A rapid microtitre plate screening method for in vitro assessment of fibrinolysis: a preliminary report

A novel and precise assay that facilitates high-throughput screening of fibrinolytic agents was developed based on the automated assessment of the euglobulin clot lysis time in microtitre plates. Euglobulin fractions from fresh plasma samples were assessed over 28 days to determine the inter-assay and intra-assay precision. The intra-assay (coefficient of variation range, 0.7-2.6%) and inter-assay precision (coefficient of variation range, 6.8-12.1%) was found to be well within limits required by the Food and Drug Administration. On day 1 and day 28, the results of the microtitre plate euglobulin clot lysis time method were compared with tissue plasminogen activator activity, plasminogen activator inhibitor activity and results produced on fibrin plates. All comparisons were found to correlate significantly. The validity of this method for assaying fibrinolytic agents was assessed by comparing dose-response curves for streptokinase produced using fibrin plates and this method. The critical influence of ambient temperature on the inter-assay reproducibility of this method was established by testing samples over a range of temperatures between 20degreesC and 40degreesC. (C) 2004 Lippincott Williams Wilkins.

Australia: new screening method for cold tolerance during the reproducitve stage in rice

Low temperature, particularly during the reproductive stage of the development of rice, limits productivity in the Riverina region of New South Wales (NSW). This study primarily examined genotypic differences in cold damage that are associated with low temperature during reproductive development. Results from experiments in temperature-controlled rooms and the cold water facility were combined with four years of field experiments, which used natural exposure to low temperature to examine the response of over 50 cultivars from diverse origins. Plants were exposed to day/night air temperatures of 27°/13°C in temperature-controlled rooms and to a constant temperature of 19°C in the cold water facility. Low temperature treatments were imposed from panicle initiation (PI) to 50% heading. In field experiments several techniques were used to increase the likelihood of inducing cold damage such as sequential sowing dates (five to eight sowing dates each year), shallow water depths (5cm) and high nitrogen rates (e.g. 300kgN ha-1). Several cultivars were identified that were more cold tolerant than Australia’s commercial cultivars.

Yield response of rice (Oryza sativa L.) genotypes to drought under rainfed lowlands 4. Vegetative stage screening in the dry season

Screening for drought resistance of rainfed lowland rice using drought score (leaf death) as a selection index has a long history of use in breeding programs. Genotypic variation for drought score during the vegetative stage in two dry season screens was examined among 128 recombinant inbred lines from four biparental crosses. The genotypic variation detected for drought score in the dry season was used to examine the reliability of the dry season screening method to estimate relative grain yield of genotypes under different types of drought stress in the wet season. Large genotypic variation for drought score existed in two experiments (A and B). However, there was no relationship between the drought scores of genotypes determined in these two experiments. Different patterns of development and severity of drought stress in these two experiments, i.e. slow development and mild plant water deficit in experiment A and fast development and severe plant water deficit in experiment B, were identified as the major factors contributing to the genotypes responding differently. Larger drought score in the dry season experiments was associated with lower grain yield under specific drought stress conditions in the wet season, but the association was weak to moderate and significant only in particular drought conditions. In most cases, a significant phenotypic and moderate genetic correlation between drought score in the dry season and grain yield in the wet season existed only when both drought score and grain yield of genotypes were affected by similar patterns and severity of drought stress in their respective experimental environments. The dry season environments used to measure genotypic variation for drought score should be managed to correspond to relevant types of drought environment that are frequent in the wet season. The efficiency of using the drought score as an indirect selection criterion for improving grain yield for drought conditions was lower than the direct selection for grain yield, and hence wet season screening with grain yield as a selection criterion would be more efficient. However, using drought score as a selection index, a larger number of genotypes can be evaluated than for wet season grain yield. Therefore, it is possible to apply higher selection intensities using the drought score system, and the selected lines can be further tested for grain yield in the wet season. (C) 2004 Elsevier B.V. All rights reserved.

