vanA 유전자와 vanC1 유전자를 동시에 가진 Enterococcus gallinarum 1예

성흥섭,윤경아,김미나,배직현 대한임상병리학회 2002 대한임상병리학회지 Vol.22 No.1

거대세포바이러스와 엡스타인바바이러스 DNA 정량검사를 위한 핵산 추출

성흥섭,황상현,고영진,김미나 대한진단검사의학회 2016 Laboratory Medicine Online Vol.6 No.3

Background: Availability of an international standard will improve the standardization of quantitative PCR (qPCR) for cytomegalovirus (CMV) and Epstein-Barr virus (EBV); however, nucleic acid extraction methods may affect qPCR results. This study was designed to determine whether routine measurement of DNA concentration and purity is required in qPCR for CMV and EBV. In addition, the performance of the automated QIASymphony DSP DNA Mini kit (Qiagen, USA) and the manual QIAamp DNA Blood Mini kit (Qiagen) in extracting DNA from whole blood samples was compared. Methods: The concentration and purity of 300 extracted DNA samples were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA). A total of 72 and 54 whole blood samples were tested by artus CMV and EBV qPCR (Qiagen), respectively. Results: No correlation was found between DNA concentration and EBV DNA load or between DNA purity and the PCR inhibition measured by ΔCq (the difference between internal control Cq of the sample and that of the negative control). Quantification of CMV and EBV DNA using the two extraction methods showed highly similar results (rho=0.946 and 0.887, respectively). Of the 29 specimens that yielded CMV DNA by both methods, however, 8 specimens (27.6%) yielded higher CMV DNA loads with QIASymphony. Conclusions: Routine measurement of DNA concentration and purity is not necessary for qPCR of CMV and EBV. The automated QIASymphony outperformed the manual QIAamp Blood Mini kit in extracting CMV and EBV DNA from whole blood samples. 배경: 거대세포바이러스(cytomegalovirus, CMV)와 엡스타인바바이러스(Epstein-Barr virus, EBV) 국제 표준물질이 이용 가능해짐으로써, CMV와 EBV 정량 PCR (qPCR)의 표준화는 향상되었지만, 핵산 추출 방법 또한 qPCR 결과에 영향을 줄 수 있다. 이 연구에서는 바이러스 qPCR 전에 DNA 농도와 순도 측정이 일상적으로 필요한지를 살펴보았으며, 수기법인 QIAamp DNA Blood Mini 키트(키트(Qiagen, USA)와 자동화법인 QIASymphony DSP DNA Mini 키트(Qiagen)를 비교하여 핵산 추출법을 도입할 때 고려해야 할 사항을 살펴보고자 하였다. 방법: DNA 정량과 순도는 QIAamp DNA Blood Mini 키트로 수기 추출한 후 EBV qPCR을 시행한 300개 전혈 DNA를 대상으로 하였으며, NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, USA)에서 측정하였다. 수기 핵산추출과 자동화된 핵산추출의 비교는 CMV qPCR이 의뢰된 72개의 전혈과 EBV qPCR이 의뢰된 54개의 전혈을 대상으로 하였다. CMV와 EBV DNA는 artus CMV 및 Artus EBV PCR로 정량하였다. 결과: DNA 농도와 EBV DNA 정량값 사이에는 일관된 상관성이 없었으며, DNA 순도와 Cq (내부대조물질의 Cq값에서 동일한 qPCR 배치에서 사용한 음성 대조검체에 첨가한 내부대조물질의 Cq값을 뺀 값) 사이에는 상관성이 없었다. 수기 핵산추출과 자동화된 핵산추출 후 CMV와 EBV qPCR값은 두 방법 간에 높은 상관 관계가 있었다(CMV, rho=0.946; EBV, rho=0.887). 두 방법 모두에서 CMV DNA가 양성이었던 29검체 중 8검체(27.6%)는 QIASymphony에서 추출한 핵산 정량값이 유의하게 높았다. 결론: 검사실에서 CMV와 EBV qPCR 전에 일상적으로 DNA 농도와 순도를 측정할 필요는 없을 것으로 판단되었다. 전혈에서 CMV와 EBV DNA를 추출할 때 자동화 기기인 QIASymphony법의 정량값이 수기법인 QIAamp DNA Blood Mini 키트보다 유의하게 높았다.

