Tenofovir disoproxil fumarate (TDF) is a nucleotide reverse transcriptase inhibitor and a prodrug of tenofovir (TFV). It is the currently recommended first line combination treatment of human immunodeficiency virus (HIV) infection in adults. Various clinical studies have associated treatment with a TDF-containing antiretroviral therapy (ART) regimen with reduced bone mineral density (BMD) and renal dysfunction. Hardly any studies to date have correlated plasma TFV concentration with markers of renal function and bone turnover (BTM). This knowledge is also unavailable in the South African public health care system. Hence, the correlations between plasma TFV concentration and renal function markers and BTM in HIV-infected women were investigated. Renal function markers and BTM in HIV-infected women were compared with those in HIV-uninfected control women. A pilot cross-sectional sub-study within the Prospective Urban and Rural Epidemiology (PURE) South Africa study was conducted. Sixty women participated, of which 30 HIV-infected women were matched for age and body mass index with 30 HIV-uninfected ones. Ethics approval was obtained from the North-West University, Human Research Ethics committee (NWU-00016-10-A1) on 12 April 2013 to conduct this sub-study and the North West Department of Health, Mmabatho on 08 August 2013 to access patient health information. A validated high-performance liquid chromatography tandem mass spectrometry method was developed to analyse TFV in plasma. Renal markers measured were the estimated glomerular filtration rate (eGFR), creatinine clearance (CrCl), albuminuria, serum creatinine (SCr), serum urea, serum uric acid, glucosuria, urine sodium (UNa) and maximum tubular reabsorption of phosphate (TmPO4/GFR). The BTM markers measured included C-terminal telopeptide (CTx), alkaline phosphatase (ALP), parathyroid hormone (PTH), total vitamin D (VitD), serum calcium (SrCa), serum phosphate (SrP) and BMD. BMD was assessed using the DTX-200 peripheral DXA system (Osteometer MediTech, Hawthorn, California, USA). Renal and bone markers were analysed on Elecsys® 2010 and COBAS INTERGRA® 400 plus (Roche Diagnostics, Switzerland). Baseline data for HIV-infected participants with regard to CD4+ cell count, SCr prior to TDF initiation, time since TDF initiation, weight prior to TDF initiation and time since HIV diagnosis were collected retrospectively from participants’ public health care files. Statistical analyses applied were linear regression, analysis of covariance, the Mann-Whitney U test, paired t-test and unpaired t-test. IBM® SPSS® Statistics software 22 was used to perform all the statistical analyses. The median and interquartile range of plasma TFV concentration was 113 (74-139.4) ng/mL (n=25) and no TFV was detected in five participants’ plasma. Adjusted analyses showed TFV concentration to be associated with albuminuria (adjusted r2 = 0.339; p = 0.001). Values of CrCl, eGFR and albuminuria (p = 0.032; p = 0.038; p = 0.048, respectively) were significantly higher in HIV-infected women compared to HIV-uninfected women. CrCl [112 (84-137) mL/min] and eGFR [134 (93-153) mL/min/1.73m2] values were abnormally high in HIV-infected women. There was also an increase in both CrCl and eGFR (p = 0.008; p < 0.001, respectively) from baseline to median follow-up of 16.6 (8.8-23.4) months in HIV-infected women. At a TFV plasma concentration of ≥ 120 ng/mL, CTx and ALP correlated positively (r = 0.704; p = 0.016). ALP (112 ± 28 U/L; p < 0.001), CTx (0.68 ± 0.4 ng/mL; p = 0.027) and PTH (56.3 ± 32 pg/mL; p = 0.050) were higher in HIV-infected women compared to HIV-uninfected women. CD4+ cell count increased from baseline to follow-up in HIV-infected women (+250 cells/mm3; p = 0.001). In HIV-infected women on a TDF-based regimen, TFV plasma concentration is associated with an increase in albuminuria, while perturbations in BTM equilibrium occur at ≥ 120 ng/mL of TFV plasma concentration. Abnormally higher CrCl and eGFR are present in HIV-infected women, seen as glomerular hyperfiltration compared with HIV-uninfected women. There was immunological improvement with TDF-based ART in HIV-infected women. Longitudinal studies with larger sample sizes are needed to confirm these findings. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2015

