A Public-Private Collaboration Model for Treatment Intervention to Improve Outcomes in Patients with Tuberculosis in the Private Sector / 결핵및호흡기질환

BACKGROUND: The treatment success rates in patients with tuberculosis are known to be lower in the private sector compared to the public sector. To improve treatment outcomes in the private sector we developed a public-private collaboration model for strengthening health education and case holding activities with public health nursing in the private sector. METHODS: We performed a prospective cohort study in new smear positive pulmonary tuberculosis patients treated at private hospitals, selected by non-randomization, with an intervention consisting of health education and case holding activities by specially trained public health nurses (intervention group) results were compared with cases treated without the intervention (conventional group). Physicians were asked to treat both groups routinely. The treatment outcomes of patients under treatment by the National Tuberculosis Programme were also analyzed for comparison. RESULTS: There were 172 cases each in the intervention and conventional groups. The mean age was 48.9+/-19.0 and 48.2+/-19.7 in the respective groups (p=0.66). The PHN interacted with the cases in the intervention group by initial face to face interview and telephone calls an average of 7.1+/-9.2 times during the initial six months. The intervention group showed a significantly higher treatment success rate, 91.6%, (Rate Ratio [RR]; 1.23, 95% Confidence Interval [CI]; 1.12~1.36), lower default, 3.6%, (RR; 0.31, 95% CI; 0.13~0.75) and transfer-out rate, 3.0%, (RR; 0.32, 95% CI; 0.12~0.86) than the conventional group where they were: 75.0%, 11.6%, 9.3%, respectively. The success rate was even higher than the rate (80.5%) of 1,027 cases treated in health centers (RR; 1.11, 95% CI; 1.05~1.17). Of the completed cases in the intervention group, 82.2% regarded the role of the public health nurse as very helpful. CONCLUSION: The treatment success rate, of tuberculosis patients in the private sector, was significantly improved by an intervention using a public-private collaboration model.

The Interval Between Initiation of Anti-tuberculosis Treatment in Patients with Culture-positive Pulmonary Tuberculosis and Receipt of Drug-susceptibility Test Results

Although mycobacterial culture and the subsequent drug-susceptibility test (DST) for anti-tuberculosis (TB) drugs take several months to complete using solid media, there are no reports on the turnaround times of these tests under clinical conditions. The aim of this study was to determine the interval between initiation of anti-TB treatment and receipt of DST requested at an outpatient clinic. We prospectively enrolled patients with culture-positive pulmonary TB at Seoul National University Hospital from September 2002 to December 2004. Patients were followed up monthly. Mycobacterial cultures were done using Ogawa media at Seoul National University Hospital. DST were performed at the Korean Institute of Tuberculosis. Of the 104 patients enrolled, 54 were male. The median age was 41 yr. The median interval from initiation of anti-TB treatment to receipt of mycobacterial culture results by clinicians was 37 days (range, 0-89 days). The median interval from initiation of treatment to confirmation of DST by requesting clinicians was 80.5 days (range, 28-145 days). Clinicians only received the results of DST more than two months after initiation of treatment when they followed up patients monthly and mycobacterial culture was performed using solid media.

Evaluation of the Broth Microdilution Method Using 2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride for Rapidly Growing Mycobacteria Susceptibility Testing

As the incidence of nontuberculous mycobacterial infection has been increasing recently in Korea, the importance of drug susceptibility test for clinical isolates of mycobacteria has become larger. In this study we determined the antimicrobial susceptibility patterns of clinical isolates of M. fortuitum and M. abscessus in Korea, and evaluated the efficacy of a modified broth microdilution method using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC), in terms of its ability to provide accurate and easy-to-read minimal inhibitory concentration (MIC) endpoints for the susceptibility testing of rapidly growing mycobacteria. Most isolates of M. fortuitum and M. abscessus in Korea are susceptible or intermediately susceptible to amikacin, cefoxitin, ciprofloxacin, and clarithromycin. Many isolates of M. fortuitum are susceptible to doxycycline, sulfamethoxazole, and imipenem, while many M. abscessus isolates are resistant to these drugs. In the present study, the modified broth microdilution method using STC was found to be reliable, easy to read, and inexpensive for M. fortuitum and M. abscessus susceptibility testing. The modified colorimetric MIC testing method using STC was proven to be a useful surrogate for RGM antibiotic susceptibility testing.

