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Atopic dermatitis (AD) is a persistent inflammatory skin condition resulting from an intricate interplay among genetic, immunological, and environmental factors. Erigeron annuus (EA), an annual winter plant belonging to the family Asteraceae, possesses anti-inflammatory, cytoprotective, and antioxidant activities. In this study, we hypothesized that Erigeron annuus extract (EAE) could be an effective agent for ameliorating AD-like symptoms. To confirm this hypothesis in vitro, we used H2O2-stimulated human keratinocytes (HaCaT cells) to demonstrate that pre-treatment with EAE protected against oxidative stress. HaCaT cells pretreated with EAE and stimulated with H2O2 showed decreased intracellular malondialdehyde content, increased superoxide dismutase activity, and reduced intracellular reactive oxygen species accumulation. To verify the in vivo hypothesis based on the intracellular results, an AD disease mouse model was induced with 1-chloro-2,4-dinitrobenzene (DNCB), and EAE was orally administered at a non-toxic concentration according to the toxicity evaluation results. The results showed that AD disease models in BALB/c mice exhibited reduced ear epidermal thickness, scratching behavior, and mast cell infiltration. In conclusion, our results indicate that EAE has the potential to improve AD by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway.
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Dermatitis Atópica , Erigeron , Humanos , Animales , Ratones , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/tratamiento farmacológico , Piel/metabolismo , Dinitroclorobenceno/toxicidad , Erigeron/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Dinitrobencenos/efectos adversos , Dinitrobencenos/metabolismo , Hemo-Oxigenasa 1/metabolismo , Peróxido de Hidrógeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ratones Endogámicos BALB C , Citocinas/metabolismoRESUMEN
BACKGROUND: Gastric cancer (GC) is among the most common types of gastrointestinal cancers and has a high incidence and mortality around the world. To suppress the progression of GC, it is essential to develop diagnostic markers. MicroRNAs regulate GC development, but a clearer insight into their role is needed before they can be applied as a molecular markers and targets. METHODS: In this study, we assessed the diagnostic value of differentially expressed microRNAs as potential diagnostic biomarkers for GC using data for 389 tissue samples from the Cancer Genome Atlas (TCGA) and 21 plasma samples from GC patients. RESULTS: The expression of hsa-miR-143-3p (also known as hsa-miR-143) was significantly downregulated in GC according to the TCGA data and plasma samples. The 228 potential target genes of hsa-miR-143-3p were analyzed using a bioinformatics tool for miRNA target prediction. The target genes correlated with extracellular matrix organization, the cytoplasm, and identical protein binding. Furthermore, the pathway enrichment analysis of target genes showed that they were involved in pathways in cancer, the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, and proteoglycans in cancer. The hub genes in the protein-protein interaction (PPI) network, were matrix metallopeptidase 2 (MMP2), CD44 molecule (CD44), and SMAD family member 3 (SMAD3). CONCLUSIONS: This study suggests that hsa-miR-143-3p may be used as a diagnostic marker for GC, contributing via the pathways involved in the development of GC.
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MicroARNs , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Fosfatidilinositol 3-Quinasas/genética , Perfilación de la Expresión Génica , MicroARNs/metabolismo , BiomarcadoresRESUMEN
This study aimed to provide basic data for the clinical application of urine samples to prevent cervical cancer due to persistent HR-HPV infection in women who refuse invasive cervical sampling. Pairs of cervical swabs and urine samples were collected from 210 asymptomatic women who visited the obstetrics and gynecology department from August to December 2020, and a total of 420 samples were collected. Using the PANA RealTyper™ HPV Screening Kit as a real-time PCR method, paired cervical swabs and random urine samples were tested. A total of 19 samples (9.1%) were both HPV positive and 177 (84.3%) were both negative. The concordance between the two types of samples was 93.3%, with κ = 0.69 (moderate, 95% CI 0.54-0.84). The HPV infection rate by age was highest in both cervical swabs and urine samples in women in their 30s, followed by those in their 20s. Thus, the HPV infection rate was high in young women under 40 at 69.2% in cervical swabs and 61.8% in urine samples. Urine samples are considered a valuable screening test for women who refuse invasive Pap tests to prevent cervical cancer caused by persistent HPV infection.
