Promotion of tumorigenesis by miR-1260b-targeting CASP8: Potential diagnostic and prognostic marker for breast cancer.

MicroRNAs are reported as promising biomarkers for the diagnosis and treatment of breast cancer. miR-1260b is identified as a tumor-associated noncoding microRNA in other cancers, although the role of miR-1260b and its clinical relevance in breast cancer remain unclear. In this study, miR-1260b as a potential prognostic biomarker was observed by univariate and multivariate Cox regression analyses in 102 breast tumor tissues. The tumorigenic role of miR-1260b in terms of proliferation, apoptosis, and migration of breast cancer cells was investigated using gain- and loss-of-function assays in vitro. Additionally, the potential early diagnosis and treatment monitoring marker of miR-1260b was validated in 129 plasma samples. We found that high miR-1260b expression was markedly associated with bulky tumor size, advanced stage, and lymph node invasion. Particularly, the high-miR-1260b-expression group showed shorter overall survival than the low-miR-1260b-expression group. The inhibition of oncogenic miR-1260b induced apoptosis and decreased migration and invasion of MDA-MB-231 cells. CASP8 was revealed as a direct target gene of miR-1260b, which is closely related to apoptosis. Furthermore, miR-1260b expression levels in plasma were significantly higher in patients with breast cancer than in healthy controls. The patients who tested positive for miR-1260b showed 16.3- and 18.2-fold higher risks in the early stage and locally advanced stage, respectively, compared with healthy controls, and the risk was decreased 6.2-fold after neoadjuvant chemotherapy. Taken together, miR-1260b may be a potential novel diagnostic, prognostic, and therapeutic target in breast cancer.

Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis.

Rapid, accurate detection of Mycobacterium tuberculosis is crucial in the diagnosis of tuberculosis (TB), but conventional diagnostic methods have limited sensitivity and specificity or are time consuming. A new highly sensitive nucleic acid amplification test, combined nested and real-time polymerase chain reaction (PCR) in a single tube (one-tube nested real-time PCR), was developed for detecting M. tuberculosis, which takes advantage of two PCR techniques, i.e., nested PCR and real-time PCR. One-tube nested real-time PCR was designed to have two sequential reactions with two sets of primers and dual probes for the insertion sequence (IS) 6110 sequence of M. tuberculosis in a single closed tube. The minimum limits of detection of IS6110 real-time PCR and IS6110 one-tube nested real-time PCR were 100 fg/µL and 1 fg/µL of M. tuberculosis DNA, respectively. AdvanSure TB/non-tuberculous mycobacteria (NTM) real-time PCR, IS6110 real-time PCR, and two-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and NTM isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR, single IS6110 real-time PCR, and one-tube nested real-time PCR were 91% (152/167), 94.6% (158/167), and 100% (167/167) for sputum specimens, respectively. In conclusion, IS6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation.

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.

Comparison of the performance of the NucliSENS EasyQ HPV E6/E7 mRNA assay and HPV DNA chip for testing squamous cell lesions of the uterine cervix.

This study aims to evaluate the clinical performance of the NucliSENS EasyQ assay and compare it with HPV DNA genotyping for the detection of high-grade squamous intraepithelial lesions (HSIL) and cancer in a Korean population. In 188 total thin prep samples, the remaining fluid after cytology slide preparation was tested with Goodgene HPV DNA chips and the NucliSENS EasyQ HPV E6/E7 messenger RNA (mRNA) assay. The sensitivity and specificity of each test were calculated with HSIL and squamous cell carcinoma (SCC) as the disease endpoint. Out of the 188 samples, 139 (74%) were positive for DNA of 14 HPV types, while 57 (30%) cases were positive for E6/E7 mRNA. The DNA test was positive in cytology cases of SCC, HSIL, and atypical squamous cell. The mRNA test yielded results of 75%, 74%, 60%, 56%, and 29% positivity in abnormal cytology cases of SCC, HSIL, atypical squamous cells - cannot exclude HSIL, atypical squamous cells of undetermined significance, and low-grade squamous intraepithelial lesion, respectively. In normal cytology cases, the positivity rates were 9% and 53% for the mRNA and DNA tests, respectively. For detection of HSIL and SCC, the sensitivity of the mRNA test was 74.36% and that of the DNA test was 100%, while the specificities of the tests were 85% and 40.83%, respectively. These findings suggest that the HPV E6/E7 mRNA assay can overcome the shortcoming of low specificity of DNA assays for clinical detection of high-grade cervical lesions and malignancies.

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).

A simple and efficient multiplex PCR assay for the identification of Mycobacterium genus and Mycobacterium tuberculosis complex to the species level.

PURPOSE: The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guérin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health. MATERIALS AND METHODS: A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates. RESULTS: All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level. CONCLUSION: The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.

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.

Molecular characterization of drug-resistant and -susceptible Mycobacterium tuberculosis isolated from patients with tuberculosis in Korea.

We investigated the causal relationship between genotype and phenotype of drug-resistant Mycobacterium tuberculosis isolates obtained from patients with pulmonary tuberculosis (TB) in Korea. Of 80 isolates tested, 17, 20, 1, and 7 isolates were mono-resistant to ethambutol (EMB), isoniazid (INH), pyrazinamide (PZA), and rifampicin (RFP), respectively, and 31 isolates (38.8%) were multidrug-resistant (MDR). Sequencing analysis showed that 78% (32/41) of RFP-resistant strains had mutations in the rifampicin resistance-determining region (RRDR) of rpoB, and the mutation at rpoB531 (59.4%) was most abundant. In 52 INH-resistant strains, mutations were found mostly at C-15T (n = 21, 40.4%) in the inhA promoter region as well as at katG315 (n = 12, 23.1%). Mutations at embB306 were mostly found in 26.7% (12/45) of EMB-resistant isolates. New mutations found here in MDR isolates include rpoB523 (Gly523Glu) and embB319 (Tyr319Ser). Consequently, mutations in the rpoB531, C-15T in the inhA promoter region, embB306, and katG315 would be a useful marker for rapid detection of MDR M. tuberculosis isolates in Korea.