Development and evaluation of real-time TaqMan® RT-PCR assays for the detection of avian nephritis virus and chicken astrovirus in chickens.

The development and preliminary evaluations of two TaqMan®-based, real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assays for the quantitative detection of avian nephritis virus (ANV) and chicken astrovirus (CAstV) RNAs are described. The assays used amplicons generated from the 3' untranslated region of the ANV genome and a conserved region of CAstV open reading frame 1b including its junction with open reading frame 2. High virus RNA levels (>10(5.99) viral copies) were detected for ANV and CAstV in 81% and 67% gut content samples from growth-retarded broiler flocks. Results from longitudinal surveys of two broiler flocks showed that ANV and CAstV RNAs were detected in most gut content and kidney samples collected at all time points from day 0 to day 35, with RNA levels of both astroviruses being higher in the gut contents than in the kidneys, and with the ANV RNA levels being greater than those of CAstV especially at early (days 7 and 14) time points. When the results obtained for the days 4/5 time-point samples from four broiler flocks with varying growth performances were compared, the two better-performing flocks had 100-fold to 1000-fold less ANV viral copies than the flocks that performed least well. Application of the rRT-PCR tests to samples collected from broiler chicks, which were experimentally infected with a crude gut content inoculum, demonstrated that ANV RNA could be detected in gut content and kidney samples at levels similar to those found at corresponding time points in longitudinal survey samples, whereas CAstV RNA was detected at lower levels than in the longitudinal survey samples, especially in kidney samples.

Nicotine and extinction of fear conditioning.

Despite known health risks, nicotine use remains high, especially in populations diagnosed with mental illnesses, including anxiety disorders and Post-Traumatic Stress Disorder (PTSD). Smoking in these populations may relate to the effects of nicotine on emotional memories. The current study examined the effects of nicotine administration on the extinction of conditioned fear memories. C57BL/6J mice were trained with two white noise conditioned stimulus (CS; 30 s, 85 dB)-foot shock (2 s, 0.57 mA) pairings. Extinction sessions consisted of six presentations of the CS (60 s) across multiple days. Mice were either tested in an AAA design, in which all stages occurred in the same context, or in an ABA design to identify if context changes alter extinction. Saline or nicotine was administered 5 min before training and/or extinction. In the AAA design, nicotine administration before training did not alter extinction. Nicotine administered prior to extinction sessions enhanced extinction and nicotine administered before training and extinction decreased extinction. In the ABA design, nicotine administered before extinction enhanced extinction and blocked context renewal of conditioned fear, while nicotine administered during training and extinction did not alter extinction but enhanced the context renewal of conditioned fear. Nicotine has a differential effect on extinction of fear conditioning depending on when it is administered. Administration during extinction enhances extinction whereas administration during training and extinction may strengthen contextual fear memories and interfere with extinction.

A seroprevalence investigation of chicken astrovirus infections.

Two genetically different isolates of chicken astrovirus (CAstV), named CAstV612 and CAstV11672, which share low levels of antigenic relatedness in cross-indirect immunofluorescence (IIF) tests, have been identified recently. In the present study, separate IIF tests for detecting antibodies to the CAstV612 and CAstV11672 isolates have been used to determine the seroprevalences of CAstV infections in four generations of flocks involved in broiler chicken production. CAstV antibodies were detected in 78% (73% CAstV612; 46% CAstV11672) of serum samples from UK broiler flocks and in all 10 flocks tested, indicating that infections were very common. Twenty-three (96%) out of 24 and 26 (93%) out of 28 broiler parent flocks, aged 23 to 26 weeks from three UK organizations, were positive for antibody to CAstV612 and CAstV11672, respectively. Of 718 samples tested from these parent flocks, 415 (53%) were positive for either CAstV612 or CAstV11672 antibody. CAstV infections were also widespread in parent flocks, with screening of pooled serum samples showing that antibodies to both CAstVs were detected in flocks from seven other UK poultry organizations and in flocks from eight other European countries. The seropositivities for CAstVs were substantially less in grandparent (28%) and great grandparent (21%) flocks. Overall, higher seropositivities were observed for CAstV612 than for CAstV11672 in broiler, parent, grandparent and great-grandparent flocks. A limited study of 99 sera from 10 turkey breeder flocks showed low-level seropositivities for CAstV612 (9%) and CAstV11672 (2%), indicating that turkeys were infected with CAstVs or antigenically related viruses.