Tenofovir disoproxil fumarate (TDF) is a nucleotide reverse transcriptase inhibitor and a prodrug of tenofovir (TFV). It is the currently recommended first line combination treatment of human immunodeficiency virus (HIV) infection in adults. Various clinical studies have associated treatment with a TDF-containing antiretroviral therapy (ART) regimen with reduced bone mineral density (BMD) and renal dysfunction. Hardly any studies to date have correlated plasma TFV concentration with markers of renal function and bone turnover (BTM). This knowledge is also unavailable in the South African public health care system. Hence, the correlations between plasma TFV concentration and renal function markers and BTM in HIV-infected women were investigated. Renal function markers and BTM in HIV-infected women were compared with those in HIV-uninfected control women. A pilot cross-sectional sub-study within the Prospective Urban and Rural Epidemiology (PURE) South Africa study was conducted. Sixty women participated, of which 30 HIV-infected women were matched for age and body mass index with 30 HIV-uninfected ones. Ethics approval was obtained from the North-West University, Human Research Ethics committee (NWU-00016-10-A1) on 12 April 2013 to conduct this sub-study and the North West Department of Health, Mmabatho on 08 August 2013 to access patient health information. A validated high-performance liquid chromatography tandem mass spectrometry method was developed to analyse TFV in plasma. Renal markers measured were the estimated glomerular filtration rate (eGFR), creatinine clearance (CrCl), albuminuria, serum creatinine (SCr), serum urea, serum uric acid, glucosuria, urine sodium (UNa) and maximum tubular reabsorption of phosphate (TmPO4/GFR). The BTM markers measured included C-terminal telopeptide (CTx), alkaline phosphatase (ALP), parathyroid hormone (PTH), total vitamin D (VitD), serum calcium (SrCa), serum phosphate (SrP) and BMD. BMD was assessed using the DTX-200 peripheral DXA system (Osteometer MediTech, Hawthorn, California, USA). Renal and bone markers were analysed on Elecsys® 2010 and COBAS INTERGRA® 400 plus (Roche Diagnostics, Switzerland). Baseline data for HIV-infected participants with regard to CD4+ cell count, SCr prior to TDF initiation, time since TDF initiation, weight prior to TDF initiation and time since HIV diagnosis were collected retrospectively from participants’ public health care files. Statistical analyses applied were linear regression, analysis of covariance, the Mann-Whitney U test, paired t-test and unpaired t-test. IBM® SPSS® Statistics software 22 was used to perform all the statistical analyses. The median and interquartile range of plasma TFV concentration was 113 (74-139.4) ng/mL (n=25) and no TFV was detected in five participants’ plasma. Adjusted analyses showed TFV concentration to be associated with albuminuria (adjusted r2 = 0.339; p = 0.001). Values of CrCl, eGFR and albuminuria (p = 0.032; p = 0.038; p = 0.048, respectively) were significantly higher in HIV-infected women compared to HIV-uninfected women. CrCl [112 (84-137) mL/min] and eGFR [134 (93-153) mL/min/1.73m2] values were abnormally high in HIV-infected women. There was also an increase in both CrCl and eGFR (p = 0.008; p < 0.001, respectively) from baseline to median follow-up of 16.6 (8.8-23.4) months in HIV-infected women. At a TFV plasma concentration of ≥ 120 ng/mL, CTx and ALP correlated positively (r = 0.704; p = 0.016). ALP (112 ± 28 U/L; p < 0.001), CTx (0.68 ± 0.4 ng/mL; p = 0.027) and PTH (56.3 ± 32 pg/mL; p = 0.050) were higher in HIV-infected women compared to HIV-uninfected women. CD4+ cell count increased from baseline to follow-up in HIV-infected women (+250 cells/mm3; p = 0.001). In HIV-infected women on a TDF-based regimen, TFV plasma concentration is associated with an increase in albuminuria, while perturbations in BTM equilibrium occur at ≥ 120 ng/mL of TFV plasma concentration. Abnormally higher CrCl and eGFR are present in HIV-infected women, seen as glomerular hyperfiltration compared with HIV-uninfected women. There was immunological improvement with TDF-based ART in HIV-infected women. Longitudinal studies with larger sample sizes are needed to confirm these findings. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2015

Background: First responders (firefighters, paramedics, and police officers) are often exposed to potentially psychologically traumatic events. When combined with insufficient social support and reduced help-seeking behaviours, such exposures may increase the risk of mental health challenges, particularly among paramedics who report the highest rates of mental disorders. Objective: The current study used the Theoretical Domains Framework (TDF) and the Consolidated Framework for Implementation Research (CFIR) to identify critical barriers and facilitators to help-seeking and accessing mental health care, and the feasibility and sustainability of a first responder clinic. Methods: Semi-structured qualitative interviews included 11 paramedics (frontline, mid-and-senior management, and union), recruited using purposive and snowball sampling. Interviews were analyzed using content and thematic analyses. The TDF and CFIR guided study design, interview content, data collection, and analysis. Results: Barriers included the complexities of stigma, confidentiality, cultural competency, and trust. Conclusions: The findings will be instrumental in developing evidence-based approaches to mental health care for paramedics.