Pulmonary Disease Caused by Mycobacterium xenopi: The First Case in Korea

Mycobacterium xenopi is a nontuberculous mycobacterium (NTM) that rarely causes pulmonary disease in Asia. Here we describe the first case of M. xenopi pulmonary disease in Korea. A 66-year-old man was admitted to our hospital with a 2-month history of productive cough and hemoptysis. His past medical history included pulmonary tuberculosis 44 years earlier, leading to a right upper lobectomy. Chest X-ray upon admission revealed cavitary consolidation involving the entire right lung. Numerous acid-fast bacilli were seen in his initial sputum, and M. xenopi was subsequently identified in more than five sputum cultures, using molecular methods. Despite treatment with clarithromycin, rifampicin, ethambutol, and streptomycin, the infiltrative shadow revealed on chest X-ray increased in size. The patient's condition worsened, and a right completion pneumonectomy was performed. The patient consequently died of respiratory failure on postoperative day 47, secondary to the development of a late bronchopleural fistula. This case serves as a reminder to clinicians that the incidence of NTM infection is increasing in Korea and that unusual NTM are capable of causing disease in non-immunocompromised patients.

Analysis of DNA fingerprints of Mycobacterium Tuberculosis Isolates from Patients Registered at Health Center in Gyeonggi Province in 2004 / 결핵및호흡기질환

BACKGROUND: IS6110 DNA fingerprint is a very useful tool for investigating the transmission of tuberculosis. The aim of this study was to identify the epidemiological situations within a given area (one province). METHODS: The 681 Mycbobacterium tuberculosis isolates from patients, who were registered at health centers in Gyeonggi Province from May to December in 2004, were subjected to IS6110 DNA fingerprinting. Patients belonging to clusters were interviewed by health-workers to determine their previous contacts or household TB history. RESULTS: The number of IS6110 copies of the 681 isolates showed diverse fingerprint patterns from 0 to 21 of which the most prevalent copy number was 10 from 120 isolates (17.6%). Thirty-three isolates (4.8%) belonged to the K strain, and 128 isolates (18.8%) belonged to the K family. There were 180 (26.4%) isolates belonged belonging to fifty clusters, of which two clusters were within household transmission. Forty-three (23.9%) out of 180 patients resided in an area under the same health center control. The rate of clusters in those aged 60-70 was higher than in any other age group ( 95% CI of RR : 1.072 ~ 1.988). CONCLUSION: This is the first report of an epidemiological survey based on a whole province using a DNA fingerprinting technique for M. tuberculosis. These results will be helpful in developing a program or policies to prevent the transmission of TB.

The Current Status of Multidrug-resistant Tuberculosis in Korea / 결핵및호흡기질환

PURPOSE: Multidrug-resistant tuberculosis (MDR-TB) is an emerging threat to human beings. However, there is little data on the current status of MDR-TB in Korea. This study investigated the current status of MDR-TB in Korea using a survey of all the data from drug susceptibility tests (DST) performed across the country over the last three years. METHOD: The DST results between Jan. 2000 and Dec. 2002 from 7 laboratories, which were in charge of all antituberculous DSTs across the country as of March 2002, were collected and analyzed to determine the actual number of drug-resistant or MDR-TB patients, annual trend, degree and pattern of resistance against anti-TB drugs, etc. RESULTS: Six laboratories used the absolute concentration method for DST and one used the proportional method. 59, 940 tests had been performed over the 3 year study period. The number of DST performed annually was 18,071, 19,950, and 21,919 in 2000-2002, respectively. The number of resistant tuberculosis patients (resistant against at least one anti-TB drug) had increased by 16.9% from 6,338 in 2000 to 7,409 in 2002. The rate of resistant tuberculosis among all DST results was 35.1% in 2000, 34.5% in 2001, and 33.8% in 2002. The number of MDR-TB patients (resistant against at least both isoniazid and rifampin) showed an increasing trend (14.5%) from 3,708 in 2000 to 4,245 in 2002. CONCLUSION: Approximately 4,000 MDR-TB cases are newly identified by DST annually and the number is showing an increasing trend. This study suggests that in order to cope with the current MDR-TB situation, the DST methods will need to be standardized and more aggressive measures will be required.

