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.

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.

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.

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.

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.

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.

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.

Clinical characteristics and genetic variation in atopic dermatitis patients with and without allergic contact dermatitis.

In patients with atopic dermatitis (AD), the risk of contact sensitization may be higher as the disrupted skin barrier may increase the penetration of contact allergens. Therefore, it is necessary to screen for concurrent allergic contact dermatitis (ACD) in AD patients. To identify the clinical characteristics and genetic variation in AD patients with concurrent ACD. In total, 281 AD subjects who underwent patch testing were included. Subjects with a positive result were classified as "AD with ACD", while the others were classified as "AD only". Clinical characteristics and prevalence of genetic variants (FLG 3321delA, FLG K4022X, KLK7, SPINK5, DEFB1, KDR, IL5RA, IL9, and IL12RB1) were compared between the two groups. Seventy-one subjects (25.3%) were found to have AD and ACD. Female sex, older age, late onset, self-reported personal or family history of ACD, and presence of prurigo nodularis were associated with concurrent ACD in AD patients. Age was useful for predicting concurrent ACD based on the receiver operating characteristic curve. However, no differences in the frequency of genetic variants were identified between the two groups. A personal or family history of ACD, late onset, and prurigo nodularis support a suspicion of concurrent ACD, although these correlations were less apparent after correcting for age and sex. Patch testing for AD in males >20 years and females >14 years may aid diagnosis of concurrent ACD with high sensitivity and specificity.

Direct Detection of Rifampin-Resistant Mycobacterium tuberculosis in Respiratory Specimens Using Quantamatrix Multiplexed Assay Platform (QMAP) System: A Multicenter Study in Korea.

Rapid and accurate detection of rifampin-resistant Mycobacterium tuberculosis (MTB) is of primary importance for infection control and selection of anti-tuberculosis drugs. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the direct detection of rifampin-resistant MTB in respiratory specimens. A total of 400 respiratory specimens collected from patients with clinically suspected tuberculosis or non-tuberculous mycobacteria (NTM) infections were tested with the culture-based conventional Mycobacterium species identification and QMAP system. Among 400 specimens, 154 samples were evaluated using phenotypic anti-tuberculosis drug susceptibility test (DST) and the QMAP system for the detection of rifampin resistance. Detection agreement rate between the culture-based conventional identification and QMAP system for MTB and NTM according to acid-fast bacillus smear positivity was as follows: 97.0% (131/135) and 93.6% (88/94) in 229 smear-positive samples and 69.4% (25/36) and 73.0% (65/89) in 171 smear-negative samples. Based on culture as the gold standard, the overall sensitivity and specificity of the QMAP system for Mycobacterium identification were 87.3 and 97.8%, respectively. The categorical agreement rate between phenotypic DST and QMAP system for rifampin was as follows: complete agreement, 92.9% (143/154); very major error, 0%; and major error, 0.6% (1/154). The overall sensitivity of the QMAP system for the detection of rifampin resistance was 97.1% (34/35). The QMAP system is a useful screening method for the early diagnosis of tuberculosis and selection of anti-tuberculosis drug, as it may detect rifampin-resistant MTB directly from respiratory specimens.

Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis Using the Quantamatrix Multiplexed Assay Platform System.

