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Find video protocols related to scientific articles indexed in Pubmed.
mRNA biomarker detection in liquid-based cytology: a new approach in the prevention of cervical cancer.
Mod. Pathol.
PUBLISHED: 09-05-2014
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Several high-risk human papillomavirus (HPV)-induced cell biomarkers have been proposed as possible candidates to identify patients harboring high-grade squamous intraepithelial lesions (HSILs) of the uterine cervix. We aimed to determine the feasibility of the detection of the mRNA of six biomarkers in cervical smear specimens obtained by liquid-based cytology and to evaluate whether this approach might be useful in the identification of patients with HSIL. One-hundred and twenty three women referred to colposcopy in the Hospital Clinic of Barcelona were included in the study. After a thorough study, including Pap test, high-risk HPV testing (Hybrid Capture 2 test), and colposcopy with directed biopsy and/or endocervical curettage, 48 patients were diagnosed with HSIL, whereas 75 were classified as negative (n=28), or harboring low-grade SIL (n=47). CDKN2A/p16, BIRC5, MMP9, TOP2A, MCM5, and MKI67 mRNA expression was analyzed by reverse transcription quantitative polymerase chain reaction in liquid-based cytology after the Pap test and Hybrid Capture 2 performance. The tissue expression of these biomarkers was analyzed by immunohistochemistry in the biopsy material. One-hundred and thirteen out of 123 (92%) liquid-based cytology yielded adequate material for mRNA analysis. TOP2A was the most sensitive (97%) biomarker for the detection of HSIL and CDKN2A/p16 the most specific (78%). The combination of TOP2A and CDKN2A/p16 showed a sensitivity of 96% (95% confidence interval (CI): 88-99) and a specificity of 71% (95% CI: 55-82). In the immunohistochemistry analysis, all biomarkers showed a high sensitivity but low specificity for HSIL, except CDKN2A/p16 which had a sensitivity of 100% and a specificity of 63%. The combination of TOP2A and CDKN2A/p16 showed a sensitivity of 100% (95% CI: 91-100) and a specificity of 43% (95% CI: 32-55). The detection of mRNA of cell biomarkers in liquid-based cytology material is feasible. The combination TOP2A and CDKN2A/p16 has a good balance between sensitivity and specificity for the detection of women with HSIL.Modern Pathology advance online publication, 5 September 2014; doi:10.1038/modpathol.2014.106.
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Strain types and antimicrobial resistance patterns of Clostridium difficile isolates from the United States, 2011 to 2013.
Antimicrob. Agents Chemother.
PUBLISHED: 04-21-2014
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We determined the PCR ribotypes and antimicrobial susceptibility patterns of 508 toxigenic Clostridium difficile isolates collected between 2011 and 2013 from 32 U.S. hospitals. Of the 29 PCR ribotypes identified, the 027 strain type was the most common (28.1%), although the rates varied by geographic region. Ribotype 014/020 isolates appear to be emerging. Clindamycin and moxifloxacin resistances (36.8% and 35.8%, respectively) were the most frequent resistance phenotypes observed. Reduced susceptibility to vancomycin was observed in 39.1% of 027 isolates.
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Detection of colonization by carbapenemase-producing Gram-negative Bacilli in patients by use of the Xpert MDRO assay.
