Hyeyoung Lee

Yonsei University, Sŏul, Seoul, South Korea

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Publications (55)130.48 Total impact

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    ABSTRACT: Background: Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide and it is responsible for most cases of cervical uterine cancer. Although HPV infections of the cervix do not always progress to cancer, 90% of cervical cancer cases have been found to be associated with high risk HPV (HR- HPV) infection. HPV DNA testing is widely used, along with Papanicolaou (Pap) testing, to screen for cervical abnormalities. However, there are no data on the prevalence of genotype-specific HPV infections assessed by measuring HPV E6/E7 mRNA in women representative of the Chinese population across a broad age range. Materials and methods: In the present study, we compared the results with the CervicGen HPV RT-qDx assay, which detects 16 HR-HPV genotypes (Alpha-9: HPV 16, 31, 33, 35, 52, and 58; Alpha-7: HPV 18, 39, 45, 51, 59, and 68; and Alpha-5, 6: HPV 53, 56, 66, and 69), and the REBA HPV-ID assay, which detects 32 HPV genotypes based on the reverse blot hybridization assay (REBA) for the detection of oncogenic HPV infection according to cytological diagnosis. We also investigated the prevalence and genotype distribution of HPV infection with a total of 324 liquid-based cytology samples collected in western Shandong province, East China. Results: The overall HPV prevalences determined by HPV DNA and HPV E6/E7 mRNA assays in this study were 79.9% (259/324) and 55.6% (180/324), respectively. Although the positivity of HPV E6/E7 mRNA expression was significantly lower than HPV DNA positivity, the HPV E6/E7 mRNA assay showed greater specificity than the HPV DNA assay (88.6% vs. 48.1%) in normal cytology samples. The prevalence of Alpha-9 (HPV 16, 31, 33, 35, 52, and 58) HPV infection among these women accounted for up to 80.3% and 76.1% of the high-grade lesions detected in the HPV mRNA and DNA tests, respectively. The HR-HPV genotype distribution, based on HPV DNA and E6/E7 mRNA expression by age group in patients with cytologically confirmed lesions, was highest in women aged 40 to 49 years (35.9% for cytologically confirmed cases, Pearson correlation r value=0.993, p<0.001) for high-grade lesions. Among the oncogenic HR-HPV genotypes for all age groups, there was little difference in the distribution of HPV genotypes between the HPV DNA (HPV -16, 53, 18, 58, and 33) and HPV E6/E7 mRNA (HPV -16, 53, 33, 58, and 18) assays. HPV 16 was the most common HPV genotype among women with high- grade lesions. Conclusions: Our results suggest that the HPV E6/E7 mRNA assay can be a sensitive and specific tool for the screening and investigation of cervical cancer. Furthermore, it may provide useful information regarding the necessity for early cervical cancer screenings and the development of additional effective HPV vaccines, such as one for HPV 53 and 58. Additionally, gaining knowledge of HPV distribution may also inform us about ecological changes in HPV after the vaccination.
    Full-text · Article · Dec 2015 · Asian Pacific journal of cancer prevention: APJCP
  • Hye-Young Wang · Hyunjung Kim · Eung Ho Choi · Hyeyoung Lee
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    ABSTRACT: Aims: The aim of this study was to evaluate the performance of a commercially available multiplex RT-PCR assay for the rapid detection and identification of dermatophytes directly from clinical samples and cultures. Methods and results: The multiplex RT-PCR assay was used to evaluate 118 clinical isolates from various specimen types and a total of 140 known specimens were compared with both conventional, commercially-available PCR-REBA, and ITS sequencing assays. In this study, multiplex RT-PCR yield significantly more positive results than culture (91.9% vs. 39.5%) and conventional methods including KOH microscopy (91.9% vs. 71.3%). Although the results among the RT-PCR, PCR-REBA, and ITS sequence analysis assays were concordant (100%) in 118 clinical isolates, concordant results between RT-PCR and culture were at 66% (78/118). The overall positive rates for the PCR-REBA, RT-PCR, and ITS sequence analysis were 98.8%, 91.9%, and 52.9%, respectively. In addition, the concordance rate of RT-PCR and the PCR-REBA assay was 93% (95% confidence interval [CI], 89.9-96.1, p<0.0001), 93.7% (95% CI, 90.5-96.4, p<0.0001) sensitivity, 100% (95% CI, 80.0-100, p<0.0001) specificity, and 99.6% positive and 81% negative predictive values, respectively. Among the 258 samples, the most frequently identified dermatophyte species were T. rubrum (n=199, 77.1%) and T. mentagrophytes (n=28, 10.9%). Conclusions: The entire RT-PCR procedure takes about 3 h, while results from culture can take up to 2 to 3 weeks. The use of the multiplex RT-PCR molecular diagnostic assay was rapid and reliable for detecting pathogen infections. Significance and impact of the study: Even though the use of molecular diagnostic technology is more expensive than conventional methods, the clinical and economic benefit of saving time relative to expense remains to be elucidated. Therefore, the RT-PCR assays may provide the essential information to accelerate therapeutic decisions for earlier and adequate antibiotic treatment in the acute phase of fungal pathogen infections. This article is protected by copyright. All rights reserved.