Polymorphisms in cytokines and growth factor genes and their association with acute rejection and recurrence of hepatitis C virus disease in liver transplantation.

Acute rejection (AR) and recurrence of hepatitis C virus (HCV) infection are complications after liver transplantation (LTx). Genetic factors play a role in cytokine production as a consequence of polymorphisms within cytokine genes. Our goal was to identify genetic factors that might be associated with AR and recurrence of HCV in liver transplant recipients (LTxRs). We studied 77 Caucasian LTxRs and 100 Caucasian healthy individuals. We studied single-nucleotide polymorphisms (SNPs) in tumor necrosis factor-alpha[TNF-alpha, interleukin-6 (IL-6), IL-10, transforming growth factor-beta1, and angiotensin-converting enzyme genes by SNaPSHOT trade mark Multiplex assay. SNPs were classified as high producers (HP), intermediate producers (IP), or low producers (LP), and their association with AR and recurrence of HCV were studied. The frequency of TNF-alpha IP and HP genotypes was significantly higher in LTxRs with AR in comparison to patients without AR (TNF-alpha HP -238: 63 vs 20%, p < 0.001; TNF-alpha HP -308: 47.4 vs 20%, p = 0.02). The frequency of IL-6 IP and HP genotypes was higher in patients with AR episodes, but the difference was not statistically significant (p = 0.14). However, when we analyzed the simultaneous presence of pro-inflammatory genotypes in the same patient, we found a significant difference between patients with and without AR, respectively (42.1 vs 14.6%, p = 0.012). Moreover, the frequency of the IL-10 LP genotype was higher in LTx patients with AR (p = 0.001) compared to patients without AR. There was an association between pro-inflammatory genotypes and HCV recurrence. Our data suggest that cytokine gene polymorphisms might play a role in AR and HCV recurrence in LTxRs.

Validation of a multiplex methylation-sensitive PCR assay for the diagnosis of Prader-Willi and Angelman's syndromes.

BACKGROUND: Prader-Willi (PWS) and Angelman's (AS) syndromes are two distinct clinical entities caused by alterations in an identical but differentially methylated region of DNA on chromosome 15q. Highly complex laboratory tests are required for diagnosis because the disorders are caused by several genetic mechanisms. Methylation-specific PCR (MSPCR) is a relatively simple alternative method to detect the methylation status of the PWS/AS region. METHODS AND RESULTS: DNA was treated with sodium bisulfite, with alterations to the published method in which a neutralization step after the alkali treatment of the modified DNA enabled the use of the modified product directly in the PCR, eliminating the need for ethanol precipitation. Multiplex MSPCR using primers to methylated and unmethylated DNA was optimized to yield equal amplification efficiency for both products. Complete concordance was observed during the clinical validation of 40 previously characterized samples, except for one patient with mosaic AS detected by fluorescence in situ hybridization. CONCLUSION: We have developed and validated a multiplex MSPCR assay with alterations of the original published protocol that is technically robust and reproducible and can be used as a screening assay to detect PWS and AS.

Detection of a novel truncated WT1 transcript in human neoplasia.

BACKGROUND: The Wilms' tumor 1 (WT1) gene encodes a transcription factor critical in urogenital development. Using a new model of prostate cancer progression that permits comparison of the cellular and molecular properties of increasingly aggressive sublines of simian virus 40 large T-antigen-immortalized human prostate epithelial cells within the same lineage, the role of WT1 in tumorigenesis was investigated. METHODS AND RESULTS: Using RT-PCR and northern blotting, we identified a novel truncated WT1 transcript in these prostate cancer cell lines. This 2.1-kb transcript consisted of the coding region of the zinc-finger domain of WT1, together with a portion of intron 5 at the 5' end of the transcript. Furthermore, two peptides were detected by western blotting using antibodies to epitopes of the COOH terminus of WT1. Using RT-PCR, the 2.1-kb transcript was also detected in leukemia cell line K562, breast cancer cell line MCF7, and blood samples from patients with acute leukemia. CONCLUSION: These novel findings in both cell lines and patient-derived specimens suggest this new WT1 gene alteration has a potential role in the development of new diagnostic assays for some human malignancies.

Randomized, placebo-controlled trial of oral phytonadione for excessive anticoagulation.

