Molecular tests for human papillomavirus (HPV), Chlamydia trachomatis and Neisseria gonorrhoeae in liquid-based cytology specimen

<p>Abstract</p> <p>Background</p> <p>Laboratory detection of Human papillomavirus (HPV), <it>Chlamydia trachomatis </it>and <it>Neisseria gonorrhoeae </it>in liquid-based cervicovaginal cytology specimens is now based on identification of the DNA sequences unique to these infectious agents. However, current commercial test kits rely on nucleotide probe hybridization to determine DNA sequences, which may lead to diagnostic errors due to cross-reactivity. The aim of this study was to find a practical approach to perform automated Sanger DNA sequencing in clinical laboratories for validation of the DNA tests for these three infectious agents.</p> <p>Methods</p> <p>A crude proteinase K digestate of 5% of the cells collected in a liquid-based cervicovaginal cytology specimen was used for the detection of DNA molecules specific for HPV, <it>C trachomatis </it>and <it>N gonorrhoeae</it>, and for preparation of materials suitable for direct automated DNA sequencing. Several sets of commercially available polymerase chain reaction (PCR) primers were used to prepare nested PCR amplicons for direct DNA sequencing.</p> <p>Results</p> <p>Some variants of HPV-16 and HPV-31 were found to share an at least 34-base long sequence homology downstream of the GP5+ binding site, and all HPV-6 and HPV-11 variants shared an upstream 34-base sequence including part of the GP5+ primer. Accurate HPV genotyping frequently required more than 34-bases for sequence alignments to distinguish some of the HPV genotype variants with closely related sequences in this L1 gene hypervariable region. Using the automated Sanger DNA sequencing method for parallel comparative studies on split samples and to retest the residues of samples previously tested positive for <it>C trachomatis </it>and/or for <it>N gonorrhoeae</it>, we also found false-negative and false-positive results as reported by two commercial nucleic acid test kits.</p> <p>Conclusion</p> <p>Identification of a signature DNA sequence by the automated Sanger method is useful for validation of HPV genotyping and for molecular testing of <it>C trachomatis </it>and <it>N gonorrhoeae </it>in liquid-based cervicovaginal Papanicolaou (Pap) cytology specimens for clinical laboratories with experience in molecular biology to increase the specificity of these DNA-based tests. However, even a highly specific test for high-risk HPV genotyping may have unacceptably low positive predictive values for precancer lesion in populations with a low cervical cancer prevalence rate.</p

Routine human papillomavirus genotyping by DNA sequencing in community hospital laboratories

<p>Abstract</p> <p>Background</p> <p>Human papillomavirus (HPV) genotyping is important for following up patients with persistent HPV infection and for evaluation of prevention strategy for the individual patients to be immunized with type-specific HPV vaccines. The aim of this study was to optimize a robust "low-temperature" (LoTemp™) PCR system to streamline the research protocols for HPV DNA nested PCR-amplification followed by genotyping with direct DNA sequencing. The protocol optimization facilitates transferring this molecular technology into clinical laboratory practice. In particular, lowering the temperature by 10°C at each step of thermocycling during <it>in vitro </it>DNA amplification yields more homogeneous PCR products. With this protocol, template purification before enzymatic cycle primer extensions is no longer necessary.</p> <p>Results</p> <p>The HPV genomic DNA extracted from liquid-based alcohol-preserved cervicovaginal cells was first amplified by the consensus MY09/MY11 primer pair followed by nested PCR with GP5+/GP6+ primers. The 150 bp nested PCR products were subjected to direct DNA sequencing. The hypervariable 34–50 bp DNA sequence downstream of the GP5+ primer site was compared to the known HPV DNA sequences stored in the GenBank using on-line BLAST for genotyping. The LoTemp™ ready-to-use PCR polymerase reagents proved to be stable at room temperature for at least 6 weeks. Nested PCR detected 107 isolates of HPV in 513 cervicovaginal clinical samples, all validated by DNA sequencing. HPV-16 was the most prevalent genotype constituting 29 of 107 positive cases (27.2%), followed by HPV-56 (8.5%). For comparison, Digene HC2 test detected 62.6% of the 107 HPV isolates and returned 11 (37.9%) of the 29 HPV-16 positive cases as "positive for high-risk HPV".</p> <p>Conclusion</p> <p>The LoTemp™ ready-to-use PCR polymerase system which allows thermocycling at 85°C for denaturing, 40°C for annealing and 65°C for primer extension can be adapted for target HPV DNA amplification by nested PCR and for preparation of clinical materials for genotyping by direct DNA sequencing. HPV genotyping is performed by on-line BLAST algorithm of a hypervariable L1 region. The DNA sequence is included in each report to the physician for comparison in following up patients with persistent HPV infection, a recognized tumor promoter in cancer induction.</p

Rotational effect on ROI's for accurate lumen quantification in bifurcated MR plaque volumes

This paper presents a use of geometric-based method integrated with classifier for lumen wall estimation using MR plaque volumes. The following are the new things the readers will observe when it comes to plaque imaging. (a) Application of three different sets of classifiers (Fuzzy, Maxkovian and Graph-based) for lumen region classification in plaque MR volumes. These classifiers axe used in multi-resolution framework. (b) Usage of rule-based region merging applied to the sub-classes of lumen region. (c) Rotational effect on region of interest in arterial bifurcation zones for accurate lumen region identification and boundary estimation

Accurate Lumen Identification, Detection, and Quantification in MR Plaque Volumes

The importance of plaque component classi .cation and vessel wall quantification has been well established by several research groups (see Refs. [1–30])

Tumor Necrosis Factor-Induced Neutrophil Adhesion Occurs Via Sphingosine Kinase-1-Dependent Activation of Endothelial α5β1 Integrin

Leukocyte recruitment plays a major role in the immune response to infectious pathogens, as well as during inflammatory and autoimmune disorders. The process of leukocyte extravasation from the blood requires a complex cascade of adhesive events between the leukocytes and the endothelium, including initial leukocyte rolling, adhesion, and finally transendothelial migration. Current research in this area aims to identify the key leukocyte subsets that initiate a given disease and to identify the trafficking molecule(s) that will most specifically inhibit those cells. Herein we demonstrate that tumor necrosis factor (TNF)α activates the integrin α5β1 without altering total expression levels of β1 integrin on human umbilical vein endothelial cells. Moreover, our studies suggest that TNFα-induced β1 activation is dependent on sphingosine kinase-1, but independent of the sphingosine-1-phosphate family of G protein-coupled receptors. We also show, using a parallel plate flow chamber assay, that neutrophil adhesion to TNFα-activated endothelium can be attenuated by blocking α5β1 or its ligand angiopoietin-2. These observations add new complexities that broaden the accepted concept of cellular trafficking with neutrophil adhesion to TNFα activated endothelial cells being sphingosine kinase-1, α5β1, and angiopoietin-2 dependent. Moreover, this work supports the notion that sphingosine kinase-1 may be the single target required for an effective broad spectrum approach to combat inflammation and immune disorders