Class of 2012 Abstract / Specific Aims: To compare and evaluate the therapies prescribed, the incidence of adverse drug events, and the time to clinical cure in transplant patients with a cytomegalovirus (CMV) infection at an academic medical center before and during the foscarnet nationwide shortage. Methods: This study was a retrospective chart review to compare CMV treatment prescribed and clinical outcomes in pediatric and adult transplant patients at an academic medical center. Transplant patients were evaluated over a 16 month time period between December 2009 and March 2011. The average dose (mg/kg) and prevalence ganciclovir, foscarnet, and cidofovir prescribed in transplant patients with CMV infection were evaluated. Additionally, the incidence of adverse drug events including acute renal dysfunction and myelosuppression were characterized. Main Results: There were 30 subjects diagnosed with CMV disease during the evalutaion period. Of all of the patients treated for CMV before the shortage, 79% received ganciclovir, 43% received foscarnet, and 21% received cidofovir. Following the shortage in September 2010, the usage of the antiviral agents changed to 100%, 25%, and 13% respectively. Overall the usage of ganciclovir increased while the usage of foscarnet decreased when there was a shortage of medication. Conclusions: The antiviral prescribing patterns changed significantly during the foscarnet shortage. The average dose and incidence of ganciclovir increased which likely contributed to serious adverse events. Due to the limited amount of patients treated for CMV and the short time frame, clinical cure could not be determined at this time. Drug shortages are a serious problem and significantly influence patient outcomes.

Class of 2012 Abstract / Specific Aims: To compare and evaluate the therapies prescribed, the incidence of adverse drug events, and the time to clinical cure in transplant patients with a cytomegalovirus (CMV) infection at an academic medical center before and during the foscarnet nationwide shortage. Methods: This study was a retrospective chart review to compare CMV treatment prescribed and clinical outcomes in pediatric and adult transplant patients at an academic medical center. Transplant patients were evaluated over a 16 month time period between December 2009 and March 2011. The average dose (mg/kg) and prevalence ganciclovir, foscarnet, and cidofovir prescribed in transplant patients with CMV infection were evaluated. Additionally, the incidence of adverse drug events including acute renal dysfunction and myelosuppression were characterized. Main Results: There were 30 subjects diagnosed with CMV disease during the evalutaion period. Of all of the patients treated for CMV before the shortage, 79% received ganciclovir, 43% received foscarnet, and 21% received cidofovir. Following the shortage in September 2010, the usage of the antiviral agents changed to 100%, 25%, and 13% respectively. Overall the usage of ganciclovir increased while the usage of foscarnet decreased when there was a shortage of medication. Conclusions: The antiviral prescribing patterns changed significantly during the foscarnet shortage. The average dose and incidence of ganciclovir increased which likely contributed to serious adverse events. Due to the limited amount of patients treated for CMV and the short time frame, clinical cure could not be determined at this time. Drug shortages are a serious problem and significantly influence patient outcomes.

No description available.

La structure des ADN polymérases (pol) du cytomégalovirus humain (CMV) et du virus herpès simplex 1 (VHS-1), appartenant tous deux à la famille des Herpesviridae, est associée à la forme d'une main droite comportant entre autres les domaines de la paume, du pouce et des doigts. L'ADN pol adopte différentes conformations (ouverte et fermée) impliquant un mouvement du domaine des doigts dans le but de faciliter l'interaction entre le nucléotide et l'ADN en cours d'élongation. Il a été montré que l'antiviral foscarnet (FOS) qui cible l'ADN pol du CMV et du VHS-1 se lierait à l'enzyme quand elle est dans sa conformation fermée et que des mutations dans le domaine des doigts conférant la résistance à cet antiviral favoriseraient une conformation plus ouverte de l'enzyme pour laquelle le FOS a une affinité plus faible. Nous avons voulu analyser si cette hypothèse s'appliquait également à des mutations qui sont localisées dans des régions du domaine NH₂-terminal et de la paume qui interagissent avec le domaine des doigts lors des changements de conformation de l'enzyme au cours de la réaction de polymérisation. Notre hypothèse est que des mutations