Predicting the role of touchless technologies within diagnostic radiography: Results of an international survey.

INTRODUCTION: The evolution of technology within healthcare is continuing at a rapid rate. Touchless technologies (i.e. those involving gestures and voice commands) are rapidly being integrated into daily life. The aim of this study was to investigate the potential role for such technologies within diagnostic radiography. METHODS: An online survey was developed, piloted and deployed using SurveyMonkey as part of an online radiology congress. Eligible respondents were radiographers or radiologic technologists, including students. The survey covered ten themes relating to the potential role of touchless technologies within diagnostic radiography. Results were analysed using descriptive and inferential statistics. RESULTS: 155 people completed the questionnaire. 100 (64.9%) were women and clinical experience ranged from 13.5 (0-40) years. The majority, 54 (35.1%), had a Bachelor's degree with respondents being from 23 different countries (five continents). 34 (21.9%) respondents did not personally own nor intended to purchase touchless technologies. 89 (84.8%) respondents saw themselves using touchless technologies, if available on current imaging equipment. 25 (16.0%) respondents reported that they currently have access to touchless technologies within their workplace. 88 (81.5%) and 67 (65.0%) respondents reported that they saw voice and gesture controls as being key in improving exam efficiency. CONCLUSION: Participants clearly perceived a role for touchless technologies within diagnostic radiography. Access to such technologies is not yet widely available within X-ray rooms. Voice activated technologies appear more appealing that gesture-based aids. The primary role for such technologies was defined by participants as focusing on improving examination efficiency. IMPLICATIONS FOR PRACTICE: Touchless technologies have been identified and as important and potentially useful in diagnostic radiography. Collaboration between healthcare institutions, industry and academia is required to design and successfully implement these technologies into practice.

Modifications to mobile chest radiography technique during the COVID-19 pandemic - implications of X-raying through side room windows.

INTRODUCTION: Modifications to common radiographic techniques have resulted from the challenges presented by the COVID-19 pandemic. Reports exist regarding the potential benefits of undertaking mobile radiography through side room windows. The aim of this study was to evaluate the impact on image quality and exposure factors when undertaking such examinations. METHODS: A phantom based study was undertaken using a digital X-ray room. Control acquisitions, using a commercially available image quality test tool, were performed using standard mobile chest radiography acquisition factors. Image quality (physical and visual), incidence surface air kerma (ISAK), Exposure Index (EI) and Deviation Index (DI) were recorded. Image quality and radiation dose were further assessed for two additional (experimental) scenarios, where a side room window was located immediately adjacent to the exit port of the light beam diaphragm. The goal of experimental scenario one was to modify exposure factors to maintain the control ISAK. The goal of experimental scenario two was to modify exposure factors to maintain the control EI and DI. Dose and image quality data were compared between the three scenarios. RESULTS: To maintain the pre-window (control) ISAK (76 µGy), tube output needed a three-fold increase (90 kV/4 mAs versus 90 kV/11.25 mAs). To maintain EI/DI a more modest increase in tube output was required (90 kV/8 mAs/ISAK 54 µGy). Physical and visual assessments of spatial resolution and signal-to-noise ratio were indifferent between the three scenarios. There was a slight statistically significant reduction in contrast-to-noise ratio when imaging through the glass window (2.3 versus 1.4 and 1.2; P = 0.005). CONCLUSION: Undertaking mobile X-ray examinations through side room windows is potentially feasible but does require an increase in tube output and is likely to be limited by minor reductions in image quality. IMPLICATIONS FOR PRACTICE: Mobile examinations performed through side room windows should only be used in limited circumstances and future clinical evaluation of this technique is warranted.

Antibodies to Epstein-Barr virus thymidine kinase: a characteristic marker for the serological detection of nasopharyngeal carcinoma.

