Subset Of Specimens Research Articles

Ordering of the Serum Angiotensin-Converting Enzyme Test in Patients Receiving Angiotensin-Converting Enzyme Inhibitor Therapy: An Avoidable but Common Error

Serum angiotensin-converting enzyme (ACE) levels may be decreased by use of ACE inhibitor (ACEI) medication. In this study, we determined how often ACE levels were measured in patients receiving ACEI therapy. ACE levels analyzed over a 54-month preintervention time period at an academic medical center were reviewed retrospectively for tests performed during ACEI therapy. These data were compared with a large, deidentified dataset of ACE levels measured at a national reference laboratory; in vitro studies of ACEI inhibition; and liquid chromatography time-of-flight mass spectrometry detection of lisinopril in a subset of clinical specimens. Over a 54-month period, 1,292 patients had ACE levels measured, with 108 patients (8.4%) receiving ACEI therapy at the time of testing. ACE levels measured for patients receiving ACEI therapy were substantially lower. In general, clinical teams did not recognize a medication effect on ACE levels. Introduction of a warning prompt in the electronic health record reduced the ordering of ACE levels in patients receiving ACEIs by > 60% in a 17-month postintervention time period. The deidentified dataset of ACE levels at a reference laboratory showed a bimodal distribution, with a peak of very low ACE levels. Using liquid chromatography time-of-flight mass spectrometry, the presence of lisinopril was confirmed in a subset of specimens with low ACE activity. In vitro studies of two different ACE assays showed significant inhibition of activity at clinically relevant concentrations. Assessment of ACE activity is often measured for patients receiving ACEIs, potentially leading to low ACE concentrations and inaccurate interpretations.

Rise (and demise?) of subspecies in the Galah (Eolophus roseicapilla), a widespread and abundant Australian cockatoo

The Galah (Eolophus roseicapilla) is a widespread Australian bird. Its geographical variation and subspecies remain poorly understood. We sampled 192 specimens from across the entire range of the species to assess its phylogeographical structure using the mitochondrially encoded NADH dehydrogenase 2 (MT-ND2) gene. A subset of specimens was examined for nuclear DNA diversity using the Amplified Fragment Length Polymorphism (AFLP) method. Weakly defined structure was evident in the mitochondrial DNA (mtDNA) data, which suggested three geographically defined haplogroups that broadly coincide with the three conventionally recognised subspecies: E. r. roseicapilla—western haplogroup; E. r. albiceps—eastern haplogroup; and E. r. kuhli—northern haplogroup. The three haplogroups all overlap and intersect broadly across the centre of the Australian continent. Geographical expansions of each haplogroup appear to have occurred before and after the Last Glacial Maximum (~20 000 years ago), the species itself probably having undergone an earlier expansion at c. 60 000 years ago. The preliminary nuclear AFLP data suggest very little structure though they likely track more recent processes of gene flow and population admixture. Expansion of the species since European settlement represents another, later expansion uncoupled from what is tracked in the mtDNA data. Future quantitative analyses of phenotypic variation will enhance the precision of how geographical variation in all datasets in this species should be interpreted.

Abstract P4-08-01: Assessing the safety and feasibility of efficient hypothesis testing in patients with metastatic triple negative breast cancer