Quantitation of BK Virus DNA for Diagnosis of BK Virus-Associated Nephropathy in Renal Transplant Recipients

성흥섭,최병후,표연정,김미나,한덕종 대한의학회 2008 Journal of Korean medical science Vol.23 No.5

Quantitative measurement of BK virus DNA (Q-BKDNA) has been used for the early diagnosis and monitoring of BK virus-associated nephropathy (BKVAN). This study was designed to determine the BKDNA cutoff for the diagnosis of BKVAN. Between June 2005 and February 2007, 64 renal transplant recipients taken renal biopsies due to renal impairment submitted plasma and urine for Q-BKDNA. Eight BKVAN patients (12.5%) had median viral loads of 6.0 log10 copies/mL in plasma and 7.3 log10 copies/mL in urine. Among 56 non-BKVAN patients, 45 were negative for Q-BKDNA; 4 were positive in plasma with a median viral load of 4.8 log10 copies/ mL, and 10 were positive in urine with a median viral load of 4.8 log10 copies/mL. Receiver operating characteristic curve analysis showed that a cutoff of 4.5 log10 copies/mL in plasma and a cutoff of 5.9 log10 copies/mL in urine had a sensitivity of 100% and a specificity of 96.4%, respectively. A combined cutoffs of 4 log10 copies/ mL in plasma and 6 log10 copies/mL in urine had better performance with a sensitivity of 100% and a specificity of 98.2% than each cutoff of urine or plasma. QBKDNA with the combined cutoffs could reliably diagnose BKVAN in renal transplant recipients.

염기서열분석을 이용한 미생물 동정의 신뢰도

성흥섭,김미나 대한감염학회 2008 Infection and Chemotherapy Vol.40 No.6

Sternal Osteomyelitis Caused by Gordonia bronchialis after Open-Heart Surgery

성흥섭 대한감염학회 2014 Infection and Chemotherapy Vol.46 No.2

We report the case of a deep sternal wound infection with sternal osteomyelitis caused by Gordonia bronchialis after open-heartsurgery. The isolate was identified as a G. bronchialis by 16S rRNA and hsp65 gene sequencing, having initially been misidentifiedas a Rhodococcus by a commercial phenotypic identification system.

디스크확산법에 의한 Helicobacter pylori의 Clarithromycin과 Amoxicillin 항균제감수성 검사

성흥섭,강정옥,이미애,이종욱,이혜경,이미경,임지훈,김미나,Helicobacter 연구회 대한임상미생물학회 2009 Annals of clinical microbiology Vol.12 No.1

Background: CLSI provides a guideline only for a agar dilution method of testing clarithromycin susceptibility for Helicobacter pylori. This study was to evaluate a disk diffusion method for clarithromycin and amoxicillin. Methods: One hundred and forty clinical isolates of H. pylori isolated from May 2005 to May 2007 were tested by the CLSI agar dilution method and a disk diffusion method using 2μg (2CLR) and 15μg (15CLR) clarithromycin disks and 2μg (2AMX) and 10μg (10AMX) amoxicillin disks. The interpretation criteria used for the disk diffusion method were established by linear regression and error rate-bounded method for disk diffusion zone of inhibition (DDZ) compared to MIC. Results: Resistance and intermediate rates to clarithromycin were 21.4% and 1.4%, respectively. A number of isolates with MIC 0.5, 1, and 2 (μg/mL) to amoxicillin were 7, 2, and 1, respectively. For 2CLR and 15CLR, the coefficients of determination (R2) between MIC and DDZ were 0.931 and 0.923 (P< 0.001), respectively, and the criteria for resistance/ susceptibility were 12/28 mm for 2CLR and 23/39 mm for 15CLR. For 2AMX and 10AMX, the R2 between MIC and DDZ were 0.478 and 0.421 (P< 0.001), respectively, and the criteria for resistance with breakpoint of 2μg/mL were 21 mm for 2AMX and 32 mm for 10AMX. All isolates had DDZ<60 mm with 2CLR and 2AMX, but 61.4% and 75.7% of the isolates had DDZ<60 mm with 15CLR and 10AMX, respectively. Conclusion: Excellent correlation and agreement between MIC and DDZ were found for clarithromycin and amoxicillin. With 2μg disks, the susceptibility breakpoints were 28 mm or less; thus, two disks could be tested in one plate.