Patients suffering from nasopharyngeal carcinoma (NPC) generally exhibit elevated serum IgA antibody titres to Epstein-Barr virus (EBV) early antigen (EA) and virus capsid antigen (VCA). This property is frequently used as a diagnostic aid. Preliminary experiments suggested that an ELISA for IgA antibodies against the EBV-encoded thymidine kinase (TK) could form the basis of a more reliable diagnostic test. Here, we describe the construction of a recombinant baculovirus that expresses the EBV TK and present a full analysis of its use in serological surveys of NPC patients. Baculovirus-derived TK was used to develop a simple ELISA for serum IgA against this antigen. ELISA reactivity was strongly associated with NPC compared with an EBV-positive, normal control population. Comparison with the existing IgA-VCA and EA assays showed that the TK ELISA had higher sensitivity whilst the specificity was similar or higher. We conclude that the TK ELISA presents a strong predictor of NPC and, in its refined form, has improved pickup rates. In addition, results from patients with chronic nasopharyngitis (CNP) suggest that individuals with both symptoms of CNP and an elevated TK ELISA value may be at increased risk for the development of head-and-neck cancer.

Ganciclovir and penciclovir, but not acyclovir, induce apoptosis in herpes simplex virus thymidine kinase-transformed baby hamster kidney cells.

The efficacies of ganciclovir (GCV), penciclovir (PCV) and acyclovir (ACV) in inducing cell death in the herpes simplex virus thymidine kinase (HSVTK) system were compared. HSVTK-transformed baby hamster kidney cells treated with GCV, PCV or ACV were monitored for growth by viable count, and for death by TUNEL assay, propidium iodide staining, detection of phosphatidyl serine translocation and detection of DNA laddering. All compounds delayed growth or reduced viability of HSVTK-transformed cells. Drug treatment reduced levels of cyclin B1 message (which normally peaks in G2/M-phase of the cell cycle) and induced a four- to fivefold upregulation of GADD45 message. Treatment with GCV or PCV induced rapid accumulation of cells in S-phase and apoptotic death. Treatment with ACV, however, was associated with sustained S-phase arrest. GCV (and to a lesser extent PCV) increased phosphatidyl serine translocation, induced positive TUNEL results with alterations in cell morphology, caused marked propidium iodide staining and induced DNA laddering. By contrast, up to 7 days' exposure to ACV did not induce DNA laddering, with very little TUNEL staining. ACV treatment had little effect on phosphatidyl serine translocation and propidium iodide staining was markedly reduced compared with treatment with the other compounds. Thus, by all criteria, GCV was the most potent inducer of cell death. The current theories regarding apoptosis or necrosis as the preferred form of cell death in prodrug gene therapy are considered and the suitability of PCV or ACV as potential alternatives to GCV in the HSVTK system is discussed.

Indolocarbazoles: potent, selective inhibitors of human cytomegalovirus replication.

In our search for new, safer anti-HCMV agents, we discovered that the natural product Arcyriaflavin A (la) was a potent inhibitor of HCMV replication in cell culture. A series of analogues (symmetrical indolocarbazoles) was synthesised to investigate structure activity relationships in this series against a range of herpes viruses (HCMV, VZV, HSV1, and 2). This identified a number of novel, selective and potent inhibitors of HCMV, 12,13-dihydro-2,10-difluoro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazol e-5,7-(6H)-dione (1d) being the best example (IC50=40 nM, therapeutic index > 1450). Compounds described in this series were generally poor inhibitors of protein kinase C betaII, and no correlation was found between the ability to inhibit HCMV and the enzyme PKC.

Synthesis and anti-herpes virus activity of 2'-deoxy-4'-thiopyrimidine nucleosides.

A series of 5-substituted 2'-deoxy-4'-thiopyrimidine nucleosides was synthesized and evaluated as potential antiviral agents. A number of analogues such as 2'-deoxy-5-propyl-4'-thiouridine (3ii), 2'-deoxy-5-isopropyl-4'-thiouridine (3iii), 5-cyclopropyl-2'-deoxy-4'-thiouridine (3iv), 2'-deoxy-4'-thio-5-vinyluridine (3viii), and 5-(2-chloroethyl)-2'-deoxy-4'-thiouridine (3xx) were found to be highly active against herpes simplex virus type-1 (HSV-1) and varicella zoster virus (VZV) in vitro with no significant cytotoxicity. The compound with the broadest spectrum of activity was 2'-deoxy-5-ethyl-4'-thiouridine (3i) which showed significant activity against HSV-1, HSV-2, and VZV.

