Hyeyoung Lee

Yonsei University, Sŏul, Seoul, South Korea

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Publications (49)115.34 Total impact

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    ABSTRACT: Human papillomavirus (HPV) infection is a major cause of premalignant dysplasia and cervical cancer. There are no data on the prevalence of genotype-specific HPV infection assessed by HPV E6/E7 mRNA in women representative of the Korean population across a broad age range. A total of 630 women aged 17-90 years were enrolled in this study and ThinPrep(®) liquid-based cytology samples were evaluated using the CervicGen HPV RT-qDx assay, which detects 16 HPV high-risk (HR) HPV genotypes (Set 1: HPV 16, 31, 33, 35, 52, and 58; Set 2: HPV 18, 39, 45, 51, 59, and 68; and Set 3: HPV 53, 56, 66, and 69). The overall prevalence of HPV infection was 209 (33.2%) and oncogenic high-risk HPV was detected in 75.9% (n=107) of 141 women with high-grade cervical lesions. HPV 16 was the most common HPV genotype among women with high-grade cervical lesions and histologically confirmed CIN2+ in the Republic of Korea (41.6%). Among women aged over 30, 182/329 (55%) patients had invasive cervical cancer and 135 (74%) of these patients were infected with oncogenic HR-HPV types (in particular 25% with HPV 16). Among patients diagnosed with CIN2+, the positivity rate of HR-HPV was the highest in women aged 40 to 49 years. Our results suggest that determination of specific HPV genotypes are very important for evaluating the potential impact of preventive measures, including the use of prophylactic vaccines, on reducing the burden of cervical cancer. Copyright © 2015. Published by Elsevier Ltd.
    International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases 06/2015; DOI:10.1016/j.ijid.2015.06.018 · 2.33 Impact Factor
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    ABSTRACT: Human papillomavirus (HPV) is a major cause of cervical cancer, which is the third most common cancer in women. Human telomerase reverse transcriptase (hTERT) and Ki67 are tumor cell markers indicating cancer cell proliferation in cancer patients, and activation of hTERT and Ki67 leads to progressive cervical carcinogenesis. In the present study, we evaluated the CervicGen HPVE6/E7 mRNA RT-qDx assay, which detects 16 HPV high-risk (HR) genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 69), and the CervicGen hTERT and Ki67 mRNA RT-qDx assay using 117 formalin-fixed paraffin-embedded (FFPE) cervical cancer tissue samples. The diagnostic validity of the CervicGen HPV RT-qDx assay for detecting histologically proven prevalent squamous cell carcinoma (SCC) was 94% sensitivity, 100% specificity, 77.8% positive predictive value (PPV), and 78.9% negative predictive value (NPV). The most common HPV genotypes detected in FFPE cervical cancer tissue samples were HPV 16 (56%) and HPV 18 (10%).
    Experimental and Molecular Pathology 03/2015; 98(3). DOI:10.1016/j.yexmp.2015.03.036 · 2.88 Impact Factor
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    ABSTRACT: Human papillomavirus (HPV) is a major cause of cervical cancer, which is the second most common cancer in women. HPV E6 initiates degradation of cellular tumor suppressor protein p53, induces human telomerase reverse transcriptase (hTERT) activity, and then leads to progressive cervical carcinogenesis. In this study, the CervicGen HPV RT-qDX assay (Optipharm, Osong, Republic of Korea), which detects 16 HPV high-risk subtypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 69), and the CervicGen hTERT RT-qDX assay (Optipharm) were evaluated using 545 ThinPrep (Hologic, Bedford, MA) Papanicolaou samples. The positivity for the HPV E6/E7 messenger RNA (mRNA) assay was 94.4%, 95.2%, 82.4%, 46.5%, 25.0%, and 1.1% in squamous cell carcinomas, high-grade squamous intraepithelial lesions (HSILs), atypical squamous cells-cannot exclude HSIL, low-grade squamous intraepithelial lesions, atypical squamous cells of undetermined significance, and normal cytology samples, respectively. Five cervical intraepithelial neoplasia grade 2+ samples were not detected by the HPV E6/E7 mRNA assay, but they exhibited positive signals in the hTERT mRNA assay. Notably, the hTERT mRNA expression level was increased in high-grade cervical lesions but was very low in all 288 normal samples. These data suggest that the combination of HPV E6/E7 and hTERT mRNA expression levels could be used in a complementary manner in diagnosing high-grade cervical lesions and malignant tumors and might be useful as a predictive marker in monitoring low-grade cervical lesions. Copyright© by the American Society for Clinical Pathology.
