Patterns of rpoC Mutations in Drug-Resistant Mycobacterium tuberculosis Isolated from Patients in South Korea / 결핵및호흡기질환

BACKGROUND: Rifampicin (RFP) is one of the principal first-line drugs used in combination chemotherapies against Mycobacterium tuberculosis, and its use has greatly shortened the duration of chemotherapy for the successful treatment of drug-susceptible tuberculosis. Compensatory mutations have been identified in rpoC that restore the fitness of RFP-resistant M. tuberculosis strains with mutations in rpoB. To investigate rpoC mutation patterns, we analyzed 93 clinical M. tuberculosis isolates from patients in South Korea. METHODS: Drug-resistant mycobacterial isolates were cultured to determine their susceptibility to anti-tubercular agents. Mutations in rpoC were identified by sequencing and compared with the relevant wild-type DNA sequence. RESULTS: In total, 93 M. tuberculosis clinical isolates were successfully cultured and tested for drug susceptibilities. They included 75 drug-resistant tuberculosis species, of which 66 were RFP-resistant strains. rpoC mutations were found in 24 of the 66 RFP-resistant isolates (36.4%). Fifteen different types of mutations, including single mutations (22/24, 91.7%) and multiple mutations (2/24, 8.3%), were identified, and 12 of these mutations are reported for the first time in this study. The most frequent mutation involved a substitution at codon 452 (nt 1356) resulting in amino acid change F452L. CONCLUSION: Fifteen different types of mutations were identified and were predominantly single-nucleotide substitutions (91.7%). Mutations were found only in dual isoniazid- and RFP-resistant isolates of M. tuberculosis. No mutations were identified in any of the drug-susceptible strains.

pncA Mutations in the Specimens from Extrapulmonary Tuberculosis / 결핵및호흡기질환

BACKGROUND: Pyrazinamide (PZA) is an effective antitubercular drug that becomes toxic to Mycobacterium tuberculosis when converted to pyrazinoic acid by pyrazinamidase (PZase), encoded by mycobacterial pncA. A strong association was noted between the loss of PZase activity and PZA resistance. The causative organisms in extrapulmonary tuberculosis are rarely cultured and isolated. To detect pncA mutations in specimens from extrapulmonary tuberculosis as confirmative diagnosis of mycobacterial infection and alternative susceptibility test to PZA. METHODS: Specimens were collected from clinically proven extrapulmonary tuberculosis. pncA was sequenced and compared with wild-type pncA. RESULTS: pncA from 30 specimens from 23 donors were successfully amplified (56.6% in specimens, 59% in donors). Six mutations in pncA were detected (20.0% in amplified specimens, 26.1% in specimen donors) at nucleotide positions of 169, 248 and 419. The mutation at position 169 results in substitution of aspartic acid for histidine, a possible allelic variation of M. bovis that have intrinsic PZA resistance. The mutation at position 248 changes proline into arginine and that at position 419, arginine into histidine. CONCLUSION: DNA-based diagnosis using pncA may be simultaneously useful for the early diagnosis of mycobacterial infection and the rapid susceptibility to PZA in extrapulmonary tuberculosis. A potential implication of pncA allelic variation at 169 might be suggested as a rapid diagnostic test for M. bovis infection or Bacille Calmette-Guerin (BCG) reactivation.

Identification of Cutaneous Mycobacterium massiliense Infections Associated with Repeated Surgical Procedures

Mycobacterium massiliense, an emerging pathogen that is increasingly reported as a causative agent in infections occurring during medical procedures, is difficult to be identified using conventional methods. Here we report the case of a cutaneous M. massiliense infection that was associated with repeated surgical procedures and that was identified via a comparative sequence analysis of rpoB and hsp65. The patient showed a substantial response to treatment with a combination of antimicrobial therapies consisting of clarithromycin, amikacin, and cefoxitin for 6 months.