The cloning, sequencing and expression of a major antigenic region from the feline calicivirus capsid protein.

RNA purified from the feline calicivirus (FCV) F9 vaccine strain was used to prepare a cDNA library in the expression vector lambda gt11. The library was screened for expression of FCV antigen using a rabbit antiserum prepared against purified FCV. A 330 bp cDNA clone was identified and used as a probe to obtain a larger overlapping clone of 1369 bp. Comparative sequence analysis with the CFI and F4 strains showed that the clones were derived from the 3' open reading frame encoding the capsid protein. The region encoded by the 330 bp clone was shown to be variable in the three strains compared, and therefore the probable location of major antigenic variation. This clone was expressed in a bacterial system and antiserum to the recombinant protein was used in immunoblots to confirm that this clone was derived from the gene encoding the capsid protein. From these immunoblots, several other capsid-related polypeptides were identified. Comparison with immunoblots using post-vaccination cat sera showed the antibody response in the cat was directed mainly against the capsid protein. Antiserum to the recombinant protein was shown to be effective in neutralizing the infectivity of FCV, indicating that at least one major neutralizing epitope had been cloned.

Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir.

Human cytomegalovirus (HCMV, a betaherpes virus) is the cause of serious disease in immunologically compromised individuals, including those with acquired immunodeficiency syndrome. One of the compounds used in the chemotherapy of HCMV infections is the nucleoside analogue 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (ganciclovir). The mechanism of action of this drug is dependent on the formation of the nucleoside triphosphate, which is a strong inhibitor of the viral DNA polymerase. Thymidine kinase, which is encoded by many of the herpesviruses, catalyses the initial phosphorylation of ganciclovir. But there is no evidence for the coding of this enzyme by HCMV, and DNA sequence analysis of the HCMV genome has shown that there is no open reading frame characteristic of a herpesvirus thymidine kinase. Here we present biochemical and immunological evidence that the HCMV UL97 open reading frame codes for a protein capable of phosphorylating ganciclovir. This protein seems to be responsible for the selectivity of ganciclovir and will be useful tool in the understanding and refinement of the antiviral activity of new selective anti-HCMV compounds.

32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase.

Alkylated nucleotides have been detected by 32P-postlabelling using the enzyme T4 polynucleotide kinase which phosphorylates the 3'-mononucleotides to give the 3',[5'-32P]bisphosphates. These may then be separated by two-dimensional TLC as the bisphosphates or the [5'-32P]monophosphates. We describe here an alternative approach using the Epstein-Barr virus (EBV) encoded thymidine kinase (TK) to directly phosphorylate adducted nucleosides to give the [5'-32P]monophosphates. Using a series of methyl, ethyl and butyl thymidines EBV-encoded TK was shown to phosphorylate a wide range of adducted thymidines with varying degrees of labelling efficiency; N3-methyl thymidine was labelled with the highest efficiency and O4-ethyl thymidine the lowest. Whereas O4-methyl thymidine was labelled at a higher efficiency than O2-methyl thymidine, O4-ethyl and O4-butyl thymidines were labelled at a much lower efficiency than the corresponding O2-alkyl thymidines. Labelling efficiency increased with pH in the range pH 7 to pH 9, but the relative labelling efficiency was ATP independent. This direct phosphorylation of adducted nucleosides offers an alternative approach to the detection of alkylated residues in DNA which may complement current postlabelling procedures.

Diagnosis of nasopharyngeal carcinoma by means of recombinant Epstein-Barr virus proteins.

The immune response of patients with nasopharyngeal carcinoma to Epstein-Barr virus (EBV) antigens is diagnostic of the tumour. Existing tests use EBV antigens produced in EBV-infected lymphoblastoid cells, but the virus replicates poorly in these cells. Serum samples from 18 patients diagnosed as having nasopharyngeal carcinoma were screened by western blot analysis, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence tests for antibodies to the EBV-coded alkaline deoxyribonuclease (DNase), thymidine kinase, and membrane antigen (gp340/220) produced in recombinant baculovirus or bovine papillomavirus systems. Each protein was a useful diagnostic marker for nasopharyngeal carcinoma, although in the gp340/220 ELISAs there was substantial overlap for both IgG and IgA antibodies between serum samples from nasopharyngeal carcinoma patients and those from healthy donors seropositive for EBV. The EBV thymidine kinase was the most sensitive predictor of nasopharyngeal carcinoma; all such samples showed both IgG and IgA antibody responses to this protein and all gave clearly distinct titres from those of the EBV-seropositive donors in the IgA test. Each of the recombinant systems described is suitable for use in large-scale screening programmes for the early diagnosis of nasopharyngeal carcinoma.

