24 efficiently expressed in recombinant BCG (rBCG), but not in E. coli. Recombinant p24 expression induced by a single heat-shock of rBCG was maintained longer than 3 weeks. Immunoblot experiments with intact rBCG did not show any distinctive positive signal, suggesting that the recombinant protein was not secreted or exposed at the surface of BCG. The guinea pigs immunized with live rBCG showed delayed type hypersensitivity (DTH) by the systemic area as well as an effective humoral immunity, suggesting that tbis rBCG is believed to elicit eKcient immune responses against p24, even though the expression is restricted only in the cytoplasm as reported previously with other antigen. These results demonstrate that BCG can be developed as a live recombinant vaccine vector against a broad spectrum of infectious disease.
Animals* ; Blotting, Western ; Clone Cells ; Cloning, Organism ; Communicable Diseases ; Cytoplasm ; Guinea Pigs ; HIV* ; HIV-1 ; Hypersensitivity ; Immunity, Humoral ; Mycobacterium bovis* ; Replication Origin

No abstract available.
Mycobacterium tuberculosis* ; Mycobacterium*

D variant of encephalomyocarditis (EMC-D) virus causes diabetes in susceptible mice by direct infection and cytolysis of pancreatic beta cells. cDNA covering the major outer capsid protein (VP1) of EMC-D virus was cloned into Mycobacterium bovis bacillus Calmette-Guerin (BCG). None of the SJL/J male mice, immunized with live recombinant BCG-VP1, became diabetic when challenged with highly diabetogenic EMC-D virus. But the control mice inoculated with normal BCG or rBCG transformed with vector alone developed diabetes in the same challenge. VP1-specific antibodies including neutralizing antibodies were markedly increased as time went on and reached to the maximum titer at week 10 after a single immunization. The plateau of the titer lasted longer than following 4 weeks. Guinea pigs immunized with the live rBCG-VP1 showed strong delayed type hypersensitivity (DTH) to the VP1of EMC-D virus. It means that the live rBCG-VP1 elicit efficient humoral and cell-mediated imrnune responses against EMC-D virus, resulting in prevention of virus-induced diabetes in susceptible mice.
Animals ; Antibodies ; Antibodies, Neutralizing ; Bacillus ; Capsid Proteins ; Clone Cells ; DNA, Complementary ; Guinea Pigs ; Humans ; Hypersensitivity ; Immunization* ; Insulin-Secreting Cells ; Male* ; Mice* ; Mycobacterium bovis*

The rpoB gene based sequencing analysis enabled not only the detection of rifampin resistant Mycobacterium tuberculosis, but also the differentiation of species in the genus Mycobacterium. In the present study, we applied the method to 68 isolates of M. tuberculosis (29 from initial treatment cases and 39 from recurrent cases) and 11 clinical isolates of nontuberculous mycobacteia (NTM) isolated from patients in Jeju island. Among rifampin resistant M. tuberculosis, two of 29 strains isolated from patients of initial cases (6.9%) and five of 39 strains isolated from patients of recurrent cases (12.8%) were confirmed to have rifampin resistant genotypes harboring mutations in rif r region of the rpoB gene. In NTM strains, M. fortuitum complex was the most frequently isolated species at the frequency of 54.5% (6/11).
Genotype ; Humans ; Mycobacterium ; Mycobacterium tuberculosis ; Rifampin ; Tuberculosis

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate BCG vaccine is urgently needed. We constructed three recombinant Mycobacterium bovis BCG (rBCG) strains over-expressing antigen (Ag) 85A, Ag85B, or both of M. tuberculosis using their own promoter and secretory sequence, or hsp60 promoter. SDS-PAGE analysis of rBCG proteins showed over-expression of Ag85A and Ag85B proteins in higher level than of those in their parental strain of BCG. In addition, rBCG(rBCG/B.FA) over-expressing Ag85A and Ag85B induced strong IFN-gamma production in splenocytes. However, there was no significant difference in protective efficacy between rBCG and their parental BCG strain. In this study, therefore, rBCG over-expressing Ag85A, Ag85B, or both failed to show enhanced protection against M. tuberculosis infection in a mouse model.
Animals ; BCG Vaccine ; Electrophoresis, Polyacrylamide Gel ; Humans ; Mice* ; Mycobacterium bovis* ; Mycobacterium tuberculosis* ; Mycobacterium* ; Parents ; Tuberculosis

The goals of this study were to identify first-line drug resistance in new and previously treated tuberculosis (TB) cases and to determine risk factors for multidrugresistant TB (MDR-TB) at a private referral center in Korea. All patients with cultureconfirmed pulmonary TB over a 2-yr period between July 2002 and June 2004 were prospectively included in this study. In total, 637 patients were included; 512 (80.4%) were new cases, and 125 (19.6%) were previously treated cases. Resistance to at least one first-line drug was identified in 11.7% of new cases and 41.6% of previously treated cases. MDR-TB was detected in 3.9% of new cases and 27.2% of previously treated cases. The proportion of extensively drug-resistant TB among MDR-TB patients was 16.7% (9/54). Factors associated with MDR-TB included age under 45 yr, previous TB treatment, and the presence of cavitation on chest radiography. Rates of first-line drug resistance are high, particularly in previously treated patients, in the private sector in Korea. This underscores the need for an improved control program, coupled with early diagnosis of MDR-TB, to reduce the spread and development of resistance.
Adolescent ; Adult ; Age Factors ; Aged ; Aged, 80 and over ; Antitubercular Agents/pharmacology/therapeutic use ; Drug Resistance, Bacterial ; Drug Resistance, Multiple ; Female ; Hospitals/statistics & numerical data ; Humans ; Korea/epidemiology ; Logistic Models ; Male ; Middle Aged ; Mycobacterium tuberculosis/*drug effects ; Prospective Studies ; Referral and Consultation ; Risk Factors ; Tuberculosis, Multidrug-Resistant/*drug therapy/epidemiology/microbiology ; Tuberculosis, Pulmonary/*drug therapy/microbiology