Simplified PCR protocols for INNO-LiPA HBV Genotyping and INNO-LiPA HBV PreCore assays.

BACKGROUND: INNO-LiPA HBV Genotyping (LiPA HBV GT) and INNO-LiPA HBV PreCore (LiPA HBV PC) are commercially available assays for hepatitis B virus (HBV) characterization. These assays are labor-intensive and may be prone to exogenous DNA contamination due to their use of nested PCR amplification procedures and lack of contamination control measures. OBJECTIVE: Standardized, single-round INNO-LiPA PCR amplification protocols incorporating uracil N-glycosylase and automated sample processing by the MagNA Pure LC instrument were evaluated. STUDY DESIGN: HBV standards containing 10,000, 1000, 100, 10, and 0 IU/mL were analyzed to determine the analytical sensitivity and reproducibility of these modified procedures. One hundred clinical serum specimens with viral titers ranging from 390 to 16,900,000 IU/mL were tested by modified LiPA HBV GT, while 34 specimens with viral titers ranging from 378 to 11,600,000 IU/mL were tested by modified LiPA HBV PC. RESULTS: Modified LiPA HBV GT and LiPA HBV PC each yielded analytical sensitivities of 100% at an HBV DNA level of 1000 IU/mL and 90% at a level of 100 IU/mL. Among clinical specimens, success rates for both INNO-LiPA procedures were > or =94%. CONCLUSIONS: Both modified INNO-LiPA procedures were sensitive and reproducible, with improved efficiency and suitability for routine laboratory use.     

ROC Study of Four LCD Displays Under Typical Medical Center Lighting Conditions

Nine observers reviewed a previously assembled library of 320 chest computed radiography (CR) images. Observers participated in four sessions, reading a different 1/4 of the sample on each of four liquid crystal displays: a 2-megapixel (MP) consumer color display, a 2-MP business color display, a 2-MP medical-grade gray display, and a 3-MP gray display. Each display was calibrated according to the DICOM Part 14 standard. The viewing application required observer login, then randomized the order of the subsample seen on the display, and timed the responses of the observer to render a 1–5 judgment on the absence or presence of ILD on chest CRs. Selections of 1–2 were considered negative, 3 was indeterminate, and 4–5 were positive. The order of viewing sessions was also randomized for each observer. The experiment was conducted under controlled lighting, temperature, and sound conditions to mimic conditions typically found in a patient examination room. Lighting was indirect, and illuminance at the display face was 195 ± 8% lux and was monitored over the course of the experiment. The average observer sensitivity for the 2 MP color consumer, 2 MP business color, 2 MP gray, and 3 MP gray displays were 83.7%, 84.1%, 85.5%, and 86.7%, respectively. The only pairwise significant difference was between the 2-MP consumer color and the 2-MP gray (P = 0.05). Effect of order within a session was not signitfficant (P = 0.21): period 1 (84.3%), period 2 (86.2%), period 3 (85.4%), period 4 (84.1%). Observer specificity for the various displays was not statistically significant (P = 0.21). Finally, a timing analysis showed no significant difference between the displays for the user group (P = 0.13), ranging from 5.3 s (2 MP color business) to 5.9 s (3 MP Gray). There was, however, a reduction in time over the study that was significant (P <<< 0.001) for all users; the group average decreased from 6.5 to 4.7 s per image. Physical measurements of the resolution, contrast, and noise properties of the displays were acquired. Most notably, the noise of the displays varied by 3.5× between the lowest and highest noise displays. Differences in display noise were indicative of observer performance. However, the large difference in the magnitude of the noise was not predictive of the small difference (3%) in the observer sensitivity for various displays. This is likely because detection of interstitial lung disease is limited by “““““anatomical noise””” rather than display or x-ray image noise.     

Acute Alcohol Consumption Attenuates Interleukin-8 (IL-8) and Monocyte Chemoattractant Peptide-1 (MCP-1) Induction in Response to Ex Vivo Stimulation