High-level expression of the Epstein-Barr virus alkaline deoxyribonuclease using a recombinant baculovirus: application to the diagnosis of nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). Infection of the insect cell line Spodoptera frugiperda (SF9) with the recombinant virus led to the expression of an enzymatically active alkaline DNase. The recombinant EBV alkaline DNase was highly soluble, and the recombinant baculovirus produced approximately 10-20 mg of EBV DNase per 1 X 10(9) cells. The recombinant enzyme activity was neutralized by specific antisera to the EBV DNase and was recognized by these sera in Western blot analysis and immunofluorescence tests. The recombinant EBV DNase was neutralized by these sera from patients with nasopharyngeal carcinoma and chronic infectious mononucleosis. Western blot analysis using these patients' sera showed that IgG and IgA antibodies to the EBV DNase could be readily detected.

Immunological studies on the Epstein-Barr virus encoded alkaline deoxyribonuclease found in virus-producing lymphoblastoid cells.

Antisera were raised against a purified recombinant form of the Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) expressed in Escherichia coli. These sera were shown to be reactive with lymphoblastoid cells expressing EBV antigens (B95-8, P3HR-1 and Raji). Immunostaining studies of cells expressing EBV antigens revealed that the DNase was a component of the restricted early antigen complex of EBV. Western blot analysis of these chemically induced cells revealed that the polypeptide associated with the EBV DNase has an Mr of approximately 55,000, slightly greater than that of the recombinant form, suggesting that the protein undergoes some form of posttranslational modification during virus replication. The DNase enzymic activities observed in B95-8, P3HR-1 and Raji cells following chemical induction were neutralized using the specific antiserum. A detailed examination of protein extracts from the nude mouse-passaged nasopharyngeal carcinoma cell line C-15 failed to detect any antigenic or biochemical evidence for the presence of the DNase. Immunostaining of biopsies of oral 'hairy' leukoplakia with the antisera against EBV DNase revealed high level expression in the more differentiated spinous layers of the epithelium, a pattern of reactivity identical to that observed for other lytic cycle antigens.

The genome of human herpesvirus 6: maps of unit-length and concatemeric genomes for nine restriction endonucleases.

More than 50 fragments resulting from complete digestion of the DNA of human herpesvirus 6 (HHV-6, strain U1102) with BamHI, EcoRI, HindIII, KpnI, NruI, SalI or SmaI have been isolated as clones in M13, plasmid, cosmid and lambda vectors. Using these clones, maps have been constructed for the fragments produced by nine restriction enzymes from unit-length virus genomes and from their concatemeric precursors. The unit-length genome is a linear, double-stranded molecule of 161.5 kbp composed of a central segment of a largely unique sequence of 141 kbp (U) with a sequence of 10 kbp duplicated in the same orientation at both 'left' and 'right' genomic termini (i.e. 'left' and 'right' copies of the direct repeat; DRL and DRR). Adopting as standard an orientation in which the major capsid protein gene is 'left' of the gene for alkaline exonuclease, then the 'right' genome termini and DRL. U junctions occur close to or within repetitive (GGGTTA)n sequences. Repetitions of short sequence motifs are present in at least two other regions of the genome. One of these regions consists of a simple repeat (TC/G) of approximately 1.5 kbp in length and is unstable as clones in bacterial vectors. The second region is stably maintained in such vectors and consists of a tandem array of at least 25 copies of a 110 bp sequence containing a single KpnI site. Comparisons of fragments arising from unit-length DNA with those from virus DNA from the nuclei of infected cells have shown that the concatemeric junctions in intracellular DNA contain head-to-tail dimers of the terminal duplications (i.e. ...U1.DRR1.DRL2.U2...). The gross structure established here for the genome from the U1102 isolate of HHV-6 resembles closely that suggested by Pellett and his colleagues for the Z29 isolate and differs from that of the five previously characterized human herpesviruses. This structure of HHV-6 DNA bears a superficial resemblance to that proposed for DNA from channel catfish virus and equine cytomegalovirus.