Abstract We hypothesize that new insights into how cancers progress and respond to treatment will come from clinical trials that i) extensively characterize the molecular features of a patient’s cancer; ii) use results to predict drug susceptibilities; iii) treat in accordance with these predictions; and iv) learn from individual patient outcomes to iterate and improve over time. To investigate the feasibility of this type of clinical study, we launched the "Intensive Trial of OMics in Cancer" (ITOMIC) for patients with metastatic triple negative breast cancer (TNBC) (Clinicaltrials.gov ID: NCT01957514). Eligible patients have metastatic TNBC, are platinum-naive, and are scheduled to receive Cisplatin. Biopsies are performed under carefully controlled conditions prior to Cisplatin – starting all subjects on a common treatment path, and uncoupling the time needed for specimen analysis from immediate therapy. Biopsies are repeated upon completion of Cisplatin and following subsequent therapies. A subset of specimens is chosen for whole Exome Sequencing, deep sequencing of a panel of cancer associated genes, and RNA-sequencing. De-identified results are placed on a web-based server for analysis and discussed at a meeting of the ITOMIC tumor board. A report describing results and potential therapies is provided to the subject’s oncologist. Treatment decisions are left to the discretion of the oncologist. If a decision is taken to pursue treatments identified in our report we offer assistance in accessing those treatments. Ten patients have been screened and seven have enrolled. Subjects range in age from 40 to 77 years and all but one has received extensive prior treatment for metastatic TNBC. All seven underwent an initial set of biopsies, targeting between two and five metastatic sites. For most metastatic sites, multiple core needle passes are performed. All subjects tolerated the biopsies well without significant adverse events, and all started treatment with Cisplatin. Three subjects completed Cisplatin and underwent a second round of biopsies. Potential targets for therapy were identified in 5 of the first 6 subjects, and three subjects have received four predicted therapies: 1) a patient with somatic loss of BRCA1 and two linked FGFR2 activating mutations, who was treated first with Veliparib through a single-patient IND and then switched to Ponatinib which produced a partial response; 2) a patient with a novel missense ROS1 mutation treated with crizotinib; and 3) a patient with CYP3A4 copy gain treated with cyclophosphamide. Conclusion: Our early experience indicates that this approach is feasible and may increase the efficiency of learning from patients with advanced cancer. Citation Format: C Anthony Blau, Colin Pritchard, Michael O Dorschner, Sibel Blau, Brigham Mecham, Elisabeth Mahen, VK Gadi, Wayne Monsky, Kimberly Burton, Arturo Ramirez, Jackie Stilwell, Eric Kladjian, Carol Collins, Jeannine S McCune, William S Noble, Julie Gralow, Frank Senecal, Linda Dhaene, Nicole Kuderer, Jennifer Specht, Chaozhong Song, Carla Grandori, Nathan Price, Mary Goldman, Aime Radenbaugh, David Haussler, Jingchun Zhu. Assessing the safety and feasibility of efficient hypothesis testing in patients with metastatic triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-08-01.

Corrosion and Fretting of a Modular Hip System: A Retrieval Analysis of 60 Rejuvenate Stems

Femoral stems with dual-taper modularity were introduced to allow independent control of length, offset, and version. Corrosion and fretting related to micromotion at the neck-stem junction are thought to stimulate an adverse local tissue reaction (ALTR). Analysis of 60 consecutively retrieved modular-neck stem implants (Rejuvenate, Stryker) revised primarily for ALTR was done to determine the variables influencing corrosion and fretting patterns at the neck-stem interface. Taper damage evaluation was performed with stereomicrocopic analysis with two observers. Evidence of fretting and corrosion was seen at the neck-stem taper in all implants, including three implants revised for periprosthetic fractures within four weeks of the index surgery indicating that this process starts early. Femoral stems paired with the long overall neck lengths had significantly higher corrosion scores. Correlation of the corrosion severity at particular locations with the length of implantation suggests that the neck-stem junction experiences cyclic cantilever bending in vivo. The positive correlation between the length of implantation and fretting/corrosion scores bodes poorly for patients who still have this implant. Scanning electron microscopy on a subset of specimens was also performed to evaluate the black corrosion material. We strongly urge frequent follow-up exams for every patient with this particular modular hip stem.

Arginine promotes fluoride uptake into artificial carious lesionsin vitro

This study focused on the interaction of arginine and fluoride on the remineralization of artificial enamel carious lesions in vitro. Human enamel blocks with artificial carious lesions were prepared and randomly divided into six treatment groups (n=30 for each group): deionized water, arginine solution, NaF solution, arginine/NaF solution, arginine-free fluoride toothpaste slurry and arginine-containing fluoride toothpaste slurry. After a pH-cycling regimen for 10 days, subsets of specimens from each group were subjected to independent analysis, including surface microhardness (n=8/30), cross-sectional microhardness (n=8/30), enamel fluoride uptake (n=8/30) and polarized light microscopy (n=6/30). The samples tested for surface microhardness were further subjected to simulated plaque acid challenge before surface microhardness was remeasured. Arginine solution promoted remineralization compared with deionized water control (p<0.05). When used in combination with fluoride, arginine significantly increased fluoride uptake compared with fluoride alone (p<0.05). In addition, lesions treated with arginine-containing toothpaste also showed superior fluoride uptake compared with those treated with conventional fluoride toothpaste (p<0.05). Arginine promoted enamel fluoride uptake when used in combination with fluoride, thereby contributing to resistance of enamel to carious demineralization.