Investigation of the Growth Rate Change in Recombinant BCG which was cloned Mycobacterium tuberculosis Adenylate Kinase Mutation Gene or Human Muscle-type Adenylate Kinase Synthetic Gene / 결핵및호흡기질환

BACKGROUND: Normal cell proliferation and viability is strongly depends on the availability of metabolic energy and the maintenance of the appropriate adenylate-nucleotide pools. Hypothetically, changes in adenylate kinase (AK) expression could therefore be associated with adaptation to altered growth characteristics or inversely altered growth characteristics of proliferating cells could drive the changes in the metabolic profile. This study investigated whether the expression of either AK1 or a Mycobacterium tuberculosis adenylate kinase mutant which has the same catalytic activity of AK1 could affect the growth rate of slow-growing BCG. METHOD: Recombinant BCGs, which were cloned the human muscle-type adenylate kinase synthetic gene (AK1) and adenylate kinase mutation gene (AKmtDM) of Mycobacterium tuberculosis into the Mycobacterium/E.coli expression vectors, were constructed. Recombinant BCGs and wild-type BCG were cultured in 7H9 media and the optical density at 600nm was measured at intervals of 2-3 days. RESULT: There wasn't the growth rate change induced by AK1 or AKmtDM expression in recombinant BCGs. CONCLUSION: The expression of AK1 or Mycobacterium tuberculosis adenylate kinase mutant in BCG does not affect the growth rate of BCG.

Evaluation of Automatic Acid-Fast Bacilli Stainer AT-2000F / 대한임상미생물학회지

BACKGROUND: Sputum smear microscopy is rapid, economic, and useful to detect patients with transmittable tuberculosis, albeit laborious. We aimed to evaluate the usefulness of an automated acid-fast bacilli stainer, which had been developed for lowering the labor and maintaining or increasing the staining quality. METHODS: One hundred sputum samples including some known positive smear specimens which were selected from clinical specimens requested for smear and culture for mycobacteria at Pusan National University Hospital, were used for evaluation. Auramine/rhodamine fluorescent acid-fast stainings were performed manually or by using the automated stainer, AT-2000F (Dagatron, Ilsan, Korea). Ziehl-Neelsen stain was also performed simultaneously. RESULTS: Concordance rate between automated and manual fluorescent stains was 98.0% and that between automated fluorescent and manual Ziehl-Neelsen stains was 88.0%. In all discordant cases, the automated stains showed one-grade higher results compared to the respective manual fluorescent or Ziehl-Neelsen stains. With the automatic stainer, all staining procedures were processed automatically except for slide loading and unloading. The process time was reduced by a half, and the slide-to-slide or day-to-day variations of staining quality were reduced compared with the manual fluorescent stain. CONCLUSION: Acid-fast bacilli stain using automated stainer AF-2000F can reduce the processing time, labor, and variations of staining quality, and enhance or maintain the detection of positive smears.

Multi-center Study on Cost Effectiveness of Anti-Tuberculosis Drug Susceptibility Test

BACKGROUND: The anti-mycobacterial susceptibility test is performed on only a small percentage of clinical isolates in Korea. The aim of this study is to propose an anti-mycobacterial susceptibility testing scheme, which is not only economic and practical but also fully informative to physicians. MATERIALS AND METHODS: The anti-mycobacterial susceptibility test results of 502 strains, isolated from five university-affiliated hospitals, were analysed. The interpretation of the results and the need for second-line drug susceptibility test were judged according to the recommendation of NCCLS M24-A guidelines. RESULTS: The isolates from 10% (38/363) of treatment-navie patients and 61% (85/139) of re- treatment patients showed resistance to at least one of the anti-mycobactial agents; 3% (11/363) and 44% (61/139) of isolates from each group were multi-drug resistant. According to the recommendation by NCCLS, the percentage of patients not needing the susceptibility test results for second-line drugs were 96% for treatment-naive and 47% for re-treatment patients. CONCLUSION: Since the susceptibility test against first-line drug is sufficient for 95% of treatment- navie patients with tuberculosis patients, susceptibility test against second-line drugs may be performed only when it is necessary. As for the re-treatment patients with tuberculosis, susceptibility test for both first-line and second-line drugs should be performed simultaneously.

