Sequence analysis of part of a large plasmid carried by Australian environmental isolate of Listeria monocytogenes strain DRDC8 has lead to identification of an islet of genes that encode proteins similar to copper binding and transport genes found in other Gram positive bacteria. Comparative sequence analysis showed that there are at least four genes (pCT0017, pCT0018, pCT0019 and ctpA) on this islet predicted to be involved in copper homeostasis. One of these, ctpA, is predicted to encode a P-type ATPase with a function analogous to CopA, a copper transporting gene in Enterococcus hirae. ORF pCT0017 is likely to be a CopY-like regulatory protein which could control the expression of ctpA. ORF pCT0019 is predicted to be a Cu²⁺ binding protein. In addition, two genes located downstream of the ctpA are predicted to encode a two component regulatory system region. The predicted function of ORF pCT0018 is not clear. A related chromosomal gene (cutR) is predicted to also encode a copper transporting P-type ATPase. To investigate the role of the protein encoded by pCT0018, the growth behavior of L. monocytogenes strain DRDC8, other strains carrying mutations within pCT0018, pCT0019, cutR and ctpA, as well as strains cured of the large plasmid, were grown under conditions of copper stress and starvation. The growth data showed that with the exception of strain DRDC8 and other strains carrying ctpA, most were unable to grow at higher copper concentration (>15 mM CuSO₄) and suggested that the copper homeostasis genes located on the large plasmid are associated with tolerance to high levels of copper. Strain DSE955PL, which carries a cutR mutation and is cured of the large plasmid, was the most sensitive (<5 mM CuSO₄). This indicated that proteins encoded by plasmid genes work synergistically to confer tolerance to copper. Of most interest was the fact that a pCT0018 mutant was more sensitive (<15 mM CuSO₄) to high levels of copper than the wild type parent DRDC8 (<20 mM CuSO₄). This suggested that ORF pCT0018 was necessary for copper tolerance. To investigate the effects of insertion mutations in pCT0017, pCT0018 and ctpA on copper uptake and export, the levels of copper accumulated by these strains was assessed using atomic absorption spectroscopy. A significant difference in copper accumulation among the bacteria strains was observed when either LEB or BHI media were used to culture the bacteria. This data suggested that the growth medium chemicals influence the levels of copper accumulated by cells. However, the effect of these media on bacteria growth rates during copper stress was not significant. Atomic absorption analysis of intracellular copper accumulation suggested that DSE955PL and DSE955 (a chromosome mutant) were able to accumulate copper (80 - 110 mg.gˉ¹ dry weight of cells), whereas DRDC8 and strains carrying mutations in pCT0018, ctpA, and strains cured of the large plasmid, were less able to accumulate copper (30 - 70 mg.gˉ¹ dry weight of cells). This data suggested that cutR may encode a copper export system and that ctpA is involved in copper uptake. To investigate the gene expression profile for pCT0018 under elevated copper, reverse transcriptase PCR was used to detect transcripts encoding pCT0017, pCT0018, pCT0019 and pCT0020 from RNA extracted from L. monocytogenes strain DRDC8 following culture at elevated levels of copper. Although transcripts for each of the target genes were detected, transcription was not responsive to copper, nor was the pattern of transcription consistent with that expected for a single operon. To directly determine whether the protein encoded by the pCT0018 open reading frame was able to bind copper, this gene was cloned in pET15b in frame with an N-terminal Histag and expressed in E. coli. The expressed protein was purified with a Ni-NTA column and shown to contain copper. Attempts to directly show that protein pCT0018 could bind copper by Cu-IMAC were unable to unequivocally show that the protein was immobilized on the column. Purified protein was used to raise a polyclonal antiserum in rabbit and the antiserum was used for Western analysis to test expression of pCT0018 by wild type L. monocytogenes DRDC8 and specific gene mutants. Although the antiserum bound to purified protein, it was not possible to demonstrate binding to native pCT0018 in cell lysates prepared from L. monocytogenes DRDC8. SDS-PAGE of cytoplasmic and cell envelope proteins isolated from L. monocytogenes strains was used to identify proteins expressed in response to copper stress and starvation. No significant differences in protein profiles for