STUDY OBJECTIVE: To compare the efficacy of managing excessive anticoagulation in the absence of bleeding by either omitting warfarin therapy alone or administering oral phytonadione in addition to omitting warfarin therapy. DESIGN: Randomized, double-blind, placebo-controlled study. SETTING: Clinical pharmacy anticoagulation service in a group model health maintenance organization. SUBJECTS: Thirty nonbleeding patients with international normalized ratios (INRs) ranging from 6.0-10.0. INTERVENTIONS: Patients were randomized to receive either a single oral dose of phytonadione 2.5 mg or placebo. Both groups omitted warfarin doses until the INR became less than or equal to 4.0. MEASUREMENTS AND RESULTS: The mean calculated time to reach an INR of 4.0 was significantly greater in the placebo than the phytonadione group (2.6 vs 1.4 days, p=0.006). Overcorrection of anticoagulation was significantly more common in patients receiving phytonadione. Overt warfarin resistance was not observed in either group after reinitiating warfarin therapy. No major bleeding or thromboembolic complications occurred, and minor bleeding episodes were similar in both groups. CONCLUSION: The addition of oral phytonadione 2.5 mg reduced the time to achieve an INR of 4.0 by approximately 1 day compared with omitting warfarin therapy alone. Adverse events did not differ between the two groups. Both strategies were effective in managing asymptomatic patients with INRs of 6.0-10.0. Oral phytonadione may be most appropriate for patients at high risk for bleeding in whom the benefit of prompt INR reduction would outweigh the thromboembolic risk associated with INR overcorrection.

Genetic changes in solid tumors.

Although most solid tumors are treated surgically, determining the genetic changes present in the tumor of an individual patient is becoming increasingly important for managing the oncology patient. Our knowledge of the genetic alterations that characterize and predispose to solid tumors continues to expand. Concurrently, the advent of newer technologies such as DNA chips has the potential to enable a more rapid and comprehensive assessment of these changes. The ultimate goal of this new information and technology is to provide sensitive and specific tests that reduce unnecessary procedures and optimize therapy. This review addresses the utility of molecular testing in evaluating cancer. A review of the current technology and hereditary cancer syndromes is also presented.

Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure.

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic-acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic-acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay.

Impact of perceived stress, major life events and pregnancy attitudes on low birth weight.

CONTEXT: While low birth weight is the leading cause of infant mortality and morbidity, the factors influencing low birth weight are not well understood. In particular, the relationship between stressful life events and birth outcomes is unclear. It is important for health care providers to better understand the impact of stress on health outcomes. METHODS: Data from a statewide case-control study of 2,378 Missouri mothers are used to examine the relationship of perceived stress, pregnancy attitudes and major life events as psychosocial risk factors on very low birth weight (i.e., birth weight lower than 1,500 g). Such births are contrasted with moderately low birth weight births (those weighing between 1,500 and 2,499 g) and normal-birth-weight infants (those weighing 2,500 g or more). A stepwise logistic regression model is used to control for all study and control variables. RESULTS: The risk of very low birth weight is one and one-half times greater if the mother perceived that she "almost always" felt stress during her pregnancy. The regression model confirms that besides perceived stress, several other factors are independently associated with an increased risk of very low or moderately low birth weight. For example, getting back with a husband or partner or experiencing a major injury accident or illness were associated with an elevated risk of low birth weight (odds ratio, 1. 7), as was pregnancy denial (1.4-1.6) and unhappiness about the pregnancy (1.3). On the other hand, a few factors (taking out a mortgage or loan, having a close relative die and having a mistimed pregnancy) appear to have reduced the odds of low birth weight (odds ratio, 0.5-0.8). CONCLUSIONS: Interventions with pregnant women, especially those assessing perceived stress and attitudes toward the pregnancy, have the potential to improve pregnancy outcomes. Additional prospective research with pregnant women on the origins and effects of stress, including the biological effects of stress, is needed.

Technology assessment: measuring the outcomes of laboratory practice.

The collision of explosive growth in biomedical technology and pressure to contain cost requires that managers of health-care services base decisions to introduce new technology on hard evidence that the benefits outweigh the costs of the new technology. Outcomes research measures the impact of new technology and changes in clinical practice on patient well-being or financial performance. Outcomes research in the clinical laboratory requires a systematic collection of data that is best accomplished in conjunction with a broader health services research effort that includes a multi-disciplinary team of laboratorians, clinicians, administrators, and statisticians. The major requirement for successful outcomes research is an integrated information system that includes clinical, demographic, administrative, claims, financial, and survey information.