Eine Monotherapie mit Tenofovir disoproxil fumarate (TDF) stellt eine hoch effiziente Therapie-option für multipel vorbehandelte Patienten mit chronischer Hepatitis-B-Virusinfektion (HBV) dar. Eine Resistenz gegen TDF wurde bislang nicht beschrieben, jedoch wird ein möglicher negativer Einfluss von Adefovir dipivoxil (ADV)-Resistenzvarianten auf die TDF-Ansprechrate diskutiert. Diese retrospektive Kohortenstudie untersucht die Dynamik von Nukleos(t)id-Analoga (NA)-Resistenzvarianten im HBV-Polymerasegen mit Fokus auf ADV-Resistenzvarianten bei 18 chronisch HBV-infizierten Patienten mit Therapieversagen auf eine vorangegangene Lamivudin (LAM)- und ADV-Therapie, sowie nur partiellem Therapieansprechen auf eine TDF-Monotherapie. Zur Detektion von NA-Resistenzvarianten wird eine HBV-Genomsequenzierung mit Tiefenpyrosequenzierung (Genome Sequencer FLX, Roche Diagnostics, Germany) (UDPS), direkte Sequenzierung (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing Sys-tem, Siemens Healthcare Diagnostic, USA) (TG) und Line Probe Assay (INNO-LiPa DRv2 und v3, Innogenetics, Belgium) (INNO-LiPA) durchgeführt. Unter TDF kommt es zu einer quantitati-ven Shift zugunsten der ADV-Resistenzvarianten mit konstant bleibendem Anteil und deutlich höher persistierender Virämie zu Monat 12 im Vergleich zu Patienten ohne ADV-Resistenzvarianten. Vor allem werden die Varianten rtA181V und rtN236T selektiert, jedoch nicht die Variante rtA181T. Die absolute Anzahl der LAM-Resistenzvarianten hingegen halbiert sich. Varianten mit einem initial per UDPS detektierten Anteil von >20% der patientenspezifi-schen HBV-Population werden meist selektiert und nehmen im Verlauf den Hauptanteil der Quasispezies ein. UDPS stellte ein potentes Medium der Detektion, Identifikation und Quantifi-zierung von HBV-Varianten dar und ist INNO-LiPa und TG überlegen. Es ergibt sich kein Hin-weis auf TDF-Resistenzvarianten, jedoch zeigt das Vorliegen von ADV-Resistenzvarianten ei-nen tendentiell negativen Einfluss auf die virale Kinetik. Weitere größere Langzeitstudien sind zur Bestätigung dieser Beobachtung notwendig. / Tenofovir disoproxil fumarate (TDF) is a highly efficient treatment option for nucleos(t)ide analogue (NA) pre-treated patients with chronic hepatitis B virus (HBV) infection. Little is known about the reasons for persistent virus replication in some rare cases. As of today, no TDF resistance variants have been identified, but a possible linkage to Adefovir dipivoxil (ADV) resistance associated variants negatively influencing HBV-DNA suppression by TDF has been suspected, based on the similarity of the chemical structure. In this retrospective cohort study the dynamics of NA resistance variants in the HBV polymerase gene with focus on ADV resistance variants were assessed. For this, we have chosen a cohort including patients with multiple failures to treatment with different NAs. Thus, data of 18 patients with previous treatment failure to LAM and ADV was analysed, showing a persistent viremia (HBV-DNA >35 copies/mL) despite switch to TDF monotherapy (median HBV-DNA at month 12 3,5±0,8 (2,1-4,9) log10 copies/mL). Sequencing analysis was performed with ultra-deep pyrosequencing (UDPS) (Genome Sequencer FLX, 454 Life Science, Roche Diagnostic, Branford, CT), direct sequencing (TG) (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) and line probe assay (INNO-LiPA) (INNO-LiPa DRv2/v3, Innogenetics, Belgium). Using TDF monotherapy, a quantitative shift in favour to ADV resistance variants was observed in this cohort. The percentage of substitutions conferring resistance to ADV at baseline (BL) and at the time of the last sequencing endpoint (EP) of the HBV genome remained constant (BL 35%, 13/37, EP 36%, 9/25). The variants rtA181V and rtN236T were mostly selected, whereas rtA181T was not selected. The total amount of substitutions conferring resistance to Lamivudin (LAM) showed a strong decline, however remained the majority part of all NA resistance variants (BL 51% (19/37), EP 40% (10/25)). The percentage of ETV resistance variants increased slightly (BL 14% (5/37), EP 24% (6/25)). Known ADV, Lam and ETV resistance variants emerged in variable abundance (1,0-99,6%) of quasispecies during TDF therapy. A homogenization of HBV quasispecies took place. Especially mutations occurring in higher abundance (>20% of viral population) were mostly selected (BL 51% (19/37), EP 80% (20/25)). No new HBV variants with possible association to resistance against TDF were identified, but patients with ADV resistance variants showed the highest HBV-DNA level at month 12 of TDF therapy (median HBV-DNA 3,57±0,72 (2,14-3,96) log10 copies/mL, not significant). A negative influence of ADV resistance variants on viral suppression with TDF monotherapy may be assumed, however more long-term studies are needed to confirm the role of ADV resistance variants in TDF therapy. UDPS is a potent medium for detection, identification and quantification of dominant to low level variants in HBV-DNA. It is superior to direct sequencing and line probe assay in the detection of variants.