Screening of obstructive and central apnoea/hypopnoea in children using variability: A preliminary study

Aim: Polysomnography (PSG) is the current standard protocol for sleep disordered breathing (SDB) investigation in children. Presently, there are limited reliable screening tests for both central (CE) and obstructive (OE) respiratory events. This study compared three indices, derived from pulse oximetry and electrocardiogram ( ECG), with the PSG gold standard. These indices were heart rate (HR) variability, arterial blood oxygen de-saturation (SaO(2)) and pulse transit time (PTT). Methods: 15 children (12 male) from routine PSG studies were recruited (aged 3 - 14 years). The characteristics of the three indices were based on known criteria for respiratory events (RPE). Their estimation singly and in combination was evaluated with simultaneous scored PSG recordings. Results: 215 RPE and 215 tidal breathing events were analysed. For OE, the obtained sensitivity was HR (0.703), SaO(2) (0.047), PTT (0.750), considering all three indices (0) and either of the indices (0.828) while specificity was (0.891), (0.938), (0.922), (0.953) and (0.859) respectively. For CE, the sensitivity was HR (0.715), SaO(2) (0.278), PTT (0.662), considering all indices (0.040) and either of the indices (0.868) while specificity was (0.815), (0.954), (0.901), (0.960) and (0.762) accordingly. Conclusions: Preliminary findings herein suggest that the later combination of these non-invasive indices to be a promising screening method of SDB in children.

Impact on perinatal mortality of missed opportunities to treat maternal syphilis in rural South Africa: baseline results from a clinic randomized controlled trial

OBJECTIVE To demonstrate the impact on perinatal mortality of inadequate treatment for maternal syphilis despite adequate screening. METHOD In 12 clinics providing antenatal care in Hlabisa, South Africa 1783 pregnant women were screened for syphilis at their first antenatal visit between June and October 1998. Pregnancy outcome was determined among those with syphilis. RESULTS A total of 158 women were diagnosed with syphilis: prevalence 9% (95% CI 8-10%). Mean gestation at first antenatal visit was 24 weeks. Thirty women (19%) received no treatment and 96 (61%) received all three recommended doses of penicillin. Among those receiving at least one dose, mean delay to the first dose was 20 days. Among those fully treated mean delay to treatment completion was 34 days. Pregnancy outcome was known for 142 women (90%) and there were 17 perinatal deaths among 15 women (11%). Eleven of 43 women (26%) who received one or fewer doses of penicillin experienced ii perinatal death whilst only four of 99 women (4%) who received two or more doses of penicillin did so (P = 0.0001). Protection from perinatal death increased with the number of doses of penicillin: linear modelling suggests that one dose reduced the risk by 41%, two doses by 65% and three doses by 79%, compared with no doses. A dose-specific, categorical model confirmed reduction in risk by 79% for all three doses. CONCLUSION Despite effective screening, many pregnant women with syphilis remain inadequately treated, resulting in avoidable perinatal mortality. Delays in starting and finishing treatment, as well as incomplete treatment occur. Near-patient syphilis testing in the antenatal clinic with early treatment could improve treatment of syphilis and reduce perinatal mortality, and a randomized trial to test this is underway.

Resistance to Fiji disease in sugar cane: Role of cultivar preference by planthopper vector Perkinsiella saccharicida (Homoptera : Delphacidae)

Fiji disease (FD) of sugar cane caused by Fiji disease virus (FDV) is transmitted by the planthopper Perkinsiella saccharicida Kirkaldy (Hemiptera: Delphacidae). FD is effectively managed by using resistant cultivars, but whether the resistance is for the vector or for the Virus is Unknown. This knowledge would help develop a rapid and reliable glasshouse-based screening method for disease resistance. Sugar cane cultivars resistant, intermediate, and susceptible to FD were screened in a glasshouse, and the relationship between vector preferences and FD incidence was studied. Cultivar preference by nymphs increased with an increase in cultivar susceptibility to FD, but the relationship between adult preference and FD resistance was not significant. There was a positive correlation between the vector population and FD incidence, and the latent period for symptom expression declined with the increase in the vector populations. FD incidence in the glasshouse trial reflected the field-resistance status of sugar cane cultivars with known FD-resistance scores. The results suggest that resistance to FD in sugar cane is mediated by cultivar preference of the plant-hopper vector.

