Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters.

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus in oysters using the quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) are well established, and various studies using RT-qPCR have detected norovirus in a considerable proportion of oyster samples, both in the UK and elsewhere. However, RT-qPCR detects viral genome, and by its nature is unable to discriminate between positive results caused by infectious viruses and those caused by non-infectious remnants including damaged virus particles and naked RNA. As a result, a number of alternative or complementary approaches to RT-qPCR testing have been proposed, including the use of infectious viral indicator organisms, most frequently F-specific RNA bacteriophage (F-RNA phage). In this study, we investigated the relationships between F-RNA phage and norovirus in digestive tissues from two sets of oyster samples, one randomly collected at retail (630 samples), and one linked to suspected norovirus illness outbreaks (nine samples). A positive association and correlation between PCR-detectable levels of genogroup II F-RNA bacteriophage (associated with human faecal contamination) and norovirus was found in both sets of samples, with more samples positive for genogroup II phage, at generally higher levels than norovirus. Levels of both viruses were higher in outbreak-related than retail samples. Infectious F-RNA phage was detected in 47.8% of all retail samples, and for a subset of 224 samples where characterisation of phage was carried out, infectious GII phage was detected in 30.4%. Infectious GII phage was detected in all outbreak-related samples. Determination of infectivity ratios by comparing levels of PCR-detectable (copies/g) and infectious GII phage (pfu/g) revealed that in the majority of cases less than 10% of virus detected by RT-qPCR was infectious. Application of these ratios to estimate infectious norovirus levels indicated that while 77.8% of outbreak-related samples contained > 5 estimated infectious norovirus/g, only 13.7% of retail samples did. Use of a combination of levels of PCR-detectable norovirus and infectious F-RNA phage showed that while only 7.0% of retail samples contained both > 100 copies/g norovirus and > 10 pfu/g F-RNA phage, these combined levels were present in 77.8% of outbreak-related samples, and 75.9% of retail samples with > 5 estimated infectious norovirus/g. We therefore suggest that combining RT-qPCR testing with a test for infectious F-RNA phage has the potential to better estimate health risks, and to better predict the presence of infectious norovirus than RT-qPCR testing alone.

Thrombolysis for stroke in Ireland: increasing access and maintaining safety in a challenging environment.

BACKGROUND: In the setting of a national audit of acute stroke services, we examined the delivery of thrombolytic therapy for ischaemic stroke and whether current practice was achieving safe outcomes and consistent delivery for patients. METHOD: Data obtained from the recent national stroke audit was compared against previous Irish audit, the most recent SSNAP UK stroke audit and the Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SITS-MOST) study. RESULTS: Thrombolysis was provided in 27 acute hospitals throughout Ireland during the period assessed with 82% (22/27) providing 24/7 access, the remaining sites using redirect policies. Decision to thrombolyse was made by stroke trained consultants in 63% (17/27) of units, with general physicians and emergency medicine consultants covering the other units. Thrombolysis rate for non-haemorrhagic stroke was 11% (n = 80/742, CI 95% ±2.23) versus a 1% rate in the 2008 audit. Sites receiving patients through a redirect policy had the highest thrombolysis rate, an average of 24%. Nearly 30% of cases were thrombolysed on the weekend. Eighty-three percent of cases were managed in a stroke unit at some time during admission versus 54% of the national total cases. Thirty-seven percent of patients were ≥80 years old. The mortality rate was 11.3% versus the national mortality rate for non-thrombolysed ischaemic strokes of 10% (p > 0.5), and this is comparable to the SITS-MOST 2007 study 3-month mortality rate of 11.3% (p > 0.5). CONCLUSION: Stroke thrombolysis is being effectively and safely provided in acute stroke services in Ireland despite regular involvement of non-specialist staff. There is still potential to improve thrombolysis rate.

A novel and non-destructive method to examine meniscus architecture using 9.4 Tesla MRI.