Made available in DSpace on 2016-08-17T18:39:47Z (GMT). No. of bitstreams: 1 5345.pdf: 1839849 bytes, checksum: 117cb76c1a10deab4b3e8c9df4d797f3 (MD5) Previous issue date: 2012-03-30 / In Brazil there was an increase of post-surgical infections by rapidly growing mycobacteria, mainly Mycobacterium massiliense, leading the Agency for Sanitary Surveillance (ANVISA) to establish standards for the mandatory notification of cases occurring nationwide. In order to deal with a major public health problem, this study aimed to determine the efficiency of using antimicrobial photodynamic inactivation (PDIa) in Mycobacterium massiliense for future use in the treatment of agent infections. The objective was to test in vitro technique the PDI to control Mycobacterium massiliense to determine the best light dose and concentration of each photosensitizer (FS). To accomplish this search were used two FS: Photogem® and salt Curcumin (N-Methyl-D-Glucominato of Curcumin, 31.8%). As a light source for radiating a BioTable ® operated at 630nm (red light) for Photogem ® and 460nm (blue light) to the salt of curcumin. After the standardized inoculum was added to the sample analyzed in triplicate to a 24 well plate which contained 500&#956;l aliquots of the inoculum +500 &#956; l of the FS for the study groups tested, and the control group containing 1 ml of inoculum, and five minutes as time incubation (protected from light). After expiration of the irradiation time in accordance with the previously analyzed fluences of the tested groups and subsequent dilution of the sample to the value of 10-5, 100mL sample were plated in triplicate on 7H10 agar + OADC 10% and maintained at 37 for about 5 days with subsequent counting of colony forming units. Based on the results, they showed that FS Photogem ® until the concentration of 1000 &#956;g/Ml was not effective in reducing the number of CFU of M. massiliense. In contrast, the FS Salt Curcumin was effective at concentrations of 1300 &#956;g/Ml with a light dose 50 J/cm2 compared to controls. Thus, these data suggest that the salt FS Curcumin in photodynamic action is a viable alternative for reducing M massiliense when tested in vitro. / No Brasil houve um crescimento de infecções pós-cirúrgicas por micobactérias de crescimento rápido, principalmente por Mycobacterium massiliense, levando a Agência de Vigilância Sanitária (ANVISA) estabelecer normas de notificação obrigatória dos casos ocorridos em todo território nacional. Tendo em vista de se tratar de um grande problema de saúde pública, o presente trabalho visou determinar a eficiência da atividade antimicrobiana utilizando Inativação Fotodinâmica (IFD) em Mycobacterium massiliense para futura utilização no tratamento de infecções por esse agente. O objetivo foi testar in vitro, a técnica da IFD no controle de Mycobacterium massiliense, para determinar a melhor dose de luz e concentração de cada fotossensibilizador (FS). Para realizar esta pesquisa foram utilizados dois FS: Photogem® e Sal de Curcumina (N-Metil-D-Glucominato de Curcumina, a 31,8%). Como fonte de luz para irradiação, uma BioTable® operou em 630nm (luz vermelha) para o Photogem® e em 460nm (luz azul) para o Sal de Curcumina. Após padronizado o inóculo, a amostra analisada foi adicionada em triplicata a uma placa de 24 poços a qual continha alíquotas de 500&#956;l do inoculo +500&#956;l do FS para os grupos de estudos testados, e o grupo controle contendo 1ml do inoculo, tendo 5 minutos como tempo de incubação (protegido da luz). Após decorrido o tempo de irradiação de acordo com as fluências previamente analisadas dos grupos testados e com posterior diluição da amostra até o valor de 10-5, 100&#956;l da amostra foram semeadas em triplicada em placas contendo Agar 7H10 + OADC 10% e mantidas a 37o por aproximadamente 5 dias com posterior contagem das unidades formadoras de colônias. Com base nos resultados obtidos, estes mostraram que o FS Photogem® até a concentração de 1000&#956;g/ml não foi efetivo na redução do número de UFC de M. massiliense. Em contraponto, o FS Sal de Curcumina foi efetivo a partir da concentração de 1300 &#956;g/ml com dose de luz a 50 J/cm2 comparado ao grupo controle. Assim, estes dados sugerem que o FS Sal de Curcumina sob ação fotodinâmica é uma alternativa viável para a redução de M. massiliense quando testadas in vitro.