    No preview · Article · Nov 2015 · Journal of Applied Microbiology
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    ABSTRACT: Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the —subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507AGC, 513GTG, 516TAT, 531ATG, and 531TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae. © 2015, The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.
    No preview · Article · Oct 2015 · The Journal of Microbiology
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    ABSTRACT: Rapid and accurate identification of a broad range of bacterial and fungal pathogens is the key to successful management of patients with bloodstream infections (BSIs). The aim of this study was to evaluate the diagnostic performance of PCR-REBA Sepsis-ID test for the detection of BSIs pathogens. EDTA anticoagulated blood for REBA Sepsis-ID assay and blood culture samples from 882 patients with suspected sepsis were simultaneously collected from January 2014 to December 2014. Of 115 patients with positive blood culture, 64 (55.7%) were gram-positive bacteria, 35 (30.4%) were gram-negative bacteria, 1 (0.9%) was Candida albicans, and 15 (13.0%) were polymicrobial infections. The concordance rate of blood culture system and PCR-REBA Sepsis ID test was 83.0% (95% confidence interval [CI], 79.8-84.8, p<0.0001). Compared to blood culture the diagnosis of bacterial proven pathogens by PCR-REBA revealed 81.0% (95% CI, 73.4-86.8, p<0.0001) sensitivity, 83.4% (95% CI, 80.0-85.4, p<0.0001) specificity, 80.9% positive and 95.8% negative predictive values, respectively. In 10 cases with PCR-REBA positive but blood culture negative, the levels of C-reactive protein were significantly elevated 18.5 mg/dL (SD±13.7, 95% CI 1.8-41.9) and six cases has been proven to have pathogen by bacterial 16S rRNA sequencing. Although the sensitivity for pathogen identification was not significantly different between PCR-REBA and blood culture (P=0.5), the combination of the two methods resulted in a significantly increased rate of pathogen detection (P=0.002). The results of this study suggested that PCR-REBA may be helpful when added to blood culture in the diagnosis and management of sepsis. PCR-REBA Sepsis-ID test is a useful tool for the rapid identification of pathogenic isolates in whole blood to ensure adequate treatment for the causative agents of BSIs. Although the cost of molecular diagnostic assays is higher than the cost of conventional methods, clinical and economic cost-benefit analysis is still needed. PCR-REBA may provide essential information for accelerating therapeutic decisions to ensure effective treatment with antibiotics in the acute phase of pathogen infection. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Aug 2015 · Journal of Applied Microbiology
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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.
    Full-text · Article · Jun 2015 · International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases
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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%).
    Full-text · Article · Mar 2015 · Experimental and Molecular Pathology
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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.
    Full-text · Article · Mar 2015 · American Journal of Clinical Pathology

  • No preview · Article · Feb 2015
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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.
    Full-text · Article · Feb 2015 · International Journal of Clinical Oncology
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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.
    Full-text · Article · Jan 2015 · The Journal of molecular diagnostics: JMD
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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.
    Preview · Article · Jan 2015 · Journal of Clinical Microbiology
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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.
    Full-text · Article · Jan 2015 · The Journal of Microbiology
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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.
