Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus

BACKGROUND: The GP5+/GP6+ PCR assay is a well-established HPV detection technique. This study has examined the effects of incorporating 'hot start' and 'touchdown' steps into the protocol. In addition, dTTP was substituted with dUTP to permit contamination control measures against carry-over PCR product. METHODS: Firstly, HPV-16 was amplified from SiHa cell DNA (0.1 ng–100 ng) diluted in a background of C-33A DNA (100 ng-2 μg). Secondly, the detection of small quantities (15ag-1.5pg) of HPV recombinant plasmids (types 16, 31, 33, 45, 51, 52, and 56) diluted in C-33A DNA was investigated. Thirdly, clinical sample DNA extracts (cervical smears, formalin-fixed vaginal lesions and breast tumors) were tested for HPV. Six different PCR protocols were assessed. HPV was detected by gel electrophoresis, and by Southern and dot blot hybridization. RESULTS: HPV detection sensitivity was dependent on the total amount of DNA in a PCR. Touchdown protocols supported HPV-16 detection from 1 ng or 0.5 ng SiHa cell DNA in a background of 2 μg or 1 μg C-33A DNA respectively, and from 0.1 ng of SiHa cell DNA (~28 copies HPV-16) in 500 ng or 100 ng background DNA. Under standard GP5+/GP6+ annealing conditions, HPV-16 went undetected when the DNA content of a PCR was 2 μg or 1 μg, and with 500 ng C-33A DNA the sensitivity limit was 1 ng SiHa cell DNA. HPV recombinant plasmids were each detected with high (albeit varying) sensitivity by a touchdown protocol. HPV-31 was better amplified under standard annealing conditions (1.5fg in 100 ng background DNA) than by a touchdown approach (15fg detection limit). HPV-52 was not amplified by the standard protocol at the dilutions tested. Seventeen different HPV types were demonstrated in 47/65 (72%) abnormal cytology samples recorded as HPV negative by standard GP5+/GP6+ conditions. Twenty-one different HPV types were recorded in 111/114 (97%) vaginal lesions. Multiple infections were also detectable using a touchdown approach. Of 26 breast tumors, 5 (19%) tested HPV positive by the standard assay and 15/26 (58%) using a touchdown protocol. CONCLUSION: Touchdown modification of the GP5+/GP6+ PCR assay enables the detection of HPV undetected under regular assay conditions. The use of standardized DNA quantities in a PCR rather than standard sample volumes containing arbitrary amounts of DNA is supported. A touchdown approach may be beneficial as an analytical test for the re-evaluation of (apparently) HPV negative abnormal cervical cytological or histological samples, and for investigating the association of HPV with disease conditions at diverse organ sites. The clinical utility of a touchdown approach for HPV detection requires further investigation as increased assay analytical sensitivity may not necessarily equate with improved clinical sensitivity or specificity

Rhodococcus Bacteremia in Cancer Patients Is Mostly Catheter Related and Associated with Biofilm Formation

Rhodococcus is an emerging cause of opportunistic infection in immunocompromised patients, most commonly causing cavitary pneumonia. It has rarely been reported as a cause of isolated bacteremia. However, the relationship between bacteremia and central venous catheter is unknown. Between 2002 and 2010, the characteristics and outcomes of seventeen cancer patients with Rhodococcus bacteremia and indwelling central venous catheters were evaluated. Rhodococcus bacteremias were for the most part (94%) central line-associated bloodstream infection (CLABSI). Most of the bacteremia isolates were Rhodococcus equi (82%). Rhodococcus isolates formed heavy microbial biofilm on the surface of polyurethane catheters, which was reduced completely or partially by antimicrobial lock solution. All CLABSI patients had successful response to catheter removal and antimicrobial therapy. Rhodococcus species should be added to the list of biofilm forming organisms in immunocompromised hosts and most of the Rhodococcus bacteremias in cancer patients are central line associated

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Hybrid capture vs. PCR screening of cervical human papilloma virus infections. Cytological and histological associations in 1270 women

<p>Abstract</p> <p>Background</p> <p>We evaluated two molecular methods of HPV detection and their correlation with cytological and histological diagnosis in a large sample of Greek women.</p> <p>Methods</p> <p>All women with liquid-based cytology performed at a University Hospital between 2000 and 2003 were included. The Hybrid Capture 2 (HC2) kit and in house Polymerase Chain Reaction (PCR) were used for HPV DNA detection. Cervical biopsy was performed for women with ASCUS+ cytology, HPV detection, or abnormal colposcopy. Positive (PLR) and negative (NLR) likelihood ratios were calculated for cytology and HPV molecular testing for the prediction of CIN2 and greater histology.</p> <p>Results</p> <p>Of the 1270 women evaluated 241 (18.5%) had abnormal cytology. Cytology diagnosed high-grade squamous intraepithelial lesion (HSIL) or invasive carcinoma in 21(1.7%) cases whereas 26 (2%) women had CIN2+ or greater histology. PCR detected HPV in 397/1270 (31.3%) and HC2 in 260/1270 (20.4%) samples. Both molecular tests exhibited high reproducibility (Cohen's kappa value 0.691, 95% CI: 0.664 - 0.718). Positive likelihood ratios (PLR) of 9.4, 3.8 and 3.4 and negative likelihood ratios of 0.13, 0.21, and 0 were noted for ≥ LSIL, any positive HC2 or any positive PCR-HPV testing, for predicting CIN2+ histology, respectively. All CIN 3+ lesions harbored high risk oncogenic HPV type infections.</p> <p>Conclusions</p> <p>HPV infection was found in a large proportion of this population and was associated with CIN 2/3 lesions and infiltrating carcinomas. Thin prep testing and HPV detection by HC2 or PCR performed very well with regards to identifying high grade lesions in an environment with experienced examiners.</p