Immunological response of nasopharyngeal carcinoma patients to the Epstein-Barr-virus-coded thymidine kinase expressed in Escherichia coli.

A bacterial expression system which produces large amounts of the Epstein-Barr-virus-coded thymidine kinase has been developed and used to produce protein for Western blot analysis of a number of human antisera. Interestingly, only sera from nasopharyngeal carcinoma (NPC) patients had any detectable IgA antibody which reacted with the EBV TK. These findings provided the basis for ELISA tests using a crude lysate of the E. coli cells expressing the EBV TK as target antigen. Sera from NPC patients showed high levels of IgA reactive antibodies in this test while other sera did not.

Identification, cloning, and expression of the major capsid protein gene of human herpesvirus 6.

DNA sequence analysis of part of the human herpesvirus 6 (HHV-6) genome led to the identification of an open reading frame with amino acid sequence homology to the major capsid proteins (MCP) of other HHVs. DIAGON analysis showed that the closest homology was with human cytomegalovirus. Plasmids were constructed which were shown to express the HHV-6 MCP as either the entire open reading frame or as portions of it, and the recombinant-produced proteins were used to raise antisera. The antisera were shown by immunofluorescence to react with HHV-6-infected lymphoblastoid cells and in Western blots with a 135-kilodalton protein specific to HHV-6-infected cells. The recombinant protein expressed from the entire HHV-6 MCP gene was detected only weakly in Western blot assays with normal HHV-6-positive human sera as a probe.

The characterization of the EBV alkaline deoxyribonuclease cloned and expressed in E. coli.

Studies of nucleic acid homology suggest the BGLF5 open reading frame of Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase) sharing some homology with that of herpes simplex virus. We report here the expression of the BGLF5 open reading frame in E. coli and the expression of high levels of a novel alkaline DNase activity in induced cells. This alkaline DNase has been purified to apparent homogeneity as a single protein species. This is the first report of the expression of a herpesvirus coded DNase in a prokaryotic system and of the purification of the EBV DNase to demonstrable purity. It has the biochemical characteristics of a typical herpesvirus alkaline exonuclease showing a high pH optimum, an absolute requirement for Mg2+ for activity and sensitivity to high salt concentrations and polyamines. The enzyme activity was neutralized by sera from patients with nasopharyngeal carcinoma and was reactive with these sera in Western blot analysis. Thus the prokaryotic expression system described here provides an economical and efficient source of the EBV DNase for biochemical and seroepidemiological analysis.

Characterization of the Epstein-Barr virus-encoded thymidine kinase expressed in heterologous eucaryotic and procaryotic systems.

The establishment of mammalian and procaryotic systems which express the Epstein-Barr virus (EBV) thymidine kinase (TK) has been reported previously (E. Littler, J. Zeuthen, A. A. McBride, E. Trøst-Sørensen, K. L. Powell, J. E. Walsh-Arrand, and J. R. Arrand, EMBO J. 5:1959-1966, 1986). The EBV TK activity expressed in both of these systems was characterized by in vitro assays and found to resemble that of the herpes simplex virus TK both in its broad range of nucleoside and nucleotide utilization and also in its ability to accept antiviral nucleoside analogs as substrates. Further results are presented which suggest that these in vitro systems may prove suitable for studying the potential anti-EBV activity of other candidate antiviral compounds.

Immunological conservation between Epstein-Barr virus and herpes simplex virus.

We have analysed Epstein-Barr virus (EBV)- and herpes simplex virus (HSV)-infected cells for evidence of antigenic conservation of virus-coded proteins. Immunofluorescence and Western blot analyses of EBV-transformed cell lines demonstrated the presence of proteins that are antigenically related to the HSV alkaline DNase, infected cell-specific protein 34/35, glycoprotein B, thymidine kinase and the major DNA-binding protein. These proteins were characterized on the basis of Mr and possible kinetic class.