    Full-text · Article · Nov 2014 · Journal of Clinical Microbiology
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    ABSTRACT: Detection of human epidermal growth factor receptor 2 gene (HER2, also known as erbB2) expression is a preparatory process to decide a treatment strategy for breast cancer patients. 20-30% of breast cancer patients have HER2 overexpression, and they usually show poor recovery rate. For detection of HER2 expression, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods are conventionally used. Although these methods are accurate and reliable, their time-consuming process and high cost need a concise method with high sensitivity and accuracy. As a complementary method to the current IHC/FISH standard techniques, PCR-based methods have been developed. Here we employed a quantitative PCR method to detect HER2 expression in one hundred ninety nine formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue samples from the patients treated over two years at the Yonsei University Severance Hospital, Republic of Korea. Relative expression of HER2 mRNA in the FFPE samples was analyzed using a quantitative RT-PCR (RT-qPCR) method and the obtained HER2 expression levels were compared with those from IHC/FISH methods. Our results show that the RT-qPCR method was highly concordant with IHC/FISH methods for detecting HER2 expression. Overall sensitivity and specificity of the BrightGen HER2 RT-qDx assay kit (Syantra, Calgary, Canada), which is a kit we used for RT-qPCR analyses, were 93.0% and 89.8% (P < 0.0001), respectively. The diagnostic cut-off value of HER2 RT-qDx for the clinical samples was determined by likelihood ratio, among which the highest likelihood ratio of relative HER2 mRNA levels was over 105.5 (AUC = 0.9466) with the highest sensitivity and specificity. Our study indicates that quantification of HER2 mRNA expression with the RT-qPCR could be an alternative method of conventional IHC/FISH methods.
    Full-text · Article · Nov 2014 · International journal of clinical and experimental pathology
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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.
    Full-text · Article · Nov 2014 · Annals of Laboratory Medicine
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    ABSTRACT: Breast cancer is a significant cause of death in women. Estrogen receptor (ER) and progesterone receptor (PR) are important prognostic factors indicating higher recovery rate in the breast cancer patients. Currently, immunohistochemical (IHC) staining is a conventional method to identify expression of ER and PR. If a breast cancer patient expresses ER or PR, a chemotherapy with estrogen inhibitors such as tamoxifen is supposed to be effective. Although IHC staining is a reliable method, it may not a useful method for continuous monitoring of ER and PR expression changes in multiple breast cancer patients. In the present study, we evaluated an alternative method of IHC for detection of ER and PR expression. A quantitative RT-PCR method called 'the BrightGen HR RT-qDx assay' was employed to detect mRNA expression of the nuclear receptors in 199 formalin-fixed paraffin-embedded (FFPE) breast cancer tissue samples. Among the ER/PR positive samples by IHC, 83 were determined positive and 16 were determined negative for the nuclear receptor mRNA by the quantitative RT-PCR method. Among the ER/PR negative samples by IHC, 37 were determined negative and 2 were determined positive by the quantitative RT-PCR method. The overall sensitivity and specificity of the quantitative RT-PCR method were 83.8% and 94.8% (P = 0.0026), respectively. We also optimized the quantitative RT-PCR method by setting up the diagnostic cut-off value using the likelihood ratio. The highest likelihood ratio was when the expression levels of the relative nuclear receptor mRNA passed 103.3 at which sensitivity and specificity was highest. These data suggest that BrightGen HR RT-qDx assay could be an alternative method for detection of the prognostic factors of nuclear receptors expressed in breast cancer patients for providing essential information for therapeutic application of tamoxifen.
    Full-text · Article · Oct 2014 · International journal of clinical and experimental pathology
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    ABSTRACT: AimsThe purpose of this study was to evaluate the usefulness of REBA Myco-ID (R) 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 mycobacterial liquid cultures that cultured from respiratory specimens including 94 acid-fast bacilli (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-Restriction fragment length polymorphism (RFLP) assays. Sensitivity of 195 MTB and 79 NTM strains determined using DNA isolated from liquid cultures was 100% and 975%, 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 gene sequence analysis. Frequently identified NTM species in the liquid cultures were Mycobacterium avium complex (n=50, 633%) and Mycobacterium abscessus complex (n=11, 139%). The PCR-REBA is able to identify mycobacterial strains more rapidly than conventional tests and does not require specialized instruments and is possible to differentiate between Myc.abscessus and Mycobacterium 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 may provide the essential information to accelerate therapeutic decisions for appropriate antibiotic treatments in the acute phase of mycobacterial diseases.
    Full-text · Article · Oct 2014 · Journal of Applied Microbiology
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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.
    Full-text · Article · Sep 2014
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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.
    Full-text · Article · Sep 2014
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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.
    Full-text · Article · Sep 2014 · Experimental and Molecular Pathology