cytoplasmic protein were observed. However, copper-immobilized metal affinity chromatography (Cu-IMAC) showed that expression of a number of copper binding proteins were differentially expressed by DRDC8 following growth in copper stress and starvation conditions. Three of these proteins were selected for amino sequence analysis by MALDI-TOFF MS. Two were confirmed to be L. monocytogenes non-heme iron-binding ferritin and a thiol peroxidase, both of which bind copper. The other protein was similar to an unknown protein from L. monocytogenes. Interestingly, no proteins directly implicated with the copper homeostasis islet were identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374407 / Thesis (M.Sc.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

Sequence analysis of part of a large plasmid carried by Australian environmental isolate of Listeria monocytogenes strain DRDC8 has lead to identification of an islet of genes that encode proteins similar to copper binding and transport genes found in other Gram positive bacteria. Comparative sequence analysis showed that there are at least four genes (pCT0017, pCT0018, pCT0019 and ctpA) on this islet predicted to be involved in copper homeostasis. One of these, ctpA, is predicted to encode a P-type ATPase with a function analogous to CopA, a copper transporting gene in Enterococcus hirae. ORF pCT0017 is likely to be a CopY-like regulatory protein which could control the expression of ctpA. ORF pCT0019 is predicted to be a Cu²⁺ binding protein. In addition, two genes located downstream of the ctpA are predicted to encode a two component regulatory system region. The predicted function of ORF pCT0018 is not clear. A related chromosomal gene (cutR) is predicted to also encode a copper transporting P-type ATPase. To investigate the role of the protein encoded by pCT0018, the growth behavior of L. monocytogenes strain DRDC8, other strains carrying mutations within pCT0018, pCT0019, cutR and ctpA, as well as strains cured of the large plasmid, were grown under conditions of copper stress and starvation. The growth data showed that with the exception of strain DRDC8 and other strains carrying ctpA, most were unable to grow at higher copper concentration (>15 mM CuSO₄) and suggested that the copper homeostasis genes located on the large plasmid are associated with tolerance to high levels of copper. Strain DSE955PL, which carries a cutR mutation and is cured of the large plasmid, was the most sensitive (<5 mM CuSO₄). This indicated that proteins encoded by plasmid genes work synergistically to confer tolerance to copper. Of most interest was the fact that a pCT0018 mutant was more sensitive (<15 mM CuSO₄) to high levels of copper than the wild type parent DRDC8 (<20 mM CuSO₄). This suggested that ORF pCT0018 was necessary for copper tolerance. To investigate the effects of insertion mutations in pCT0017, pCT0018 and ctpA on copper uptake and export, the levels of copper accumulated by these strains was assessed using atomic absorption spectroscopy. A significant difference in copper accumulation among the bacteria strains was observed when either LEB or BHI media were used to culture the bacteria. This data suggested that the growth medium chemicals influence the levels of copper accumulated by cells. However, the effect of these media on bacteria growth rates during copper stress was not significant. Atomic absorption analysis of intracellular copper accumulation suggested that DSE955PL and DSE955 (a chromosome mutant) were able to accumulate copper (80 - 110 mg.gˉ¹ dry weight of cells), whereas DRDC8 and strains carrying mutations in pCT0018, ctpA, and strains cured of the large plasmid, were less able to accumulate copper (30 - 70 mg.gˉ¹ dry weight of cells). This data suggested that cutR may encode a copper export system and that ctpA is involved in copper uptake. To investigate the gene expression profile for pCT0018 under elevated copper, reverse transcriptase PCR was used to detect transcripts encoding pCT0017, pCT0018, pCT0019 and pCT0020 from RNA extracted from L. monocytogenes strain DRDC8 following culture at elevated levels of copper. Although transcripts for each of the target genes were detected, transcription was not responsive to copper, nor was the pattern of transcription consistent with that expected for a single operon. To directly determine whether the protein encoded by the pCT0018 open reading frame was able to bind copper, this gene was cloned in pET15b in frame with an N-terminal Histag and expressed in E. coli. The expressed protein was purified with a Ni-NTA column and shown to contain