localisées dans l'hélice K (domaine NH₂-terminal) et la région II (domaine de la paume) qui participent aux changements de conformation de l'enzyme pourraient favoriser une conformation plus ouverte des ADN pol virales, et par conséquent, réduire la sensibilité des virus au FOS. Nous avons donc sélectionné des substitutions théoriques en utilisant une stratégie basée sur l'alignement des séquences en acides aminés des ADN pol du CMV et du VHS-1 (sensibles au FOS) avec celles des bactériophages RB69 et T4 (résistantes au FOS). Nous avons tenté de générer les virus recombinants CMV et VHS-1 possédant les différentes substitutions théoriques sélectionnées. Cependant, l'introduction de certaines substitutions [Q578P, R581T, L587F (hélice K), P712Y, F718L (région II) pour le CMV et Q617P, R620T, L626F (hélice K), F718L (région II) pour le VHS-1] étaient délétères pour les ADN pol, ce qui empêchait les virus recombinants de se répliquer en culture cellulaire. Parmi les substitutions sélectionnées dans l'hélice K, la substitution I619K confère une résistance du VHS-1 au FOS. Au sein de l'hélice K, nous avons également caractérisé la substitution théorique Q579I qui conférait une hypersensibilité du CMV au FOS. Nous avons caractérisé cette substitution en la comparant à la mutation K805Q localisée dans l'hélice P (domaine des doigts) déjà connue pour induire une hypersensibilité du CMV au FOS. Dans la région II, les substitutions V715S et A719T confèrent une résistance des deux virus au FOS. La substitution Q697P du CMV confère une résistance du CMV au FOS mais pas pour le VHS-1. Les profils de sensibilité des virus recombinants au FOS ont également été confirmés par des tests enzymatiques dans lesquels nous avons déterminé l'inhibition de l'activité des ADN pol recombinantes mutées par cet antiviral. Nous avons également constaté une diminution des capacités réplicatives des CMV et VHS-1 recombinants possédant ces substitutions par rapport à celle des virus sauvages correspondants. Des analyses tri-dimensionnelles ont été réalisées et ont suggéré que les substitutions conférant une résistance au FOS seraient associées à une déstabilisation de la conformation fermée des ADN pol et favoriseraient une conformation plus ouverte pour laquelle l'antiviral a une plus faible affinité. D'autre part, les modélisations tri-dimensionnelles des protéines possédant les substitutions conférant une hypersensibilité au FOS ont montré que les ADN pol favorisaient une conformation plus fermée pour laquelle le FOS a une plus grande affinité. La caractérisation de la substitution théorique V715S du CMV et du VHS-1 (FOSᴿ/GCVᴿ et FOSᴿ/ACVᴿ, respectivement) a été comparée aux substitutions V715G du VHS-1 (FOSᴿ/ACVᴿ), V715M du CMV et du VHS-1 (FOSᴿ/GCV[exposant S] et FOS[exposant S]/ACVᴿ, respectivement), déjà décrites dans la littérature. Brièvement, nous avons montré que l'introduction de ces différentes substitutions induisaient des changements au niveau de l'environnement hydrophobe de la valine à la position 715 influençant ainsi les phénotypes de sensibilité aux antiviraux observés. L'ensemble de ces résultats nous ont permis d'appuyer notre hypothèse selon laquelle les mutations localisées dans l'hélice K et la région II peuvent influencer la sensibilité des virus au FOS en modifiant la structure de la protéine et en interférant avec les changements de conformation de l'enzyme. / The structure of the human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) DNA polymerase (pol), belonging to the Herpesviridae family, is associated to a right hand with palm, thumb and fingers domains. The viral DNA pol adopts different conformations (open and closed) that implies a move of the fingers domain to facilitate the interaction between the nucleotide and the elongating DNA. It has been shown that the antiviral foscarnet (FOS) which targets the HCMV and HSV-1 DNA pol binds to the enzyme in its closed conformation and mutations confering resistance to this antiviral and localised in the fingers domain would favor a more open conformation of the enzyme for which FOS has a lower affinity. The aim of this thesis was to analyse whether this hypothesis could be extended to mutations localised in the NH₂-terminal and the palm domains which interact with the fingers domain during the conformational changes of the enzyme during the polymerization process. Our hypothesis is that mutations localized in the helix K (NH₂-terminal domain) and region II (palm domain) that participate in the conformational changes of the enzyme could favor a more open conformation of the viral DNA pol, and thus, decrease the susceptibility of viruses to FOS. We selected theoretical