OBJECTIVE: To investigate the ability of high-field (9.4 T) magnetic resonance (MR) imaging to delineate porcine knee meniscal tissue structure and meniscal tears. MATERIALS AND METHODS: Porcine knees were obtained from a local abattoir, and eight medial menisci with no visible defects were dissected. Lesions simulating longitudinal tears were created on two of the menisci. MR images of the menisci were obtained at 9.4 T using a three-dimensional (3D)-FLASH sequence. A detailed 3D internal architecture of the intact and injured menisci was demonstrated on high-resolution MR images. RESULTS: High-resolution 3D MR imaging allowed visualisation of internal architecture of the meniscus and disruption to the internal structural network in damage models. The architecture of the porcine knee meniscus revealed by the MR scans appeared similar to the structures visualised by histology in previously reported studies. CONCLUSION: High-field MRI is a non-destructive technique to examine the internal structural components and damage/wear of meniscal tissue. It has tremendous potential in the field of functional cartilage/meniscus biomechanics and biotribology.

The effect of glycosaminoglycan depletion on the friction and deformation of articular cartilage.

Glycosaminoglycans (GAGs) have been shown to be responsible for the interstitial fluid pressurization of articular cartilage and hence its compressive stiffness and load-bearing properties. Contradictory evidence has been presented in the literature on the effect of depleting GAGs on the friction properties of articular cartilage. The aim of this study was to investigate the effect of depleting GAGs on the friction and deformation characteristics of articular cartilage under different tribological conditions. A pin-on-plate machine was utilized to measure the coefficient of friction of native and chondroitinase ABC (CaseABC)-treated articular cartilage under two different models: static (4 mm/s start-up velocity) and dynamic (4 mm/s sliding velocity; 4 mm stroke length) under a load of 25 N (0.4 MPa contact stress) and with phosphate-buffered saline as the lubricant. Indentation tests were carried out at 1 N and 2 N loads (0.14 MPa and 0.28 MPa contact stress levels) to study the deformation characteristics of both native and GAG-depleted cartilage samples. CaseABC treatment rendered the cartilage tissue soft owing to the loss of compressive stiffness and a sulphated-sugar assay confirmed the loss of GAGs from the cartilage samples. CaseABC treatment significantly increased (by more than 50 per cent) the friction levels in the dynamic model (p < 0.05) at higher loading times owing to the loss of biphasic lubrication. CaseABC treatment had no effect on friction in the static model in which the cartilage surfaces did not have an opportunity to recover fluid because of static loading unlike the cartilage tissue in the dynamic model, in which translation of the cartilage surfaces was involved, ensuring effective biphasic lubrication. Therefore the depletion of GAGs had a smaller effect on the coefficient of friction for the static model. Indentation tests showed that GAG-depleted cartilage samples had a lower elastic modulus and higher permeability than native tissue. These results corroborate the role of GAGs in the compressive and friction properties of articular cartilage and emphasize the need for developing strategies to control GAG loss from diseased articular cartilage tissue.

A pilot study of attention deficits, balance control and falls in the subacute stage following stroke.

OBJECTIVES: To (1) identify and monitor changes in deficits of attention and balance in the subacute stage following stroke, (2) investigate fall events, (3) explore relationships between deficits of attention, balance and falling post stroke. DESIGN: A prospective observational study. SETTING: Hospitalized patients with stroke were recruited from a rehabilitation ward. SUBJECTS: Fourteen subjects were recruited, 13 subjects (age 21-80 years) completed all assessments. Ten had right and three had left hemispheric lesions. Eight subjects were male. MAIN OUTCOME MEASURES: Subjects were assessed on two occasions during a six-week period. Standardized tests were used to measure unilateral visual neglect, sustained and selective attention and balance. Fall histories were collected by chart review and semi-structured interview. RESULTS: At the initial assessment six subjects had sustained auditory attention deficit, eight had deficits of auditory selective attention, 12 had visual selective attention deficits and seven subjects presented with visual inattention. Scores for visual inattention and visual selective attention improved over time (p = 0.006, p = 0.026 respectively) as did scores for balance control (p = 0.001). Auditory selective attention scores at the first assessment were found to correlate with measures of balance (p < 0.01), subjects with normal attention scores achieved better balance scores. Four subjects fell at least once during the six weeks. Scores for attention and balance of fallers were not found to be significantly different from the scores of nonfallers. CONCLUSIONS: The sample size was small but high levels of attention deficit were identified. Findings suggest a weak or no relationship between attention deficits and falls. Further studies are required to explore the extent of attention deficits post stroke and its impact on balance control and falling.

Initial biomechanical properties of anterior cruciate ligament reconstruction autografts.