Pulmonary tuberculosis misdiagnosed as lung Metastasis in childhood cancer patients / 소아과

PURPOSE: The differential diagnosis for a pulmonary nodule is intriguing in cancer patients. Metastasis might be a preferential diagnosis, and yet possibilities of other medical conditions still exist. Pulmonary tuberculosis should be enlisted in the differential diagnosis for a pulmonary nodule in cancer patients in Korea. This study was aimed at analyzing the incidence and clinical features of pulmonary tuberculosis that were misdiagnosed as pulmonary metastasis during radiologic follow-up in pediatric cancer patients. METHODS: We retrospectively studied 422 cancer patients less than 18 years old in the Korea Cancer Center Hospital from January 2001 to June 2007. We collected episodes of lung metastasis of primary tumor and tuberculosis during treatment or follow-up, and analyzed medical records. RESULTS: There were 5 cases of tuberculosis confirmed after surgery which were initially regarded as cancer. Two patients had respiratory symptoms such as cough and sputum but the other 3 patients did not. One patient had a family history of tuberculosis. Acid-fast M. tuberculosis was found in one case upon tissue specimen analysis. Two cases were Mantoux positive and the sputum examination was negative in all cases. The polymerase chain reaction for tuberculosis on a pathologic specimen was used to differentiate M. tuberculosis from non-tuberculosis mycobacterium (NTM). It was positive in one case. Lung lesions in one case showed a concurrence of tuberculosis along with lung metastasis. One of these patients died after cancer recurrence. CONCLUSION: It is necessary to consider the possibility of tuberculosis when a lung mass is newly detected during treatment or follow-up in patients with childhood cancer.

Rapid Identification of Rickettsiae using the Real-Time PCR

In this study, new real-time PCR method based on the groEL gene was developed and investigated. Four spotted fever group (SFG) strains, four typhus group (TG) strains, and four scrub typhus group (STG) strains were easily differentiated as a distinct entity. This PCR assay was applied to detect Rickettsia DNA from 100 ticks. Twelve Haemaphysalis longicornis ticks were found positive and identified as spotted fever group Rickettsia. This real-time PCR method could simultaneously perform the rapid identification of rickettsiae and the differential diagnosis of SFG, TG, and STG in a single reaction.

A Case of Mycobacterium genavense Infection that Manifested as an Erosive Lesion of Duodenum in a Patient with Advanced HIV Infection

Mycobacterium genavense, first identified in 1990, is known as a pathogen that mimics disseminated Myocobacterium avium-intracellulare complex (MAC) infection with particular propensity for the gastrointestinal tract. In Korea, no case with the organism has been reported. Herein we report a case of Mycobacterium genavense infection that manifested with erosive lesion of duodenum in a patient with acquired immune deficiency syndrome. The patient presented with epigastric pain and fever, diarrhea. Duodenal biopsy showed histiocytic infiltration with numerous acid-fast bacilli. Identification of the mycobacterial isolate by the polymerase chain reaction restriction analysis of 16S rRNA gene revealed Mycobacterium genavense.

A Case of Mycobacterium genavense Infection that Manifested as an Erosive Lesion of Duodenum in a Patient with Advanced HIV Infection

Mycobacterium genavense, first identified in 1990, is known as a pathogen that mimics disseminated Myocobacterium avium-intracellulare complex (MAC) infection with particular propensity for the gastrointestinal tract. In Korea, no case with the organism has been reported. Herein we report a case of Mycobacterium genavense infection that manifested with erosive lesion of duodenum in a patient with acquired immune deficiency syndrome. The patient presented with epigastric pain and fever, diarrhea. Duodenal biopsy showed histiocytic infiltration with numerous acid-fast bacilli. Identification of the mycobacterial isolate by the polymerase chain reaction restriction analysis of 16S rRNA gene revealed Mycobacterium genavense.