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Gastric cancer (GC) is one of the most common cancers and a leading cause of cancer deaths around the world. Chemotherapy is one of the most effective treatments for cancer patients, and has remarkably enhanced survival rates. However, it has many side effects. Recently, microRNAs (miRNAs) have been intensively studied as potential biomarkers for cancer diagnosis and treatment monitoring. However, definitive biomarkers in chemotherapy-induced peripheral neuropathy (CIPN) are still lacking. The aim of this study was to identify the factors significant for neurological adverse events in GC patients receiving XELOX (oxaliplatin and capecitabine) chemotherapy. The results show that XELOX chemotherapy induces changes in the expression of hsa-miR-200c-3p, hsa-miR-885-5p, and hsa-miR-378f. Validation by qRT-PCR demonstrated that hsa-miR-378f was significantly downregulated in CIPN. Hsa-miR-378f was identified as showing a statistically significant correlation in GC patients receiving XELOX chemotherapy according to the analysis of differentially expressed (DE) miRNAs. Furthermore, 34 potential target genes were predicted using a web-based database for miRNA target prognostication and functional annotations. The identified genes are related to the peptidyl-serine phosphorylation and regulation of alternative mRNA splicing with enrichment in the gastric cancer, neurotrophin, MAPK, and AMPK signaling pathways. Collectively, these results provide information useful for developing promising strategies for the treatment of XELOX-chemotherapy-induced peripheral neuropathy.
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Antineoplásicos , MicroARN Circulante , MicroARNs , Enfermedades del Sistema Nervioso Periférico , Neoplasias Gástricas , Antineoplásicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica , Biomarcadores , Capecitabina/efectos adversos , MicroARN Circulante/genética , Perfilación de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Oxaloacetatos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genéticaRESUMEN
Early diagnosis increases the treatment success rate for active tuberculosis (ATB) and decreases mortality. MicroRNAs (miRNAs) have been studied as blood-based markers of several infectious diseases. We performed miRNA profiling to identify differentially expressed (DE) miRNAs using whole blood samples from 10 healthy controls (HCs), 15 subjects with latent tuberculosis infection (LTBI), and 12 patients with ATB, and investigated the expression of the top six miRNAs at diagnosis and over the treatment period in addition to performing miRNA-target gene network and gene ontology analyses. miRNA profiling identified 84 DE miRNAs in patients with ATB, including 80 upregulated and four downregulated miRNAs. Receiver operating characteristic curves of the top six miRNAs exhibited excellent distinguishing efficiency with an area under curve (AUC) value > 0.85. Among them, miR-199a-3p and miR-6886-3p can differentiate between ATB and LTBI. Anti-TB treatment restored the levels of miR-199b-3p, miR-199a-3p, miR-16-5p, and miR-374c-5p to HC levels. Furthermore, 108 predicted target genes were related to the regulation of cellular amide metabolism, intrinsic apoptotic signaling, translation, transforming growth factor beta receptor signaling, and cysteine-type endopeptidase activity. The DE miRNAs identified herein are potential biomarkers for diagnosis and therapeutic monitoring in ATB.
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Human papillomavirus (HPV) infection in males is associated with various cancers, including cervical cancer in women and penile and bladder cancers in men. However, there is limited research on the prevalence and prevention of male HPV infection. Moreover, a rapid test that can prevent the increase in HPV infection is needed. In this study, the prevalence of sexually transmitted pathogen (STP) and HPV infection was analyzed using real-time polymerase chain reaction assay in random urine samples collected from asymptomatic male sexual partners of women with sexually transmitted diseases. Among 130 men, 65 (50.0%) had STP and 12 (9.23%) had HPV infection. There was no association between STP and HPV infection. Among 12 cases of HPV infection, three were HPV-16 single infections, six were multiple infections, including HPV-16, and three of other high-risk HPV infections. Our results suggest the need for STP testing, including HPV testing, in sexual partners of high-risk women with sexually transmitted diseases, even in men without clinical symptoms (asymptomatic). Further research should be conducted by diversifying urine samples. We report the most convenient method for HPV detection, and it is expected to be widely applied to prevent sexually transmitted diseases in men and women.