Detection and characterisation of novel bocavirus (genus Bocaparvovirus) and gastroenteritis viruses from asymptomatic pigs in Ireland

BackgroundLivestock animals have been the assumed source of several human epidemics in recent years, for example, influenza H1N1, rotavirus G8/G9, and MERS-CoV. Surveillance of novel viruses in animals is essential to evaluate the risk to human and animal health and to determine any economic impact, for example, failure to thrive. There is a paucity of data regarding detection and characterisation of gastroenteritis viruses, particularly novel viruses, in porcines in Ireland. Recently, a number of small novel porcine DNA viruses have emerged globally, for example, torque teno sus virus, porcine bocavirus, and parvoviruses 2 & 4, and little is known about the biology and potential pathogenicity of these viruses. Bocaparvovirus is a genetically distinct group of viruses which has been recently detected in humans and animals.MethodsIn this study, the presence of gastroenteritis viruses (rotavirus A, porcine circovirus, adenovirus, and porcine bocavirus) was investigated in a selection of archived faecal samples from asymptomatic piglets from a commercial farm in Ireland. A total of 104 specimens were pooled and screened using conventional molecular techniques (PCR and RT-PCR), a subset of specimens (n=44) were then examined individually. Viral diversity was then investigated using statistical and phylogenetic techniques.ResultsInitial screening showed a high prevalence of PBoV in this farm, with the formation of three distinct groups in phylogenetic analysis. Other viruses were also investigated in this study with the first report of PCV, PAdV and lineage I G5 RVA in Ireland. Some specimens contained >1 virus, with statistical analysis indicating a strong correlation for mixed infections of PBoV and PAdV on this farm.ConclusionInvestigating the diversity of circulating enteric viruses on Irish porcine farms is important to improve the prophylactic tools available and to facilitate the early detection of changes in circulating viruses.

Multi-center evaluation of the adenovirus R-gene US assay for the detection of adenovirus in respiratory samples

BackgroundAdenoviruses (AdV) cause a variety of upper and lower respiratory tract infections, with the potential for severe outcomes, especially in persons with immune suppression or other underlying diseases. The ADENOVIRUS US R-gene (AdV R-gene, Argene/bioMérieux) is a FDA cleared real-time PCR assay that utilizes primers and fluorescent probes that target a conserved region of the hexon gene and an internal control DNA. ObjectivesThis prospective multi-center study evaluated the clinical performance of AdV R-gene for AdV detection in respiratory specimens from symptomatic patients of all ages. Study designNucleic acids from nasopharyngeal washes/aspirates (NPW/A; n=393) and NP flocked swabs (NPS, Copan) (n=1183) were extracted using NucliSENS easyMAG (bioMérieux) and AdV R-gene PCR was performed using the SmartCycler (Cepheid). AdV R-gene results were compared to R-Mix culture (Quidel/Diagnostic Hybrids). For a subset of samples (n=946) AdV R-gene and R-Mix results were also compared to A549 cell culture. ResultsIn first intention analysis for NPS the AdV R-gene positive percent agreement (PPA), and negative percent agreement (NPA) were 91.7% and 96.2%, respectively, and for NPW/A were 100% and 94.4%, respectively, compared to R-Mix culture. In second intention analysis, discordant samples only were tested with an AdV real-time PCR assay (Viracor-IBT Labs) and amplicon sequencing. For NPS, the sensitivity, specificity, PPV and NPV for AdV R-gene were 98.9%, 100%, 100%, and 99.9%, respectively and for R-Mix culture were 51.7%, 99.7%, 93.8%, and 96.3%, respectively. For NPW/A, the sensitivity, specificity, PPV and NPV for AdV R-gene were 100%, 99.7%, 97.6%, and 100%, respectively, and for R-Mix culture were 52.5%, 100%, 100%, and 94.9%, respectively. Overall, AdV was detected by AdV R-gene and R-Mix in 7.4% and 4.1% NPS, respectively, and in 10.7% and 5.3% NPW/A, respectively. Children 5yr and younger had the highest rates of AdV infections. In a subset of specimens (n=946) the sensitivity of AdV R-gene, R-Mix, and A549 cell culture were 95.0%, 55.4% and 66.3%. ConclusionsAdV R-gene is sensitive and specific for the detection of AdV in NPW/A and NPS samples. AdV R-gene is simple to use and provides a rapid time to results (within 2.5–3h).

