TGF-beta1 expression was studied in 25 patients with tuberculosis (lung, 9 cases and lymph node, 16 cases) using a polyclonal antibody in formalin-fixed paraffin embedded tissue. Nineteen cases (76.0%) out of 25 cases showed TGF-beta1 expression. TGF-beta1 was present in cytoplasm of epithelioid cells and Langhans' giant cells. Pulmonary tuberculosis and tuberculous lymphadenitis showed different patterns of staining. Five of 9 cases of pulmonary tuberculosis were positive for TGF-beta1: four of acid-fast bacilli positive cases (4/5, 80.0%) and one of acid-fast bacilli negative cases (1/4, 25.0%). However, high expression of TGF-beta1 was detected in tuberculous lymphadenitis of both acid-fast bacilli positive group (3/4, 75.0%) and acid-fast bacilli negative group (11/12, 91.7%). TGF-beta1 was also expressed in all of 6 cases of BCG-induced tuberculous lymphadenitis: 2 acid-fast bacilli positive and 4 acid-fast bacilli negative cases. TGF-beta1 expression was shown in 19 cases (86.4%) of 22 in active tuberculosis, while no TGF-beta1 expression was detected in any cases of inactive, healed tuberculosis (p<0.008). This study supports that the TGF-beta1 expression of epithelioid cells may alter their function resulting in the impaired antimycobacterial activity. Thus the increased production of TGF-beta1 may be one of the important mechanisms by which Mycobacterium tuberculosis avoids destruction by host macrophages.
Cytoplasm ; Epithelioid Cells ; Giant Cells ; Granuloma* ; Humans ; Lymph Nodes ; Macrophages* ; Mycobacterium tuberculosis ; Paraffin ; Transforming Growth Factor beta1* ; Tuberculosis ; Tuberculosis, Lymph Node ; Tuberculosis, Pulmonary

BACKGROUND: The combined use of liquid media and solid media is recommended for mycobacterial culture. We evaluated diagnostic performance of combination of BACTEC Mycobacteria Growth Indicator Tube (MGIT; Becton Dickinson, USA) and 2% Ogawa media (Korean Institute of Tuberculosis, Korea) for recovery of mycobacteria. METHODS: In September 2007, 1,764 specimens from 1,059 patients were cultured with MGIT and Ogawa. Acid fast bacilli (AFB) smear was fluorochrome-stained. The isolates were identified into Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) with PCR using Seeplex TB Detection Kit (Seegene, Korea). Recovery rate, time to detection (TTD), contamination rate, mixed growth rate and species distribution were analyzed. RESULTS: Two hundred thirty-five specimens (13.3%) from 165 patients (15.6%) were positive for mycobacterial culture. Recovery rates of mycobacteria from the group using both media, MGIT only, and Ogawa only were 13.3%, 12.1%, and 7.8%, respectively. While MGIT recovered 98.9% of MTB and 79.7% of NTM, Ogawa recovered 65.9% of MTB and 54.1% of NTM. TTDs of total mycobacteria/MTB/NTM in MGIT and Ogawa were 10.6/11.4/9.7 days and 31/29/33 days, respectively. MGIT TTDs of total mycobacteria/MTB/NTM from AFB-positive specimens were significantly shorter than those of AFB-negative specimens; 8.2/9.5/4.4 days vs 11.6/12.7/10.7 days. Contamination and mixed growth rate of MGIT were 9.6% and 3.7%. Primary culture of Ogawa recovered 1 MTB and 1 NTM among the 170 MGIT-contaminated specimens and 38 mycobacteria among 66 specimens that showed mixed cultures of MGIT. CONCLUSIONS: MGIT warrants sensitive and rapid isolation of mycobacteria. However, the combination of MGIT and Ogawa is more desirable to recover mycobacteria in the case of contaminations or mixed cultures.
*Culture Media ; False Positive Reactions ; Humans ; Mycobacterium/*growth & development/isolation & purification ; Mycobacterium Infections/*diagnosis/microbiology ; Mycobacterium tuberculosis/*growth & development/isolation & purification ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Sputum/microbiology ; Time Factors

BACKGROUND: PCR is a widely used method for rapid and accurate diagnosis of mycobacteriosis. The sensitivity and specificity of a real time PCR kit newly developed in Korea were evaluated for detecting mycobacteria in respiratory specimens. METHODS: One hundred twenty nine Mycobacterium tuberculosis (TB) culture positive respiratory specimens (82 AFB stain positive and 47 stain negative specimens) were used for evaluation of the sensitivity. Nine non-tuberculous mycobacteria (NTM) culture positive specimens were also included. For evaluation of the specificity, 48 AFB stain and culture negative respiratory specimens from patients who were initially not fully excluded from mycobacterial diseases (specificity group 1) were used. Other 51 respiratory specimens from patients who were not suspected of mycobacterial diseases were also included (specificity group 2). Real time PCR was performed by using AdvanSure TB/NTM real time PCR Kit (LG Lifescience, Korea) and SLAN real time PCR detection system (LG Lifescience). The target genes of TB and NTM were IS6110 and rpoB, respectively. RESULTS: Among 129 TB culture positive specimens, 82 of 82 AFB stain positive specimens (100%) and 35 of 47 (74.5%) stain negative specimens revealed real time PCR positivity for TB, resulting in sensitivity of 90.7%. Five of nine NTM culture positive specimens resulted in real time PCR positivity for NTM (55.6%). Forty seven of 48 specimens (97.9%) and all 51 specimens (100%) of the specificity group 1 and 2, respectively, were real time PCR negative for TB and NTM. CONCLUSIONS: AdvanSure TB/NTM real time PCR Kit should be useful for detecting TB in respiratory specimens with high sensitivity and specificity.
DNA, Bacterial/analysis ; Humans ; Mycobacterium tuberculosis/genetics/growth & development/*isolation & purification ; Polymerase Chain Reaction/*methods ; Reagent Kits, Diagnostic ; Respiratory System/microbiology ; Sensitivity and Specificity ; Specimen Handling ; Tuberculosis/*diagnosis