J. Clin. Microbiol.
PUBLISHED: 09-04-2013
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Detecting colonization of patients with carbapenemase-producing bacteria can be difficult. This study compared the sensitivity and specificity of a PCR-based method (Xpert MDRO) for detecting blaKPC, blaNDM, and blaVIM carbapenem resistance genes using GeneXpert cartridges to the results of culture with and without a broth enrichment step on 328 rectal, perirectal, and stool samples. The culture method included direct inoculation of a MacConkey agar plate on which a 10-?g meropenem disk was placed and plating on MacConkey agar after overnight enrichment of the sample in MacConkey broth containing 1 ?g/ml of meropenem. Forty-three (13.1%) samples were positive by PCR for blaKPC and 11 (3.4%) were positive for blaVIM; none were positive for blaNDM. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay for blaKPC were 100%, 99.0%, 93.0%, and 100%, respectively, compared to broth enrichment culture and sequencing of target genes. The sensitivity, specificity, PPV, and NPV of the assay for blaVIM were 100%, 99.4%, 81.8%, and 100%, respectively. Since none of the clinical samples contained organisms with blaNDM, 66 contrived stool samples were prepared at various dilutions using three Klebsiella pneumoniae isolates containing blaNDM. The PCR assay showed 100% positivity at dilutions from 300 to 1,800 CFU/ml and 93.3% at 150 CFU/ml. The Xpert MDRO PCR assay required 2 min of hands-on time and 47 min to complete. Rapid identification of patients colonized with carbapenemase-producing organisms using multiplex PCR may help hospitals to improve infection control activities.
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Alternative processing of the U2 small nuclear RNA produces a 19-22nt fragment with relevance for the detection of non-small cell lung cancer in human serum.
PLoS ONE
PUBLISHED: 02-21-2013
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RNU2 exists in two functional forms (RNU2-1 and RNU2-2) distinguishable by the presence of a unique 4-bases motif. Detailed investigation of datasets obtained from deep sequencing of five human lung primary tumors revealed that both forms express at a high rate a 19-22nt fragment (miR-U2-1 and -2) from its 3 region and contains the 4-bases motif. Deep sequencing of independent pools of serum samples from healthy donors and lung cancer patients revealed that miR-U2-1 and -2 are pervasively processed in lung tissue by means of endonucleolytic cleavages and stably exported to the blood. Then, microarrays hybridization experiments of matched normal/tumor samples revealed a significant over-expression of miR-U2-1 in 14 of 18 lung primary tumors. Subsequently, qRT-PCR of miR-U2-1 using serum from 62 lung cancer patients and 96 various controls demonstrated that its expression levels identify lung cancer patients with 79% sensitivity and 80% specificity. miR-U2-1 expression correlated with the presence or absence of lung cancer in patients with chronic obstructive pulmonary disease (COPD), other diseases of the lung - not cancer, and in healthy controls. These data suggest that RNU2-1 is a new bi-functional ncRNA that produces a 19-22nt fragment which may be useful in detecting lung cancer non-invasively in high risk patients.
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Genome-Wide Sequencing of Cellular microRNAs Identifies a Combinatorial Expression Signature Diagnostic of Sepsis.
PLoS ONE
PUBLISHED: 01-01-2013
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Sepsis is a common cause of death in the intensive care unit with mortality up to 70% when accompanied by multiple organ dysfunction. Rapid diagnosis and the institution of appropriate antibiotic therapy and pressor support are therefore critical for survival. MicroRNAs are small non-coding RNAs that play an important role in the regulation of numerous cellular processes, including inflammation and immunity.
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Characterization of nasal and blood culture isolates of methicillin-resistant Staphylococcus aureus from patients in United States Hospitals.
Antimicrob. Agents Chemother.
PUBLISHED: 12-12-2011
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A total of 299 nares and 194 blood isolates of methicillin-resistant Staphylococcus aureus (MRSA), each recovered from a unique patient, were collected from 23 U.S. hospitals from May 2009 to March 2010. All isolates underwent spa and staphylococcal cassette chromosome mec element (SCCmec) typing and antimicrobial susceptibility testing; a subset of 84 isolates was typed by pulsed-field gel electrophoresis (PFGE) using SmaI. Seventy-six spa types were observed among the isolates. Overall, for nasal isolates, spa type t002-SCCmec type II (USA100) was the most common strain type (37% of isolates), while among blood isolates, spa type t008-SCCmec type IV (USA300) was the most common (39%). However, the proportion of all USA100 and USA300 isolates varied by United States census region. Nasal isolates were more resistant to tobramycin and clindamycin than blood isolates (55.9% and 48.8% of isolates versus 36.6% and 39.7%, respectively; for both, P < 0.05). The USA300 isolates were largely resistant to fluoroquinolones. High-level mupirocin resistance was low among all spa types (<5%). SCCmec types III and VIII, which are rare in the United States, were observed along with several unusual PFGE types, including CMRSA9, EMRSA15, and the PFGE profile associated with sequence type 239 (ST239) isolates. Typing data from this convenience sample suggest that in U.S. hospitalized patients, USA100 isolates of multiple spa types, while still common in the nares, have been replaced by USA300 isolates as the predominant MRSA strain type in positive blood cultures.