MP66-15 ADVANCES IN THE ULTRA-RAPID VITRIFICATION OF HUMAN SPERMATAZOA WITH A MICROCAPILLARY SYSTEM

You have accessJournal of UrologyInfertility: Basic Research, Physiology & Pathophysiology1 Apr 2014MP66-15 ADVANCES IN THE ULTRA-RAPID VITRIFICATION OF HUMAN SPERMATAZOA WITH A MICROCAPILLARY SYSTEM Joseph W. McQuaid, Yahir Santiago-Lastra, Diane Wright, Mehmet Toner, Thomas L. Toth, and Cigdem Tanrikut Joseph W. McQuaidJoseph W. McQuaid More articles by this author , Yahir Santiago-LastraYahir Santiago-Lastra More articles by this author , Diane WrightDiane Wright More articles by this author , Mehmet TonerMehmet Toner More articles by this author , Thomas L. TothThomas L. Toth More articles by this author , and Cigdem TanrikutCigdem Tanrikut More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2062AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Freezing low concentrations of sperm from severely oligospermic patients while maintaining adequate post-thaw motility remains a challenge. We previously reported the feasibility of ultra-rapid vitrification using a microcapillary system. In an attempt to improve these results, several modifications were introduced including a larger capillary system and lower concentrations of non-toxic cryoprotectants: trehalose and propanediol. METHODS Using an IRB-approved protocol, 27 fresh ejaculate samples from healthy men were obtained and prepared via a swim-up protocol in human tubal fluid (HTF). 15 samples with an average concentration of 77 million/mL and pre-thaw total motility of 86% were included in our modified protocol. These samples were diluted in a drop-wise, 1:1 fashion with HTF media containing trehalose (0.05M) and propanediol (PrOH, 0.75M). After loading the samples into a series of 200 μm capillaries with heat-sealed ends, they were divided into two equal groups: 1) Control, from which the samples were immediately expelled, washed, then assessed via computer-assisted semen analysis, and 2) Experimental, which was vitrified in a super-cooled, liquid nitrogen bath, stored for up to 24 hours, then thawed, washed, and analyzed. A subset of specimens from the experimental group was used for viability staining. RESULTS Consistent post-thaw motility was achieved using this protocol. While a total motility of 48.6% was recorded in the control, the experimental group demonstrated an overall post-thaw motility of 10.5%. Subset analysis allowed stratification of samples based upon their ability to withstand this process. Of the highest-performing specimens, defined as those with a post-thaw motility >15%, we were able to conserve 22% of the sham control motility in our vitrified samples. CONCLUSIONS Building upon our prior experience, we sought to enhance sperm survival and improve post-thaw motility. Modifications included use of a larger capillary to reduce shear stress, conversion to a less viscous and less toxic media, and incorporation of a wash prior to final analysis. Gains were achieved in post-thaw motility and confirmed the feasibility of this system. However, it is clear from viability stains and subset analysis that there are yet undiscovered intrinsic and extrinsic variables affecting optimal recovery of specimens with this process. We will continue efforts to optimize this system for use in severely oligoospermic patients. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e746 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Joseph W. McQuaid More articles by this author Yahir Santiago-Lastra More articles by this author Diane Wright More articles by this author Mehmet Toner More articles by this author Thomas L. Toth More articles by this author Cigdem Tanrikut More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

In vitro biomechanical evaluation of fibular movement in below knee amputations

BackgroundIn below knee amputations, the remaining fibula may be subjected to abnormal forces resulting in problematic tibia-fibular movement. The purpose of the current work was to examine the effect of amputation length and interosseous membrane integrity on fibular movement when subjected to unopposed biceps femoris muscle tension. MethodsForty embalmed cadaveric specimens were subjected to a below knee amputation with fibular lengths of 5cm and 10cm. A subset of specimens (n=20) was further modified by sectioning the interosseous membrane. The tibias were mounted in a material testing machine and the biceps femoris was sutured to the actuator. Position-controlled tensile cyclic loading was applied (initial displacement of 4mm for 100cycles at 0.5Hz with increments of 2mm up to 20mm) to the biceps femoris. The kinematics of the fibula with respect to the tibia was analyzed for three degrees of freedom: abduction, flexion and rotation. FindingsThere was no interaction between below knee amputation length and interosseous membrane integrity on the degree of abduction, flexion, and rotation. However, below knee amputations with a sectioned interosseous membrane are abducted to a significantly greater degree than intact interosseous membrane below knee amputations. Furthermore, although embalmed specimens were tested here, embalming was consistent across specimens and it is unlikely that this confounded the findings. InterpretationUnderstanding the cause of fibular abduction in below knee amputation will lead to recommendations for preventive surgical and rehabilitative measures.