Chronic alcohol use is associated with impaired immunity and host defense. Even acute ethanol treatment both in vitro and in vivo has been shown to result in decreased inflammatory cytokine production. However, the potential immunoregulatory effects of acute, moderate alcohol use are yet to be fully explored. Here we show that in vitro acute alcohol treatment of normal blood monocytes resulted in a significant, dose-dependent (25–100 mM) attenuation of staphylococcus enterotoxin B (SEB), phytohemagglutinin (PHA), or IFN-induced monocyte IL-8 and MCP-1 production (P < 0.01). Likewise, ethanol (100 mM) in vitro reduced MCP-1 levels in response to SEB, PHA, or IFN stimulation in mononuclear cells (31–62% reduction). Furthermore, acute alcohol consumption (0.85 g/kg body weight) significantly attenuated SEB- or LPS-induced IL-8 and MCP-1 levels in whole-blood samples obtained 4 hr after alcohol consumption from normal nonalcoholic individuals (P < 0.01). RANTES and MIP-1 were only minimally inhibited (16–25% inhibition) by in vitro ethanol (100 mM) in mononuclear cells, suggesting that ethanol may have a selective effect on the regulation of various chemokines. These results demonstrate that acute alcohol, in vivo as well as in vitro, attenuates monocyte-derived chemokine production in response to a subsequent immune challenge. Our data show for the first time that activation of nuclear regulatory factor B (NF-B), a common regulator binding in the promoter region of IL-8 and MCP-1 genes, is inhibited by acute ethanol (25 mM) treatment in SEB-stimulated human monocytes. These results imply that inhibition of NF-B activation may be one of the intracellular mechanisms for the ethanol-induced inhibition of IL-8 and MCP-1 production in monocytes. Thus, impaired chemokine (particularly MCP-1 and IL-8) induction upon an immune challenge is likely to contribute to compromised host defense after acute alcohol consumption and may also affect progression of diseases such as atherosclerosis or HIV infection where chemokines contribute to progression of the disease.     

Effect of Automated Image Registration on Radiologist Interpretation

In this study, we present preliminary data on the effect of automated 3D image alignment on the time to arrive at a decision about an imaging finding, the agreement of multiple of multiple observers, the prevalence of comparison examinations, and technical success rates for the image alignment algorithm. We found that automated image alignment reduced the average time to make a decision by 25% for cases where the structures are rigid, and when the scanning protocol is similar. For cases where these are not true, there is little or no benefit. In our practice, 54% of cases had prior examinations that could be automatically aligned. The overall benefit seen in our department for highly similar exams might be 20% for neuro and 10% for body; the benefit seen in other practices is likely to vary based on scanning practices and prevalence of prior examinations.     

Inhibition of antigen-presenting cell functions by alcohol: implications for hepatitis C virus infection.

The mechanisms of alcohol-induced immunosuppression include defects in innate and adaptive immune responses. Monocytes and dendritic cells (DCs) link innate and adaptive immune responses as they recognize viral antigens and induce antigen-specific T-cell activation. We investigated the effects of alcohol on antigen-presenting cell functions. Acute alcohol consumption by healthy volunteers (vodka, 2 ml/kg) resulted in significantly reduced antigen-presenting cell function of monocyte-derived DCs. Reduced allostimulatory capacity of DCs treated with alcohol in vitro correlated with decreased co-stimulatory molecule (B7.1 and B7.2) expression, as well as with reduced interleukin (IL)-12 and increased IL-10 concentrations, in mixed lymphocyte cultures. Dendritic cells recognize viral antigens in hepatitis C virus (HCV) infection, and HCV disease is accelerated in alcohol-dependent individuals. For patients with chronic HCV infection, we found reduced allostimulatory capacity of myeloid DCs. Furthermore, DC function was reduced by in vitro alcohol treatment of DCs obtained from HCV-infected patients, supporting the suggestion that viral factors and alcohol may interact to doubly suppress DC functions. We found that induction of maturation with lipopolysaccharide could not fully ameliorate the reduced DC allostimulatory capacity caused by alcohol treatment, HCV infection, or their combination. In addition, soluble factors in the supernatants obtained from mixed lymphocyte cultures containing alcohol-treated DCs or DCs obtained from HCV-infected patients could transfer inhibition of T-cell proliferation in cultures containing DCs obtained from healthy volunteers. Anti-IL-10 neutralizing antibody ameliorated the reduced mixed lymphocyte reaction containing DCs obtained from HCV-infected patients, whereas exogenous IL-12, but not anti-IL-10, treatment ameliorated the reduced T-cell proliferation induced by alcohol treatment of DCs obtained from healthy volunteers. Our results support the suggestion that both acute alcohol intake and in vitro alcohol treatment inhibit DC antigen-presenting cell function and support the hypothesis that viral factors interact with alcohol to reduce DC functions in HCV infection.     

