• Tuberculosis and Respiratory Diseases
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• v.43 no.2
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• pp.128-137
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• 1996

Background : We can diagnose pulmonary tuberculosis with sputum AFB smear and culture, but sputum AFB smear has low sensitivity and culture needs long period, and they are not available in the patients who can not expectorate effectively. Recently developed, PCR is a fast diagnostic tool in tuberculosis, but false positive and false negative are important problems. So, we studied the diagnostic value of bronchoalveolar lavage fluid AFB smear, culture, PCR through the bronchoscopy. Methods : The 67 pulmonary tuberculosis patients and 43 non-pulmonary tuberculosis patients were analyzed with their sputum specimen AFB smear and culture. Also, bronchoscopy and bronchoalveolar lavage were done, and bronchoalveolar lavage fluid AFB smear, culture and PCR were done. Results: 1) In the cases of pulmonary tuberculosis, the sensitivity of sputum AFB smear and culture were 32.8% and 57.4%, respectively. And the sensitivity of bronchoalveolar lavage fluid AFB smear and culture were 47.8% and 80.6%. respectively. 2) In the cases of pulmonary tuberculosis, the sensitivity and the positive predictive value(for predicting a positive culture) of PCR were 80.6% and 81.5%, respectively. 3) In the cases of sputum AFB smear-negative and culture-negative pulmonary tuberculosis, the sensitivity of bronchoalveolar lavage fluid AFB smear, culture, PCR, and the positive predictive value(for predicting a positive culture) of PCR were 23.1%, 100%, 88.5%, and 82.4%, respectively. 4) The specificity of bronchoalveolar lavage fluid PCR was 77.0%. 5) The median number of days between obtaining a specimen and starting therapy was 5 days for sputum AFB smear, 9 days for bronchoalveolar lavage fluid AFB smear, 26 days for bronchoalveolar lavage fluid PCR, 32 days for sputum culture, 56 days for bronchoalveolar lavage fluid culture. Conclusion : The sensitivity of bronchoalveolar lavage fluid AFB smear and culture are higher than sputum AFB smear and culture. So, the bronchoscopy must be considered for evaluating suspected cases of pulmonary tuberculosis in patients from whom smears of expectorated sputum do not reveal mycobacteria or from whom no sputum can be obtained. Especially, combined with PCR, it is expected that pulmonary tuberculosis can be diagnosed more rapidly and more accurately, so bronchoalveolar lavage fluid APB smear and PCR can be helpful in the early treatment of pulmonary tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.44 no.3
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• pp.470-478
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• 1997

Background : Phagocytosis is probably the first step for mycobacteria to be virulent in host because virulent strains are more readily phagocytosed by macrophage than attenuated strains. According to the traditional concept, multi-drug resistant strains have been regarded as less virulent. However, this concept has been challenged, since recent studies(reported) showed that the degree of virulence and drug-resistance is not related. The purpose of this study is to evaluate whether the phagocytic activity of M.tuberculosis by peripheral blood mononuclear cells(PBMC) is different according to drug-resistance or host factor. To evaluate this, we estimated the difference of phagocytic activity of drug-resistant and drug-sensitive M.tuberculosis and also estimated the phagocytic activity of PBMC from intractable tuberculosis patients and healthy controls. Methods : PBMC from ten intractable tuberculosis patients and twelve healthy control, and three different strains of heat-killed M.tuberculosis, ie, ADS(all drug sensitive), MDR(multi-drug resistant), and ADR(all drug resistant) were used. After incubation of various strains of M.tuberculosis with PBMC, the phagocytic activity was evaluated by estimating proportion of PBMC which have phagocytosed M.tuberculosis. Results : Drug-resistant strains of M.tuberculosis were phagocytosed easily than drug sensitive strains(Percentage of PBMC phagocytosed M.tuberculosis in healthy control : ADS : $32.3{\pm}2.9%$, ADR : $49.6{\pm}3.4%$, p = 0.0022, Percentage of PBMC phagocytosed M.tuberculosis in intractable tuberculosis patients : ADS : $34.9{\pm}3.6%$, ADR : $50.7{\pm}4.5%$, p = 0.0069). However, there was no difference in phagocytic activity of PBMC from healthy control and intractable tuberculosis patients. Conclusion : Drug-resistant strains of M.tuberculosis were phagocytosed easily than drug sensitive strains and host factors does not seems to influence the phagocytosis of M.tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.45 no.2
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• pp.271-279
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• 1998