Clinical Validation of a PCR Assay for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer: Retrospective Testing of Specimens from the EURTAC Trial

The EURTAC trial demonstrated that the tyrosine kinase inhibitor (TKI) erlotinib was superior to chemotherapy as first-line therapy for advanced non-small cell lung cancers (NSCLC) that harbor EGFR activating mutations in a predominantly Caucasian population. Based on EURTAC and several Asian trials, anti-EGFR TKIs are standard of care for EGFR mutation-positive NSCLC. We sought to validate a rapid multiplex EGFR mutation assay as a companion diagnostic assay to select patients for this therapy. Samples from the EURTAC trial were prospectively screened for EGFR mutations using a combination of laboratory-developed tests (LDTs), and tested retrospectively with the cobas EGFR mutation test (EGFR PCR test). The EGFR PCR test results were compared to the original LDT results and to Sanger sequencing, using a subset of specimens from patients screened for the trial. Residual tissue was available from 487 (47%) of the 1044 patients screened for the trial. The EGFR PCR test showed high concordance with LDT results with a 96.3% overall agreement. The clinical outcome of patients who were EGFR-mutation detected by the EGFR PCR test was very similar to the entire EURTAC cohort. The concordance between the EGFR PCR test and Sanger sequencing was 90.6%. In 78.9% of the discordant samples, the EGFR PCR test result was confirmed by a sensitive deep sequencing assay. This retrospective study demonstrates the clinical utility of the EGFR PCR test in the accurate selection of patients for anti-EGFR TKI therapy. The EGFR PCR test demonstrated improved performance relative to Sanger sequencing.

Which specimens from a museum collection will yield DNA barcodes? A time series study of spiders in alcohol.

We report initial results from an ongoing effort to build a library of DNA barcode sequences for Dutch spiders and investigate the utility of museum collections as a source of specimens for barcoding spiders. Source material for the library comes from a combination of specimens freshly collected in the field specifically for this project and museum specimens collected in the past. For the museum specimens, we focus on 31 species that have been frequently collected over the past several decades. A series of progressively older specimens representing these 31 species were selected for DNA barcoding. Based on the pattern of sequencing successes and failures, we find that smaller-bodied species expire before larger-bodied species as tissue sources for single-PCR standard DNA barcoding. Body size and age of oldest successful DNA barcode are significantly correlated after factoring out phylogenetic effects using independent contrasts analysis. We found some evidence that extracted DNA concentration is correlated with body size and inversely correlated with time since collection, but these relationships are neither strong nor consistent. DNA was extracted from all specimens using standard destructive techniques involving the removal and grinding of tissue. A subset of specimens was selected to evaluate nondestructive extraction. Nondestructive extractions significantly extended the DNA barcoding shelf life of museum specimens, especially small-bodied species, and yielded higher DNA concentrations compared to destructive extractions. All primary data are publically available through a Dryad archive and the Barcode of Life database.

Identification of early HIV infections using the fourth generation Abbott ARCHITECT HIV Ag/Ab Combo chemiluminescent microparticle immunoassay (CIA) in San Diego County

BackgroundHIV screening assays have gone through several generations of development in an effort to narrow the “window period” of detection. Utilizing a fourth generation HIV screening assay has the potential to detect earlier HIV infection, thus reducing HIV-1 transmission. ObjectiveTo identify acute infections to decrease HIV transmission in San Diego County. Study designSerum specimens were collected from clients seen by multiple submitters in San Diego County. All acceptable specimens were screened using the 4th Gen Combo Assay. Initially reactive specimens were repeated in duplicate and if repeatedly reactive, were confirmed by HIV-1 Immunofluorescent Antibody Assay (IFA).IFA negative/inconclusive specimens were sent for HIV-1 NAT and HIV-2 antibody testing to referral laboratories. BioRad Multispot HIV-1/HIV-2 Rapid Test was also performed on a subset of specimens. ResultsOf 14,559 specimens received in 20 months, 14,517 specimens were tested. Of the 14,517 specimens that were tested, a total of 279 (1.9%) specimens were CIA repeatedly reactive and 240 of the 279 confirmed by HIV-1 IFA. Thirty-nine gave IFA negative/inconclusive result and 30 were further tested for HIV-1 NAT and 36 for HIV-2 antibody. Thirteen specimens were considered false positives by CIA and 17 specimens were classified as acute infections. Eleven of 39 IFA negative/inconclusive specimens were further tested by Multispot. Five of the 11 were positive by Multispot. ConclusionThe fourth generation Abbott ARCHITECT HIV Ag/Ab Combo Assay identified 17 patients who may have been missed by the prior HIV-1 screening assay used at San Diego County Public Health Laboratory.