vanA 유전자와 vanC1 유전자를 동시에 가진 Enterococcus gallinarum 1예

성흥섭,윤경아,김미나,배직현 대한진단검사의학회 2002 Annals of Laboratory Medicine Vol.22 No.1

저자들은 중환자실 감시배양을 통해 3년간 치료받던 환자에서vanA 와 vanC1 유전자를 동시에 가진 E. gallinarum을 분리하였다. E. gallinarum, SI04는 반코마이신에 고도내성(MIC of ≥256 g/mL) 이었으며, teicoplanin에도 고도내성(MIC of ≥256g/mL)이었다. vanA, vanB, vanC1, vanC2/3 내성형 결정을위하여 multiplex PCR을 시행한 결과 SI04에서 vanA 와 vanC1두 가지 유전자가 동시에 관찰되었다. 본 증례는 국내에서 처음으로 VanC형의 반코마이신 내성 장구균에서vanA 유전자가 존재함을 보여 주어, 반코마이신 내성 장구균의 임상적 의의를 판단하는데 내성 유전자의 검출이 필요하다는 것을 시사하였다.

Sezary 증후군 1예

성흥섭,서을주,박찬정,지현숙,허주령,이규형 大韓血液學會 2000 大韓血液學會誌 Vol.35 No.3

Rhinovirus, Human Metapneumovirus, Coronavirus 검출을 위한SeeplexTM RV Detection 키트의 평가

성흥섭,박숙자,우영대,최병후,김미나 대한진단검사의학회 2008 Annals of Laboratory Medicine Vol.28 No.2

Background : Direct antigen test (DAT) and culture are primary tests to diagnose infections by respiratory viruses, but are mainly available for the traditional viral pathogens such as respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), and adenovirus in clinical laboratories. The objective of this study was to evaluate a multiplex reverse transcriptase-PCR method using SeeplexTM RV Detection kit (Seegene, Korea) for the detection of rhinovirus, coronavirus, and human metapneumovirus (hMPV). Methods : From January to May 2007, nasopharyngeal aspirates (NPAs) from pediatric patients negative for culture and DAT of traditional viral pathogens were tested with SeeplexTM. All the amplicons were directly sequenced and homology of the sequences was searched in the National Center for Biotechnology Information (NCBI) database. Patients’ medical records were reviewed for clinical and demographic features. Results : Forty-seven (26.4%) of 178 NPAs were positive: 18 rhinovirus, 15 hMPV, 4 RSV A, 3 coronavirus OC43, 3 influenza virus A, 2 adenovirus, 1 coronavirus NL63, and 1 RSV B. Based on maximum identity, each of the sequences indicating rhinovirus, hMPV, and coronavirus OC43 matched to the corresponding viruses with homology of 94-98%, 96-99%, and 98-100%, respectively. SeeplexTMpositive patients were 0-11 yr old with a male:female ratio of 1.5:1. Clinical diagnoses included 9 pneumonia, 6 bronchiolitis, 2 cold, 1 asthma exacerbation for rhinovirus; 10 pneumonia, 4 bronchiolitis, and 1 clinical sepsis for hPMV; and 1 pneumonia, 2 croup, and 1 cold for coronavirus. Conclusions : Multiplex reverse transcriptase-PCR method using SeeplexTM RV Detection kit is a reliable test to detect rhinovirus, hMPV, and coronavirus. It may improve the diagnostic sensitivity for RSV, influenza virus, PIV, and adenovirus. (Korean J Lab Med 2008;28:109-17)

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR

성흥섭,용동은,기창석,김재석,성문우,이혁민,김미나 대한진단검사의학회 2016 Annals of Laboratory Medicine Vol.36 No.5

Background: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. Methods: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). Results: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). Conclusions: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.