copper. Attempts to directly show that protein pCT0018 could bind copper by Cu-IMAC were unable to unequivocally show that the protein was immobilized on the column. Purified protein was used to raise a polyclonal antiserum in rabbit and the antiserum was used for Western analysis to test expression of pCT0018 by wild type L. monocytogenes DRDC8 and specific gene mutants. Although the antiserum bound to purified protein, it was not possible to demonstrate binding to native pCT0018 in cell lysates prepared from L. monocytogenes DRDC8. SDS-PAGE of cytoplasmic and cell envelope proteins isolated from L. monocytogenes strains was used to identify proteins expressed in response to copper stress and starvation. No significant differences in protein profiles for cytoplasmic protein were observed. However, copper-immobilized metal affinity chromatography (Cu-IMAC) showed that expression of a number of copper binding proteins were differentially expressed by DRDC8 following growth in copper stress and starvation conditions. Three of these proteins were selected for amino sequence analysis by MALDI-TOFF MS. Two were confirmed to be L. monocytogenes non-heme iron-binding ferritin and a thiol peroxidase, both of which bind copper. The other protein was similar to an unknown protein from L. monocytogenes. Interestingly, no proteins directly implicated with the copper homeostasis islet were identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374407 / Thesis (M.Sc.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

Objetivo: Verificar graus de concordância diagnóstica pela Angio-TC entre médicos da emergência, residentes de radiologia e radiologistas torácicos em casos suspeitos de tromboembolismo pulmonar (TEP). Material e Métodos: Foram retrospectivamente estudados 123 pacientes consecutivos com suspeita de TEP, de maço/2012 a fevereiro/2013, os quais realizaram Angio-TC pulmonar, obtida com colimação por multidetector 64x0,5. As imagens foram inicialmente interpretadas isoladamente por dois médicos da Emergência e por dois médicos residentes da Radiologia, e subsequentemente por dois Radiologistas Torácicos em consenso, verificando-se o grau de concordância interobservador entre eles quanto à presença de TEP. Resultados: O grau de concordância entre os Residentes da radiologia e os Radiologistas torácicos foi muito bom (Índice Kappa de 0,82 e 0,81); entre os Médicos da Emergência e os Radiologistas torácicos foi baixo ou moderado (Kappa de 0,37 e 0,42.), com um índice de 40,0% de relatos falso-positivos, aproximadamente. Aproximadamente 40,0% das interpretações falso-positivas correlacionaram-se principalmente com casos de TEP lobar e segmentar, enquanto que as falso-negativas ocorreram mais vezes com casos de TEP segmentar e subsegmentar. O grau de concordância geral entre todos os observadores mostrou-sei também moderado (Kappa de 0,50). Conclusão: Usando a Angio-TC no diagnóstico do TEP, o grau de concordância entre os radiologistas torácicos e os residentes da radiologia foi muito bom; entretanto, a concordância entre os médicos da emergência com os radiologistas torácicos foi baixa, com tendência a superestimar o diagnóstico da condição. / Purpose: To assess interobserver agreement rates between the Radiology resident, the thoracic radiologist and the Emergency physician for diagnosing pulmonary embolism (PE) in Computed Tomography Pulmonary Angiography (CTPA) examinations. Methods: Two Emergency physicians and two Radiology residents retrospectively evaluated 123 CTPA scans at our institution, and reported the presence of PE or not, individually. Two thoracic radiologists then reviewed the images, and a consensus was reached. Statistical analysis was performed, in order to provide the interobserver agreement. Results were expressed in kappa values for subsequent comparison. Results: Very good agreement in CTPA reading was observed between Radiology residents and thoracic radiologists (kappa index of 0.82 and 0.81). Fair and moderate agreement (kappa index of 0.39 and 0.42) was demonstrated between Emergency physicians and thoracic radiologists, with a rate of 40% false-positive reports, approximately, mainly in cases of both lobar and segmental involvement, whereas false-negative occurred more times in cases of segmental and subsegmental PE. The overall agreement was also moderate (kappa index of 0.50). Conclusion: The inter-observer agreement in CTPA examinations between radiology residents and thoracic radiologists in PE diagnoses was good, but it was low between the emergency physicians and the radiologists, with a tendency of overestimating that diagnoses.