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Evolutionary systematics of the AustralianEocyzicusfauna (Crustacea: Branchiopoda: Spinicaudata) reveals hidden diversity and phylogeographic structure

Although ‘large branchiopods’ are an important faunal element of the temporary water bodies in Australia's vast (semi)arid regions, knowledge of their diversity, distribution and ecology is still poor. Here, on the basis of one mitochondrial [cytochrome oxidase subunit I (COI)] and three nuclear (EF1α, ITS2 and 28S) markers, we present new data relating to the diversity and phylogeography of eastern and central Australian Eocyzicus (Spinicaudata) fauna. Using a combination of phylogenetic, haplotype network and DNA barcoding analyses of COI, 312 individuals were grouped into eleven main lineages. To infer whether these lineages are reproductively isolated from each other (the prerequisite for species delineation according to the Biological or Hennigian Species Concepts), separate analyses of each nuclear marker were performed on a subset of specimens. Although some lineages are non-monophyletic in the analysis of one nuclear marker, this is mostly attributed to processes such as incomplete lineage sorting rather than ongoing reproduction. The eleven lineages translate into at least seven species whose reproductive isolation is additionally indicated by sympatry, including both Australian Eocyzicus species previously described. Another three lineages may constitute further species, but their clear allopatric distribution rendered the test for reproductive isolation inapplicable. One lineage appears not to be reproductively isolated and is therefore considered a genetically distinct lineage within one of the other species, and one divergent lineage within E. argillaquus may constitute an additional species. Although sympatry is very common – six species occur in the central Paroo River catchment in eastern Australia, for instance – syntopic occurrence is rare. It is possible that a combination of differing habitat preferences and priority effects inhibits the presence of more than one Eocyzicus species per water body. There is little to no genetic differentiation between certain populations of the species found in eastern and central Australia (e.g. the Murray–Darling Basin, the Bulloo River catchment and the eastern and northern Lake Eyre Basin; LEB), suggesting high dispersal rates within this large area. Between the central Australian populations themselves, however (e.g. those inhabiting the central and western LEB), genetic differentiation is pronounced, probably as a result of the lack of abundance of important dispersal vectors (aquatic birds) and the lower diversity and density of suitable habitats in the area. The most prominent biogeographical break exists towards north-eastern Australia (north-east LEB), which does not share species with any other region studied.

Indeterminate tcdB using a Clostridium difficile PCR assay: a retrospective cohort study

BackgroundC. difficile (CD) real-time polymerase chain reaction (PCR) for toxin B gene (tcdB) is more sensitive, and reduces turnaround time when compared to toxin immunoassay. We noted typical amplification curves with high tcdB cycle thresholds (Ct) and low endpoints (Ept) that are labeled negative by the Xpert® C. difficile assay (Cepheid) and undertook this study to determine their significance.MethodsWe defined an indeterminate CD assay result as detection of a typical PCR amplification curve with an Ept >10 that was interpreted as negative by the Xpert® assay. Samples with indeterminate Xpert® result were collected for 5 months and retested by Xpert®, cultured for toxigenic CD, and isolates subjected to PCR ribotyping, detection of toxin genes and multilocus variable-number tandem repeat analysis (MLVA) typing. Chart reviews were completed to assess if patients met the Society of Healthcare Epidemiology of America and the Infectious Diseases Society of America CD infection (CDI) clinical case definition. Illness severity was compared with tcdB Ct and culture results.ResultsDuring the 5-month study period, 48/3620 (1%) of specimens were indeterminate and 387/3620 (11%) were positive. Of the 48 patients with indeterminate results, 39 (81%) met the clinical case definition of CDI, and 7 of these (18%) met criteria for severe CDI. Toxigenic stool cultures were positive for 86% (6/7) of patients with severe CDI, 19% (6/32) of patients with non-severe CDI, and 44% (4/9) of patients who did not meet the clinical case definition of CDI (p = 0.002). Lower tcdB Ct and higher Ept were associated with greater likelihood of toxigenic culture positivity (p = 0.03) and more severe symptoms (p = 0.06). Indeterminate results were not associated with a particular technologist or instrument module, or CD strain type.ConclusionsA subset of specimens (1%) using the Xpert® C. difficile assay have typical amplification curves and are interpreted as negative. At least one-third of these results are associated with positive CD culture. The mechanism of these indeterminate results is not technique-related, equipment-related, or due to particular CD strains. Clinicians should be aware that even PCR testing has the potential to miss CDI cases and further highlights the importance of clinical context when interpreting results.