Interferon regulatory factor 3 and type I interferons are protective in alcoholic liver injury in mice by way of crosstalk of parenchymal and myeloid cells

Alcoholic liver disease (ALD) features increased hepatic exposure to bacterial lipopolysaccharide (LPS). Toll-like receptor-4 (TLR4) recognizes LPS and activates signaling pathways depending on MyD88 or TRIF adaptors. We previously showed that MyD88 is dispensable in ALD. TLR4 induces Type I interferons (IFNs) in an MyD88-independent manner that involves interferon regulatory factor-3 (IRF3). We fed alcohol or control diets to wild-type (WT) and IRF3 knock-out (KO) mice, and to mice with selective IRF3 deficiency in liver parenchymal and bone marrow-derived cells. Whole-body IRF3-KO mice were protected from alcohol-induced liver injury, steatosis, and inflammation. In contrast to WT or bone marrow-specific IRF3-KO mice, deficiency of IRF3 only in parenchymal cells aggravated alcohol-induced liver injury, associated with increased proinflammatory cytokines, lower antiinflammatory cytokine interleukin 10 (IL-10), and lower Type I IFNs compared to WT mice. Coculture of WT primary murine hepatocytes with liver mononuclear cells (LMNC) resulted in higher LPS-induced IL-10 and IFN-β, and lower tumor necrosis factor alpha (TNF-α) levels compared to LMNC alone. Type I IFN was important because cocultures of hepatocytes with LMNC from Type I IFN receptor KO mice showed attenuated IL-10 levels compared to control cocultures from WT mice. We further identified that Type I IFNs potentiated LPS-induced IL-10 and inhibited inflammatory cytokine production in both murine macrophages and human leukocytes, indicating preserved cross-species effects. These findings suggest that liver parenchymal cells are the dominant source of Type I IFN in a TLR4/IRF3-dependent manner. Further, parenchymal cell-derived Type I IFNs increase antiinflammatory and suppress proinflammatory cytokines production by LMNC in paracrine manner. CONCLUSION: Our results indicate that IRF3 activation in parenchymal cells and resulting type I IFNs have protective effects in ALD by way of modulation of inflammatory functions in macrophages. These results suggest potential therapeutic targets in ALD.     

Evaluation of the COBAS TaqMan HCV test with automated sample processing using the MagNA pure LC instrument

The COBAS TaqMan HCV Test (TaqMan HCV; Roche Molecular Systems Inc., Branchburg, N.J.) for hepatitis C virus (HCV) performed on the COBAS TaqMan 48 Analyzer (Roche Molecular Systems) currently relies on a manual sample processing method. Implementation of an automated sample processing method would facilitate the clinical use of this test. In this study, we evaluated the performance characteristics of TaqMan HCV following automated sample processing by the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, Ind.). The analytical sensitivity of TaqMan HCV following sample processing by MP was 8.1 IU/ml (95% confidence interval, 6.1 to 15.2). The assay showed good linearity (R(2) = 0.99) across a wide range of HCV RNA levels (25 to 5 x 10(6) IU/ml), with coefficients of variation ranging from 10% to 46%. Among 83 clinical specimens, the sensitivity and specificity of TaqMan HCV were 100% and 95%, respectively, when compared to the COBAS AMPLICOR hepatitis C virus test, version 2.0 (COBAS AMPLICOR; Roche Molecular Systems), with TaqMan HCV detecting two more HCV RNA-positive specimens than COBAS AMPLICOR. Both specimens were confirmed to be HCV RNA positive by the VERSANT HCV RNA qualitative test (Bayer HealthCare LLC, Tarrytown, N.Y.). There was also strong correlation (R(2) = 0.95) and good agreement between the results from TaqMan HCV and the VERSANT HCV RNA 3.0 assay (bDNA) (Bayer HealthCare LLC) among a group of 93 clinical specimens. The MP is a versatile, labor-saving sample processing platform suitable for reliable performance of TaqMan HCV.     

Factor instability of clinical teaching assessment scores among general internists and cardiologists

CONTEXT: We are unaware of studies examining the stability of teaching assessment scores across different medical specialties. A recent study showed that clinical teaching assessments of general internists reduced to interpersonal, clinical teaching and efficiency domains. We sought to determine the factor stability of this 3-dimensional model among cardiologists and to compare domain-specific scores between general internists and cardiologists. METHODS: A total of 2000 general internal medicine and cardiology hospital teaching assessments carried out from January 2000 to March 2004 were analysed using principal factor analysis. Internal consistency and inter-rater reliability were calculated. Mean item scores were compared between general internists and cardiologists. RESULTS: The interpersonal and clinical teaching domains previously demonstrated among general internists collapsed into 1 domain among cardiologists, whereas the efficiency domain remained stable. Internal consistency of domains (Cronbach's alpha range 0.89-0.93) and inter-rater reliability of items (range 0.65-0.87) were good to excellent for both specialties. General internists scored significantly higher (P<0.05) than cardiologists on most items except for 4 items that more accurately assessed the cardiology teaching environment. CONCLUSIONS: We observed factor instability of clinical teaching assessment scores from the same instrument administered to general internists and cardiologists. This finding was attributed to salient differences between these specialties' educational environments and highlights the importance of validating assessments for the specific contexts in which they are to be used. Future research should determine whether interpersonal domain scores identify superior teachers and study the reasons why interpersonal and clinical teaching domains are unstable across different educational settings.