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A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 08-13-2011
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The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy.
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Comparison of strain typing results for Clostridium difficile isolates from North America.
J. Clin. Microbiol.
PUBLISHED: 03-09-2011
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Accurate strain typing is critical for understanding the changing epidemiology of Clostridium difficile infections. We typed 350 isolates of toxigenic C. difficile from 2008 to 2009 from seven laboratories in the United States and Canada. Typing was performed by PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) of whole-cell DNA. The Cepheid Xpert C. difficile test for presumptive identification of 027/NAP1/BI isolates was also tested directly on original stool samples. Of 350 isolates, 244 (70%) were known PCR ribotypes, 224 (68%) were 1 of 8 common REA groups, and 187 (54%) were known PFGE types. Eighty-four isolates typed as 027, NAP1, and BI, and 83 of these were identified as presumptive 027/NAP1/BI by Xpert C. difficile. Eight additional isolates were called presumptive 027/NAP1/BI by Xpert C. difficile, of which three were ribotype 027. Five PCR ribotypes contained multiple REA groups, and three North American pulsed-field (NAP) profiles contained both multiple REA groups and PCR ribotypes. There was modest concordance of results among the three methods for C. difficile strains, including the J strain (ribotype 001 and PFGE NAP2), the toxin A-negative 017 strain (PFGE NAP9 and REA type CF), the 078 animal strain (PFGE NAP7 and REA type BK), and type 106 (PFGE NAP11 and REA type DH). PCR-ribotyping, REA, and PFGE provide different but overlapping patterns of strain clustering. Unlike the other methods, the Xpert C. difficile 027/NAP1/BI assay gave results directly from stool specimens, required only 45 min to complete, but was limited to detection of a single strain type.
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Activity of ACHN-490 against meticillin-resistant Staphylococcus aureus (MRSA) isolates from patients in US hospitals.
Int. J. Antimicrob. Agents
PUBLISHED: 02-18-2011
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The activity of ACHN-490 was evaluated against 493 meticillin-resistant Staphylococcus aureus (MRSA) isolates collected in 2009-2010 from 23 US hospitals. The MIC(50) and MIC(90) values (minimal inhibitory concentrations for 50% and 90% of the organisms, respectively) for ACHN-490 were 1 and 2 ?g/mL compared with 8 and 32 ?g/mL for amikacin, 0.5 and 1 ?g/mL for gentamicin and 2 and >16 ?g/mL for tobramycin. The gene encoding the aminoglycoside-modifying enzyme APH(2?)-Ia/AAC(6)-Ie was present in 12% of the subset of 84 isolates examined by polymerase chain reaction (PCR), whilst the gene encoding ANT(4)-Ia was present in 89% of isolates. ACHN-490 activity was not affected by either enzyme.
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Laboratory diagnosis of Clostridium difficile infection can molecular amplification methods move us out of uncertainty?