substitutions using a strategy based on amino acid sequences alignement of the DNA pol of HCMV and HSV-1 (susceptible to FOS) with those of RB69 and T4 bacteriophages (naturally resistants to this antiviral). We tried to generate recombinant HCMV and HSV-1 containing the different theoretical substitutions that we selected. However, the introduction of some theoretical substitutions [Q578P, R581T, L587F (helix K), P712Y, F718L (region II) for HCMV and Q617P, R620T, L626F (helix K), F718L (region II) for HSV-1] was so detrimental for the DNA pol that recombinant viruses were not able to grow in cell culture. Among the substitutions selected in the helix K, the substitution I619K confers resistance of HSV-1 to FOS. In the helix K, we also characterized the theoretical Q579I substitution that confers hypersusceptibility of HCMV to FOS. We compared this substitution with the K805Q substitution located in the helix P (fingers domain), already known to induce hypersusceptibility of HCMV to FOS. In region II, substitutions V715S and A719T confer resistance of both viruses to FOS whereas the Q697P substitution was associated with resistance of HCMV to FOS but not for HSV-1. The susceptibility profiles of recombinant viruses to FOS were confirmed by enzymatic assays that allowed us to determine the inhibition of the recombinant mutated DNA pol activity by this antiviral compound. We observed a decrease of the replicative capacities of recombinant HCMV and HSV-1 harboring these mutations compared to their wild-type counterparts. Tri-dimensional modeling was also performed to better understand the impact of these substitutions on the DNA pol of HCMV and HSV-1. On the one hand, substitutions confering resistance to FOS were associated to a destabilization of the closed conformation of the DNA pol and would favor a more open conformation for which the antiviral has a lower affinity. On the other hand, substitutions associated to a hypersusceptibility profile would favor a more closed conformation of the DNA pol for which FOS has a higher affinity. The characterization of the theoretical substitution V715S of HCMV and HSV-1 (FOSᴿ/GCVᴿ and FOSᴿ/ACVᴿ, respectively) was compared to the substitutions V715G of HSV-1 (FOSᴿ/ACVᴿ), V715M of HCMV and HSV-1 (FOSᴿ/GCV[superscript S] and FOS[superscript S]/ACVᴿ, respectively), already described in the literature and that were, thus, associated with different antiviral susceptibility phenotypes compared to those of V715S. Briefly, we showed that the introduction of these different substitutions could induce varying changes of the hydrophobic environment of the valine at position 715 influencing the antiviral susceptibility profile. Altogether, these results support our hypothesis that substitutions in helix K and region II could influence the susceptibility of HCMV and HSV-1 to FOS by modifiying the protein structure and impacting the correct conformational changes of the enzyme.

Copies of author's previously published works inserted. / Bibliography: leaves 230-282. / 291 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Covers clinical data relating to trials with two antiretroviral agents, didanosine and foscarnet, conducted at St Stephen's clinic, London, discussing aspects of their therapetic efficacy, effect on survival, clinical and laboratory tolerability. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1995

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Aspects of bovine herpesvirus 5 (BoHV-5) pathogenesis and experimental therapies against BoHV-1 and BoHV-5 were investigated in vitro and in inoculated rabbits. In chapter 1, we investigated the role of nitric oxide (NO), a component of innate immunity against pathogens, in the neurological disease by BoHV-5 in rabbits. Spectrophotometry for NO products revealed that NO levels were significantly increased in several regions of the brain of rabbits with neurological disease [F(4, 40)=3.33; P<0.02]. Quantification of NO levels in the brain at different time points after virus inoculation revealed a gradual increase [F(12, 128)=2.82; P<0,003], correlated spatially and temporally with virus dissemination within the brain and preceding the development of neurological signs. Thus, we propose that the overproduction of NO in the brain of BoHV-5-infected rabbits may participate in the pathogenesis of neurological disease. In chapter 2, the activity of three anti-herpetic drugs was tested against BoHV-1, BoHV-2 and BoHV-5 in vitro by plaque reduction assay. Acyclovir was moderately active against the three viruses; Gancyclovir was moderately effective against BoHV-2, and to a lesser extent against BoHV-5, being poorly active against