PURPOSE: Many medical centers routinely culture bronchoscopy samples for Mycobacterium tuberculosis, even when tuberculosis is not strongly suspected. The value of this practice, however, is controversial. We evaluated the role of that procedure in the diagnosis of pulmonary tuberculosis in an intermediate tuberculosis-burden country. PATIENTS AND METHODS: A prospective, observational study was conducted in a tertiary referral center and included 733 consecutive patients who underwent bronchoscopy examination. RESULTS: M. tuberculosis was isolated in 47 patients (6.4%). According to radiographic features, the rate of positive culture for M. tuberculosis was relatively high in patients with atelectasis (5/33, 15.2%) and those with pulmonary infiltrations of suspicious infections (26/183, 14.2%). M. tuberculosis was isolated even in patients with pulmonary masses (9/266, 3.4%) and those with pulmonary nodules (5/175, 2.9%). In 16/47 (34.0%) patients with positive cultures for M. tuberculosis, active pulmonary tuberculosis was not suspected at the time of bronchoscopy. CONCLUSION: These results suggest that routinely culturing for M. tuberculosis during bronchoscopy is still useful in the diagnosis of pulmonary tuberculosis in an intermediate tuberculosis-burden country.
Adult ; Aged ; Bacteriological Techniques/methods ; Bronchoscopy ; Female ; Humans ; Lung/microbiology/pathology ; Lung Neoplasms/microbiology ; Male ; Middle Aged ; Mycobacterium tuberculosis/growth & development/*isolation & purification ; Prospective Studies ; Pulmonary Atelectasis/microbiology ; Reproducibility of Results ; Sensitivity and Specificity ; Tuberculosis, Pulmonary/*diagnosis/microbiology

BACKGROUND: The purpose of this study was to compare the turnaround time for liquid culturing and primary anti-tuberculous drug susceptibility testing (DST) performed using the mycobacteria growth indicator tube (MGIT) 960 system (Becton Dickinson, USA) with that for conventional culturing and DST (by the absolute concentration method) performed using solid culture medium and to determine the concordance rates of DST results obtained using these 2 methods. METHODS: In this retrospective study, we compared the turnaround times from receiving the request for mycobacterial culture to reporting the DST results before and after the introduction of the MGIT 960 system. Further, we determined the concordance between DST results for isoniazid and rifampin for Mycobacterium tuberculosis isolates obtained using the MGIT 960 system and the absolute concentration method, which was conducted at the Korean Institute of Tuberculosis. RESULTS: The overall turnaround time for mycobacterial culturing and DST was 27 days for liquid culturing and DST using the MGIT 960 system versus approximately 70 days for culturing on solid medium and DST with the absolute concentration method (P<0.001). There was a good concordance between findings of DST obtained with the 2 methods (97.2%, kappa coefficient=0.855 for rifampin; and 95.6%, kappa coefficient=0.864 for isoniazid), for 1,083 clinical isolates. CONCLUSIONS: The automated MGIT 960 system for culturing and DST of M. tuberculosis was successfully introduced in a hospital laboratory setting in Korea with significant shortening of the turnaround time.
Antitubercular Agents/*pharmacology ; Automation ; *Drug Resistance, Multiple, Bacterial/drug effects ; Humans ; Isoniazid/pharmacology ; *Microbial Sensitivity Tests/instrumentation/methods ; Mycobacterium tuberculosis/*drug effects/growth & development/isolation & purification ; Retrospective Studies ; Rifampin/pharmacology ; Time Factors ; Tuberculosis/*diagnosis

BACKGROUNDIsocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage.

METHODSMTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique.

RESULTSRT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS.

CONCLUSIONSMTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

Animals ; Apoptosis ; genetics ; physiology ; Cell Line ; In Situ Nick-End Labeling ; Interferon-gamma ; metabolism ; Isocitrate Lyase ; genetics ; metabolism ; Macrophages ; cytology ; metabolism ; microbiology ; Microbial Viability ; Microscopy, Fluorescence ; Mycobacterium smegmatis ; enzymology ; genetics ; growth & development ; Mycobacterium tuberculosis ; enzymology ; genetics ; Nitric Oxide ; metabolism ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transformation, Genetic