Analytical performance evaluation of the HPV OncoCheck assay for detection of high-risk HPV infection in liquid-based cervical samples.

Recent studies have demonstrated low specificity (false positive) of human papillomavirus (HPV) DNA testing for the screening and diagnosis of cervical samples. Therefore, we evaluated the performance of the HPV OncoCheck assay, which is an HPV E6/E7 mRNA-based assay, for the detection of 16 high-risk (HR)-HPVs including HPV 16 and HPV 18 genotypes in cervical samples using multiplex reverse transcriptase-quantitative PCR. In the present study, the analytical performance of the assay was evaluated using 16 HPV single strand DNAs. Clinical evaluation was performed using 319 Thinprep® liquid-based cytology samples obtained from women with cervical diseases, and the HPV OncoCheck assay results were compared with those of cytological diagnosis and sequence analysis. All 16 types of HPVs were detected with a minimum detection sensitivity of 100 copies per reaction and high specificity was observed. The sensitivity and specificity of the HPV OncoCheck assay for detecting high-grade lesions were 94.1% (95% confidence interval (CI), 0.875-0.975; p < .0001) and 95.4% (95% CI, 0.868-0.989; p < .0001), and sequence analysis were 99.4% (95% CI, 0.965-0.999; p < .0001), and 98% (95% CI, 0.939-0.996; p < .0001), respectively. Moreover, the agreement between the HPV OncoCheck assay and sequence analysis for the detection of HR-HPV was 98.8% (κ = 0.98, 95% CI 0.967-0.996; p < .0001). The results of this study showed high agreement and specificity with cytological diagnoses and sequence analysis. Future studies with histological follow- up are needed to determine whether use of the HPV OncoCheck assay in cervical screening may aid detection of the most significant cervical disease while reducing false-positive results.

MiR-9, miR-21, and miR-155 as potential biomarkers for HPV positive and negative cervical cancer.

BACKGROUND: Cervical cancer is the second leading cause of death among female patients with cancer in the world. High risk human papillomavirus has causal roles in cervical cancer initiation and progression by deregulating several cellular processes. However, HPV infection is not sufficient for cervical carcinoma development. Therefore, other genetic and epigenetic factors may be involved in this complex disease, and the identification of which may lead to better diagnosis and treatment. Our aim was to analyze the expression of microRNAs in cervical cancer cases positive or negative for HPV E6/E7 mRNA, and to assess their diagnostic usefulness and relevance. METHODS: The expression of three different microRNAs (miR-9, miR-21, and miR-155) in 52 formalin-fixed paraffin-embedded (FFPE) primary cervical cancer tissue samples and 50 FFPE normal cervical tissue samples were evaluated. RESULTS: MiR-9, miR-21, and miR-155 were significantly overexpressed in cervical cancer tissues compared to normal tissues (P < 0.001). MiR-21 and miR-155 expression combined with the HPV E6/E7 mRNA assay in HPV E6/E7 negative cervical cancer showed increased AUC of 0.7267 and 0.7000, respectively (P = 0.01, P = 0.04), demonstrating their potential as diagnostic tools. Moreover, miR-21 and miR-155 were predictors showing a 7 fold and 10.3 fold higher risk for HPV E6/E7 negative patients with cervical cancer (P = 0.024 and P = 0.017, respectively) while miR-155 was a predictor showing a 27.9 fold higher risk for HPV E6/E7 positive patients with cervical cancer (P < 0.0001). CONCLUSIONS: There is a strong demand for additional, alternative molecular biomarkers for diagnosis and management of precancer patients. MiR-21 and miR-155 may be helpful in the prediction of both HPV positive and HPV negative cases of cervical cancer.

Comparison of FFPE histological versus LBP cytological samples for HPV detection and typing in cervical cancer.