Mutations of katG and inhA in MDR M. tuberculosis / 결핵및호흡기질환

BACKGROUNDS: Mutations of katG and inhA (ORF and promoter) are known to be related to isoniazid (INH) resistance of Mycobacterium tuberculosis. Because reports on these mutations in Korean isolates are limited (i.e. only the frequency of katG codon 463 was evaluated.), we tried to know the kinds of mutations of two genes and their frequencies in INH resistant Korean M. tuberculosis strains. METHODS: PCR was performed to amplify katG (2,223 bp), inhA ORF (-77~897, 975 bp), and inhA promoter (-168~80, 248 bp) from 29 multidrug resistant M. tuberculosis (MDR-TB) DNAs prepared by bead beater-phenol method. Their sequences were determined and analyzed by ABI PRISM 3730 XL Analyzer and MegAlign package program, respectively. RESULTS: All of the isolates had more than one mutation in katG or inhA gene. Twenty seven (93%) of 29 tested strains had katG mutations, which suggests that katG is a critical gene determining INH resistance of M. tuberculosis. Amino acid substitutions, such as Arg463Leu and Ser315Thr, due to point mutations of the katG were the most frequent (62.1% and 55.2%) mutations. In addition, deletion of the katG gene was frequently observed (17.2%). Analyzed Korean MDR-TB isolates also had variable inhA mutations. Point mutation of inhA promoter region, such as -15 (C-->T) was frequently found. Substitution of amino acid (Lsy8Asn) due to point mutation (AAA-->AAC) of inhA ORF was found in 1 isolate. Interestingly, 14 point mutated types that were not previously reported were newly found. While four types resulted in amino acid change, the others were silent mutations. CONCLUSIONS: Although it is not clear that the relationship of these newly found mutations with INH resistance, they show marked diversity in Korean MDR-TB strains. It also suggests their feasibility as a molecular target to supplement determining the INH resistance of clinical isolates because of the possible existence of low-level INH resistant strains.

Mutations of katG and inhA in MDR M. tuberculosis / 결핵및호흡기질환

BACKGROUNDS: Mutations of katG and inhA (ORF and promoter) are known to be related to isoniazid (INH) resistance of Mycobacterium tuberculosis. Because reports on these mutations in Korean isolates are limited (i.e. only the frequency of katG codon 463 was evaluated.), we tried to know the kinds of mutations of two genes and their frequencies in INH resistant Korean M. tuberculosis strains. METHODS: PCR was performed to amplify katG (2,223 bp), inhA ORF (-77~897, 975 bp), and inhA promoter (-168~80, 248 bp) from 29 multidrug resistant M. tuberculosis (MDR-TB) DNAs prepared by bead beater-phenol method. Their sequences were determined and analyzed by ABI PRISM 3730 XL Analyzer and MegAlign package program, respectively. RESULTS: All of the isolates had more than one mutation in katG or inhA gene. Twenty seven (93%) of 29 tested strains had katG mutations, which suggests that katG is a critical gene determining INH resistance of M. tuberculosis. Amino acid substitutions, such as Arg463Leu and Ser315Thr, due to point mutations of the katG were the most frequent (62.1% and 55.2%) mutations. In addition, deletion of the katG gene was frequently observed (17.2%). Analyzed Korean MDR-TB isolates also had variable inhA mutations. Point mutation of inhA promoter region, such as -15 (C-->T) was frequently found. Substitution of amino acid (Lsy8Asn) due to point mutation (AAA-->AAC) of inhA ORF was found in 1 isolate. Interestingly, 14 point mutated types that were not previously reported were newly found. While four types resulted in amino acid change, the others were silent mutations. CONCLUSIONS: Although it is not clear that the relationship of these newly found mutations with INH resistance, they show marked diversity in Korean MDR-TB strains. It also suggests their feasibility as a molecular target to supplement determining the INH resistance of clinical isolates because of the possible existence of low-level INH resistant strains.

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.

Prevalence of Spotted Fever Group Rickettsia from Haemaphysalis Ticks in Chungju Province

A total of 190 ticks collected from the Chungju area of Korea was examined for the presence of Spotted Fever Group(SFG) Rickettsia using a PCR assay. Twenty-five (13.2%) Haemaphysalis ticks were found positive of the groEL gene of SFG Rickettsia. The prevalence rate of R. japonica in these 25 Haemaphysalis ticks was 72% (18 out of 25 SFG Rickettsia). The prevalence rate of R. conorii and new SFG rickettsia in these 25 Haemaphysalis ticks was 4% (1 out of 25 SFG Rickettsia) and 24% (6 out of 25 SFG Rickettsia), respectively. These results suggest that R. japonica was the highest infection frequency among in Haemaphysalis ticks SFG Rickettsia, and that R. conorii and new SFG Rickettsia are also present in the Chungju area.