J Mol Diagn
PUBLISHED: 01-27-2011
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The laboratory diagnosis of Clostridium difficile infection (CDI) continues to be challenging. Recent guidelines from professional societies in the United States note that enzyme immunoassays for toxins A and B do not have adequate sensitivity to be used alone for detecting CDI, yet the optimal method for diagnosing this infection remains unclear. Nucleic acid amplification tests (NAATs) that target chromosomal toxin genes (usually the toxin B gene, tcdB) show high sensitivity and specificity, provide rapid results, and are amenable to both batch and on-demand testing, but these tests were not universally recommended for routine use in the recent guidelines. Rather, two-step algorithms that use glutamate dehydrogenase (GDH) assays to screen for C. difficile in stool specimens, followed by either direct cytotoxin testing or culture to identify toxin-producing C. difficile isolates, were recommended in one guideline and either GDH algorithms or NAATs were recommended in another guideline. Unfortunately, neither culture nor direct cytotoxin testing is widely available. In addition, this two-step approach requires 48 to 92 hours to complete, which may delay the initiation of therapy and critical infection control measures. Recent studies also show the sensitivity of several GDH assays to be <90%. This review considers the role of NAATs for diagnosing CDI and explores their potential advantages over two-step algorithms, including shorter time to results, while providing comparable, if not superior, accuracy.
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Evaluation of a rapid and completely automated real-time reverse transcriptase PCR assay for diagnosis of enteroviral meningitis.
J. Clin. Microbiol.
PUBLISHED: 12-15-2010
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Nucleic acid amplification tests (NAATs) for enterovirus RNA in cerebrospinal fluid (CSF) have emerged as the new gold standard for diagnosis of enteroviral meningitis, and their use can improve the management and decrease the costs for caring for children with enteroviral meningitis. The Xpert EV assay (Cepheid, Sunnyvale, CA) is a rapid, fully automated real-time PCR test for the detection of enterovirus RNA that was approved by the U.S. Food and Drug Administration for in vitro diagnostic use in March 2007. In this multicenter trial we established the clinical performance characteristics of the Xpert EV assay in patients presenting with meningitis symptoms relative to clinical truth. Clinical truth for enteroviral meningitis was defined as clinical evidence of meningitis, the absence of another detectable pathogen in CSF, and detection of enterovirus in CSF either by two reference NAATs or by viral culture. A total of 199 prospectively and 235 retrospectively collected specimens were eligible for inclusion in this study. The overall prevalence of enteroviral meningitis was 26.04%. The Xpert EV assay had a sensitivity of 94.69% (90% confidence interval [CI] = 89.79 to 97.66%), specificity of 100% (90% CI = 99.07 to 100%), positive predictive value of 100%, negative predictive value of 98.17, and an accuracy of 98.62% relative to clinical truth. The Xpert EV assay demonstrated a high degree of accuracy for diagnosis of enteroviral meningitis. The simplicity and on-demand capability of the Xpert EV assay should prove to be a valuable adjunct to the evaluation of suspected meningitis cases.
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Rapid molecular detection of tuberculosis and rifampin resistance.
N. Engl. J. Med.
PUBLISHED: 09-10-2010
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Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect.
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Impact of strain type on detection of toxigenic Clostridium difficile: comparison of molecular diagnostic and enzyme immunoassay approaches.
J. Clin. Microbiol.
PUBLISHED: 08-11-2010
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A multicenter clinical trial assessed the performance of the Cepheid Xpert C. difficile assay on stool specimens collected from patients suspected of having Clostridium difficile infection (CDI). A total of 2,296 unformed stool specimens, collected from seven study sites, were tested by Xpert C. difficile enrichment culture followed by cell culture cytotoxicity testing of the isolates (i.e., toxigenic culture with enrichment) and the study sites standard C. difficile test methods. The methods included enzyme immunoassay (EIA), direct cytotoxin testing, and two- and three-step algorithms using glutamate dehydrogenase (GDH) screening followed by either EIA or EIA and an in-house PCR assay. All C. difficile strains were typed by PCR-ribotyping. Compared to results for toxigenic culture with enrichment, the sensitivity, specificity, and positive and negative predictive values of the Xpert assay were 93.5, 94.0, 73.0, and 98.8%, respectively. The overall sensitivity of the EIAs compared to that of enrichment culture was 60.0%, and the sensitivity of combined GDH algorithms was 72.9%; both were significantly lower than that of Xpert C. difficile (P < 0.001 and P = 0.03, respectively). The sensitivity of the EIA was significantly lower than that of the Xpert C. difficile assay for detection of ribotypes 002, 027, and 106 (P < 0.0001, P < 0.0001, and P = 0.004, respectively, Fishers exact test), and the sensitivity of GDH algorithms for ribotypes other than 027 was lower than that for Xpert C. difficile (P < 0.001). The Xpert C. difficile assay is a simple, rapid, and accurate method for detection of toxigenic C. difficile in unformed stool specimens and is minimally affected by strain type compared to EIA and GDH-based methods.