    American Journal of Clinical Pathology 03/2015; 143(3):344-51. DOI:10.1309/AJCPF2XGZ2XIQYQX · 3.01 Impact Factor
  • 02/2015; 29(1):47-55. DOI:10.11001/jksww.2015.29.1.047
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    ABSTRACT: Breast cancer is a highly prevalent disease among women worldwide. While the expression of certain proteins within breast cancer tumors is used to determine the prognosis and select therapies, additional markers need to be identified. Circulating tumor cells (CTCs) are constituent cells that have detached from a primary tumor to circulate in the bloodstream. CTCs are considered the main source of breast cancer metastases; therefore, detection of CTCs could be a promising diagnostic method for metastatic breast cancer. In this study, the CircleGen CTC RT-qDx assay was used to analyze the mRNA expression levels of six CTC-specific markers including EpCAM, CK19, HER2, Ki67, hTERT, and vimentin with a total of 692 peripheral whole blood samples from 221 breast cancer patients and 376 healthy individuals. This assay showed high specificity with multiple markers; none of the healthy controls were detected positive, whereas 21.7 and 14 % of breast cancer patients were positive for EpCAM and CK19, respectively. Of the 221 breast cancer patients, 84 (38 %), 46 (20.8 %), 83 (37.6 %), and 39 (17.6 %) were positively for HER2, Ki67, hTERT, and vimentin mRNA, respectively. Of the 84 patients who were HER2 positive, nine (4 %) were also positive for EpCAM, CK19, Ki67, hTERT, and vimentin. Of the 139 breast cancer patients who were HER2 negative, 65 (29.1 %) were negative for EpCAM, CK19, Ki67, hTERT, and vimentin. Furthermore, the EpCAM-positive population decreased from 21.5 to 8.3 % after completion of anti-tumor treatment (TP4). Similarly, the CK19, HER2, hTERT, and vimentin positives also decreased from 13.9 to 9.5 %, from 37.7 to 21.4 %, from 37.2 to 33.3 %, and from 17.5 to 14.3 %, respectively, after completion of anti-tumor treatment. In contrast, the Ki67 positives increased from 20.6 to 41.7 % after completion of anti-tumor treatment. mRNA overexpression of six CTC-specific markers was detected by the CircleGen CTC RT-qDx assay with high specificity, and the obtained mRNA expression levels of CTC-specific markers might provide useful criteria to select appropriate anti-tumor treatment for breast cancer patients.
    International Journal of Clinical Oncology 02/2015; DOI:10.1007/s10147-015-0798-3 · 2.17 Impact Factor
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    ABSTRACT: Interferon (IFN)-γ release assays have limited sensitivity and cannot differentiate between active tuberculosis (TB) disease and latent TB infection (LTBI). Numerous cytokines and regulator factors have been implicated in the pathogenesis and control of Mycobacterium tuberculosis infection. Additional cytokines and chemokines associated with M. tuberculosis infection may improve the performance of IFN-γ release assays. We developed a real-time RT-PCR TaqMan assay for targeting levels of eight human targets [IFN-γ, tumor necrosis factor (TNF)-α, IL-2R, IL-4, IL-10, CXCL9, CXCL10, and CXCL11] and evaluated the assay with three different study groups. Results showed that the sensitivity of TNF-α, IL-2R, and CXCL10 in the active pulmonary tuberculosis (PTB) group was 96.43%, 96.43%, and 100%, respectively. The sensitivity of IL-2R and CXCL10 in the latent tuberculosis infection group was 86.36% and 81.82%, respectively. Statistical results showed that TNF-α and CXCL9 were the best individual markers for differentiating between the PTB, LTBI, and non-TB groups. For optimal sensitivity and differentiation of M. tuberculosis infection status, the simultaneous detection of multiple targets was attempted. The combination of IFN-γ, TNF-α, and IL-2R, and the combination of TNF-α, IL-2R, CXCL9, and CXCL10 showed the best performance for detecting active PTB (both 100% positivity) and LTBI (86.36% and 81.82% positivity, respectively). These results imply that the combination of suitable markers is useful in efficiently diagnosing TB and differentiating M. tuberculosis infection status. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