Molecular predictors of outcome and response to bevacizumab (BEV) based on analysis of RTOG 0825, a phase III trial comparing chemoradiation (CRT) with and without BEV in patients with newly diagnosed glioblastoma (GBM).

LBA2010 Background: RTOG 0825 evaluated the addition of BEV to standard CRT in the treatment of GBM and included molecular stratification that assessed the degree of mesenchymal (MES) gene enrichment. We investigated the ability of the MES signature to predict response to BEV. Methods: Sufficient FFPE tissue for molecular analysis was available for 650 registered, eligible patients. TaqMan PCR was performed prospectively using the molecular stratifier on all patients and an expanded 43 member MES set on 234 cases. A subset of specimens was subjected to whole genome expression profiling (GEP). Predictive models were evaluated for their ability to predict survival (overall, OS, and progression-free, PFS) in the training cohort of the BEV arm after adjusting for prognostic factors and treatment arm. Unsupervised clustering of GEP data was used to identify molecular subsets and gene set enrichment analysis (GSEA) performed to evaluate for MES enrichment. Results: We observed a significant association between increasing MES signature and worse PFS and OS in the BEV arm (p=0.036 and p=0.032, respectively). Based on the association between high MES expression and poor outcome in the BEV arm, we sought to optimize a predictor using an expanded set of MES genes. Unbiased gene selection from a total of 43 genes followed by radial basis machine modeling identified a 10-gene predictor of outcome in the BEV arm (p&lt;0.001/HR 4.04 for OS and p&lt;0.001/HR 2.21 for PFS). To further support the association of MES enrichment and poor outcome in the BEV arm, we performed GEP. Unsupervised clustering identified a subtype of tumors with MES enrichment associated with the 10-gene predictor survival probability (p=0.0124, t-test). Ongoing studies will determine the extent to which this represents a predictive marker for BEV. Conclusions: We developed a 10-gene predictor specific to BEV treatment and suitable for FFPE that may serve to identify subsets of patients with newly diagnosed GBM who benefit from BEV. Supported by RTOG grant U10 CA21661, CCOP grant U10 CA37422, and Brain SPORE P50 CA127001 from the NCI and Genentech. Clinical trial information: NCT00884741.

A Geographic Information Systems (GIS)-based analysis of modern South African rodent distributions, habitat use, and environmental tolerances

Goals of this study were to: (1) develop distributional maps of modern rodent genera throughout the countries of South Africa, Lesotho, and Swaziland by georeferencing museum specimens; (2) assess habitat preferences for genera by cross-referencing locality position with South African vegetation; and (3) identify mean annual precipitation and temperature range where the genera are located. Conterminous South Africa including the countries of Lesotho and Swaziland Digital databases of rodent museum specimens housed in the Ditsong National Museum of Natural History, South Africa (DM), and the Division of Mammals, National Museum of Natural History, Smithsonian Institution, United States (NMNH), were acquired and then sorted into a subset of specimens with associated coordinate data. The coordinate data were then used to develop distributional maps for the rodent genera present within the study area. Percent habitat occupation and descriptive statistics for six climatic variables were then determined for each genus by cross-referencing locality positions with vegetation and climatic maps. This report presents a series of maps illustrating the distribution of 35 rodent genera based on 19,471 geo-referenced specimens obtained from two major collections. Inferred habitat use by taxon is provided for both locality and specimen percent occurrence at three hierarchical habitat levels: biome, bioregion, and vegetation unit. Descriptive statistics for six climatic variables are also provided for each genus based on locality and specimen percent incidence. As rodent faunas are commonly used in paleoenvironmental reconstructions, an accurate assessment of rodent environmental tolerance ranges is necessary before confidence can be placed in an actualistic model. While the data presented here represent only a subset of the modern geographic distributions for many of the taxa examined, a wide range of environmental regimes are observed, suggesting that more research is necessary in order to accurately reconstruct an environmental signature when these taxa are found in the fossil record.