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Eine Monotherapie mit Tenofovir disoproxil fumarate (TDF) stellt eine hoch effiziente Therapie-option für multipel vorbehandelte Patienten mit chronischer Hepatitis-B-Virusinfektion (HBV) dar. Eine Resistenz gegen TDF wurde bislang nicht beschrieben, jedoch wird ein möglicher negativer Einfluss von Adefovir dipivoxil (ADV)-Resistenzvarianten auf die TDF-Ansprechrate diskutiert. Diese retrospektive Kohortenstudie untersucht die Dynamik von Nukleos(t)id-Analoga (NA)-Resistenzvarianten im HBV-Polymerasegen mit Fokus auf ADV-Resistenzvarianten bei 18 chronisch HBV-infizierten Patienten mit Therapieversagen auf eine vorangegangene Lamivudin (LAM)- und ADV-Therapie, sowie nur partiellem Therapieansprechen auf eine TDF-Monotherapie. Zur Detektion von NA-Resistenzvarianten wird eine HBV-Genomsequenzierung mit Tiefenpyrosequenzierung (Genome Sequencer FLX, Roche Diagnostics, Germany) (UDPS), direkte Sequenzierung (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing Sys-tem, Siemens Healthcare Diagnostic, USA) (TG) und Line Probe Assay (INNO-LiPa DRv2 und v3, Innogenetics, Belgium) (INNO-LiPA) durchgeführt. Unter TDF kommt es zu einer quantitati-ven Shift zugunsten der ADV-Resistenzvarianten mit konstant bleibendem Anteil und deutlich höher persistierender Virämie zu Monat 12 im Vergleich zu Patienten ohne ADV-Resistenzvarianten. Vor allem werden die Varianten rtA181V und rtN236T selektiert, jedoch nicht die Variante rtA181T. Die absolute Anzahl der LAM-Resistenzvarianten hingegen halbiert sich. Varianten mit einem initial per UDPS detektierten Anteil von >20% der patientenspezifi-schen HBV-Population werden meist selektiert und nehmen im Verlauf den Hauptanteil der Quasispezies ein. UDPS stellte ein potentes Medium der Detektion, Identifikation und Quantifi-zierung von HBV-Varianten dar und ist INNO-LiPa und TG überlegen. Es ergibt sich kein Hin-weis auf TDF-Resistenzvarianten, jedoch zeigt das Vorliegen von ADV-Resistenzvarianten ei-nen tendentiell negativen Einfluss auf die virale Kinetik. Weitere größere Langzeitstudien sind zur Bestätigung dieser Beobachtung notwendig.:INHALTSVERZEICHNIS 2 ABBILDUNGSVERZEICHNIS 5 TABELLENVERZEICHNIS 6 1 BIBLIOGRAPHISCHE ZUSAMMENFASSUNG 8 ABKÜRZUNGSVERZEICHNIS 9 2 EINFÜHRUNG 10 2.1 Epidemiologie der chronischen Hepatitis-B-Virusinfektion 10 2.2 Aufbau, Replikation und Resistenzentwicklung des Hepatitis-B-Virusgenoms 10 2.3 Antivirale Therapie 13 2.4 Sequenziermethoden 14 3 AUFGABENSTELLUNG 15 4 MATERIAL UND METHODE 16 4.1 Studiendesign und Beschreibung der Kohorte 16 4.2 Evaluation der TDF-Monotherapie und Resistenzanalyse 17 4.3 Statistische Auswertung 18 4.4 Verbrauchsmaterialien und Reagenzien 18 4.5 Puffer 22 4.6 Geräte 22 4.7 Durchführung der Laborarbeiten 24 4.8 HBV-DNA Quantifizierung und Bestimmung biochemischer Parameter 24 4.9 Extraktion von Nukleinsäuren aus Serumproben 24 4.10 Tiefenpyrosequenzierung mittels Genome Sequencer FLX System (454 Life Science, Roche Diagnostic, Branford, CT) 25 4.10.1 GS FLX HBV-DNA-Library 25 4.10.2 GS FLX Emulsions-PCR 31 4.10.3 GS FLX Sequenzierung 37 4.10.4 GS FLX Datenauswertung 41 4.11 Direkte Sequenzierung mittels TRUGENETM HBV Genotyping Kit (OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) 42 4.11.1 PCR-Amplifikation 42 4.11.2 CLIP-Amplifikations-Reaktion 42 4.11.3 Genotyp-und Variantenanalyse 44 4.12 Sequenzierung mittels Line Probe Assay INNO-LiPa HBV DRv2 und v3 (Innogenetics, Belgium) 44 4.12.1 HBV-DNA Amplifikation 45 4.12.2 Denaturierung und Hybridisierung 46 4.12.3 Farbentwicklung 46 5 ERGEBNISSE 47 5.1 Patientencharakteristika zur Baseline 47 5.2 Virologisches Ansprechen 48 5.3 Biochemisches Ansprechen 49 5.4 Serologisches Ansprechen 50 5.5 Therapie-Adhärenz und medikamentöse Verträglichkeit 50 5.6 Ergebnisse der Tiefenpyrosequenzierung mit Genome Sequencer FLX System (454 Life Science, Roche Diagnostic, Branford, CT) 50 5.6.1 Verschiedene Einzelverläufe der NA-Resistenzvarianten 55 5.6.2 Kombiniert oder isoliert auftretenden NA-Resistenzvarianten 57 5.6.3 Entwicklung der ADV-Resistenzvarianten 61 5.6.4 Entwicklung der LAM-Resistenzvarianten 66 5.6.5 Entwicklung der ETV-Resistenzvarianten 68 5.6.6 Shift der NA-Resistenzvarianten und Auswirkung auf die Dynamik der HBV-DNA 70 5.6.7 Entwicklung der potentiellen NA-Resistenzvarianten 72 5.6.8 Entwicklung der HBsAg-Varianten 75 5.6.9 Entwicklung der HBV-Quasispezies 77 5.7 Ergebnisse der direkten Sequenzierung mit TRUGENETM HBV Genotyping Kit (OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) 80 5.8 Ergebnisse des Line Probe Assays mit INNO-LiPa HBV DRv2 und v3 (Innogenetics, Belgium) 81 5.9 Vergleich der Sequenziermethoden 81 6 DISKUSSION 83 6.1 Patientenkohorte und Ansprechen auf die TDF-Monotherapie 83 6.2 Entwicklung und Einfluss der ADV-Resistenzvarianten unter TDF-Monotherapie 84 6.3 Entwicklung und Einfluss der LAM-Resistenzvarianten unter TDF-Monotherapie 90 6.4 Entwicklung und Einfluss der ETV-Resistenzvarianten unter TDF-Monotherapie 92 6.5 Entwicklung und Einfluss der HBV-Wildtyp-Varianten unter TDF-Monotherapie 93 6.6 Entwicklung und Einfluss der potentiellen NA-Resistenzvarianten 94 6.7 Entwicklung und Einfluss der HBsAg-Varianten 96 6.8 Einfluss von multiplen Vortherapien unter TDF-Monotherapie 98 6.9 Entwicklung und Einfluss der HBV-DNA-Serumkonzentration 98 6.10 Entwicklung und Einfluss von HBV-Quasispezies unter TDF-Monotherapie 100 6.11 Vergleich der Sequenziermethoden 101 7 ZUSAMMENFASSUNG DER ARBEIT 106 8 LITERATURVERZEICHNIS 109 9 EIDESSTATTLICHE VERSICHERUNG 114 10 CURRICULUM VITAE 115 11 ANTEILSERKLÄRUNG AN ERFOLGTEN PUBLIKATIONEN 116 12 DANKSAGUNG 117 / Tenofovir disoproxil fumarate (TDF) is a highly efficient treatment option for nucleos(t)ide analogue (NA) pre-treated patients with chronic hepatitis B virus (HBV) infection. Little is known about the reasons for persistent virus replication in some rare cases. As of today, no TDF resistance variants have been identified, but a possible linkage to Adefovir dipivoxil (ADV) resistance associated variants negatively influencing HBV-DNA suppression by TDF has been suspected, based on the similarity of the chemical structure. In this retrospective cohort study the dynamics of NA resistance variants in the HBV polymerase gene with focus on ADV resistance variants were assessed. For this, we have chosen a cohort including patients with multiple failures to treatment with different NAs. Thus, data of 18 patients with previous treatment failure to LAM and ADV was analysed, showing a persistent viremia (HBV-DNA >35 copies/mL) despite switch to TDF monotherapy (median HBV-DNA at month 12 3,5±0,8 (2,1-4,9) log10 copies/mL). Sequencing analysis was performed with ultra-deep pyrosequencing (UDPS) (Genome Sequencer FLX, 454 Life Science, Roche Diagnostic, Branford, CT), direct sequencing (TG) (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) and line probe assay (INNO-LiPA) (INNO-LiPa DRv2/v3, Innogenetics, Belgium). Using TDF monotherapy, a quantitative shift in favour to ADV resistance variants was observed in this cohort. The percentage of substitutions conferring resistance to ADV at baseline (BL) and at the time of the last sequencing endpoint (EP) of the HBV genome remained constant (BL 35%, 13/37, EP 36%, 9/25). The variants rtA181V and rtN236T were mostly selected, whereas rtA181T was not selected. The total amount of substitutions conferring resistance to Lamivudin (LAM) showed a strong decline, however remained the majority part of all NA resistance variants (BL 51% (19/37), EP 40% (10/25)). The percentage of ETV resistance variants increased slightly (BL 14% (5/37), EP 24% (6/25)). Known ADV, Lam and ETV resistance variants emerged in variable abundance (1,0-99,6%) of quasispecies during TDF therapy. A homogenization of HBV quasispecies took place. Especially mutations occurring in higher abundance (>20% of viral population) were mostly selected (BL 51% (19/37), EP 80% (20/25)). No new HBV variants with possible association to resistance against TDF were identified, but patients with ADV resistance variants showed the highest HBV-DNA level at month 12 of TDF therapy (median HBV-DNA 3,57±0,72 (2,14-3,96) log10 copies/mL, not significant). A negative influence of ADV resistance variants on viral suppression with TDF monotherapy may be assumed, however more long-term studies are needed to confirm the role of ADV resistance variants in TDF therapy. UDPS is a potent medium for detection, identification and quantification of dominant to low level variants in HBV-DNA. It is superior to direct sequencing and line probe assay in the detection of variants.:INHALTSVERZEICHNIS 2 ABBILDUNGSVERZEICHNIS 5 TABELLENVERZEICHNIS 6 1 BIBLIOGRAPHISCHE ZUSAMMENFASSUNG 8 ABKÜRZUNGSVERZEICHNIS 9 2 EINFÜHRUNG 10 2.1 Epidemiologie der chronischen Hepatitis-B-Virusinfektion 10 2.2 Aufbau, Replikation und Resistenzentwicklung des Hepatitis-B-Virusgenoms 10 2.3 Antivirale Therapie 13 2.4 Sequenziermethoden 14 3 AUFGABENSTELLUNG 15 4 MATERIAL UND METHODE 16 4.1 Studiendesign und