A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples

Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNIA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADM), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type I virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.

Improving precision and reducing bias in biological surveys: estimating false-negative error rates

The use of presence/absence data in wildlife management and biological surveys is widespread. There is a growing interest in quantifying the sources of error associated with these data. We show that false-negative errors (failure to record a species when in fact it is present) can have a significant impact on statistical estimation of habitat models using simulated data. Then we introduce an extension of logistic modeling, the zero-inflated binomial (ZIB) model that permits the estimation of the rate of false-negative errors and the correction of estimates of the probability of occurrence for false-negative errors by using repeated. visits to the same site. Our simulations show that even relatively low rates of false negatives bias statistical estimates of habitat effects. The method with three repeated visits eliminates the bias, but estimates are relatively imprecise. Six repeated visits improve precision of estimates to levels comparable to that achieved with conventional statistics in the absence of false-negative errors In general, when error rates are less than or equal to50% greater efficiency is gained by adding more sites, whereas when error rates are >50% it is better to increase the number of repeated visits. We highlight the flexibility of the method with three case studies, clearly demonstrating the effect of false-negative errors for a range of commonly used survey methods.

The inhibitory effect of pentobarbitone on reverse transcription-PCR

Pentobarbitone sodium (Sodium 5-ethyl-5[1-methylbutyl]-pentobarbitone) is a short-acting barbiturate that is commonly used to euthanase animals. As part of our studies into the molecular genetics of copper toxicosis in Bedlington terrier dogs, reverse-transcription (RT)-PCR was noted to always fail on RNA samples collected from livers of dogs sacrificed by pentobarbitone injection. When samples were collected without pentobarbitone, however, RTPCR was always successful. We suspected the possible inhibition by pentobarbitone sodium of either reverse transcriptase or Taq polymerase. In vitro studies showed that pentobarbitone interference of PCR occurred at >4 mug/mul. To identify if pentobarbitone produced competitive inhibition, each components (Taq polymerase, MgCl2, dNTP, etc.) of the PCR was individually altered. However, inhibition still persisted, suggesting that multiple PCR components may be affected. Also it was shown that pentobarbitone interference was not dependent on the PCR product size. Simple dilution of pentobarbitone contaminated DNA solutions, and the addition of bovine serum albumin (BSA) to the PCR mix overcame pentobarbitone interference. In vivo, PCR by pentobarbitone was found to be compounded by high DNA concentration and pentobarbitone contamination. In addition, both high DNA concentration and pentobarbitone contamination could be overcome through dilution and the addition of BSA. Further work is required to quantify pentobarbitone concentration in the liver-extracted DNA and RNA samples before this inhibition effect on PCR can be fully elucidated. (C) 2004 Elsevier B.V. All rights reserved.

Attentional bias toward fear-related stimuli: An investigation with nonselected children and adults and children with anxiety disorders

Research investigating anxiety-related attentional bias for emotional information in anxious and nonanxious children has been equivocal with regard to whether a bias for fear-related stimuli is unique to anxious children or is common to children in general. Moreover, recent cognitive theories have proposed that an attentional bias for objectively threatening stimuli may be common to all individuals, with this effect enhanced in anxious individuals. The current study investigated whether an attentional bias toward fear-related pictures could be found in nonselected children (n = 105) and adults (n = 47) and whether a sample of clinically anxious children (n = 23) displayed an attentional bias for fear-related pictures over and above that expected for nonselected children. Participants completed a dot-probe task that employed fear-related, neutral, and pleasant pictures. As expected, both adults and children showed a stronger attentional bias toward fear-related pictures than toward pleasant pictures. Consistent with some findings in the childhood domain, the extent of the attentional bias toward fear-related pictures did not differ significantly between anxious children and nonselected children. However, compared with nonselected children, anxious children showed a stronger attentional bias overall toward affective picture stimuli. (C) 2004 Elsevier Inc. All rights reserved.

Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas

Koala retrovirus (KoRV) is a newly described endogenous retrovirus and is unusual in that inserts comprise a full-length replication competent genome. As koalas are known to suffer from an extremely high incidence of leukaemia/lymphoma, the association between this retrovirus and disease in koalas was examined. Using quantitative real-time reverse transcriptase PCR it was demonstrated that KoRV RNA levels in plasma are significantly increased in animals suffering from leukaemia or lymphoma when compared with healthy animals. Increased levels of KoRV were also seen for animals with clinical chlamydiosis. A significant positive association between viral RNA levels and age was also demonstrated. Real-time PCR demonstrated as much as 5 log variation in KoRV proviral DNA levels in genomic DNA extracted from whole blood from different animals. Taken together these data indicate that KoRV is an active endogenous retrovirus and suggests that it may be causally linked to neoplastic disease in koalas.

Differences in mail and telephone responses to self-rated health: use of multiple imputation in correcting for response bias

Objectives: To estimate differences in self-rated health by mode of administration and to assess the value of multiple imputation to make self-rated health comparable for telephone and mail. Methods: In 1996, Survey 1 of the Australian Longitudinal Study on Women's Health was answered by mail. In 1998, 706 and 11,595 mid-age women answered Survey 2 by telephone and mail respectively. Self-rated health was measured by the physical and mental health scores of the SF-36. Mean change in SF-36 scores between Surveys 1 and 2 were compared for telephone and mail respondents to Survey 2, before and after adjustment for socio-demographic and health characteristics. Missing values and SF-36 scores for telephone respondents at Survey 2 were imputed from SF-36 mail responses and telephone and mail responses to socio-demographic and health questions. Results: At Survey 2, self-rated health improved for telephone respondents but not mail respondents. After adjustment, mean changes in physical health and mental health scores remained higher (0.4 and 1.6 respectively) for telephone respondents compared with mail respondents (-1.2 and 0.1 respectively). Multiple imputation yielded adjusted changes in SF-36 scores that were similar for telephone and mail respondents. Conclusions and Implications: The effect of mode of administration on the change in mental health is important given that a difference of two points in SF-36 scores is accepted as clinically meaningful. Health evaluators should be aware of and adjust for the effects of mode of administration on self-rated health. Multiple imputation is one method that may be used to adjust SF-36 scores for mode of administration bias.