Objetivo: Verificar graus de concordância diagnóstica pela Angio-TC entre médicos da emergência, residentes de radiologia e radiologistas torácicos em casos suspeitos de tromboembolismo pulmonar (TEP). Material e Métodos: Foram retrospectivamente estudados 123 pacientes consecutivos com suspeita de TEP, de maço/2012 a fevereiro/2013, os quais realizaram Angio-TC pulmonar, obtida com colimação por multidetector 64x0,5. As imagens foram inicialmente interpretadas isoladamente por dois médicos da Emergência e por dois médicos residentes da Radiologia, e subsequentemente por dois Radiologistas Torácicos em consenso, verificando-se o grau de concordância interobservador entre eles quanto à presença de TEP. Resultados: O grau de concordância entre os Residentes da radiologia e os Radiologistas torácicos foi muito bom (Índice Kappa de 0,82 e 0,81); entre os Médicos da Emergência e os Radiologistas torácicos foi baixo ou moderado (Kappa de 0,37 e 0,42.), com um índice de 40,0% de relatos falso-positivos, aproximadamente. Aproximadamente 40,0% das interpretações falso-positivas correlacionaram-se principalmente com casos de TEP lobar e segmentar, enquanto que as falso-negativas ocorreram mais vezes com casos de TEP segmentar e subsegmentar. O grau de concordância geral entre todos os observadores mostrou-sei também moderado (Kappa de 0,50). Conclusão: Usando a Angio-TC no diagnóstico do TEP, o grau de concordância entre os radiologistas torácicos e os residentes da radiologia foi muito bom; entretanto, a concordância entre os médicos da emergência com os radiologistas torácicos foi baixa, com tendência a superestimar o diagnóstico da condição. / Purpose: To assess interobserver agreement rates between the Radiology resident, the thoracic radiologist and the Emergency physician for diagnosing pulmonary embolism (PE) in Computed Tomography Pulmonary Angiography (CTPA) examinations. Methods: Two Emergency physicians and two Radiology residents retrospectively evaluated 123 CTPA scans at our institution, and reported the presence of PE or not, individually. Two thoracic radiologists then reviewed the images, and a consensus was reached. Statistical analysis was performed, in order to provide the interobserver agreement. Results were expressed in kappa values for subsequent comparison. Results: Very good agreement in CTPA reading was observed between Radiology residents and thoracic radiologists (kappa index of 0.82 and 0.81). Fair and moderate agreement (kappa index of 0.39 and 0.42) was demonstrated between Emergency physicians and thoracic radiologists, with a rate of 40% false-positive reports, approximately, mainly in cases of both lobar and segmental involvement, whereas false-negative occurred more times in cases of segmental and subsegmental PE. The overall agreement was also moderate (kappa index of 0.50). Conclusion: The inter-observer agreement in CTPA examinations between radiology residents and thoracic radiologists in PE diagnoses was good, but it was low between the emergency physicians and the radiologists, with a tendency of overestimating that diagnoses.