Human papillomavirus (HPV) infection is closely associated with cervical cancer. This study analyzed HPV genotype prevalence in 75 cases of formalin-fixed paraffin embedded (FFPE) tissue samples from patients diagnosed with cervical cancer. Genotype prevalence was assessed using Reverse Blot Assay (REBA) and quantitative polymerase chain reaction (qPCR), which target the HPV L1 and HPV E6/E7 genes, respectively. HPV DNA chip tests were also performed using liquid based preparation (LBP) cytological samples from the same patients who provided the FFPE histological samples. We observed a slight difference in HPV genotype distribution as assessed by DNA chip versus REBA. One possible explanation for this difference is that normal regions could be mixed with lesion regions when cytological samples are extracted from each patient with cancer. For the detection of moderate dysplasia, the main target of diagnosis, this difference is anticipated to be greater. We also made several unexpected observations. For example, HPV multi-infection was not detected. Moreover, the rate of HPV positivity varied radically depending on the cancer origin, e.g. squamous cell carcinoma versus adenocarcinoma. Our results imply that it is important to determine whether cytological specimens are suitable for HPV genotyping analysis and cervical cancer diagnosis. Future research on the mechanisms underlying cervical cancer pathogenesis is also necessary.

Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples.

BACKGROUND: Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods. METHODS: In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples. RESULTS: The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001). CONCLUSION: Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression.

Skin Barrier Function Is Not Impaired and Kallikrein 7 Gene Polymorphism Is Frequently Observed in Korean X-linked Ichthyosis Patients Diagnosed by Fluorescence in Situ Hybridization and Array Comparative Genomic Hybridization.

X-linked ichthyosis (XLI) is a recessively inherited ichthyosis. Skin barrier function of XLI patients reported in Western countries presented minimally abnormal or normal. Here, we evaluated the skin barrier properties and a skin barrier-related gene mutation in 16 Korean XLI patients who were diagnosed by fluorescence in situ hybridization and array comparative genomic hybridization analysis. Skin barrier properties were measured, cytokine expression levels in the stratum corneum (SC) were evaluated with the tape stripped specimen from skin surface, and a genetic test was done on blood. XLI patients showed significantly lower SC hydration, but normal basal trans-epidermal water loss and skin surface pH as compared to a healthy control group. Histopathology of ichthyosis epidermis showed no acanthosis, and levels of the pro-inflammatory cytokines in the corneal layer did not differ between control and lesional/non-lesional skin of XLI patients. Among the mutations in filaggrin (FLG), kallikrein 7 (KLK7), and SPINK5 genes, the prevalence of KLK7 gene mutations was significantly higher in XLI patients (50%) than in controls (0%), whereas FLG and SPINK5 prevalence was comparable. Korean XLI patients exhibited unimpaired skin barrier function and frequent association with the KLK7 gene polymorphism, which may differentiate them from Western XLI patients.

Diagnostic performance of HPV E6/E7, hTERT, and Ki67 mRNA RT-qPCR assays on formalin-fixed paraffin-embedded cervical tissue specimens from women with cervical cancer.

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%). The positivity rate of hTERT and Ki67 mRNA expressions in FFPE cervical cancer tissue samples on RT-qPCR was 65% and 93% respectively. Moreover, the positivity rates were 92% for a combination of HPV E6/E7 and hTERT mRNA expressions, 97% for HPV E6/E7 and Ki67 mRNA expressions, and 99% (99/100) for the combination of HPV E6/E7, hTERT, and Ki67 mRNA expressions. These data showed that SSC FFPE cervical cancer tissue samples correlated more strongly with high Ki67 mRNA expressions than with hTERT mRNA expressions. Notably, hTERT and Ki67 mRNA expression level was increased in high-grade cervical lesions, but was very low in normal samples. Our findings suggest that the combination of HPV E6/E7, hTERT, and Ki67 mRNA expression levels could be used in a complementary manner in diagnosing high-grade cervical lesions. Further studies are required to evaluate these assays as a useful predictive tool for screening low-grade cervical lesions.