Hazard detection in older adult drivers

Older adults make up an increasing propordon of automobile drivers in Australia. Despite the fact that older drivers generally drive much less than younger drivers, there is a disdnct increase in accidents, fatalides and injuries in drivers over age 65 (per actual kilometres driven). Accurate means of screening older adults to idendfy those at increased risk of motor vehicle accidents have proved elusive. Neuropsychological assessment and clinical examinadon are not well-correlated with accident risk. On-road tesdng, which is more highly correlated with accident risk, is expensive and dme-consuming, as well as being less suitable as a screening process. Hazard percepdon methods have been used as an effecdve screening method for idendfying younger adults at increased risk of accidents. A brief video-based hazard percepdon screening test involving footage of genuine traffic hazards for use on older individuals will be presented.

A comparison of telepaediatric activity at two regional hospitals in Queensland

We conducted a 15-month feasibility study of telepaediatrics. A novel service was offered to two hospitals in Queensland (Mackay and Hervey Bay). We used data from all other hospitals throughout the state as the control group. Although both intervention hospitals were provided with the same service, the telepaediatric activity generated and the effect on admissions and outpatient activity were markedly different. There was a significant decrease in the number of patient admissions to Brisbane from the Mackay region. In addition, there was an increase in the number of Mackay patients treated locally (as outpatients). In contrast, little change was observed in Hervey Bay. We assessed whether the observed differences between the two hospitals were due to various factors which influenced the use of the telepaediatric service. These factors included the method of screening patients before transfer to the tertiary centre and the physical distance between each facility and the tertiary centre. We believe that the screening method used for patient referrals was the most important determinant of the use of the telepaediatric service.

A PCR-based method of screening individuals of all ages, from neonates to the elderly, for familial hyperaldosteronism type I

Aim: Unless specifically treated (glucocorticoids in low doses), Familial Hyperaldosteronism Type I(FH-I) may result in early death from stroke. We report the successful application of a rapid, polymerase chain reaction (PCR)-based method of detecting the 'hybrid' 11 beta-hydroxylase (11 beta-OHase)/aldosterone synthase (AS) gene as a screening test for FH-I. Methods: 'Long-PCR' was used to amplify, concurrently, a 4 kb fragment of AS gene (both primers AS-specific) and a 4 kb fragment of the hybrid gene (5' primer 11 beta-OHase-specific, 3'primer AS-specific) from DNA extracted from blood either collected locally or transported from elsewhere. Sample collection and transport were straightforward. This 4 kb fragment contains all the currently recognised hybrid gene 'crossover' points. Results: Within a single family, long-PCR identified all 21 individuals known to have FH-I. Hypertension was corrected in all 11 treated with glucocorticoids. Nine with normal blood pressure are being closely followed for development of hypertension. Long-PCR cord blood analysis excluded FH-I in three neonates born to affected individuals. Long-PCR newly identified two other affected families: (1) a female (60 years) with a personal and family history of stroke and her normotensive daughter (40 years), and (2) a female (51 years) previously treated for primary aldosteronism with amiloride, her two hypertensive sons (14 and 16 years) and her hypertensive mother (78 years). No false negative or false positive results have yet been encountered. At least seven other centres have successfully performed this test. Conclusion: Long-PCR is a reliable method of screening individuals of all ages for FH-I.

A PCR method for the identification of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs

Aim: The aim of this study was to assess the discriminatory power and potential turn around time ( TAT) of a PCR-based method for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs. Methods: Screening swabs were examined using the current laboratory protocol of direct culture on mannitol salt agar supplemented with oxacillin (MSAO-direct). The PCR method involved pre-incubation in broth for 4 hours followed by a multiplex PCR with primers directed to mecA and nuc genes of MRSA. The reference standard was determined by pre-incubation in broth for 4 hours followed by culture on MSAO (MSAO-broth). Results: A total of 256 swabs was analysed. The rates of detection of MRSA using MSAO-direct, MSAO-broth and PCR were 10.2, 13.3 and 10.2%, respectively. For PCR, the sensitivity, specificity, positive predictive value and negative predictive values were 66.7% (95% CI 51.9 - 83.3%), 98.6% ( 95% CI 97.1 - 100%), 84.6% ( 95% CI 76.2 - 100%) and 95.2% ( 95% CI 92.4 - 98.0%), respectively, and these results were almost identical to those obtained from MSAO-direct. The agreement between MSAO-direct and PCR was 61.5% ( 95% CI 42.8 - 80.2%) for positive results, 95.6% ( 95% CI 93.0 - 98.2%) for negative results and overall was 92.2% ( 95% CI 88.9 - 95.5%). Conclusions: ( 1) The discriminatory power of PCR and MSAO-direct is similar but the level of agreement, especially for true positive results, is low. ( 2) The potential TAT for the PCR method provides a marked advantage over conventional methods. ( 3) Further modifications to the PCR method such as increased broth incubation time, use of selective broth and adaptation to real-time PCR may lead to improvement in sensitivity and TAT.