    The Journal of molecular diagnostics: JMD 01/2015; 17(1):90-99. DOI:10.1016/j.jmoldx.2014.08.005 · 3.96 Impact Factor
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    ABSTRACT: This study aimed to evaluate the serodiagnostic potential of immunoglobulin G (IgG) responses to Mycobacterium tuberculosis (M. tb) antigens in pulmonary tuberculosis (TB) patients, recent TB contacts with latent TB infection (LTBI), and healthy subjects. Infections were assessed using tuberculin skin tests, QuantiFERON-TB Gold In-Tube tests, drug susceptibility testing, and molecular genotyping of clinical isolates, and serum IgG responses to selective M. tb antigens including the 38 kDa and 16 kDa antigens, lipoarabinomannan (LAM), and recombinant early secreted antigen target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) were determined. We found that serum IgG responses to all antigens could differentiate between active TB and LTBI, with LAM having the highest diagnostic value (AUC=0.7756, P<0.001). Recurrent TB cases showed significantly higher IgG responses to 38 kDa, CFP-10 (P<0.01), and LAM (P<0.05) than new cases, and male patients had higher levels of antigen-specific IgG, as compared with females (P<0.05). Conversely, drug resistance and patient body mass index did not affect IgG responses (P>0.05). LAM-specific IgG responses differentiated between AFB-smear positive and negative patients (P<0.01), whereas antigen-specific IgG responses did not vary with M. tb genotype (P>0.05). Significantly higher IgG responses to 38 kDa and 16 kDa were observed in AFB-smear negative patients, as compared with controls. These results suggest that assessment of serum IgG responses to selective purified M. tb antigens may help improve diagnosis of active TB, particularly for sputum-smear negative patients or recurrent cases, and these may also help to differentiate between active TB and LTBI. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Journal of Clinical Microbiology 01/2015; 53(3). DOI:10.1128/JCM.03050-14 · 4.23 Impact Factor
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    ABSTRACT: Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. The diagnosis of diseases caused by NTM is difficult because NTM species are prevalent in the environment and because they have fastidious properties. In the present study, we evaluated 279 clinical isolates grown in liquid culture provided by The Catholic University of Korea, St. Vincent's Hospital using real-time PCR based on mycobacterial rpoB gene sequences. The positive rate of real-time PCR assay accurately discriminated 100% (195/195) and 100% (84/84) between MTB and NTM species. Comparison of isolates identified using the MolecuTech REBA Myco-ID(®) and Real Myco-ID® were completely concordant except for two samples. Two cases that were identified as mixed infection (M. intracellulare-M. massiliense and M. avium-M. massiliense co-infection) by PCRREBA assay were only detected using M. abscessus-specific probes by Real Myco-ID(®). Among a total of 84 cases, the most frequently identified NTM species were M. intracellulare (n=38, 45.2%), M. avium (n=18, 23.7%), M. massiliense (n=10, 13.2%), M. fortuitum (n=5, 6%), M. abscessus (n=3, 3.9%), M. gordonae (n=3, 3.9%), M. kansasii (n=2, 2.4%), M. mucogenicum (n=2, 2.4%), and M. chelonae (n= 1, 1.2%). Real Myco-ID(®) is an efficient tool for the rapid detection of NTM species as well as MTB and sensitive and specific and comparable to conventional methods.