Multi-center Study on Cost Effectiveness of Anti-Tuberculosis Drug Susceptibility Test

BACKGROUND: The anti-mycobacterial susceptibility test is performed on only a small percentage of clinical isolates in Korea. The aim of this study is to propose an anti-mycobacterial susceptibility testing scheme, which is not only economic and practical but also fully informative to physicians. MATERIALS AND METHODS: The anti-mycobacterial susceptibility test results of 502 strains, isolated from five university-affiliated hospitals, were analysed. The interpretation of the results and the need for second-line drug susceptibility test were judged according to the recommendation of NCCLS M24-A guidelines. RESULTS: The isolates from 10% (38/363) of treatment-navie patients and 61% (85/139) of re- treatment patients showed resistance to at least one of the anti-mycobactial agents; 3% (11/363) and 44% (61/139) of isolates from each group were multi-drug resistant. According to the recommendation by NCCLS, the percentage of patients not needing the susceptibility test results for second-line drugs were 96% for treatment-naive and 47% for re-treatment patients. CONCLUSION: Since the susceptibility test against first-line drug is sufficient for 95% of treatment- navie patients with tuberculosis patients, susceptibility test against second-line drugs may be performed only when it is necessary. As for the re-treatment patients with tuberculosis, susceptibility test for both first-line and second-line drugs should be performed simultaneously.

Mycobacterium kansasii Pulmonary Diseases in Korea

Mycobacterium kansasii is one of the most common cause of pulmonary diseases due to nontuberculous mycobacteria. We investigated the changing in the number of isolation of M. kansasii and the clinical characteristics of M. kansasii pulmonary disease in Korea. Through searching the database of the Korean Institute of Tuberculosis, we identified the cases of isolated M. kansasii from 1992 to 2002. The number of M. kansasii isolation had increased from once in 1992 to 62 in 2002. Fifteen patients with M. kansasii pulmonary disease were identified during the period January 1997 to December 2002. Twelve patients (80%) were male and fourteen (93%) were from highly industrialized areas. The most common symptom was a cough. Seven patients (47%) had a cavitary lesion and right upper lobe was most commonly involved. Patients responded well to isoniazid and rifampicin based regimens both bacteriologically and radiographically. In conclusion, M. kansasii isolation has increased, especially in highly industrialized areas, as well as other nontuberculous mycobacteria in Korea.

Cross Resistance of Fluoroquinolone Drugs on gyrA Gene Mutation in Mycobacterium tuberculosis / 결핵및호흡기질환

BACKGROUND: Fluoroquinolone drugs are an important anti-tuberculous agent for the treatment of multi-drug resistant tuberculosis. However, many drugs belonging to the fluoroquinolones have different cross resistance to each other. METHODS: Sixty-three ofloxacin (OFX) resistant and 10 pan-susceptible M. tuberculosis isolates were selected, and compared for their cross resistance using a proportion method on Lowenstein-Jensen media, containing ofloxacin (OFX), ciprofloxacin (CIP), levofloxacin (LVX), moxifloxacin (MXF), gatifloxacin (GAT) and sparfloxacin (SPX), at concentrations ranging from 0.5 to 3microgram/ml. DNA extracted from the isolates was directly sequenced after amplifying from the gyrA and gyrB genes. RESULTS: The 63 OFX resistant M. tuberculosis isolates showed complete cross resistance to CIP, but only 90.5, 44.4, 36.5 and 46.0% to LVX, MXF, GAT, and to SPX, respectively. Fifty-one of the isolates (81.0%) had point mutations in codons 88, 90, 91 and 94 in gyrA, which are known to be correlated with OFX resistance. The Gly88Ala, Ala90Valand Asp94Ala mutations in gyrA showed a tendency to be susceptible to MXF, GAT and SPX. Only 4 isolates had mutations in the gyrB gene, which did not affect the OFX resistance. CONCLUSION: About 60% of the OFX resistant M. tuberculosis isolates were susceptible to GAT, SPX and MXF. These fluoroquinolones may be useful in the treatment of TB patients showing OFX resistance.

The Proportion of Rifabutin-susceptible Strains among Rifampicin- resistant Isolates and Its Specific rpoB Mutations / 결핵및호흡기질환