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Alphapapillomavirus , Infecciones por Papillomavirus , Enfermedades de Transmisión Sexual , ADN Viral , Femenino , Humanos , Masculino , Papillomaviridae/genética , Infecciones por Papillomavirus/epidemiología , Prevalencia , Factores de Riesgo , Parejas Sexuales , Enfermedades de Transmisión Sexual/epidemiologíaRESUMEN
A dusty thermal vacuum chamber (DTVC) containing a regolith simulant bed is essential for testing equipment and techniques related to lunar surface exploration. Space agencies have been reluctant to operate a DTVC because of the challenge of controlling soil disturbance of the lunar regolith simulant bed during pumping down or depressurization, which may contaminate or even damage the chamber and vacuum equipment. There appears to be no previously available solution to this problem, or how to avoid it. We investigated the mechanism of soil disturbance during depressurization and established a criterion for evaluating its occurrence. The proposed criterion was validated by extensive experiments and numerical modelling to simulate air evacuation from soil voids. There is a critical pressure difference (CPD) between the top and bottom of the lunar regolith simulant bed that causes soil disturbance during depressurization. We found a simple equation estimating the CPD and further provided guideline on the optimum depressurization rate to avoid soil disturbance before the target vacuum level is achieved under varying soil conditions.
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Human papilloma virus (HPV) is known to be a major cause of cervical cancer. In Korea, although the mortality of cervical cancer has decreased, HPV infection rates are increasing rapidly in young women. One of the reasons for a high rate of human immunodeficiency virus (HIV) infection appears to be associated with a low frequency to visit gynecology clinics because of the uncomfortable sampling process for HPV testing. Therefore, it is necessary to develop a non-invasive method, such as urine testing to diagnose cervical cancer rather than use of the existing invasive method. This study aimed to test validity of HPV DNA detection in urine specimens that can be easily collected from women. Paired vaginal discharge and urine samples were collected prospectively from 203 women who visited the local hospital between January and August 2018 in Busan, Korea. By using the Virocheck® assay kit (Optipharm), we found that 17.2% (35/203) of vaginal discharge samples were HPV positive and 82.8% (168/203) were HPV negative. In urine samples, 15.8% (32/203) were HPV positive and 84.2% (171/203) were HPV negative. The co-incident rate for HPV DNA detection was 84.8% in both vaginal discharge and urine samples. These results suggest that the HPV DNA detection using urine samples might be an alternative way to diagnose HPV infection in a non-invasive way. This analytical approach can be utilized as a screening test to identify HIV-infected patients who need a follow-up process by using urine samples.
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Interaction between electrons has long been a focused topic in condensed-matter physics since it has led to the discoveries of astonishing phenomena, for example, high-Tc superconductivity and colossal magnetoresistance (CMR) in strongly-correlated materials. In the study of strongly-correlated perovskite oxides, Nb-doped SrTiO3 (Nb:SrTiO3) has been a workhorse not only as a conducting substrate, but also as a host possessing high carrier mobility. In this work, we report the observations of large linear magnetoresistance (LMR) and the metal-to-insulator transition (MIT) induced by magnetic field in heavily-doped Nb:STO (SrNb0.2Ti0.8O3) epitaxial thin films. These phenomena are associated with the interplay between the large classical MR due to high carrier mobility and the electronic localization effect due to strong spin-orbit coupling, implying that heavily Nb-doped Sr(Nb0.2Ti0.8)O3 is promising for the application in spintronic devices.
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We investigated the magnetotransport properties of Bi2Te3 films grown on GaAs (001) substrate by a cost-effective metallo-organic chemical vapor deposition (MOCVD). We observed the remarkably high carrier mobility and the giant linear magnetoresistance (carrier mobility â¼ 22 000 cm(2) V(-1) s(-1), magnetoresistance â¼ 750% at 1.8 K and 9 T for a 100 nm thick film) that depends on the film thickness. In addition, the Shubnikov-de Haas oscillation was observed, from which the effective mass was calculated to be consistent with the known value. From the thickness dependence of the Shubnikov-de Haas oscillation, it was found that a two dimensional electron gas with the conventional electron nature coexists with the topological Dirac fermion states and dominates the carrier transport in the Bi2Te3 film with thickness higher than 300 nm. These results are attributed to the intrinsic nature of Bi2Te3 in the high-mobility transport regime obtained by a deliberate choice of the substrate and the growth conditions.