Molecular Taxonomic Survey of Mycobacteria Clinical Isolates from Patients in Jeju Island by rpoB Gene Based Molecular Biological Methods

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).

groEL Gene Analysis of Borrelia afzelii Isolated in Korea

Eleven Borrelia afzelii strains, isolated from Ixodes nipponensis and Apodemus agrarius in Korea, were characterized by groEL gene analysis. Results from previous studies suggested that the groEL gene, which encodes the 60-kDa heat shock protein GroEL, was useful for the differentiation of B. burgdorferi sensu lato. The B. afzelii isolates could be divided into two groups by the phylogenetic tree constructed by UPGMA method and Tsp509 I PCR-RFLP analysis. The result suggested that the groEL gene is useful for identification and characterization of B. burgdorferi sensu lato though a short DNA fragment (310 bp) of the gene was sequenced and compared each other, and that Korean B. afzelii strains are heterogeneous genotypically.

groEL Gene Analysis of Borrelia afzelii Isolated in Korea

Eleven Borrelia afzelii strains, isolated from Ixodes nipponensis and Apodemus agrarius in Korea, were characterized by groEL gene analysis. Results from previous studies suggested that the groEL gene, which encodes the 60-kDa heat shock protein GroEL, was useful for the differentiation of B. burgdorferi sensu lato. The B. afzelii isolates could be divided into two groups by the phylogenetic tree constructed by UPGMA method and Tsp509 I PCR-RFLP analysis. The result suggested that the groEL gene is useful for identification and characterization of B. burgdorferi sensu lato though a short DNA fragment (310 bp) of the gene was sequenced and compared each other, and that Korean B. afzelii strains are heterogeneous genotypically.

Development of Rickettsia Specific Nested PCR Method Based on groEL Gene Sequences

To detect Rickettsia, we have developed a nested PCR method amplifying the groEL gene. Rickettsia strains were successfully amplified by this PCR method but the microorganisms causing other febrile diseases, such as Orientia tsutsugamushi, Coxiella burnetii, Ehrlichia sennetsu, Borrelia burgdorferi sensu lato, Borrelia hermsii, and Leptospira interrogans were not amplified. This PCR assay was applied to detect Rickettsia DNA from 100 ticks. Sixteen Haemaphysalis longicornis ticks were positive by this PCR assay. These results suggest that the new nested PCR method might be sensitive and useful for discrimination between Rickettsia and other febrile disease-causing microorganisms.

Human Cytomegalovirus Immediate-Early 2Prote in Transactivates c-jun Promoter Through ATF and MEF2 Site / 감염

BACKGROUND: Human cytomegalovirus (HCMV) has the ability to activate the expression of many viral and cellular genes. The c-jun proto-oncogene has known to be induced at immediate early time of HCMV infection, however, the mechanism of up-regulation of the gene was not known. We found HCMV immediate-early (IE) 2 expression transactivate the c-jun promoter in human embryonal lung cell (HEL). METHODS: The c-jun promoter region between -117 and -59 contains binding sites for the transcription factors Sp1, CAAT, AP-1 like (ATF/CREB), and MEF2. We tried to map the sequences in the c-jun promoter responsible for activation of the promoter by HCMV IE2 expression. Transient expression assays were performed using various reporter plasmids containing the c-jun promoter-regulatory region linked to the luciferase gene and a plasmid expressing HCMV IE2 gene. RESULTS: Deletional and point mutational analysis showed that ATF, MEF2, and another down stream elements were involved in the up-regulation of c-jun promoter. Gel mobility shift assay showed that there are several factors in HEL cell nuclear extracts that specifically bind to these sites and in vitro translated IE2 could not move or supershift the specific bands. CONCLUSION: This study delineate the mechanism of c-jun up-regulation in HCMV infection and would give the clue for the possible contribution of HCMV in tumorigenesis.