BACKGROUND: Rifabutin (ansamycin) is a spiro-piperidyl rifamycin, which is highly active against Mycobacterium tuberculosis. It has been found that some clinical isolates of tubercle bacilli that are resistant to rifampicin are susceptible to rifabutin, with some patients with multi-drug resistant pulmonary tuberculosis having shown favorable clinical and bacteriological responses to the rifabutin. This study was conducted to find the proportion of rifabutin- susceptible strains among rifampicin-resistant isolates from Korean MDR-TB patients, and investigate the presence of specific rpoB mutations, which may confer resistance to rifampicin, but not to rifabutin. METHODS: 201 rifampicin-resistant and 50 pan-susceptible M. tuberculosis isolates were randomly selected for this study. The isolates were retested at rifampicin and rifabutin concentrations of 0, 20, 40 and 80 microgram/ml, respectively. The isolates that grew at and/or over a rifabutin concentration of 20 microgram/ml were judged rifabutin-resistant. The rpoB gene was extracted from the isolates, and then amplified for direct sequencing to investigate specific rpoB mutations that conferred rifabutin- susceptibility but rifampicin-resistance. RESULTS: Out of the 201 rifampicin-resistant M. tuberculosis, 41 strains (20.4%) were susceptible to rifabutin using the absolute concentration method on Lowenstein-Jensen media. The rpoB mutation types that showed susceptibility to rifabutin were Leu511Pro, Ser512Arg, Gln513Glu, Asp516Ala, Asp516Gly, Asp516Val, Asp516Tyr, Ser522Leu, His526Asn, His526Leu, His526Cys, Arg529Pro and Leu533Pro. A reverse hybridization technique was able to detect 92.5% of the rifabutin-susceptible isolates, with a specificity of 96.1% among 195 M. tuberculosis isolates with the rpoB mutation. CONCLUSIONS: Around 20% of the rifampicin-resistant isolates in Korea showed susceptibility to rifabutin, which was associated with some specific mutations of rpoB. Rifabutin could be used for the treatment of MDR-TB patients, especially when drug susceptibility testing reveals susceptibility to rifabutin.

Implication of embB Gene Mutation in Ethambutol-Susceptible Clinical Isolates of Mycobacterium tuberculosis / 결핵및호흡기질환

BACKGROUND: Ethambutol(EMB) is one of the first-line drugs included in short-course anti-tuberculosis therapy. The point mutations in embB gene have been speculated to be associated EMB resistance. However, detection of embB mutations at these positions have been observed in both EMB-susceptible isolates; thus, it remains controversial whether these mutations are associated with EMB resistance METHODS: The 36 M. tuberculosis isolates were selected from clinical isolates which tested susceptible to EMB and resistant to at least one drug. DNA extracted from the isolates was analyzed by amplifying embB gene. The PCR products were purified and directly sequenced. We reviewed the history of past drug susceptibility test results. RESULTS: Out of 36 EMB-susceptible strains, 3 strains (8.3%) had a mutation in codon 306 or 406 of the embB gene. These three strains had at least isoniazid resistance. They grew at 1.0 mcg/ml of EMB in Lowenstein-Jensen media. The patients of the strains were continuously smear-positive for over 3 years despite taking TB therapy. One strain had been EMB-resistant in past drug susceptibility tests. CONCLUSION: EMB-susceptible strains containing embB mutation may be caused by decreased viability in vitro test not by itself.

Detection of embB Gene Mutation of Mycobacterium tuberculosis by Reverse Hybridization Assay / 결핵및호흡기질환

BACKGROUND: Ethambutol (EMB) is one of important first-line drug in the treatment of tuberculosis. Molecular techniques to detect embB gene mutations have been considered as an method to define the EMB resistance. We investigated the mutation rate within embB gene among EMB resistant strains using reverse hybridization techniques. METHODS: We made 11 probes that had wild or mutated sequences containing codons 306, 406, or 497 within embB gene respectively. These probes were reverse-hybridized with PCR products amplified from embB gene which were isolated from 149 ethambutol resistant strains and 50 pan-susceptible strains. RESULTS: Out of 149 ethambutol resistant strains, one hundred (67.1%) had mutation at least one base at codon 306, 406, or 497 in embB gene. Mutation at codon 306, 406, 497 were demonstrated in 75 (50.3%), 16 (10.7%), and 13 strains (8.7%) respectively. There were four strains that showed multi-mutation at codon 306 and codon 406 simultaneously. A high proportion (8.1%) had single mutation at codon 406. There was no mutation observed in embB gene among 50 pan-susceptible strains. CONCLUSION: Reverse hybridization will be useful technique for detection of gene mutation correlated to ethambutol resistance.

Protective Efficacy of Recombinant Proteins Adenylate Kinase, Nucleoside Diphosphate Kinase, and Heat-Shock Protein 70 against Mycobacterium tuberculosis Infection in Mice / 결핵및호흡기질환

BACKGROUND: Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. METHOD: M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and IFN-gamma after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. RESULT: Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and IFN-gamma responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. CONCLUSION: Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant.