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Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology.
J. Clin. Microbiol.
PUBLISHED: 10-28-2009
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Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert Systems MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assays buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.
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Prevalence and management of invalid GeneXpert enterovirus results obtained with cerebrospinal fluid samples: a 2-year study.
J. Clin. Microbiol.
PUBLISHED: 07-15-2009
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A total of 525 cerebrospinal fluid (CSF) samples submitted during the 2007 and 2008 enteroviral seasons were included in a study to determine the prevalence of and potential risk factors for invalid Cepheid GeneXpert enterovirus assay (GXEA) results, as well as possible solutions for the problem. The invalid GXEA results were reported for 43 (8.2%) specimens and correlated with increased visibility of red blood cells (P < 0.0001) but not with CSF xanthochromia and clotting. Invalid GXEA result rates were markedly diminished by 82.1% and 96.0% and test sensitivities were minimally decreased by 1.7% and 3.6% when these specimens were tested at a 1:5 dilution and after a freeze-thaw cycle, respectively.
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Democratizing molecular diagnostics: the GeneXpert(®) enterovirus assay.
Expert Opin Med Diagn
PUBLISHED: 01-01-2009
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Background: Few clinical scenarios are as diagnostically demanding as the workup of acute meningitis in the emergency room setting. In humans, most cases of meningitis are due to enteroviral infection, which usually resolves with supportive care and does not generally require hospitalization. The difficulty in evaluating meningitis is often related to its nonspecific presentation; rapid identification of the underlying etiologic agent is critical to selection of the most appropriate course of therapy. Objective: To develop a rapid, automated polymerase chain reaction-based test for enteroviral meningitis that can be run on demand, and to make the test easy enough to operate that most hospital laboratories can offer it on an immediate basis in order to provide maximum medical impact. Methods: A sensitive and specific test on the GeneXpert was developed for detection of enteroviral RNA in cerebrospinal fluid. The automated test was carried out in a disposable GeneXpert cartridge, which conducts the steps of nucleic acid purification, amplification and detection of enteroviral genomic RNA in ? 2 h. Preclinical and clinical studies showed sensitivities of 97.1 and 98%, respectively, and specificities of 100 and 97.1%, respectively. In February 2006, the test received FDA clearance as an aid in the diagnosis of viral meningitis. Conclusions: Rapid and accurate molecular diagnostic testing for enterovirus infection provides clinicians with information they need to make the best patient decisions in managing patients with meningitis. The GeneXpert enterovirus assay features on-demand availability and a level of simplicity that allows nearly any hospital laboratory to offer a sophisticated test that was once the exclusive domain of reference laboratories.
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Antimicrobial-resistant strains of Clostridium difficile from North America.
Antimicrob. Agents Chemother.
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A total of 316 toxigenic Clostridium difficile clinical isolates of known PCR ribotypes from patients in North America were screened for resistance to clindamycin, metronidazole, moxifloxacin, and rifampin. Clindamycin resistance was observed among 16 different ribotypes, with ribotypes 017, 053, and 078 showing the highest proportions of resistance. All isolates were susceptible to metronidazole. Moxifloxacin resistance was present in >90% of PCR-ribotype 027 and 053 isolates but was less common among other ribotypes. Only 7.9% of the C. difficile isolates were resistant to rifampin. Multidrug resistance (clindamycin, moxifloxacin, and rifampin) was present in 27.5% of PCR-ribotype 027 strains but was rare in other ribotypes. These results suggest that antimicrobial resistance in North American isolates of C. difficile varies by strain type and parallels rates of resistance reported from Europe and the Far East.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.