Detection of circulating tumor cell-specific markers in breast cancer patients using the quantitative RT-PCR assay.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream 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 Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., 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 with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene.

Evaluation of a quantitative RT-PCR assay to detect HER2 mRNA overexpression for diagnosis and selection of trastuzumab therapy in breast cancer tissue samples.

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.

Detection of circulating tumor cells in patients with breast cancer using the quantitative RT-PCR assay for monitoring of therapy efficacy.

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 reverse transcription PCR (RT-qPCR). A total of 153 (69.2%) blood samples of 221 patients with breast cancer were found to be positive for at least one of the cancer-associated marker gene before treatment. After chemotherapy, no CTCs 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 this increased to 7 (14%) cases 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.

Performance of HPV E6/E7 mRNA RT-qPCR for screening and diagnosis of cervical cancer with ThinPrep Pap test samples.

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 91% 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.

Real-time PCR TaqMan assay for rapid screening of bloodstream infection.

BACKGROUND: 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. METHODS: The Real-GP®, -GN®, and -CAN® 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. RESULTS: 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. CONCLUSIONS: The Real-GP®, -GN®, and -CAN® real-time PCR kits could be useful tools for the rapid and accurate screening of bloodstream infections (BSIs).

PCR-reverse blot hybridization assay for screening and identification of pathogens in sepsis.

Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specific and 13 species-specific probes; it uses additional probes for antibiotic resistance genes, i.e., the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) and the vanA and vanB genes of vancomycin-resistant enterococci (VRE). The REBA Sepsis-ID test successfully identified clinical isolates and blood culture samples as containing Gram-positive bacteria, Gram-negative bacteria, or fungi. The results matched those obtained with conventional microbiological methods. For the REBA Sepsis-ID test, of the 115 blood culture samples tested, 47 (40.8%) and 49 (42.6%) samples were identified to the species and genus levels, respectively, and the remaining 19 samples (16.5%), which included five Gram-positive rods, were identified as Gram-positive bacteria, Gram-negative bacteria, or fungi. The antibiotic resistances of the MRSA and VRE strains were identified using both conventional microbiological methods and the REBA Sepsis-ID test. In conclusion, the REBA Sepsis-ID test developed for this study is a fast and reliable test for the identification of Gram-positive bacteria, Gram-negative bacteria, fungi, and antibiotic resistance genes (including mecA for MRSA and the vanA and vanB genes for VRE) in bloodstream infections.

Comparison of Multiplex Real-Time PCR and PCR-Reverse Blot Hybridization Assays for the Direct and Rapid Detection of Porcine Circovirus Type 2 Genotypes.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), poses a serious economic threat for the swine industry. Currently, PCV2 is classified into five major genotypes: PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. The aim of this study is to evaluate the performance of two commercially available methods, multiplex real-time PCR assay and PCR-reverse blot hybridization assay (REBA), for the rapid detection of PCV2 and direct identification of PCV2 genotypes from clinical samples as well as to compare the results with that of sequence analysis. Molecular diagnostic methods were used to evaluate a total of 180 samples, including tissues and blood samples from pigs that were suspected of PCVAD infection. The results of this study showed that the detection rate for positive PCV2 was 48.3% (n = 87) in both multiplex real-time PCR and PCR-REBA methods. Using sequence analysis, which is the gold standard, and multiplex real time PCR assay, the sensitivity, specificity, positive predictive value, and negative predictive value of PCV2 genotyping were found to be 97.1% (n = 67, 95% CI 0.894-0.998, p < 0.001), 100% (n = 93, 95% CI 0.966-1.000, p < 0.001), 100% (95% CI 0.953-1.000, p < 0.001), 97.9% (95% CI 0.921-0.998, p < 0.001), respectively. The results of PCR-REBA were found to be consistent with those of sequence analysis for all the samples and showed good agreement (κ = 1). The most prevalent genotypes detected in this study were PCV2d (n = 53, 60.9%), followed by PCV2a (n = 17, 19.5%), PCV2b (n = 14, 16.1%), and PCV2a/b co-infection (n = 3, 3.5%). Both the methods required ~3 h for completion. Therefore, we conclude that two molecular methods are rapid and reliable for the characterization of the causative pathogen with PCV2 genotypes.