To provide more information to consider when selecting a reconstruction technique, we did a side-by-side comparison of some of the initial biomechanical properties of currently accepted reconstruction methods. Our research hypotheses were that a quadrupled, woven semitendinosus and gracilis graft is as strong as any of the other commonly used graft materials and that quadrupling and weaving the hamstring graft may increase the stiffness of the overall construct Using lower extremity cadaveric specimens harvested from young donors, we fashioned seven each of seven types of graft: 9-mm, 10-mm, and 11-mm-wide patellar tendon graft (PTG); 10-mm-wide central quadriceps tendon graft; doubled semitendinosus graft; tripled semitendinosus graft; and quadrupled, woven semitendinosus and gracilis graft. Specimens were stripped of remaining soft tissue, and anterior cruciate ligament (ACL) constructs were created for biomechanical testing. The tibia was translated anteriorly on the femur, mimicking a pivot shift maneuver, andfailure strength, failure mechanism, and construct stiffness were recorded. No differences in mean strength were detected. The quadrupled, woven graft was significantly stiffer than the doubled semitendinosus graft and no less stiff than any of the PTG constructs. All grafts showed similar and adequate initial absolute strength to reconstruct the ACL. Quadrupling and weaving the semitendinosus and gracilis graft increases the stiffness of the reconstructed specimen to a level statistically similar to that of specimens reconstructed with a PTG.

Enhancement of the functional repertoire of the rat parietal peritoneal mesothelium in vivo: directed expression of the anticoagulant and antiinflammatory molecule thrombomodulin.

We have used our previously described ex vivo mesothelial cell (MC)-mediated gene therapy strategy (Gene Ther. 2:393-401, 1995) to modify the functional properties of the rat parietal peritoneal mesothelium in vivo by expression of a membrane-bound recombinant protein on the MC surface. Rat primary MCs were stably transfected (using strontium phosphate DNA coprecipitation) with a plasmid containing the gene for rat thrombomodulin (TM), a transmembrane glycoprotein that functions as an essential cofactor for the physiological activation of the anticoagulant protein C by the enzyme thrombin. As demonstrated by immunohistochemistry and by direct equilibrium binding with radiolabeled thrombin, genetically modified MCs expressed high levels of TM antigen on their surface in vitro. As judged by a thrombin-dependent protein C activation assay, such MC membrane-bound TM was biologically active. Once reseeded on the denuded parietal peritoneal surface of syngeneic recipients, these TM-transfected MCs continued to express TM antigen in vivo for at least 90 days. Moreover, the recombinant TM expressed on the reconstituted parietal mesothelium retained its ability to activate protein C in a thrombin-dependent manner. Our data indicate that MC-mediated expression of TM can be used to augment the anticoagulant properties of the parietal peritoneal surface. In general, our results suggest that ex vivo MC-mediated gene therapy can be used to deliver other therapeutic transmembrane proteins to the MC surface to enhance the functional repertoire of the parietal mesothelium in vivo.

Graft fixation strength with arthroscopic anterior cruciate ligament reconstruction. Two-incision rear entry technique compared with one-incision technique.

To compare the fixation strengths of two arthroscopic anterior cruciate ligament reconstruction techniques, we harvested 10-mm bone-patellar tendon-bone grafts with 25-mm bone plugs from 12 fresh-frozen cadaveric knees. One knee of each pair was fixed using Acufex instruments and the two-incision technique; one knee was fixed using Arthrotek instruments and the one-incision technique. We used cannulated 9 x 25 mm Kurosaka screws for femoral and tibial tunnel fixation. All knees were mounted on the Instron Test System and were stressed to failure by recreating a pivot shift maneuver. The one-incision technique graft fixation was significantly stronger (mean failure, 695 N) than that with the two-incision technique (mean failure, 593 N). In all one-incision technique knees, either the patellar tendon graft avulsed off a bone plug or the plug broke. In five of six two-incision technique knees, the tibia bone plug pulled out around the interference screw. Patellar tendon graft length ranges from 90 to 105 mm, and the average two-incision technique tunnel length is 120 mm. The interference screw compressed the femoral bone plug into conical bone but compressed the tibial bone plug into cancellous bone in the two-incision technique, while in the one-incision technique the bone plug was compressed into cortical bone on both sides.