    The Journal of Microbiology 01/2015; 53(1):38-46. DOI:10.1007/s12275-015-4495-8 · 1.53 Impact Factor
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    ABSTRACT: Mycobacterium tuberculosis (Mtb) is the major causative agent of tuberculosis (TB). The IFN-γ release assay (IGRA) has been widely used to diagnose TB by testing cell-mediated immune responses but has no capacity for distinguishing between active TB and latent TB infection (LTBI). This study aims to identify a parameter that will help to discriminate active TB and LTBI. Whole blood samples from 33 active TB patients and 20 individuals with LTBI, and 26 non-TB controls were applied to the commercial IFN-γ release assay QuantiFERON-TB Gold In Tube, and the plasma was analyzed for IL-2, IL-6, IL-8, IL-10, IL-13, TNF-α, IFN-γ, MIG, IP-10, I-TAG, and MCP-1 by using a commercial cytometric bead array. Mtb antigen-specific production of most of the assayed cytokines and chemokines was higher in active TB than in LTBI. Mitogen-induced responses were lower in the active TB than in LTBI group. When the ratio of TB-specific to mitogen-induced responses was calculated, IL-2, IL-6, IL-10, IL-13, TNF-α, IFN-γ, MIG, and IP-10 were more useful in discriminating active TB from LTBI. In particular, most patients showed higher IP-10 production to Mtb antigens than to mitogen at individual level, and the ratio for IP-10 was the strongest indicator of active infection vs. LTBI with 93.9% sensitivity and 90% specificity. In conclusion, the ratio of TB-specific to mitogen-induced IP-10 responses showed the most promising accuracy for discriminating active TB vs. LTBI, and should be further studied whether it can serve as a biomarker that can help clinicians administer appropriate treatments. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Journal of Clinical Microbiology 11/2014; 53(2). DOI:10.1128/JCM.02758-14 · 4.23 Impact Factor
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    ABSTRACT: Background The aim of this study was to evaluate a newly developed PCR-based reverse blot hybridization assay (PCR-REBA), REBA Sepsis-ID (M&D, Wonju, Korea), to rapidly detect the presence of bacteremia and antimicrobial resistance gene in blood culture samples. Methods One thousand four hundred consecutive blood culture samples from patients with a delta neutrophil index greater than 2.7% were selected from March to July in 2013. Three hundred positive and 1,100 negative for bacterial growth in blood culture bottles samples were tested by conventional and real-time PCR-REBA, respectively. Results The overall agreement between the conventional identification test and the REBA Sepsis-ID test was 95.3% (286/300). Agreement for gram-positive bacteria, gram-negative bacteria, fungi, and polymicrobials was 94.5% (190/201), 97.3% (71/73), 100% (14/14), and 91.7% (11/12), respectively. The detection rate of the mecA gene from methicillin-resistant Staphylococcus isolates was 97.8% (90/92). The vanA gene was detected in one blood culture sample from which vancomycin-resistant Enterococcus was isolated. When the cycle threshold for real-time PCR was defined as 30.0, 2.4% (26/1,100) of negative blood culture samples tested positive by real-time PCR. Conclusions The REBA Sepsis-ID test is capable of simultaneously and quickly detecting both causative agents and antimicrobial resistance genes, such as mecA and van, in blood culture positive samples.
    Annals of Laboratory Medicine 11/2014; 34(6):446-55. DOI:10.3343/alm.2014.34.6.446 · 1.48 Impact Factor
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    ABSTRACT: AimsThe purpose of this study was to evaluate the usefulness of REBA Myco-ID® assay based on the principle of reverse blot hybridization assay (REBA) for the rapid detection of Mycobacterium tuberculosis (MTB), the differentiation of MTB from nontuberculous mycobacteria (NTM), and the identification of NTM species in liquid cultures.Methods and ResultsA total of 274 mycobacteiral liquid cultures that cultured from respiratory specimens including 94 AFB smear-positives and 180 AFB smear-negatives were used in this study. The results of PCR-REBA were compared with those of real-time PCR and PCR-RFLP assays. Sensitivity of 195 MTB and 79 NTM strains was determined using DNA isolated from liquid cultures was 100% and 97.5%, respectively. However, two of the liquid culture NTM specimens were not detected by PCR-REBA, and were identified as Rhodococcus jostii and R. erythropolis by PRA Myco-ID and rpoB sequence analysis. Frequently identified NTM species in the liquid cultures were M. avium complex (n = 50, 63.3%) and M. abscessus complex (n =11, 13.9%). The PCR-REBA is able to identify mycobacteiral strains more rapidly than conventional tests, and does not require specialized instruments and is possible to differentiate between M. abscessus and M. massiliense strains.ConclusionsPCR-REBA assay showed rapid, highly sensitive, and specific results for the identification of MTB and NTM, and discriminated between NTM species from mycobacterial liquid cultures.Significance and Impact of the StudyEven though the use of molecular diagnostic technologies is more expensive than the use of conventional methods, the clinical and economic benefit of saving time in regard to expenses remains to be elucidated. Therefore, the PCR-REBA assay may provide the essential information to accelerate therapeutic decisions for appropriate antibiotic treatments in the acute phase of mycobacterial diseases.This article is protected by copyright. All rights reserved.