One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples.

Porcine cytomegalovirus (PCMV) is a pathogen that must be removed from pigs for use as organ donors in xenotransplantation. Recently, it has been found that when donor pigs are infected with PCMV, a pig-to-non-human-primate xenotransplantation lower transplant survival by 2-3 times. Therefore, highly sensitive methods are needed to maintain designated pathogen free (DPF) pig and screen for xenografts. The purpose of this study was to evaluate the performance of commercially available method with one-tube nested real-time PCR assay to quickly detect PCMV infection in clinical samples and compare the results with those of sequence analysis. Molecular diagnostic methods were used to evaluate 127 samples, including tissues and blood samples from pigs suspected of PCMV infection. The detection rate for positive PCMV was 38.6% (n = 49), 23.6% (n = 30), and 12.6% (n = 16) in one-tube nested real-time PCR, nested PCR, and conventional PCR methods, respectively. All PCMV-positive samples in conventional PCR or nested PCR methods were also positive in the one-tube nested real-time PCR assay. All the PCR products in the three methods were checked for amplification of PCMV gene by PCR and subsequent direct sequencing. The results of one-tube nested real-time PCR were found to be consistent with those of sequence analysis for all the samples and showed good agreement (κ = 1). Our study found that the one-tube nested real-time PCR assay is more sensitive than the other two methods. This assay required approximately 1.5 h for completion. Therefore, we concluded that one-tube nested real-time PCR assay is a fast and reliable method for the characterizing pathogen responsible for PCMV infection.

Diagnostic performance of the E6/E7 mRNA-based Optimygene HR-HPV RT-qDx assay for cervical cancer screening.

OBJECTIVES: Pap smear and high-risk human papillomavirus (HR-HPV) DNA testing are the most widely applied methods for cervical cancer screening, but both methods are limited by their low specificity and lack of association with patient prognoses. The aim of this study was to compare the clinical and prognostic significance of HPV E6/E7 mRNA as an early biomarker with cytology and HPV DNA detection in cervical cancer screening. METHODS: This study evaluated the performance of the Optimygene HR-HPV RT-qDx assay, which is an HPV E6/E7 mRNA-based assay, to detect 16 HR-HPV subtypes: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 69. The clinical evaluation was conducted using 563 ThinPrep liquid-based cytology samples and the results were compared to those of cytological and histological diagnoses and HPV DNA testing. RESULTS: The clinical sensitivity and specificity of the Optimygene HR-HPV RT-qDx assay for the detection of high-grade lesions, according to cervical cytology, were 92.4% (95% confidence interval (CI) 0.9167-0.9972, p<0.0001) and 96.9% (95% CI 0.8632-0.9524, p<0.0001), respectively; they were 85.9% (95% CI 0.7631-0.9211, p<0.0001) and 82.5% (95% CI 0.7491-0.8825, p<0.0001), respectively, for CIN2+. This assay showed a higher specificity and positive predictive value for cytological and histological diagnosis than HPV DNA testing. Overall, the agreement between the Optimygene HR-HPV RT-qDx assay and HPV DNA testing in cytological and histological diagnosis was 87.9% (κ=0.76, 95% CI 0.7054-0.8128, p<0.0001) and 90.5% (k=0.81, 95% CI 0.7338-0.8878, p<0.0001), respectively. In this study, the most frequently detected HPV genotypes among HR-HPV-positive women were HPV 16 (37.9%), HPV 33-58 (21.5%), and HPV 18 (11.4%). CONCLUSIONS: These findings suggest that the higher specificity and positive predictive value of the Optimygene HR-HPV RT-qDx assay are valuable for predicting insignificant HPV DNA infections among patients with a borderline cytological diagnosis. This assay could be used to prevent unnecessary biopsy procedures and the over-referral of patients with transient HPV infections, as well as reduce patient anxiety during the follow-up period.

