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.
Base Sequence ; Codon ; Drug Resistance, Multiple ; Drug Therapy ; Drug Therapy, Combination ; Humans ; Korea* ; Mycobacterium tuberculosis* ; Mycobacterium* ; Rifampin ; Tuberculosis ; Tuberculosis, Multidrug-Resistant

Diagnostic Errors ; Female ; Humans ; Hysterectomy ; Leiomyoma ; diagnosis ; surgery ; Middle Aged ; Tomography, X-Ray Computed ; Ultrasonography ; Uterine Neoplasms ; diagnosis ; surgery ; Uterus ; blood supply ; diagnostic imaging ; Venous Thrombosis ; diagnosis ; surgery

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.
Amidohydrolases ; Antitubercular Agents ; Arginine ; Aspartic Acid ; Diagnostic Tests, Routine ; Early Diagnosis ; Histidine ; Humans ; Mycobacterium bovis ; Mycobacterium tuberculosis ; Proline ; Pyrazinamide ; Tissue Donors ; Tuberculosis

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.
Diagnosis, Differential ; DNA ; Fever ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Rickettsia ; Scrub Typhus ; Ticks ; Typhus, Epidemic Louse-Borne

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.
Chungcheongbuk-do* ; Fever* ; Korea ; Polymerase Chain Reaction ; Prevalence* ; Rickettsia* ; Ticks*

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.
Animals ; Borrelia burgdorferi Group* ; Borrelia* ; DNA ; Heat-Shock Proteins ; Ixodes ; Korea* ; Murinae ; Population Characteristics

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.
Animals ; Borrelia burgdorferi Group* ; Borrelia* ; DNA ; Heat-Shock Proteins ; Ixodes ; Korea* ; Murinae ; Population Characteristics

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.
Borrelia ; Borrelia burgdorferi Group ; Coxiella burnetii ; Discrimination (Psychology) ; DNA ; Leptospira interrogans ; Neorickettsia sennetsu ; Orientia tsutsugamushi ; Polymerase Chain Reaction* ; Rickettsia* ; Ticks

Cyclosporine(CsA) has been known to cause an endothelial dysfunction following its use as an immunosuppressive agent. On the other hand, the vascular endothelium has been recognized as an endocrine organ in its own right, i.e., it releases vasoactive factors such as nitric oxide(NO) and hyperpolarizing factor(EDHF). NO is synthesized by at least three isoforms of NO synthases(NOS), among which ecNOS is constitutively expressed in the endothelium. The principle of EDHF has not been determined. The present study was aimed at further investigating the mechanisms underlying the CsA-induced vasculopathy. Rats were treated with CsA (25mg/kg/day, subcutaneous) for one week and their thoracic aortae were isolated. Their changes of iso-metric tension in responses to acetylcholine, diazoxide, and high concentrations of calcium were recorded. The expression of ecNOS mRNA and protein was determined by reverse transcription-polymerase reaction and Western blot analysis, respectively. The acetylcholine-induced relaxation of the aortic rings was significantly diminished follawing the CsA-treatment, which was prevented by L-arginine supplemented along with the CsA-treatment. The relaxation of the thoracic aorta in response to either diazoxide or high concentrations of extracellular calcium was not affected by CsA-treatrnent. The vascular tissue contents of NO metabolites were significantly decreased following the CsA-treatment, which was also prevented by L-arginine-supple-mentation. Neither ecNOS mRNA nor protein expression was significantly altered following the CsA-treatment. It is suggested that CsA induces an endothelial dysfunction, which cannot be attributed to an altered role of EDHF, but to an impairment in L-arginine/NO pathway at the steps beyond NOS protein expression.
Acetylcholine ; Animals ; Aorta, Thoracic ; Arginine ; Blotting, Western ; Calcium ; Diazoxide ; Endothelium ; Endothelium, Vascular ; Hand ; Protein Isoforms ; Rats* ; Relaxation ; RNA, Messenger

The gene encoding the 66 kilodalton (kDa) protein of Borrelia hermsii HS1 was cloned and sequenced. Chromosomal DNA was prepared from purified B. hermsii and used in construction of genomic library. The library was screened for positive clones by 314 bp DIG-labeled probe synthesized on the basis of the part of the sequence of B. hermsii. Positive clone was subcloned into p2ErO vector and was designated as pBH11. pBH11 were subcloned into pBluscript vector and were designated as pBH11-1 (500 bp), pBH11-2 (800 bp), pBH11-3 (600 bp) and pBH11-4 (800 bp). The plasmids were sequenced and determined the nucleotide sequence of p66. The open reading frame of the p66 consisted of 1803 base pairs coding for 600 amino acid protein. The basic information on the p66 gene of B. hermsii HS1 obtained from this study will be useful for further analysis and experiment of pathogenesis of the borrelia.
Base Pairing ; Base Sequence ; Borrelia* ; Clinical Coding ; Clone Cells* ; Cloning, Organism* ; DNA ; Genomic Library ; Open Reading Frames ; Plasmids