Background: Diagnosis by direct microscopy and/or by culture of the Mycobacterium tuberculosis from body fluids or biopsy specimens is "Gold standard". However, the sensitivity of direct microscopy after Ziehl-Neelsen staining is relatively low and culture of mycobacteria is time consuming. Detection of mycobacterial DNA in clinical samples by the polymerase chain reaction is highly sensitive but laborious and expensive. Therefore, rapid, sensitive and readily applicable new tests need to be developed. So we had evaluated the clinical significance of serologic detection of antibody to 38 kDa antigen, which is known as the most specific to the M. tuberculosis complex, and culture filtrate antigen by ELISA in sputum AFB smear negative patients. Method: In this study, culture tests for acid fast bacilli with sputa or bronchial washing fluids of 183 consecutive patients who were negative of sputum AFB smear were performed. Simultaneously serum antibodies to 38 kDa antigen and unheated culture filtrate of M. tuberculosis were detected by an ELISA method. Results: The optical densities of ELISA test with 38 kDa and culture filtrate antigen were significantly higher in active pulmonary tuberculosis cases than in non tuberculous pulmonary diseases (p<0.05), but in patients with active pulmonary tuberculosis, those of the sputum culture positive patients for M. tuberculosis were not significantly different from those of the sputum culture negative cases(p>0.05). In the smear-negative active pulmonary tuberculosis patients, the sensitivity of the ELISA using 38 kDa antigen and culture filtrate was 20.0% and 31.4%. respectively. The specificity was 95.3% and 93.9%. respectively. Conclusion : In active pulmonary tuberculosis but smear negative, the serologic detection of antibody to 38 kDa antigen and culture filtrate by ELISA cannot substitute traditional diagnostic tests and does not have clinically significant role to differenciate the patient with active pulmonary tuberculosis from other with non-tuberculous pulmonary diseases.

• Tuberculosis and Respiratory Diseases
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• v.50 no.1
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• pp.29-41
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• 2001

Background : The appearance of multiple-drug-resistant Mycobacterium tuberculosis strains has been seriously compromising successful control of tuberculosis. Rifampin-resistance, caused by mutations in the rpoB gene, can be indicative of multiple-drug-resistance, and its detection is of great importance. The present study aimed to develop an oligonucleotide chip for accurate and convenient screening of drug-resistance. Methods : In order to detect point mutations in the rpoB gene, an oligonucleotide chip was prepared by immobilizing specific probe DNA to a microscopic slide glass by a chemical reaction. The probe DNA that was selected from the 81 bp core region of the rpoB gene was designed to have mutation sites at the center. A total of 17 mutant probes related to rifampin-resistance including 8 rifabutin-sensitive mutant probes were used in this study. For accurate determination, wild type probes were prepared for each mutation position with an equal length, which enabled a direct comparison of the hybridization intensities between the mutant and wild type. Results : Mycobacterial genomic DNA from clinical samples was tested with the oligonucleotide chip and the results were compared with those of the drug-susceptibility test in addition to sequencing and INNO-LiPA Rif. TB kit test in some cases. Out of 15 samples, the oligonucleotide chip results of 13 samples showed good agreement with the rifabutin-sensitivity results. The two samples with conflicting result also showed a discrepancy between the other tests, suggesting such possibilities as existence of mixed strains and difference in drug-sensitivity. Further verification of these samples in addition to more case studies are required before the final evaluation of the oligonucleotide chip can be made. Conlcusion : An oligonucleotide chip was developed for the detection of rpoB gene mutations related to drugsusceptibility. The results to date show the potential for using the oligonucleotide chip for accurate and convenient screening of drug-resistance to provide useful information in antituberculosis drug therapy.