Identification of Mycobacterium tuberculosis and non-tuberculous mycobacteria from cutaneous sarcoidosis lesions by reverse blot hybridization assay.

While the etiology of sarcoidosis remains uncertain, mycobacteria have been suggested as a causative infectious agent. To investigate the causal relationship between mycobacteria and sarcoidosis, we performed a reverse blot hybridization assay (REBA) to identify mycobacteria from the skin samples of nine patients with sarcoidosis. Six of the nine samples were shown to be positive for mycobacteria by REBA, including Mycobacterium tuberculosis and non-tuberculous mycobacteria. This is the first study to identify mycobacteria from the skin samples of sarcoidosis patients using REBA, and our results could strengthen the etiologic association between mycobacteria and sarcoidosis.

Clinical Characteristics and Genetic Variations in Early-Onset Atopic Dermatitis Patients.

BACKGROUND: Hereditary factors contribute to atopic dermatitis (AD) development. We developed the reverse blot hybridization assay (REBA) kit to simultaneously detect variations in skin barrier- and immune response-related genes prevalent in Korean AD patients. OBJECTIVE: To identify genetic variations and clinical characteristics that could predict early AD development. METHODS: We compared AD-related genetic variations between early-onset AD subjects and non-AD controls, and clinical characteristics and genetic variations between early- and late-onset AD subjects. We compared 28 early-onset AD subjects and 57 non-AD controls from a birth cohort and 108 early- (age ≤3 years) and 90 late-onset AD subjects and 189 non-AD controls from a university hospital. Genetic variations were detected via REBA. RESULTS: There were no differences in AD-related genetic variation between early-onset AD subjects and non-AD controls in the birth cohort. When the birth cohort and hospital populations were combined, early-onset AD subjects and non-AD controls showed different frequencies of genetic variations of KLK7, SPINK5 1156, DEFB1, IL5RA, IL12RB1a, and IL12RB1b. No differences in the frequency of genetic variations were observed between early- and late-onset AD subjects. Immunoglobulin E positivity for house dust mites was prevalent in late-onset AD subjects. A family history of atopic diseases was associated with early-onset AD. CONCLUSION: No AD-related genetic variations could predict early AD development in Koreans, even though neonates with a family history of atopic diseases are likely to develop AD at ≤3 years of age. Environmental exposure may be more important than genetic variation in determining the onset age of AD.

HPV E6/E7, hTERT, and Ki67 mRNA RT-qPCR Assay for Detecting High-Grade Cervical Lesion with Microscope Slides.

After breast and colon cancer, cervical cancer is the third most common cancer of women worldwide. Since human papillomavirus (HPV) infection is known to be the predominant cause of cervical cancer, molecular HPV screening is currently used along with cytological and histological examination methods for precancer diagnosis. Nevertheless, the sensitivity of the current HPV test is less than 80%; thus, many cervical cancer cases are not able to be diagnosed by HPV screening alone, and likewise, patients with cervical cancer are often determined to be HPV-negative by the current screening methods. Therefore, human telomerase reverse transcriptase (hTERT) and Ki67 previously identified as cancer markers were attempted. And cervical exfoliated cells of high-grade squamous intraepithelial lesion (HSIL), the most severe precancerous lesion of cancer, were used in the study. However, it takes a long time to collect enough specimens to conduct statistical analysis. Therefore, in the present study, microscope slides, cervical exfoliated cells on glass slides, were attempted. The results of the analysis demonstrated that hTERT and Ki67 expression levels were useful in distinguishing between cancerous and normal specimens, exhibiting a higher sensitivity and specificity than conventional HPV E6/E7 testing. And the study suggests clinical slide cell samples could be effectively used in the context of retrospective studies to identify novel biomarkers.