Beschreibung der Kohorte 16 4.2 Evaluation der TDF-Monotherapie und Resistenzanalyse 17 4.3 Statistische Auswertung 18 4.4 Verbrauchsmaterialien und Reagenzien 18 4.5 Puffer 22 4.6 Geräte 22 4.7 Durchführung der Laborarbeiten 24 4.8 HBV-DNA Quantifizierung und Bestimmung biochemischer Parameter 24 4.9 Extraktion von Nukleinsäuren aus Serumproben 24 4.10 Tiefenpyrosequenzierung mittels Genome Sequencer FLX System (454 Life Science, Roche Diagnostic, Branford, CT) 25 4.10.1 GS FLX HBV-DNA-Library 25 4.10.2 GS FLX Emulsions-PCR 31 4.10.3 GS FLX Sequenzierung 37 4.10.4 GS FLX Datenauswertung 41 4.11 Direkte Sequenzierung mittels TRUGENETM HBV Genotyping Kit (OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) 42 4.11.1 PCR-Amplifikation 42 4.11.2 CLIP-Amplifikations-Reaktion 42 4.11.3 Genotyp-und Variantenanalyse 44 4.12 Sequenzierung mittels Line Probe Assay INNO-LiPa HBV DRv2 und v3 (Innogenetics, Belgium) 44 4.12.1 HBV-DNA Amplifikation 45 4.12.2 Denaturierung und Hybridisierung 46 4.12.3 Farbentwicklung 46 5 ERGEBNISSE 47 5.1 Patientencharakteristika zur Baseline 47 5.2 Virologisches Ansprechen 48 5.3 Biochemisches Ansprechen 49 5.4 Serologisches Ansprechen 50 5.5 Therapie-Adhärenz und medikamentöse Verträglichkeit 50 5.6 Ergebnisse der Tiefenpyrosequenzierung mit Genome Sequencer FLX System (454 Life Science, Roche Diagnostic, Branford, CT) 50 5.6.1 Verschiedene Einzelverläufe der NA-Resistenzvarianten 55 5.6.2 Kombiniert oder isoliert auftretenden NA-Resistenzvarianten 57 5.6.3 Entwicklung der ADV-Resistenzvarianten 61 5.6.4 Entwicklung der LAM-Resistenzvarianten 66 5.6.5 Entwicklung der ETV-Resistenzvarianten 68 5.6.6 Shift der NA-Resistenzvarianten und Auswirkung auf die Dynamik der HBV-DNA 70 5.6.7 Entwicklung der potentiellen NA-Resistenzvarianten 72 5.6.8 Entwicklung der HBsAg-Varianten 75 5.6.9 Entwicklung der HBV-Quasispezies 77 5.7 Ergebnisse der direkten Sequenzierung mit TRUGENETM HBV Genotyping Kit (OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) 80 5.8 Ergebnisse des Line Probe Assays mit INNO-LiPa HBV DRv2 und v3 (Innogenetics, Belgium) 81 5.9 Vergleich der Sequenziermethoden 81 6 DISKUSSION 83 6.1 Patientenkohorte und Ansprechen auf die TDF-Monotherapie 83 6.2 Entwicklung und Einfluss der ADV-Resistenzvarianten unter TDF-Monotherapie 84 6.3 Entwicklung und Einfluss der LAM-Resistenzvarianten unter TDF-Monotherapie 90 6.4 Entwicklung und Einfluss der ETV-Resistenzvarianten unter TDF-Monotherapie 92 6.5 Entwicklung und Einfluss der HBV-Wildtyp-Varianten unter TDF-Monotherapie 93 6.6 Entwicklung und Einfluss der potentiellen NA-Resistenzvarianten 94 6.7 Entwicklung und Einfluss der HBsAg-Varianten 96 6.8 Einfluss von multiplen Vortherapien unter TDF-Monotherapie 98 6.9 Entwicklung und Einfluss der HBV-DNA-Serumkonzentration 98 6.10 Entwicklung und Einfluss von HBV-Quasispezies unter TDF-Monotherapie 100 6.11 Vergleich der Sequenziermethoden 101 7 ZUSAMMENFASSUNG DER ARBEIT 106 8 LITERATURVERZEICHNIS 109 9 EIDESSTATTLICHE VERSICHERUNG 114 10 CURRICULUM VITAE 115 11 ANTEILSERKLÄRUNG AN ERFOLGTEN PUBLIKATIONEN 116 12 DANKSAGUNG 117

Bakgrund: Nationellt eftersträvas en digital omställning där digital och fysisk vård integreras inom hälso- och sjukvården. Incitament för denna omställning är att ge invånare ökad tillgänglighet till vård men också att nå en ökad delaktighet för invånare. Den digitala omställningen av vården och därmed integrering av digitala vårdkontakter är inom regionalt driven psykiatri ännu i sin linda. Detta trots växande evidens, framsteg i utveckling och tillgång till digitala verktyg, en pandemi som forcerat barriärer och ökad samhällelig acceptans för vård på distans. En förutsättning för att digitala vårdtjänster integreras som en legitim del av vård och behandling vid psykisk ohälsa är att vårdpersonal integrerar digitala arbetssätt och vårdkontakter i yrkesvardagen.  Idag är lite känt om hur vårdpersonal inom specialiserad öppenvårdspsykiatri i Sverige uppfattar en sådan omställning.   Syfte: Studiens huvudsyfte är att undersöka möjligheter och hinder som vårdpersonal inom regionalt driven öppenvårdspsykiatri upplever vid omställning där patienter erbjuds kombination av fysisk och digital vård.  Metod: Ett webbaserat frågeformulär med öppna och slutna frågor utformades. Insamlade data analyserades med kvalitativ innehållsanalys utifrån en deduktiv ansats utifrån Theoretical Domains Framework (TDF). Deltagare i studien var vårdpersonal verksamma i regionalt driven psykiatrisk öppenvård för vuxna i en region i Södra Sverige. Resultat: Baserat på TDF ramverket identifierades områden som påverkar vårdpersonalen genom att utgöra möjligheter eller hinder för att använda digitala vårdformer som arbetssätt.  