Clinical utility of a quantitative polymerase chain reaction for diagnosis of cytomegalovirus disease in solid organ transplant patients.

BACKGROUND: Accurate and rapid diagnosis of human cytomegalovirus (HCMV) disease in solid organ transplant patients remains a challenge. We evaluated the clinical utility of a quantitative polymerase chain reaction (QPCR) method to diagnose transplant patients with HCMV disease. METHODS: A total of 429 plasma samples from 121 solid organ transplant patients were prospectively collected and evaluated for HCMV using a QPCR assay. To enhance the sensitivity of the QPCR assay, plasma samples were centrifuged in a manner designed to concentrate the virions before nucleic acid extraction. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) that contained the same primer sequences as for HCMV. Polymerase chain reaction products were detected by hybridization to 96-well microtiter plates coated with either a HCMV- or an IS-specific probe. RESULTS: A total of 103 patients had all samples negative by QPCR. None of the 103 patients developed HCMV disease during the study. In contrast, 18 patients showed at least 1 sample positive by the QPCR assay, but only 8 of these developed HCMV disease. The mean viral load value for patients without HCMV disease was 93 viral copies (vc) per ml of plasma (range: 35-325 vc/ml plasma) and for the 8 patients with HCMV disease was 67,686 vc/ml plasma (range: 167-1,325,000 vc/ml plasma) (P<0.001). Using a cut-off value of 100 vc/ml plasma and clinical diagnosis of HCMV disease, the QPCR assay showed a sensitivity of 100% and specificity of 99.1%. CONCLUSION: HCMV viral load may be useful in the diagnosis of HCMV disease in solid organ transplant patients.

What families know about funeral-related costs: implications for social work practice.

Social workers provide essential services in the area of end-of-life care to individuals who are dying and their families. Results reported here suggest that social work's role be expanded to provide basic information about local final arrangement (funeral and burial) options and costs. This study was undertaken to determine the knowledge and experience level of people responsible for funeral and cemetery arrangements and to investigate factors affecting familiarity with final costs. Survey responses from 163 survivors of older adults in Kansas City showed that adult children play an important role in the final arrangements of a parent and that half the survivors responsible for final arrangements had no idea what to expect in terms of costs.

The role of perceived stress on prenatal care utilization: implications for social work practice.

Data from the National Institute of Child Health and Human Development/Missouri Maternal and Infant Health Survey on about 2,828 mothers were used to examine the relationship between perceived stress and prenatal care utilization. Major life events that contribute to stress also were examined in relation to adequacy of prenatal care. Women who received inadequate prenatal care were more likely to have reported that they almost always felt stress during pregnancy. Odds ratios were statistically significant for women who were not black and Medicaid recipients but not for black women and women who were not covered by Medicaid. Social work intervention for stress reduction on behalf of pregnant women has the potential to contribute to improved prenatal care utilization, but further analysis of the kinds of stress women experience will enhance social work's ability to target specific interventions.

Role of medical, technical, and administrative leadership in the human resource management life cycle: a team approach to laboratory management.

We believe the team approach to laboratory management achieves the best outcomes. Laboratory management requires the integration of medical, technical, and administrative expertise to achieve optimal service, quality, and cost performance. Usually, a management team of two or more individuals must be assembled to achieve all of these critical leadership functions. The individual members of the management team must possess the requisite expertise in clinical medicine, laboratory science, technology management, and administration. They also must work together in a unified and collaborative manner, regardless of where individual team members appear on the organizational chart. The management team members share in executing the entire human resource management life cycle, creating the proper environment to maximize human performance. Above all, the management team provides visionary and credible leadership.

Going home: community reentry of light care nursing facility residents age 65 and over.

Issues of consumer choice, and rising public expenditures of nursing facility care for the rapidly increasing elderly population have fueled interest in community reentry of nursing facility residents. The Minimum Data Set Plus (MDS+) contains a wealth of information which can be used to provide a better understanding of nursing facility residents including those who discharge. This study employs the Andersen model of health services utilization and logistical regression on MDS+ data to examine characteristics of higher functioning nursing facility residents age 65 and over related to community reentry in one midwestern state. Findings include having Medicaid as a payer source significantly decreased the likelihood of discharge. In contrast, being younger than 85, retaining decision making responsibilities, and having no cognitive impairments were found to increase the likelihood of discharge. Policy and program implications related to identifying and assisting nursing facility residents in resuming community living are discussed.