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Point contact Andreev reflection (PCAR) has become a standard method for measuring the spin polarization (P) of spintronic materials due to its unique simplicity and the firm physical ground, but it is still challenging to achieve a clean point contact between a superconductor (SC) and a metal (N) for implementing PCAR. In this work, we suggest a much simpler method for PCAR measurement, where a point contact between SC and N is provided by a metallic filament in a transition-metal oxide generated by electrical bias. This method has been successfully demonstrated using a structure composed of Nb/NiO/Pt, where P of the Ni filament was estimated to be about 40%, consistent with the known value of the bulk Ni. In addition, we investigated the dependence of the conductance spectrum on the measurement temperature and the magnetic field. We found that the superconductivity is not fully suppressed until 9 T far above the critical field of Nb, which is associated with the nm-sized constriction of our SC/N junction, much smaller than the coherence length of the SC.
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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.
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Antígenos Bacterianos/farmacología , Células Sanguíneas/efectos de los fármacos , Quimiocina CXCL9/genética , Tuberculosis Latente/diagnóstico , Tuberculosis Pulmonar/diagnóstico , Factor de Necrosis Tumoral alfa/genética , Adulto , Anciano , Células Sanguíneas/inmunología , Células Sanguíneas/microbiología , Células Cultivadas , Quimiocina CXCL10/genética , Quimiocina CXCL10/inmunología , Quimiocina CXCL9/inmunología , Diagnóstico Diferencial , Femenino , Expresión Génica , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Tuberculosis Latente/genética , Tuberculosis Latente/inmunología , Tuberculosis Latente/microbiología , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/fisiología , ARN Mensajero/genética , ARN Mensajero/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/inmunología , Sensibilidad y Especificidad , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
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.
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Mycobacterium tuberculosis/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Tuberculosis/microbiología , Humanos , Límite de Detección , Mycobacterium tuberculosis/aislamiento & purificación , Micobacterias no Tuberculosas/genética , Reacción en Cadena de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Esputo/microbiología , Tuberculosis/diagnósticoRESUMEN
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.
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Prueba de Papanicolaou/métodos , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Pruebas de ADN del Papillomavirus Humano/métodos , Humanos , Persona de Mediana Edad , Proteínas Oncogénicas Virales/genética , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y EspecificidadRESUMEN
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.
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Alphapapillomavirus/genética , ADN Viral , Pruebas de ADN del Papillomavirus Humano/métodos , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/complicaciones , Lesiones Intraepiteliales Escamosas de Cuello Uterino/diagnóstico , Lesiones Intraepiteliales Escamosas de Cuello Uterino/virología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Genotipo , Pruebas de ADN del Papillomavirus Humano/normas , Humanos , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virología , Adulto JovenRESUMEN
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).
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Bacteriemia/diagnóstico , Candidemia/diagnóstico , Tamizaje Masivo/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sepsis/diagnóstico , Candida/clasificación , Candida/aislamiento & purificación , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Grampositivas/clasificación , Bacterias Grampositivas/aislamiento & purificación , Humanos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
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.
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Reacción en Cadena de la Polimerasa Multiplex/métodos , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium/aislamiento & purificación , Animales , Bovinos , Clasificación/métodos , Cartilla de ADN , Genes Bacterianos , Humanos , Mycobacterium/clasificación , Mycobacterium/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Especificidad de la EspecieRESUMEN
Rapid diagnosis of multidrug-resistant tuberculosis (MDR-TB) is essential for the prompt initiation of effective second-line therapy to improve treatment outcome and limit transmission of this obstinate disease. A variety of molecular methods that enable the rapid detection of mutations implicated in MDR-TB have been developed. The sensitivity of the methods is dependent, in principle, on the repertoire of mutations being detected, which is typically limited to mutations in the genes rpoB, katG and the promoter region of inhA. In this study, a new reverse hybridization assay, REBA MTB-MDR (M&D), that probes mutations in the oxyR-ahpC intergenic region, in addition to those in rpoB, katG and the inhA promoter region, was evaluated. A set of 240 Mycobacterium tuberculosis clinical isolates from patients receiving retreatment regimens was subjected to conventional phenotypic drug-susceptibility testing (DST) and the REBA MTB-MDR assay. The nucleotide sequences of the loci known to be involved in drug resistance were determined for comparison. In brief, the results showed that the REBA MTB-MDR assay efficiently recognized nucleotide changes in the oxyR-ahpC intergenic region as well as those in rpoB, katG and the inhA promoter region with higher sensitivity, resulting in an 81.0â% detection rate for isoniazid resistance. Inclusion of the oxyR-ahpC intergenic region in the REBA MTB-MDR assay improved the overall sensitivity of molecular DST for MDR-TB from 73.1 to 79.9â%.