BACKGROUND: The increasing prevalence of drug-resistant tuberculosis (TB) infection represents a global public health emergency. We evaluated the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform [QMAP], QuantaMatrix, Seoul, Korea) to rapidly identify the Mycobacterium tuberculosis complex (MTBC) and detect rifampicin (RIF) and isoniazid (INH) resistance-associated mutations. METHODS: A total of 200 clinical isolates from respiratory samples were used. Phenotypic anti-TB drug susceptibility testing (DST) results were compared with those of the QMAP system, reverse blot hybridization (REBA) MTB-MDR assay, and gene sequencing analysis. RESULTS: Compared with the phenotypic DST results, the sensitivity and specificity of the QMAP system were 96.4% (106/110; 95% confidence interval [CI] 0.9072-0.9888) and 80.0% (72/90; 95% CI 0.7052-0.8705), respectively, for RIF resistance and 75.0% (108/144; 95% CI 0.6731-0.8139) and 96.4% (54/56; 95% CI 0.8718-0.9972), respectively, for INH resistance. The agreement rates between the QMAP system and REBA MTB-MDR assay for RIF and INH resistance detection were 97.6% (121/124; 95% CI 0.9282-0.9949) and 99.1% (109/110; 95% CI 0.9453-1.0000), respectively. Comparison between the QMAP system and gene sequencing analysis showed an overall agreement of 100% for RIF resistance (110/110; 95% CI 0.9711-1.0000) and INH resistance (124/124; 95% CI 0.9743-1.0000). CONCLUSIONS: The QMAP system may serve as a useful screening method for identifying and accurately discriminating MTBC from non-tuberculous mycobacteria, as well as determining RIF- and INH-resistant MTB strains.

Differences in Genetic Variations Between Treatable and Recalcitrant Atopic Dermatitis in Korean.

PURPOSE: Variations in barrier- or immune response-related genes are closely related to the development of atopic dermatitis (AD). This study was designed to identify genetic variations and clinical features to predict 'recalcitrant AD.' METHODS: AD patients were classified as treatable and recalcitrant. Treatable AD patients showed satisfactory clinical improvement with basic and topical treatments. Recalcitrant AD patients used systemic immune-suppressants for over 4 weeks as they had not shown clinical improvement with basic and topical treatments. The frequency of gene variations in barrier- (FLG 3321delA, FLG K4022X, KLK7, SPINK 1156, SPINK 1188, SPINK 2475) and immune response- (DEFB1, KDR, IL-5RA, IL-9, and IL-12RB1a, b) related genes were compared between each AD group and the controls. RESULTS: Of all, 249 treatable AD and 32 recalcitrant AD were identified. Heterozygous mutations (Hetero) in KLK7 was more frequent in recalcitrant AD patients than treatable AD, without statistical significance. Hetero in DEFB1 was more frequent in treatable AD patients. However, no other significant genetic differences between treatable and recalcitrant AD was observed. Instead, higher initial Eczema Area Severity Index (EASI) score, serum immunoglobulin E (IgE) level, allergen specific IgE for house dust mites, and family history of atopic diseases were associated with recalcitrant AD with statistical significance. CONCLUSIONS: According to our study, no genetic variation to predict recalcitrant AD was identified, suggesting that clinical manifestation, rather than genetic variations of AD patients is more likely to be an important factor in predicting the prognosis of AD. Further large-scale studies on the correlation between genetic variation and recalcitrant AD are needed.

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.

Performance of the Quantamatrix Multiplexed Assay Platform system for the differentiation and identification of Mycobacterium species.

PURPOSE: The purpose of this study was to evaluate the usefulness of a new diagnostic multiplexed bead-based bioassay (Quantamatrix Multiplexed Assay Platform; QMAP) system with shape-encoded silica microparticles for the rapid and accurate detection and identification of 23 mycobacterial species/groups, including Mycobacterium tuberculosis complex (MTBC). METHODOLOGY: A total of 295 mycobacterial clinical isolates cultured from respiratory specimens were used for identification of MTBC and non-tuberculous mycobacteria (NTM) using the QMAP system and the results were confirmed with PCR-restriction fragment length polymorphism (RFLP) analysis of the rpoB gene, rpoB sequence analysis and PCR-reverse blot hybridization assay (REBA).Results/Key findings. The Mycobacterium genus-specific probe of the QMAP system was positive for all 46 Mycobacterium reference strains and negative for 59 non-Mycobacterium strains. Based on 295 liquid culture-positive samples, both the culture-based conventional identification method and the QMAP system identified each 212 and 81 isolates as MTB and NTM species. The concordance rates for the identification of NTM species between the QMAP system and molecular assays were 92.8 % (77/83), 97.6 % (81/83) and 100 % (83/83) for PCR-RFLP, the rpoB sequence analysis and PCR-REBA, respectively. CONCLUSION: The QMAP system yielded rapid, highly sensitive and specific results for the identification of MTBC and NTM and accurately discriminated between NTM species within 3 h.

Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria.

BACKGROUND: The differentiation of Mycobacterium tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) is of primary importance for infection control and the selection of anti-tuberculosis drugs. Up to date data on rifampicin (RIF)-resistant tuberculosis (TB) is essential for the early management of multidrug-resistant TB. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the rapid differentiation of 23 Mycobacterium species including MTBC and RIF-resistant strains. METHODS: A total of 314 clinical Mycobacterium isolates cultured from respiratory specimens were used in this study. RESULTS: The sensitivity and specificity of the QMAP system for Mycobacterium species were 100% (95% CI 99.15-100%, p<0.0001) and 97.8% (95% CI 91.86-99.87%, p<0.0001), respectively. The results of conventional drug susceptibility testing and the QMAP Dual-ID assay were completely concordant for all clinical isolates (100%, 95% CI 98.56-100%). Out of 223 M. tuberculosis (MTB) isolates, 196 were pan-susceptible and 27 were resistant to RIF according to QMAP results. All of the mutations in the RIF resistance-determining region detected by the QMAP system were confirmed by rpoB sequence analysis and a REBA MTB-Rifa reverse blot hybridization assay. The majority of the mutations (n=26, 96.3%), including those missing wild-type probe signals, were located in three codons (529-534, 524-529, and 514-520), and 17 (65.4%) of these mutations were detected by three mutation probes (531TTG, 526TAC, and 516GTC). CONCLUSIONS: The entire QMAP system assay takes about 3h to complete, while results from the culture-based conventional method can take up to 48-72h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples.

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.

Development of a Rapid Reverse Blot Hybridization Assay for Detection of Clinically Relevant Antibiotic Resistance Genes in Blood Cultures Testing Positive for Gram-Negative Bacteria.

Rapid and accurate identification of the causative pathogens of bloodstream infections is crucial for the prompt initiation of appropriate antimicrobial therapy to decrease the related morbidity and mortality rates. The aim of this study was to evaluate the performance of a newly developed PCR-reverse blot hybridization assay (REBA) for the rapid detection of Gram-negative bacteria (GNB) and their extended-spectrum ß-lactamase (ESBL), AmpC ß-lactamase, and carbapenemase resistance genes directly from the blood culture bottles. The REBA-EAC (ESBL, AmpC ß-lactamase, carbapenemase) assay was performed on 327 isolates that were confirmed to have an ESBL producer phenotype, 200 positive blood culture (PBCs) specimens, and 200 negative blood culture specimens. The concordance rate between the results of REBA-EAC assay and ESBL phenotypic test was 94.2%. The sensitivity, specificity, positive predictive value, and negative predictive value of the REBA-EAC assay for GNB identification in blood culture specimens were 100% (95% CI 0.938-1.000, P < 0.001), 100% (95% CI 0.986-1.000, P < 0.001), 100% (95% CI 0.938-1.000, P < 0.001), and 100% (95% CI 0.986-1.000, P < 0.001), respectively. All 17 EAC-producing GNB isolates from the 73 PBCs were detected by the REBA-EAC assay. The REBA-EAC assay allowed easy differentiation between EAC and non-EAC genes in all isolates. Moreover, the REBA-EAC assay was a rapid and reliable method for identifying GNB and their ß-lactamase resistance genes in PBCs. Thus, this assay may provide essential information for accelerating therapeutic decisions to achieve earlier appropriate antibiotic treatment during the acute phase of bloodstream infection.

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.

Comparison of multiplex real-time PCR and PCR-reverse blot hybridization assay for the direct and rapid detection of bacteria and antibiotic resistance determinants in positive culture bottles.

The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; P<0.001), 100 % (95 % CI, 0.983-1.000; P<0.001), 100 % and 99 %, respectively. However, positivity of the Real-methicillin-resistant Staphylococcusaureus multiplex real-time PCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.