Awareness and memory function during paediatric anaesthesia.

BACKGROUND: Previous research indicates a much higher incidence of awareness during anaesthesia in children than in adults. The present study is the first large-scale, intraoperative assessment of awareness during paediatric anaesthesia using the isolated forearm technique, and the first large-scale study of memory function during paediatric anaesthesia. METHODS: One hundred and eighty-four children, 5-18 yr, underwent the isolated forearm technique during the first 17 min of surgery while receiving volatile anaesthesia. The isolated forearm technique was modified to accommodate brief or no paralysis. Bispectral index was monitored in a subset of 54 patients. Sixteen neutral words were played 20 times during surgery and, on recovery, implicit memory for these words was tested with a word identification task. Explicit memory for the surgical period was tested with a structured interview. Behavioural changes were assessed with age-appropriate questionnaires. RESULTS: No child had explicit recall of intraoperative events on recovery, and there was no evidence of implicit memory for words presented during anaesthesia. Two of 184 children made unambiguous and verified responses on the modified isolated forearm technique, an incidence of intraoperative awareness of 1.1%. One of these children reported that he was uncomfortable and not completely unconscious during surgery. Neither child had implicit memory for the neutral words, or adverse behaviour change. CONCLUSIONS: The incidence of awareness during surgery in children is approximately eight times that measured in adults by postoperative recall. In contrast to adults, there is no evidence for preserved memory priming during anaesthesia.

Development of a real-time RT-PCR assay for the detection of potato spindle tuber viroid.

Potato spindle tuber viroid (PSTVd) is a quarantine pathogen in the European Union and causes damaging diseases of solanaceous crops. Under the EU Plant Health directive 2000/29/EC, countries must have the ability to detect and identify accurately and rapidly the introduction of harmful organisms in plants or plant products; furthermore, if the quarantine pathogen is found, be able to survey extensively for it. In this respect, PSTVd poses an interesting technical problem, since its RNA does not code for any proteins and thus any diagnostic method must be based on the detection of the RNA and be suitable for scaling up to testing large sample numbers. With this in mind a one-tube real-time RT-PCR assay based on TaqMan chemistry was developed. Investigations were carried out into various aspects of the assay relevant to the efficient amplification of targets that have a significant amount of secondary structure such as viroids. Thus comparisons were made of reverse transcription temperature, concentration and type of reverse transcriptase, RNA denaturation, sample purity and single versus two-tube reaction format. The assay developed was shown to be able to detect a wide range of isolates of PSTVd and in comparison with a chemi-luminescent hybridisation system was shown to be 1000-fold more sensitive. A further significant advantage of this assay format compared with hybridisation is that it is suitable for scaling up to large sample numbers using robotic liquid handling systems.

Detection of different strains of Potato virus Y and their mixed infections using competitive fluorescent RT-PCR.

A competitive fluorescent RT-PCR assay (CF RT-PCR) was developed for the rapid and reliable detection and discrimination of the two most common strains of Potato virus Y (PVY) found in potato (necrotic and ordinary). The assay incorporates two strain specific primers labelled with fluorescent labels, used in conjunction with a universal PVY primer. The strain specific primers compete for the same annealing site which further increases specificity. Discrimination is conferred by the fluorescent labels; green PCR products for PVY(O) and red for PVY(N), whilst mixed infections are detected as orange PCR products without the need for staining agarose gels. The assay can be scaled up for the processing of 96 samples simultaneously, with the detection of PCR products directly using a fluorescent microtitre plate reader. The assay successfully discriminated between 20 isolates of PVY tested, and could be used for the direct detection of PVY in potato tubers.

Strain specific recombinant antibodies to potato virus Y potyvirus.

Single chain Fv antibody fragments have been selected from a synthetic phage-antibody library following three and four rounds of affinity selection with purified potato virus Y, common strain (PVY(O)). The selected fragments were highly specific for PVY and detected seven out of nine isolates of PVY(O) whilst failing to detect three isolates of PVY(N) and 12 isolates of PVY(NTN). Nucleotide sequence of the scFv genes showed the variable heavy fragments belonged to the human VH4 family, whilst the variable light fragments belonged to the Vlambda1 family. The fragments were used in ELISA to detect virus at concentrations of 50 ng/ml in plant sap and in comparisons with commercially available PVY monoclonal antibodies were shown to have similar limits of detection. This is the first report of the selection of a scFv specific for a member of the potyviridae, and its use in detecting and differentiating strains of PVY in infected plant sap. The results highlight the potential of the technology for the selection of strain specific antibodies with an avidity equivalent to traditional monoclonal antibodies raised against viral pathogens and their use for viral diagnosis.

Pharmacokinetics of lignocaine in children after infiltration for cleft palate surgery.

We have studied the pharmacokinetics of lignocaine in children after local infiltration for cleft palate surgery. After induction of anaesthesia, lignocaine 2.5 mg kg-1 with adrenaline 1:200,000 was injected into the palate. Blood samples were collected before and at 2, 5, 10, 15, 20, 30, 60 and 120 min after infiltration. Plasma concentrations of lignocaine were measured by a gas-liquid chromatographic technique. There were no signs of systemic toxicity on routine monitoring of the patients and the peak plasma concentrations were less than the accepted toxic values. Mean half-life was 72.9 (SEM 9.9) min, similar to that found previously in adults and children. However differences in mean clearance (24.6 (2.04) ml kg-1 min-1) and volume of distribution (0.80 (0.07) litre kg-1) were found between this and previous studies.

Molecular analysis of a resistance-breaking strain of potato virus X.

Full-length cDNA clones of potato virus X (PVX) strains PVXUK3 and PVXHB have been constructed in plasmid vectors to allow in vitro transcription of infectious PVX RNA. In both instances the transcript-derived virus infected tobacco and potato identically to the respective progenitor strains: in tobacco and susceptible potato cultivars both strains infected systemically, producing symptomless or mild mosaic symptoms. In potato carrying the Rx or Nx resistance genes, the virus derived from the PVXHB cDNA infected systemically, whereas the virus derived from the PVXUK3 cDNA failed to infect the Rx plants or induced apical necrosis, characteristic of a hypersensitive response of the Nx gene. Three hybrid viral genomes were constructed at the cDNA level to localize the resistance breaking determinants of PVXHB. Transcripts of all three hybrids were infectious on tobacco. On potato cultivars with either the Rx or Nx resistance genes, the hybrid viruses infected in the same way as PVXHB, rather than PVXUK3. The common feature of these hybrid viruses, the coat protein gene, is therefore the determinant of Nx and Rx resistance breaking of PVXHB.

I.v. sedation for conservative dentistry. A comparison of midazolam and diazepam.

Midazolam and the emulsion formulation of diazepam were compared in a cross-over study in 50 patients undergoing out-patient conservative dentistry, with particular regard to sedation and the quality of recovery. Both agents proved effective, but sedation was achieved more rapidly with midazolam (P = 0.001) and was more effective (P less than 0.02). Significantly greater anterograde amnesia for the dental procedure (P less than 0.001) and a more rapid return to normal activities (P less than 0.02) were found with midazolam. Psychometric testing, however, failed to show any objective differences between the treatments. A mean dose of midazolam 0.14 mg kg-1 was required to achieve sedation equating to 0.29 mg kg-1 of diazepam, although there was considerable variation between individual patients.