Quantum effect induced reverse kinetic molecular sieving in microporous materials

We report kinetic molecular sieving of hydrogen and deuterium in zeolite rho at low temperatures, using atomistic molecular dynamics simulations incorporating quantum effects via the Feynman-Hibbs approach. We find that diffusivities of confined molecules decrease when quantum effects are considered, in contrast with bulk fluids which show an increase. Indeed, at low temperatures, a reverse kinetic sieving effect is demonstrated in which the heavier isotope, deuterium, diffuses faster than hydrogen. At 65 K, the flux selectivity is as high as 46, indicating a good potential for isotope separation.

A 5 '-nuclease real-time reverse transcriptase-polymerase chain reaction assay for the detection of a broad range of influenza A subtypes, including H5N1

A 5'-nuclease real-time reverse transcriptase-polymerase chain reaction assay was developed for the detection of influenza type A and was validated using a range of influenza A subtypes, including avian strains, and 126 nasopharyngeal aspirate samples. The results show the assay is suitable for screening for influenza A infections, particularly in regions where avian strains may be circulating. (c) 2005 Elsevier Inc. All rights reserved.

Lifetime prevalence estimates of major depression: An indirect estimation method and a quantification of recall bias

The measurement of lifetime prevalence of depression in cross-sectional surveys is biased by recall problems. We estimated it indirectly for two countries using modelling, and quantified the underestimation in the empirical estimate for one. A microsimulation model was used to generate population-based epidemiological measures of depression. We fitted the model to 1-and 12-month prevalence data from the Netherlands Mental Health Survey and Incidence Study (NEMESIS) and the Australian Adult Mental Health and Wellbeing Survey. The lowest proportion of cases ever having an episode in their life is 30% of men and 40% of women, for both countries. This corresponds to a lifetime prevalence of 20 and 30%, respectively, in a cross-sectional setting (aged 15-65). The NEMESIS data were 38% lower than these estimates. We conclude that modelling enabled us to estimate lifetime prevalence of depression indirectly. This method is useful in the absence of direct measurement, but also showed that direct estimates are underestimated by recall bias and by the cross-sectional setting.

Attentional bias to pictures of fear-relevant animals in a dot probe task

Attentional bias to fear-relevant animals was assessed in 69 participants not preselected on self-reported anxiety with the use of a dot probe task showing pictures of snakes, spiders, mushrooms, and flowers. Probes that replaced the fear-relevant stimuli (snakes and spiders) were found faster than probes that replaced the non-fear-relevant stimuli, indicating an attentional bias in the entire sample. The bias was not correlated with self-reported state or trait anxiety or with general fearfulness. Participants reporting higher levels of spider fear showed an enhanced bias to spiders, but the bias remained significant in low scorers. The bias to snake pictures was not related to snake fear and was significant in high and low scorers. These results indicate preferential processing of fear-relevant stimuli in an unselected sample.

Correcting for numerator/denominator bias when assessing changing inequalites in occupational class mortality, Australia 1981-2002

Objective Comparisons of the changing patterns of inequalities in occupational mortality provide one way to monitor the achievement of equity goals. However, previous comparisons have not corrected for numerator/denominator bias, which is a consequence of the different ways in which occupational details are recorded on death certificates and on census forms. The objective of this study was to measure the impact of this bias on mortality rates and ratios over time. Methods Using data provided by the Australian Bureau of Statistics, we examined the evidence for bias over the period 1981-2002, and used imputation methods to adjust for this bias. We compared unadjusted with imputed rates of mortality for manual/non-manual workers. Findings Unadjusted data indicate increasing inequality in the age-adjusted rates of mortality for manual/non-manual workers during 1981-2002, Imputed data suggest that there have been modest fluctuations in the ratios of mortality for manual/non-manual workers during this time, but with evidence that inequalities have increased only in recent years and are now at historic highs. Conclusion We found that imputation for missing data leads to changes in estimates of inequalities related to social class in mortality for some years but not for others. Occupational class comparisons should be imputed or otherwise adjusted for missing data on census or death certificates.

Examination of emotional priming among children and young adolescents: Developmental issues and its association with anxiety

An affective priming task was used to examine bias in the processing of threat-related material in 25 clinically anxious compared to 25 matched, non-anxious control children and young adolescents. No significant differences were found between anxious and non-anxious children in terms of priming effects. However, age-related differences were found depending upon the valence of the target, independent of anxiety status. Both younger (7-10 years) and older (11-14 years) children showed faster response times to pleasant targets when they were preceded by a congruent compared to incongruent stimulus, consistent with a traditional priming effect. For threat target stimuli, older children showed no difference in response latency according to the congruency of the prime-target valence. Younger children, in contrast, showed a reverse priming effect for threat target stimuli, with slower response times for threat-congruent trials than for threat targets preceded by a pleasant prime. Possible explanations for developmental differences in the processing of threat-related material are discussed.