    Journal of Applied Microbiology 10/2014; DOI:10.1111/jam.12670 · 2.39 Impact Factor
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    ABSTRACT: Accurate and rapid diagnosis of dermatophytosis, a disease whose prevalence has been steadily increased, is important for successful treatment. Current laboratory methods for diagnosing dermatophytosis rely on KOH mount and fungal culture method. However, these methods have low sensitivity and are time-consuming (2~4 weeks to diagnosis). In our previous study, a rapid molecular diagnostic assay (PCR-reverse blot hybridization assay, REBA) was developed to identify the following 6 main species of dermatophytes: Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Microsporum canis, M. gypseum, and Epidermophyton floccosum. However, the REBA required more evaluation to validate its use in clinical examinations. The aim of the present study was to evaluate and validate the ability of the PCR-REBA to successfully identify dermatophytes in clinical isolates from dermatophytosis patients. Both conventional identification methods and the PCR-REBA were used to assess the presence of species of dermatophytes in 148 clinical isolates. The results of the two approaches were compared, and discrepancies between the two approaches were resolved by fungal ITS1 sequence analysis. T. rubrum was the most prevalent dermatophyte identified by conventional identification methods (118/148, 79.7%) and the PCR-REBA (131/148, 88.4%). The overall rate of consistency between conventional identification methods and the PCR-REBA was 79.0% (117/148 samples). Fungal ITS1 sequence analysis showed that PCR-REBA results were correct for 93.5% (29/31) of the discrepant samples. The PCR-REBA is rapid, sensitive, and highly specific compared with conventional identification methods. Thus, the PCR-REBA is a potentially useful tool for identifying dermatophytes in clinical settings.
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    Sunghyun Kim, Sangjung Park, Hyeyoung Lee
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    ABSTRACT: Tuberculosis (TB) is one of the major global health problems and it has been estimated that in 5~10% of Mycobacterium tuberculosis (MTB)-infected individuals, the infection progresses to an active disease. Numerous cytokines and chemokines regulate immunological responses at cellular level including stimulation and recruitment of wide range of cells in immunity and inflammation. In the present study, the mRNA expression levels of eight host immune markers containing of IFN-γ, TNF-α, IL-2R, IL-4, IL-10, CXCL9, CXCL10, and CXCL11 in whole blood cells from active pulmonary TB patients were measured after T-cell mitogen (PHA) and MTB specific antigens (ESAT-6, CFP-10, and TB7.7). Among the TH1-type factors, IFN-γ mRNA expression was peaked at 4 h, TNF-α and IL-2R mRNA expression was significantly high at the late time points (24 h) in active TB patients, TH2-type cytokine (IL4 and IL10) mRNA expression levels in both active TB and healthy controls samples did not changed significantly, and the mRNA expression of the three IFN-γ-induced chemokines (CXCL9, CXCL10, and CXCL11) were peaked at the late time points (24 h) in active TB patients after MTB specific antigen stimulation. In conclusion, the mRNA expression patterns of the TB-related immune markers in response to the T-cell mitogen (PHA) differed from those in response to MTB specific antigens and these findings may helpful for understanding the relationship between MTB infection and host immune markers in a transcripts level.
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    ABSTRACT: Breast cancer patients who have a positive result for HER2 overexpression are commonly treated with Herceptin, a HER2-targeted therapy. In the present study, the BrightGen HER2 RT-qDx (Syantra, Calgary, Canada), which is based on a one-tube nested RT-qPCR method that detects HER2 mRNA overexpression, was clinically evaluated in a total of 237 formalin-fixed paraffin-embedded (FFPE) tissue samples from breast cancer patients. Among the 38 HER2 positive samples, which were determined via IHC/FISH methods, 13 samples out of 16 (81.3%) that were IHC2 +/FISH + and 22 samples out of 22 (100%) that were IHC3 + have been decided positive for HER2 expression via the RT-qPCR method. The true positivity and false positivity results for the RT-qPCR were 92% (35/38) and 2% (1/65), respectively. The concordance between RT-qPCR and IHC results and RT-qPCR and IHC/FISH was 87.2% and 92.1%, respectively. Conclusively, the BrightGen HER2 RT-qDx may be a reliable and convenient method that can supplement traditional IHC and FISH methods for efficient use of trastuzumab.