Objetivo: Verificar graus de concordância diagnóstica pela Angio-TC entre médicos da emergência, residentes de radiologia e radiologistas torácicos em casos suspeitos de tromboembolismo pulmonar (TEP). Material e Métodos: Foram retrospectivamente estudados 123 pacientes consecutivos com suspeita de TEP, de maço/2012 a fevereiro/2013, os quais realizaram Angio-TC pulmonar, obtida com colimação por multidetector 64x0,5. As imagens foram inicialmente interpretadas isoladamente por dois médicos da Emergência e por dois médicos residentes da Radiologia, e subsequentemente por dois Radiologistas Torácicos em consenso, verificando-se o grau de concordância interobservador entre eles quanto à presença de TEP. Resultados: O grau de concordância entre os Residentes da radiologia e os Radiologistas torácicos foi muito bom (Índice Kappa de 0,82 e 0,81); entre os Médicos da Emergência e os Radiologistas torácicos foi baixo ou moderado (Kappa de 0,37 e 0,42.), com um índice de 40,0% de relatos falso-positivos, aproximadamente. Aproximadamente 40,0% das interpretações falso-positivas correlacionaram-se principalmente com casos de TEP lobar e segmentar, enquanto que as falso-negativas ocorreram mais vezes com casos de TEP segmentar e subsegmentar. O grau de concordância geral entre todos os observadores mostrou-sei também moderado (Kappa de 0,50). Conclusão: Usando a Angio-TC no diagnóstico do TEP, o grau de concordância entre os radiologistas torácicos e os residentes da radiologia foi muito bom; entretanto, a concordância entre os médicos da emergência com os radiologistas torácicos foi baixa, com tendência a superestimar o diagnóstico da condição. / Purpose: To assess interobserver agreement rates between the Radiology resident, the thoracic radiologist and the Emergency physician for diagnosing pulmonary embolism (PE) in Computed Tomography Pulmonary Angiography (CTPA) examinations. Methods: Two Emergency physicians and two Radiology residents retrospectively evaluated 123 CTPA scans at our institution, and reported the presence of PE or not, individually. Two thoracic radiologists then reviewed the images, and a consensus was reached. Statistical analysis was performed, in order to provide the interobserver agreement. Results were expressed in kappa values for subsequent comparison. Results: Very good agreement in CTPA reading was observed between Radiology residents and thoracic radiologists (kappa index of 0.82 and 0.81). Fair and moderate agreement (kappa index of 0.39 and 0.42) was demonstrated between Emergency physicians and thoracic radiologists, with a rate of 40% false-positive reports, approximately, mainly in cases of both lobar and segmental involvement, whereas false-negative occurred more times in cases of segmental and subsegmental PE. The overall agreement was also moderate (kappa index of 0.50). Conclusion: The inter-observer agreement in CTPA examinations between radiology residents and thoracic radiologists in PE diagnoses was good, but it was low between the emergency physicians and the radiologists, with a tendency of overestimating that diagnoses.

BACKGROUND: South Africa bares a significant burden of HIV and imaging is commonly performed as part of the workup for respiratory distress. OBJECTIVES: The aim of this study was to document the prevalence of pulmonary thrombo-embolic disease (PTED) and other findings in HIV-infected patients referred for CT pulmonary angiography (CTPA) for suspected PTED. METHOD: Forty CTPA studies of documented HIV-infected individuals investigated for suspected PTED during a 1-year period were retrieved, anonymised and interpreted by three consultant radiologists. Inter-reader reliability was calculated using Free Marginal multi-rater Kappa. RESULTS: Fourteen of the forty cases (35%) were positive for PTED. In the pulmonary embolism (PE)-positive group, 57.14% had peripheral disease and 42.86% had both peripheral and central disease. Associated findings in the PE-positive cases were pulmonary infarcts (17.5%), mosaic attenuation (17.5%) and linear atelectasis (7.5%). The most common incidental findings were solid pulmonary nodules (52.5%), non-wedge-shaped consolidation (45%), cardiomegaly (52.5%) and enlarged intra-thoracic lymph nodes (52.5%). Thirty per cent of the study population had findings related directly to the presence of PTED, whilst most cases in the study (77.5%) had pulmonary findings unrelated to PTED. In the PE-negative cases, 55% reported emergent findings that warranted immediate or urgent medical attention. CONCLUSION: Computed tomography pulmonary angiography imaging is critical for diagnosing PE. However, further investigation into the judicious application of CTPA in HIV-infected patients with suspected PTED is required, as CTPA findings in most of the cases in this study were unrelated to PE.