A method for screening the temperature dependence of three-dimensional crystal formation

Temperature is an important parameter controlling protein crystal growth. A new temperature-screening system (Thermo-screen) is described consisting of a gradient thermocycler fitted with a special crystallization-plate adapter onto which a 192-well sitting-drop crystallization plate can be mounted (temperature range 277-372 K; maximum temperature gradient 20 K; interval precision 0.3 K). The system allows 16 different conditions to be monitored simultaneously over a range of 12 temperatures and is well suited to conduct wide (similar to 20 K) and fine (similar to 3 K) temperature-optimization screens. It can potentially aid in the determination of temperature phase diagrams and run more complex temperature-cycling experiments for seeding and crystal growth.

A novel method for selection of chymotrypsin inhibitors from a phage peptide library

A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library. In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates. After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing. These peptides share a consensus sequence motif as (S/T)RVPR(R/H). Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin. The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure.

The clinical utility of the Hopkins Verbal Learning Test as a screening test for mild dementia

Objective. The purpose of this study was to determine whether the Hopkins Verbal Learning Test (HVLT) could be used as a valid and reliable screening test for mild dementia in older people, and to compare its performance to that of the Mini-Mental State Examination (MMSE). Method. Using a cross-sectional design, we studied three groups of older subjects recruited from a district geriatric psychiatry service: (1) 26 patients with DSM-IV dementia and MMSE scores of 18 or better; (2) 15 patients with psychiatric diagnoses other than dementia; and (3) 15 normal controls. The relationship of each potential cutting point on the HVLT and the MMSE was examined against the independently ascertained DSM-IV diagnoses of dementia using a Receiver Operating Characteristic (ROC) analysis. Results. The subjects consisted of 21 (37.5%) males and 35 (62.5%) females with a mean age of 74.7 (SD 6.1) years and a mean of 8.5 (SD 1.8) years of formal education. ROC analysis indicated that the optimal cutting point for detecting mild dementia in this group of subjects using the HVLT was 18/19 (sensitivity = 0.96, specificity = 0.80) and using the MMSE was 25/26 (sensitivity = 0.88, specificity = 0.93). Conclusions. The HVLT can be recommended as a valid and reliable screening test for mild dementia and as an adjunct in the clinical assessment of older people. The HVLT had better sensitivity than the MMSE in detecting patients with mild dementia, whereas the MMSE had better specificity. Copyright (C) 2000 John Wiley & Sons, Ltd.

Cervical screening in migrants to Australia

Objective: To examine differentials and time trends in self-reported Pap test rates by migrant status from the 1989/90 and 1995 Australian National Health Surveys (NHS). Method: Unit record data for females with the variables of interest were extracted from the 1989/90 and 1995 NHS and combined. The dichotomous outcome variables were 'ever had a Pap test and 'had a Pap test within three years'. The principal study factor was country-of-birth, but language spoken at home (English or not) was also examined. The indirect age-standardised screening ratio was used to calculate proportions of 'ever had a Pap test' and 'had a Pap test within three years' and differences were tested statistically using logistic regression analysis for each year of survey by migrant status. Results: Odds ratios for rates of reporting 'ever had a Pap test' were significantly lower in women born in southern Europe, Italy, other countries, southern Asia, Middle East, Greece and South-East Asia compared with Australian-born. Reported rates of 'ever had a Pap test' were significantly higher in the 1995 NHS (p

Novel miniaturized systems in high-throughput screening

High-throughput screening (HTS) using high-density microplates is the primary method for the discovery of novel lead candidate molecules. However, new strategies that eschew 2D microplate technology, including technologies that enable mass screening of targets against large combinatorial libraries, have the potential to greatly increase throughput and decrease unit cost. This review presents an overview of state-of-the-art microplate-based HTS technology and includes a discussion of emerging miniaturized systems for HTS. We focus on new methods of encoding combinatorial libraries that promise throughputs of as many as 100 000 compounds per second.