    Experimental and Molecular Pathology 09/2014; 97(3). DOI:10.1016/j.yexmp.2014.09.011 · 2.88 Impact Factor
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    ABSTRACT: Circulating tumor cells (CTCs) are an independent prognostic factor for patients with breast cancer. However, the role of CTCs in early breast cancer management is not yet clearly defined. The aim of this study was to isolate and characterize CTCs in blood sample of a breast cancer patient as a biomarker for monitoring treatments efficacy. In this study, 692 blood samples from 221 breast cancer patients and 376 healthy individuals was used to detect CTCs with multiple markers including epithelial cell adhesion molecule (EpCAM), cytokeratin (CK) 19, human epidermal growth factor (HER) 2, Ki67, human telomerase reverse transcriptase (hTERT), and vimentin using quantitative real-time PCR (RT-qPCR). A total of 153 (69.2%) blood samples of 221 patient with breast cancer were found to be positive signal for at least one of the cancer-associated marker gene before treatment. After chemotherapy, no CTC were found in 28 (33.3%) of the 84 blood samples analyzed for the presence of CTCs using the RT-qPCR, whereas 56 (66.7%) blood samples were still found to be positive for at least one of the markers. After completing the therapy, the CTC positivity rate decreased to 7 (20.6%) in the neoadjuvant group, whereas 7 (14%) cases increased in the adjuvant group. There was no statistically significant relationship between TNM stage and detection of CTC-related markers. Data from this study suggest that RT-qPCR assay for the detection of CTC markers might be useful in selecting appropriate therapeutics and for monitoring treatment efficacy in breast cancer patients.
    Experimental and Molecular Pathology 09/2014; 97(3). DOI:10.1016/j.yexmp.2014.09.003 · 2.88 Impact Factor
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    ABSTRACT: Rapid, accurate detection of Mycobacterium tuberculosis is crucial in the diagnosis of tuberculosis (TB), but conventional diagnostic methods have limited sensitivity and specificity or are time consuming. A new highly sensitive nucleic acid amplification (NAA) test, combined nested and real-time PCR in a single tube (one-tube nested real-time PCR), was developed for detecting M. tuberculosis, which takes advantages of two PCR techniques, i.e., nested PCR and real-time PCR. One-tube nested real-time PCR was designed to have two sequential reactions with two sets of primers and dual probes for the IS6110 sequence of M. tuberculosis in a single closed tube. The minimum limits of detection of IS6110 real-time PCR and IS6110 one-tube nested real-time PCR were 100 fg/μL and 1 fg/μL of M. tuberculosis DNA, respectively. AdvanSure TB/NTM real-time PCR, IS6110 real-time PCR, and one-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and non-tuberculous mycobacteria (NTM) isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR, single IS6110 real-time PCR, and one-tube nested real-time PCR were 91% (152/167), 94.6% (158/167), and 100% (167/167) for sputum specimens, respectively. In conclusion, IS6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation.
    Diagnostic Microbiology and Infectious Disease 08/2014; 80(4). DOI:10.1016/j.diagmicrobio.2014.08.009 · 2.57 Impact Factor
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    ABSTRACT: Recent research has shown that oncogenic human papillomavirus (HPV) DNA, which is currently used in the screening and diagnosis of cervical cancer, can be detected not only in high-grade cervical lesions, but also in low-grade cervical lesions and normal tissues. For this reason, HPV tests targeting the E6 and E7 mRNA of five oncogenic HPV strains (HPV genotypes 16, 18, 31, 33, and 45), which are known to be responsible for the oncogenesis of cervical cancer, have been commercialized using a real-time nucleic acid sequence based amplification (NASBA) assay. Previous data has shown that the real-time NASBA assay has higher clinical specificity than HPV DNA testing (97.1% vs. 53.7%). However, the sensitivity of the real-time NASBA assay was lower than that of HPV DNA testing (41.1% vs. 100%). Despite the fact that there are more than 16 oncogenic HPV genotypes known to cause cervical cancer (HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 69), the commercialized real-time NASBA kit was designed to detect only five genotypes (16, 18, 31, 33, and 45). Therefore, in the present study, CervicGen HPV RT-qDX (Optipharm), a commercial diagnostic kit targeting a HPV E6/E7 mRNA based on RT-qPCR assay was evaluated with RNA extracted from ThinPrep® Pap samples, and the results were compared to real-time NASBA data. The sensitivity and specificity of the RT-qPCR assay were 92% and 98.6%, respectively, for the detection of cervical intraepithelial neoplasia CIN2(+) high-grade cervical lesions. Therefore, the CervicGen HPV RT-qDX assay showed a significantly higher sensitivity (91.1%) compared to the real-time NASBA assay (41.1%). In normal cytohistology cases, the specificity was 98.6% and 53.7% for HPV mRNA RT-qPCR and HPV DNA testing, respectively. These results demonstrate that HPV mRNA RT-qPCR better reflects clinical diagnosis. In conclusion, it is suggested that HPV mRNA RT-qPCR overcomes the shortcomings of lower specificity seen in the DNA assay and the lower sensitivity of the commercialized HPV mRNA real-time NASBA assay when testing from ThinPrep® Pap samples.