Inledning: Lungemboli (LE) är den tredje vanligaste dödsorsaken bland hjärt- och kärlsjukdomar. Den dominerande strategin för kliniskt diagnostikstöd vid misstanke om LE är Wells kriterier och det senaste årtiondet har datortomografisk pulmonell angiografi (CTPA) varit den bilddiagnostiska guldstandarden. Dubbel energi (DE) CTPA kan göra det möjligt att urskilja patologi och använda datauppsättningar för att konstruera jodkartor som kan nyttjas i diagnostiken. Syfte: Granska bildkvalitet, stråldos och diagnostisk noggrannhet vid DE CTPA jämfört med enkel energi (SE) CTPA vid diagnostisering av LE. Metod: Studien genomfördes som en allmän litteraturöversikt och behandlar tolv vetenskapliga artiklar av kvantitativ retrospektiv- och prospektiv metod. Resultat: DE ger en hög bildkvalitet som är likvärdig eller bättre än SE. Det finns ingen signifikant skillnad mellan DE och SE gällande den diagnostiska noggrannheten men i kombination med jodkartor och monokromatiska bilder från DE kan det detekteras LE som inte syns med SE. DE kan ge både högre och lägre stråldos jämfört med SE. Slutsats: CTPA med DE ger en hög bildkvalitet som är jämförbar eller bättre än SE. Den diagnostiska noggrannheten ökar med jodkartor och monokromatiska bilder. Beroende på metod och modell av DT varierar stråldosen mellan DE och SE.

Background: After the first COVID-19 infection wave, a constant increase of pulmonary embolism (PE) hospitalizations not linked with active PCR-confirmed COVID-19 was observed, but potential contributors to this observation are unclear. Therefore, we analyzed associations between changes in PE hospitalizations and (1) the incidence of non-COVID-19 pneumonia, (2) the use of computed tomography pulmonary angiography (CTPA), (3) volume depletion, and (4) preceding COVID-19 infection numbers in Germany. Methods: Claims data of Helios hospitals in Germany were used, and consecutive cases with a hospital admission between May 6 and December 15, 2020 (PE surplus period), were analyzed and compared to corresponding periods covering the same weeks in 2016–2019 (control period). We analyzed the number of PE cases in the target period with multivariable Poisson general linear mixed models (GLMM) including (a) cohorts of 2020 versus 2016–2019, (b) the number of cases with pneumonia, (c) CTPA, and (d) volume depletion and adjusted for age and sex. In order to associate the daily number of PE cases in 2020 with the number of preceding SARS-CoV-2 infections in Germany, we calculated the average number of daily infections (divided by 10,000) occurring between 14 up to 90 days with increasing window sizes before PE cases and modeled the data with Poisson regression. Results: There were 2,404 PE hospitalizations between May 6 and December 15, 2020, as opposed to 2,112–2,236 (total 8,717) in the corresponding 2016–2019 control periods (crude rate ratio [CRR] 1.10, 95% CI 1.05–1.15, P < 0.01). With the use of multivariable Poisson GLMM adjusted for age, sex, and volume depletion, PE cases were significantly associated with the number of cases with pneumonia (CRR 1.09, 95% CI 1.07–1.10, P < 0.01) and with CTPA (CRR 1.10, 95% CI 1.09–1.10, P < 0.01). The increase of PE cases in 2020 compared with the control period remained significant (CRR 1.07, 95% CI 1.02–1.12, P < 0.01) when controlling for those factors. In the 2020 cohort, the number of preceding average daily COVID-19 infections was associated with increased PE case incidence in all investigated windows, i.e., including preceding infections from 14 to 90 days. The best model (log likelihood −576) was with a window size of 4 days, i.e., average COVID-19 infections 14–17 days before PE hospitalization had a risk of 1.20 (95% CI 1.12–1.29, P < 0.01). Conclusions: There is an increase in PE cases since early May 2020 compared to corresponding periods in 2016–2019. This surplus was significant even when controlling for changes in potential modulators such as demographics, volume depletion, non-COVID-19 pneumonia, CTPA use, and preceding COVID-19 infections. Future studies are needed (1) to investigate a potential causal link for increased risk of delayed PE with preceding SARS-CoV-2 infection and (2) to define optimal screening for SARS-CoV-2 in patients presenting with pneumonia and PE.