Dysplastic Squamous Cells Are Frequently Present in Urine Cytology Specimens of Patients with High-Grade Urothelial Carcinoma

Background: In urothelial carcinoma (UCA), squamous differentiation (SqD) occurs mainly in high-grade UCA with invasion. Therefore, we sought to determine the frequency of dysplastic squamous cells (DSC) in urine specimens obtained from patients with high-grade UCA asking if DSC could serve as a surrogate marker for high-grade UCA. Design: We searched for cases with a histologic diagnosis of high-grade UCA and available concurrent cytology, yielding 93 surgical specimens (including 71 biopsies, 12 cystectomies, 5 nephrectomies, 4 ureterectomies, and 1 urethrectomy) from 68 patients with 98 urine cytology samples. Both cytology and histologic specimens were evaluated for the presence of any SqD on histology and the presence of DSC on cytology besides urothelial cells. Results: Forty-three of 68 patients (63%) had a cytologic diagnosis of ‘positive/suspicious’. Twenty-one patients (30%) had surgical specimens that showed SqD. Seventeen patients had urine cytology specimens showing DSC (25%). Thirteen of these 17 patients showed DSC with concurrent malignant urothelial cells, while 4 patients displayed only isolated DSC. Conclusion: SqD is common in patients with high-grade UCA. DSC were detected in a subset of specimens from patients with high-grade UCA. In some instances, isolated DSC on cytology may represent the only evidence of an unsampled high-grade malignancy.

Heterogeneous Glycation of Cancellous Bone and Its Association with Bone Quality and Fragility

Non-enzymatic glycation (NEG) and enzymatic biochemical processes create crosslinks that modify the extracellular matrix (ECM) and affect the turnover of bone tissue. Because NEG affects turnover and turnover at the local level affects microarchitecture and formation and removal of microdamage, we hypothesized that NEG in cancellous bone is heterogeneous and accounts partly for the contribution of microarchitecture and microdamage on bone fragility. Human trabecular bone cores from 23 donors were subjected to compression tests. Mechanically tested cores as well as an additional 19 cores were stained with lead-uranyl acetate and imaged to determine microarchitecture and measure microdamage. Post-yield mechanical properties were measured and damaged trabeculae were extracted from a subset of specimens and characterized for the morphology of induced microdamage. Tested specimens and extracted trabeculae were quantified for enzymatic and non-enzymatic crosslink content using a colorimetric assay and Ultra-high Performance Liquid Chromatography (UPLC). Results show that an increase in enzymatic crosslinks was beneficial for bone where they were associated with increased toughness and decreased microdamage. Conversely, bone with increased NEG required less strain to reach failure and were less tough. NEG heterogeneously modified trabecular microarchitecture where high amounts of NEG crosslinks were found in trabecular rods and with the mechanically deleterious form of microdamage (linear microcracks). The extent of NEG in tibial cancellous bone was the dominant predictor of bone fragility and was associated with changes in microarchitecture and microdamage.

Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

The KIT gene has been shown to have multiple functions in hematopoiesis, melanogenesis, and gametogenesis. In addition, mutations of this gene cause pigmentation disorders in humans and mice and are responsible for coat color differences in pigs. While characterizing polymorphisms in the porcine KIT gene, we detected alternative splicing (AS) of the NAGNAG splice acceptor site at the boundary of intron 4 and exon 5. This AS event generated the E and I isoforms, characterized by insertion or deletion, respectively, of CAG at the borders of coding sequence. AS patterns measured in tissue samples from two randomly selected animals did not identified any tissue-specific outcomes. Analysis of AS patterns using three breeds demonstrated that Landrace and Large White pigs expressed both the E and I isoforms. In contrast, a subset of specimens from Korean Native Pigs (KNP) yielded a single I isoform. Alignment of the sequence from several species revealed that the region between the branch point sequence (BPS) and 3′ acceptor site is conserved. However, it is appeared that the selection of either the proximal or distal splice site varied between species. To test the breed specificity the NAGNAG splice acceptor site, we constructed two lineages of minigenes from KNP and Landrace pigs harboring breed-specific mutations. The minigene splicing assay demonstrated that both types of minigenes expressed both the E and I isoforms in two host cell lines, and no differences were detected in the AS pattern between the two breeds. We conclude that the AS at the NAGNAG splice acceptor site on intron 4/exon 5 in the porcine KIT gene is the result of noise selection at the splice site by the splicing machinery. Therefore, this AS event in the porcine KIT gene is unlikely to have any relationship with the coat color variations of Landrace and KNP breeds.