• Tuberculosis and Respiratory Diseases
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• v.53 no.5
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• pp.497-509
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• 2002

Background : The mechanisms through which cellular activation results in intracellular mycobacterial killing is only partially understood. However, in vitro studies of human immunity to Mycobacterium tuberculosis have been largely modeled on the work reported by Crowle, which is complicated by several factors. The whole blood culture is simple and allows the simultaneous analysis of the relationship between bacterial killing and the effect of effector cells and humoral factors. In this study, we attempted to determine the extent to which M. tuberculosis is killed in a human whole blood culture and to explore the role of the host and microbial factor in this process. Methods : The PPD positive subject were compared to the umbilical cord blood and patients with tuberculosis, diabetes and lung cancer. The culture is performed using heparinized whole blood diluted with a culture medium and infected with a low number of M. avium or M. tuberculosis $H_{37}Ra$ for 4 days by rotating the culture in a $37^{\circ}C$, 5% $CO_2$ incubator. In some experiments, methlprednisolone- or pentoxifyline were used to inhibit the immune response. To assess the role of the T-cell subsets, CD4+, CD8+ T-cells or both were removed from the blood using magnetic beads. The ${\Delta}$ log killing ratio was defined using a CFU assay as the difference in the log number of viable organisms in the completed culture compared to the inoculum. Results : 1. A trend was noted toward the improved killing of mycobacteria in PPD+ subjects comparing to the umbilical cord blood but there was no specific difference in the patients with tuberculosis, diabetes and lung cancer. 2. Methylprednisolone and pentoxifyline adversely affected the killing in the PPD+ subjects umbilical cord blood and patients with tuberculosis. 3. The deletion of CD4+ or CD8+ T-lymphocytes adversely affected the killing of M. avium and M. tuberculosis $H_{37}Ra$ by PPD+ subjects. Deletion of both cell types had an additive effect, particularly in M. tuberculosis $H_{37}Ra$. 4. A significantly improved mycobacterial killing was noted after chemotherapy in patients with tuberculosis and the ${\Delta}$ logKR continuously decreased in a 3 and 4 days of whole blood culture. Conclusion : The in vitro bactericidal assay by human whole blood culture model was settled using a CFU assay. However, the host immunity to M. tuberculosis was not apparent in the human whole blood culture bactericidal assay, and patients with tuberculosis showed markedly improved bacterial killing after anti-tuberculous chemotherapy compared to before. The simplicity of a whole blood culture facilitates its inclusion in a clinical trial and it may have a potential role as a surrogate marker in a TB vaccine trial.

• Tuberculosis and Respiratory Diseases
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• v.54 no.1
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• pp.33-44
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• 2003

Background : Mycobacterium avium complex(MAC) is the most common respiratory pathogen in nontuberculous mycobacterial pulmonary disease. This study described the clinical characteristics of the patients with pulmonary disease caused by MAC. Materials and Methods : The clinical characteristics of 24 patients with pulmonary disease caused by the MAC, who fulfilled the 1997 American Thoracic Society diagnostic criteria for nontuberculous mycobacterial pulmonary disease, were retrospectively analyzed. Results : Fourteen patients(58%) were male and the median age at diagnosis was 61 years(range 46-75). Of the 24 patients, 16(67%) had a M. intracellulare infection, 7(29%) had a M. avium infection and one patient was not identified. Coughing (92%) and sputum (88%) were most frequently observed symptoms. The sputum smear for acid-fast bacilli was positive in 17(71%) patients. Fourteen(58%) patients had the upper lobe cavitary form and 10(42%) patients had the nodular bronchiectatic form. In a comparison between the patients with the upper lobe cavitary form and those with the nodular bronchiectatic form, significant differences were found according to sex(male 86% vs. 20%, p=0.003), smoking history(79% vs. 10%, p=0.008), the presence of an underlying disease(64% vs. 20%, p=0.036), the pulmonary function(% forced vital capacity, median 71% vs. 88%, p=0.022; % forced expiratory volume in one second, median 69% vs. 89%, p=0.051) and bilateral disease at chest radiography(29% vs. 90%, p=0.005). The time from the onset of symptoms to diagnosis was longer in those with the nodular bronchiectatic form(median 72 months, range 8-132) than those with the upper lobe cavitary form(median 22 months, range 6-60) Conclusions : MAC pulmonary disease occurs in two distinct populations with two distinct clinical presentations. For a correct diagnosis of MAC pulmonary disease, knowledge of the diverse clinical and radiological findings is essential.