Resultatets höga överensstämmelse med tidigare forskning antyder att identifierade TDF- domäner kan användas för att stärka acceptansen och implementeringen av digitala vårdformer inom ramen för psykiatrisk öppenvård. / Background:  In Sweden, a digital transition is sought where digital and physical care, Digi-physical healthcare, are integrated into public health care. A digital transition is one approach to meet the increasing need for health services that significantly challenges the health system but also aims to achieve increased patient participation. The digital transformation of care and the integration of e-mental health is still in its infancy in public psychiatry. This is despite growing evidence, progress in development, and access to digital tools, a pandemic that has pushed through barriers and increased acceptance of Digi-physical healthcare. A prerequisite for the integration of Digi-physical healthcare as a routine in public psychiatry depends on the acceptance of health care professionals. Aim: This study aims to investigate barriers and facilitators, as perceived by health care professionals, for implementing a combination of physical and digital care in Swedish public psychiatry.   Methods: Participants were health care professionals active in public outpatient psychiatry in a region in Southern Sweden. A web-based questionnaire with open and closed questions was designed. Collected data was analyzed with qualitative content analysis based on a deductive approach using the Theoretical Domains Framework (TDF). Results: Based on the TDF framework, barriers and enablers that influence mental health care professionals to apply the distance format as a way of working were identified. The result`s high agreement with previous research, suggests that identified TDF domains can be used to strengthen the acceptance and implementation of Digi-physical healthcare within psychiatric outpatient care.

Tortilla sales are projected to exceed 9.5 billion by 2014. However, currently no wheat cultivars have been identified that possess the intrinsic quality attributes needed for the production of optimum quality tortillas. Tortillas made with refined wheat flour low in dietary fiber (DF) are popular in the United States due to their sensory properties. This study explored the use of wheat lines (WL) possessing variations in high molecular weight glutenin allele sub-units (HMW-GS) for production of tortillas and also investigated the use of corn based resistant starches (RS), type II (RS2) and wheat based RS type IV (RS4) to increase DF in tortillas. Tortillas were made with 0-15 percent RS and 100 percent whole white wheat (WW). Flour protein profiles, dough, and tortilla properties were evaluated to determine the effects of the allelic variations and RS substitution on tortilla quality. Sensory properties of tortillas with RS were determined. Variations in HMW-GS composition significantly affected the protein quality and tortilla properties. Flour from WL possessing allelic combinations (2*, 17+18, 7, 2+12), (1, 17+18, 5+10), (2*, 17, 2+12) and (1, 2*, 17+18, 2+12) had 12.8-13.3 percent protein. These WL had extensible doughs and produced large diameter tortillas with superior (greater than or equal to 3.0) flexibility after 16 days compared to control. However, WL with (17+18 and 5+10) and (2*, 17+7, 5) produced extensible doughs, large, but less flexible, tortillas compared to control. WL with (2*,17+18,5+10) and (1,2*,7+9,5+10) produced smaller diameter tortillas, but with superior flexibility compared to control. RS2, WW, and cross-linked-pre-gelatinized RS4 (FiberRite) produced hard, less-extensible doughs and thinner tortillas compared to control, due to high water absorption. Cross-linked RS4 (Fibersym) dough and tortillas were comparable to control. 15 percent of RS2 and RS4 increase DF in control to 6 and 14 percent respectively, compare to control (2.8 percent DF). WW tortillas were less acceptable than control in appearance, flavor and texture, while tortillas with 15 percent Fibersym had higher overall acceptability than control. RS2 negatively affected dough machinability and tortilla shelf stability. However, 15 percent RS4 improved the DF in refined flour tortillas to meet FDA's "good source of fiber claim," without negatively affecting dough/tortilla quality.