Tumor associated PD-L1 expression pattern in microscopically tumor positive sentinel lymph nodes in patients with melanoma

Background: Characterization of PD-L1 expression within clinically/radiologically negative but microscopically tumor positive sentinel lymph nodes (SLN) is important to our understanding of the relevance of this immune checkpoint pathway for adjuvant therapy. Methods: Patients included had primary cutaneous melanoma, Breslow thickness of 2.01-4.0 or >4mm with or without tumor ulceration (T3a, T3b, T4a, T4b). All patients had microscopically tumor positive SLN. Hematoxylin and eosin (H&E) staining was performed, followed by PD-L1 immunohistochemical (IHC) staining using a preliminary IHC assay with anti-PD-L1 antibody clone 22C3. The slides were separately evaluated by two pathologists (JY and CG). Samples containing metastatic melanoma lesions were scored separately for PD-L1 expression in intratumoral and peritumoral locations, by utilizing two scoring methods. Results: Twenty-four patients where metastatic melanoma presence in the SLN was confirmed by H&E review of the cut sections were included in the final analysis of PD-L1 expression. SLN tumor size ranged from 1 to 2mm. For three patients, the melanin content was too high to confidently assign a PD-L1 score. For the remaining 21 patients, all had some evidence of either intratumoral or peritumoral PD-L1 expression. The frequency of intratumoral tumor-associated PD-L1 expression was: 0% of tumor cells (3pts, 14%); 10% (7pts, 33%). Conclusions: Tumor-associated PD-L1 expression is readily detectable within melanoma micrometastases in the SLN of the majority of patients. These results support the testing of a therapeutic role for PD1/PD-L1 inhibition in the adjuvant setting, targeting melanoma micrometastases

Measurement of VH, H → b b ¯ production as a function of the vector-boson transverse momentum in 13 TeV pp collisions with the ATLAS detector

Cross-sections of associated production of a Higgs boson decaying into bottom-quark pairs and an electroweak gauge boson, W or Z, decaying into leptons are measured as a function of the gauge boson transverse momentum. The measurements are performed in kinematic fiducial volumes defined in the `simplified template cross-section' framework. The results are obtained using 79.8 fb-1 of proton-proton collisions recorded by the ATLAS detector at the Large Hadron Collider at a centre-of-mass energy of 13 TeV. All measurements are found to be in agreement with the Standard Model predictions, and limits are set on the parameters of an effective Lagrangian sensitive to modifications of the Higgs boson couplings to the electroweak gauge bosons

Measurement of the t¯tZ and t¯tW cross sections in proton-proton collisions at √s=13 TeV with the ATLAS detector

A measurement of the associated production of a top-quark pair (t¯t) with a vector boson (W, Z) in proton-proton collisions at a center-of-mass energy of 13 TeV is presented, using 36.1  fb−1 of integrated luminosity collected by the ATLAS detector at the Large Hadron Collider. Events are selected in channels with two same- or opposite-sign leptons (electrons or muons), three leptons or four leptons, and each channel is further divided into multiple regions to maximize the sensitivity of the measurement. The t¯tZ and t¯tW production cross sections are simultaneously measured using a combined fit to all regions. The best-fit values of the production cross sections are σt¯tZ=0.95±0.08stat±0.10syst pb and σt¯tW=0.87±0.13stat±0.14syst pb in agreement with the Standard Model predictions. The measurement of the t¯tZ cross section is used to set constraints on effective field theory operators which modify the t¯tZ vertex

Search for heavy neutral Higgs bosons produced in association with b-quarks and decaying into b-quarks at root s=13 TeV with the ATLAS detector

A search for heavy neutral Higgs bosons produced in association with one or two b -quarks and decaying to b -quark pairs is presented using 27.8  fb − 1 of √ s = 13  TeV proton-proton collision data recorded by the ATLAS detector at the Large Hadron Collider during 2015 and 2016. No evidence of a signal is found. Upper limits on the heavy neutral Higgs boson production cross section times its branching ratio to b ¯ b are set, ranging from 4.0 to 0.6 pb at 95% confidence level over a Higgs boson mass range of 450 to 1400 GeV. Results are interpreted within the two-Higgs-doublet model and the minimal supersymmetric Standard Model