    Experimental and Molecular Pathology 08/2014; DOI:10.1016/j.yexmp.2014.08.004 · 2.88 Impact Factor
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    ABSTRACT: This study aims to evaluate the clinical performance of the NucliSENS EasyQ assay and compare it with HPV DNA genotyping for the detection of high-grade squamous intraepithelial lesions (HSIL) and cancer in a Korean population. In 188 total Thin Prep samples, the remaining fluid after cytology slide preparation was tested with Goodgene HPV DNA chips and the NucliSENS EasyQ HPV E6/E7 mRNA assay. The sensitivity and specificity of each test were calculated with HSIL and squamous cell carcinoma (SCC) as the disease endpoint. Out of the 188 samples, 139 (74%) were positive for DNA of 14 HPV types, while 57 (30%) cases were positive for E6/E7 mRNA. The DNA test was positive in cytology cases of SCC, HSIL, and atypical squamous cell. The mRNA test yielded results of 75%, 74%, 60%, 56%, and 29% positivity in abnormal cytology cases of SCC, HSIL, atypical squamous cells – cannot exclude HSIL (ASC-H), atypical squamous cells of undetermined significance (ASC-US), and low-grade squamous intraepithelial lesion (LSIL), respectively. In normal cytology cases, the positivity rates were 9% and 53% for the mRNA and DNA tests, respectively. For detection of HSIL and SCC, the sensitivity of the mRNA test was 74.36% and that of the DNA test was 100%, while the specificities of the tests were 85% and 40.83%, respectively. These findings suggest that the HPV E6/E7 mRNA assay can overcome the shortcoming of low specificity of DNA assays for clinical detection of high-grade cervical lesions and malignancies.
    Diagnostic Microbiology and Infectious Disease 08/2014; 79(4). DOI:10.1016/j.diagmicrobio.2014.04.004 · 2.57 Impact Factor
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    ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of blood stream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA® and -MRCoNS® multiplex real-time PCR assay (M&D, Republic of Korea) uses the following TaqMan® probes: 16S rRNA for Staphylococcus species, the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10(3) CFU/mL per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous-monitoring blood-culture system. The results obtained by the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility test methods except for one organism. Out of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166), 97.2% (35/36), and 99.2% (117/118) for 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA® and -MRCoNS® multiplex real-time PCR assay is very useful for the rapid and accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA® and -MRCoNS® multiplex real-time PCR assay could have an important impact on choosing the appropriate antimicrobial therapy based on detection of the mecA gene.
    Journal of clinical microbiology 03/2014; 52(6). DOI:10.1128/JCM.00389-14 · 4.23 Impact Factor
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    ABSTRACT: Sepsis is one of the main causes of mortality and morbidity. The rapid detection of pathogens in blood of septic patients is essential for adequate antimicrobial therapy and better prognosis. This study aimed to accelerate the detection and discrimination of Gram-positive (GP) and Gram-negative (GN) bacteria and Candida species in blood culture samples by molecular methods. The Real-GPR, -GNR, and -CANR real-time PCR kit (M&D, Wonju, Republic of Korea) assays use the TaqMan probes for detecting pan-GP, pan-GN, and pan-Candida species, respectively. The diagnostic performances of the real-time PCR kits were evaluated with 115 clinical isolates, 256 positive and 200 negative blood culture bottle samples, and the data were compared to results obtained from conventional blood culture. Eighty-seven reference strains and 115 clinical isolates were correctly identified with specific probes corresponding to GP-bacteria, GN-bacteria and Candida, respectively. The overall sensitivity and specificity of the real-time PCR kit with blood culture samples were 99.6% and 89.5%, respectively. The Real-GPR, -GNR, and -CANR real-time PCR kits could be useful tools for the rapid and accurate screening of bloodstream infections (BSIs).
    Annals of Clinical Microbiology and Antimicrobials 01/2014; 13(1):3. DOI:10.1186/1476-0711-13-3 · 1.51 Impact Factor