A Trend in Acquired Drug Resistances of Tuberculosis Patients Registered in Health Centers from 1981 to 2004 / 결핵및호흡기질환

BACKGROUND: The drug resistance rate in tuberculosis patients with history of chemotherapy is an important indicator of for evaluation of appropriateness of treatment regimens and compliance of patients. This study examined the long-term changes in the drug resistance rates among TB patients failed in treatment or reactivated. METHODS: The results of drug susceptibility testing data from patients registered in health centers from 1981 to 2004 were analyzed. RESULTS: The rate of resistance to isoniazid decreased from 90% to 20%, and the resistance to ethambutol decreased from 45% to 6%. The rate of resistance to rifampicin varied from 13% to 28% and the resistance to pyrazinamide was 5% to 10%. Multidrug resistance was about 2-3% lower than any rifampicin resistance rates. The second-line drug resistance was ranged from 1% to 3%. There was no difference between patients' genders. Patient numbers per 100,000 population increased with age. The regional distribution was even at 4-6 patients per 100,000 population, and drug resistance rates were significantly lower in big city areas than in small towns and rural areas. CONCLUSION: The rates of resistance of Mycobacterium tuberculosis isolated from TB patients with history of chemotherapy to isoniazid, rifampin, ethambutol, and isoniazid plus rifampin were significantly decreased during over two decades.

Construction of Recombinant BCGs Overexpressing Antigen 85 Complex and Their Protective Efficacy against Mycobacterium tuberculosis Infection in a Mouse Model / 결핵및호흡기질환

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.

Trial for Drug Susceptibility Testing of Mycobacterium tuberculosis with Live and Dead Cell Differentiation / 결핵

BACKGROUND: The resurgence of tuberculosis and outbreaks of multidrug resistant (MDR) tuberculosis have increased the emphasis for the development of new susceptibility testing of the Mycobacterium tuberculosis for the effective treatment and control of the disease. Conventional drug susceptibility testings, such as those using egg-based or agar-based media have some limits, such as the time required and difficulties in determining critical inhibitory concentrations, but these are still being used in many diagnostic laboratories because of no better alternatives, considering cost and accuracy. To overcome these limits, a rapid and simple method for new susceptibility testing, using live and dead assays, was applied for a bacterial cell viability assay to distinguish dead from live bacterial cells based on two-color fluorescence. MATERIALS AND METHODS STRAINS: Forty strains were used in this study, 20 susceptible to all antituberculosis drugs and the other 20 resistant to the four first line antituberculosis drugs isoniazid, rifampicin, streptomycin and ethambutol. ANTIBIOTICS: The four antibiotics were dissolved in 7H9 broth to make the following solutions: 0.1micro gram isoniazid(INH)/ml, 0.4micro gram rifampicin(RMP)/ml, 4.0micro gram streptomycin(SM)/ml and 4.0micro gram ethambutol(EMB)/ml. RESULTS: Live and dead Mycobacterium tuberculosis cells fluoresced green and red with the acridin (Syto 9) and propidium treatments, respectively. These results are very well accorded with conventional drug susceptibility testing by proportional method on Lowensen-Jensen media (L-J) containing 4 drugs (INH, RMP, EMB and SM), showing a 93.7 % accordance rate in susceptible strains and 95% in resistant strains. CONCLUSION: The results of the drug susceptibility testing using the live and dead bacterial cell assay showed high accordance rates compared with the conventional proportion method on L-J. This finding suggests that the live and dead bacterial cell assay can be used as an alternative to conventional drug susceptibility testing for M. tuberculosis strains.

Analysis of the Cell Lysate Proteome of a Korean Mycobacterium tuberculosis Isolate K01 with H37Rv and H37Ra Strains

Despite recent economic prosperity, Korea still has high prevalence of tuberculosis. Molecular biologic characterization of Korean Mycobacterium tuberculosis strains might provide a deeper understanding of the forces contributing to the spread of tuberculosis in Korea. Therefore, we analyzed the cell lysate proteome of a representative Korean Mycobacterium tuberculosis isolate (K01) in comparison with laboratory reference strains H37Rv and H37Ra. Seven spots were strongly expressed only in K01 strain compared with M. tuberculosis H37Rv and H37Ra. Through continuous MALDI-MS analysis, these spots were identified as hypothetical protein Rv3849, secreted immunogenic protein Mpt64, Acetyl/propionyl-CoA Carbpxylase (AccD1), alkyl hydroperoxide reductase C (AhpC), N-acetylmuramyl-L-alanine amidase, a putative UDP glucose epimerase, and a transposase. A deeper study of these proteins may provide a clue in the development of effective new anti-tuberculosis vaccines against Korean M. tuberculosis isolates.