Differentiation of Mycobacterium avium Complex Isolated in Korea by DT1-BT6 PCR

Recently, selective PCR method using DT1 and DT6 sequences was introduced to identify and differentiate the Mycobacterium avium complex (MAC) into M. intracellulare and M. avium. We applied this method to 49 MAC clinical isolates identified by biochemical tests. They were differentiated into 39 strains of M. intracelluare and 10 strains of M. avium. Compared to those results obtained by 16S rDNA sequencing, DT1-DT6 PCR method showed 100% specificity. While the sensitivity of DT6 PCR for M. avium was 100%, that of DT1 PCR for M. intracellulare was 84.6%. These results show heterogeneity of M intracellulare Korea clinical isolates from Korea. In conclusion, although the in-house DTl-DT6 PCR is an easy and convenient method in differentiating MAC members, other methods such as 16S rDNA sequencing analysis should be performed for the correct identification, especially of M intracellulare.

Identification of Fastidious Mycobacteria other than M. tuberculosis ( MOTT ) by Comparative Sequence Analysis of rpoB and 16S rDNA

Conventional tests for the identification of mycobacteria may frequently result in erroneous identification and underestimate the diversity within the genus Mycobacterium. However, this problem can be overcome by molecular approach like as 16S rRNA gene (rDNA) or RNA polymerase gene (rpoB) sequence analysis. In this study, a molecular approach analyzing partial sequence of 16S rDNA and rpoB gene was applied to mycobacteria other than M tuberculosis (MOTT) isolates that had not been definitely identified by conventional physical and biochemical tests. Among the eighteen isolates included in this study, twelve isolates could be identified to the species level and six were identified to the complex level. Compared with the results by 16S rDNA analysis, the rpoB analysis could di6erentiate some of the strains into the subspecies level.

Gene Cloning of the Epstein-Barr Virus (EBV) Antigen Reactive with the Serum from EBV-infected Patients / 감염

BACKGROUND: Epstein-Barr virus (EBV) is causative agent of infectious mononucleosis and nasopharyngeal carcinoma and associated with Burkitt lymphoma and other tumors. The recombinant protein is needed for the rapid and sensitive serodiagnosis of EBV infection. METHODS: EBV gene encoding the protein reactive with the sera of EBV-infected patient was cloned and characterized with lambda gt11 expression library of cDNA of EBV B95-8 strain. RESULTS: The recombinant proteins from clone 12, 15 and 21 were expressed as 120, 118, 160 kDa-usion protein with beta-galactosidase, respectively, which were reactive with IgG anti-EBV antibody-positive sera, but not with anti-EBV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with EBV B95-8 sequences revealed that those were located at 61716~62087, 61898~62085, and 102128~103158, respectively. These positions correspond to BFRF3, BFRF3, and BZLF1, respectively, which were reported as immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. All the patients' sera were reactive with clone 12 protein, but only 5 out of 9 patients' sera were reactive with clone 21 protein. CONCLUSION: Clone 21 protein expressing BFRF3 fragment was immunoreactive in patient sera from natural EBV infection and was regarded as useful candidate for the serodiagnosis of EBV infection.

A Case Report of Mycobacterium Abscessus Septicemia Due to Chemoport Infection / 감염

Mycobacterium abscessus, a rapidly growing mycobacterium, is an opportunistic pathogen which causes a wide variety of clinical symptoms. Recently non-tuberculous mycobacterial infections are increasing among immunocompromised patients and made 4% of total cases of mycobacterial infection. To our knowledge, there has been no report of systemic infection caused by rapidly growing mycobacterium in Korea. We experienced a case of M. abscessus septicemia due to chemoport infection in a 47-year old female who was diagnosed as ovarian cancer stage IIIc and was in the immunocompromised state after systemic chemotherapy. The patient manifested with fever, chilling, headache, and nausea, though, there were no abnormalities on physical examination. When the patient was receiving empirical antibiotic therapy, a rapidly growing mycobacterium was detected in repeated blood cultures. She was improved with not only systemic an-tibiotic treatment but also removing the chemoport. But short course (4 weeks) of antibiotic therapy caused incomplete treatment and made multiple skin abscess. After incision and drainage of the lesions and administration of prolonged antibiotic therapy, no additional infection was observed. Based on our experience and the review of the literatures, catheter-related bacteremia due to rapidly growing mycobacterium, including M. abscessus, should be treated with catheter removal and appropriate antibiotic therapy for at least 3 to 6 months based on in vitro susceptibility testing. When a patient in neutropenic state presents sustained fever after treatment with conventional antibiotics, non-tuberculous mycobacterial infection should be considered.