Hyungki Lee

Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens

ABSTRACT We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.

Naturally occurring hepatitis B virus X deletions and insertions among Korean chronic patients.

Deletions and insertions in the hepatitis B virus (HBV) X region have been associated with severe forms of liver disease, including hepatocellular carcinoma (HCC). However, the molecular epidemiologic features of this virus have been described rarely. Deletions and insertions in the X region were determined by direct sequencing in a Korean cohort of 267 patients with different clinical statuses. Deletions and insertions were observed in two sets of six patients each (2.2%, 6/267). The prevalence of deletions or insertions was significantly higher in patients with severe liver disease, HCC, or cirrhosis of the liver (7.2%, 10/132) compared to patients who were carriers or had chronic hepatitis (1.5%, 2/135) (P = 0.017). All deletions in six strains were concentrated at the C terminal end of HBx, encompassing the 113th to 154th codons. A total of four novel types of insertions (PKLL, GM, FFN, and tt) were observed in six patients. Of particular interest, all six strains with insertions were accompanied by double mutations in the basal core promoter (BCP). In conclusion, these results suggest that deletions or insertions in the X region may contribute to disease progression in Korean patients with genotype C infection. J. Med. Virol. 83:65–70, 2011.

Mycobacterium paraterrae sp. nov. recovered from a clinical specimen: novel chromogenic slow growing mycobacteria related to Mycobacterium terrae complex

A previously unidentified, slowly growing scotochromogenic Mycobacterium was isolated from a Korean patient with symptomatic pulmonary infection. Phenotypically, this strain was generally similar to Mycobacterium terrae complex strains, however it uniquely produced orange pigmentation. Unique mycolic acid profiles and phylogenetic analyses based on three alternative chronometer molecules, 16S rRNA gene, hsp65 and rpoB, confirmed the taxonomic status of this strain as a novel species. These results support that this strain represents a novel Mycobacterium species. The name Mycobacterium paraterrae sp. nov. is proposed. The type strain is 05‐2522 (= DSM 45127 = KCTC 19556).

Molecular characterization of Mycobacterium intracellulare-related strains based on the sequence analysis of hsp65, internal transcribed spacer and 16S rRNA genes

We investigated the molecular epidemiological features of 94 Mycobacterium intracellulare-related strains, isolated from Korean patients, using sequence analysis targeting 3 independent chronometer molecules, hsp65, the internal transcribed spacer 1 region and the 16S rRNA gene. By collective consideration of these three gene-based approaches, the 94 strains were divided into 5 groups (INT1, INT2, INT3, INT4 and INT5). The frequencies of genotype INT1, 2, 3, 4 and 5 in the 94 isolates were 57.4 % (54), 27.7 % (26), 6.4 % (6), 5.3 % (5) and 3.2 % (3), respectively. When correlations between genotypes and clinical parameters (age, sex, radiological type and the presence of a cavity) were analysed in 78 patients with non-tuberculous mycobacteria pulmonary diseases, no relationships were observed with respect to age, sex and radiological type, but genotype and the presence of a cavity tended to be related (P=0.051).

Identification of Mycobacterium species in direct respiratory specimens using reverse blot hybridisation assay.

BACKGROUND The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. OBJECTIVE To evaluate the performance of the reverse blot hybridisation assay (REBA) Myco-ID for the rapid identification of Mycobacterium species in acid-fast bacilli (AFB) smear-positive respiratory specimens. DESIGN A total of 148 AFB smear-positive respiratory specimens were used for the identification of Mycobacterium species using polymerase chain reaction (PCR) REBA, and the results were compared with the gold standard method as well as culture, PCR-restriction fragment length polymorphism analysis (PRA) and rpoB sequence analysis. RESULTS The results of this assay showed that 59/148 samples were positive for M. tuberculosis and 87 were positive for non-tuberculous mycobacteria (NTM); one sample had mixed infection with both M. tuberculosis and NTM. One case was negative for both M. tuberculosis and NTM and was identified as Nocardia farcinica using PRA and sequence analysis. Of a total of 89 cases, the most frequently identified NTM species were M. intracellulare (n = 28, 31.5%), M. avium (n = 21, 23.6%), M. massiliense (n = 19, 21.3%) and M. abscessus (n = 16, 18.0%). CONCLUSION The PCR-REBA assay is an efficient tool for the rapid identification of the main Mycobacterium species in clinical specimens. The PCR-REBA assay can therefore provide useful information to physicians for appropriate treatment by clearly identifying Mycobacterium species.

First Case of Disseminated Mycobacterium bolletii Infection in a Young Adult Patient

ABSTRACT Mycobacterium bolletii is a rapidly growing nontuberculous mycobacterium first characterized in 2006. Here, we report a case of disseminated infection caused by M. bolletii in a young adult patient. To our knowledge, this is the first case of disseminated M. bolletii infection in an otherwise healthy young adult.

Cellular mechanisms for trazodone-induced cardiotoxicity

The second-generation selective 5-HT2 receptor antagonists and reuptake inhibitors (SARIs) class antidepressants are known to have fewer cardiovascular side effects than the older ones. However, several case reports showed that trazodone, one of the second-generation SARIs, induces QT prolongation, cardiac arrhythmia, and ventricular tachycardia. Although these clinical cases suggested trazodone-induced cardiotoxicity, the toxicological actions of trazodone on cardiac action potentials (APs) beyond the human ether-a-go-go related gene (hERG) remain unclear. To elucidate the cellular mechanism for the adverse cardiac effects of trazodone, we investigated its effects on cardiac APs and ion channels using whole-cell patch clamp techniques in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and transiently transfected human embryonic kidney cells (HEK293) with cardiac ion channel complementary DNA. Trazodone dose-dependently decreased the maximum upstroke velocity (Vmax) and prolonged the AP duration, inducing early after depolarizations at 3 and 10 μM that triggered ventricular arrhythmias in hiPSC-CMs. Trazodone also inhibited all of the major ion channels (IKr, IKs, INa, and ICa), with an especially high inhibitory potency on hERG. These data indicate that the prolonged AP duration and decreased Vmax due to trazodone are mainly the result of hERG and sodium ion inhibition, and its inhibitory effects on cardiac ion channels can be exhibited in hiPSC-CMs.

The Development of a Novel Mycobacterium-Escherichia coli Shuttle Vector System Using pMyong2, a Linear Plasmid from Mycobacterium yongonense DSM 45126T

The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.