BoHV-1. Foscarnet (PFA) exhibited the most pronounced antiviral activity, being the only drug that, at the concentration of 100 ìg/mL, completely inhibited plaque formation by all three viruses. In chapter 3, we report the activity of PFA in rabbits inoculated with BoHV-1 or BoHV-5. Rabbits inoculated with BoHV-5 and treated with 100 mg/kg of PFA presented mortality rates (11/22 or 50%) statistically lower than non-treated controls (21/22 ou 95.4%) (P<0.0008). A significant reduction in the mean virus titers was observed at day 3 pi, the peak of virus shedding [F(9,108) = 2,23; P<0.03]. Reduction in virus shedding, frequency, severity and duration of ocular signs were also observed in rabbits inoculated with BoHV-1 into the conjunctival sac, comparing to the controls. The prolonged incubation period and the reduction in the duration of the clinical course of the PFA-treated group was significant (P<0.005 and P<0.04, respectively). Therefore, the activity of PFA in vivo against BoHV-1 and BoHV-5 may be exploited in further experimental therapies. In chapter 4, we investigated the effect of the inhibition of the inducible isoform of nitric oxide synthase (iNOS), associated or not with PFA treatment, on neurological infection by BoHV-5 in rabbits. Groups of BoHV-5-inoculated rabbits were treated with the iNOS inhibitor aminoguanidine (AG); with PFA; with both drugs; or maintained as virus controls. Morbidity and mortality rates were 100% (6/6) in the groups AG and CV, 66.7% (4/6) in the group PFA and 83.3% (5/6) in the group AG+PFA. The incubation period was significantly lower (P<0.05) and the onset of neurological disease occurred earlier and was more severe in the group AG. These results demonstrate that treatment with PFA reduced morbidity and mortality rates associated to BoHV-5 infection, that AG treatment anticipated the development of neurological signs, and that the development of neurolocial disease was delayed in the group treated with both drugs. Taken together, these results contribute to the knowledge of the pathogenesis of BoHV-5 neurological disease and pave the way for other experimental pathogenesis and therapy studies. / Aspectos da patogenia da infecção neurológica pelo herpesvírus bovino 5 (BoHV-5) e terapias experimentais contra o BoHV-1 e BoHV-5 foram estudados in vitro e em coelhos inoculados. O capítulo 1 relata a investigação do papel do óxido nítrico (NO), um componente da imunidade inata contra patógenos, na doença neurológica produzida pelo BoHV-5 em coelhos. Espectrofotometria para os produtos de degradação do NO revelou um aumento significativo nos seus níveis em várias regiões do encéfalo de coelhos infectados (F(4, 40)=3.33; P<0,02). A quantificação do NO no encéfalo nos dias seguintes à inoculação viral revelou um aumento gradativo (F(12, 128)=2.82; P<0,003), correlacionado temporal e espacialmente com a invasão e disseminação viral, e precedendo o desenvolvimento de sinais neurológicos. Sugere-se, assim, que a produção aumentada de NO em resposta à infecção possa participar da patogenia dessa doença neurológica. No capítulo 2, investigou-se a atividade de três fármacos antivirais frente ao BoHV-1, BoHV-2 e BoHV-5 in vitro pelo teste de redução do número de placas. O Aciclovir foi moderadamente ativo frente aos três vírus; o Ganciclovir apresentou atividade moderada frente ao BoHV-2 e, em menor grau, contra o BoHV-5, sendo ineficaz frente ao BoHV-1. O Foscarnet (PFA) apresentou a atividade antiviral mais pronunciada, sendo o único fármaco que, na concentração de 100 μg/mL, inibiu completamente a produção de placas pelos três herpesvírus bovinos. No capítulo 3, investigou-se a atividade do PFA em coelhos inoculados com o BoHV-1 ou BoHV-5. Coelhos inoculados com o BoHV-5 e tratados com 100 mg/kg do PFA apresentaram índices de mortalidade (11/22; 50%) estatisticamente inferiores aos controles não-tratados (21/22; 93,7%) (P<0,0008). Uma redução significativa no título médio de vírus foi observada no dia 3 pi, pico da excreção viral [F(9,108) = 2,23; P<0,03]. Em coelhos inoculados no saco conjuntival com o BoHV-1 e tratados com o PFA, foram observadas reduções na excreção viral, na frequência, severidade comparando-se com o grupo controle. O período de incubação prolongado e a redução na duração do curso clínico no grupo tratado foi significante (P<0,005 e P<0,04, respectivamente). A atividade antiviral do PFA in vivo contra o BoHV-1 e BoHV-5 abre a perspectiva para outras terapias experimentais. No capítulo 4, investigou-se o efeito da inibição da isoforma induzível da enzima óxido nítrico sintase (iNOS), associada ou não ao tratamento com o PFA, na infecção neurológica pelo BoHV-5 em coelhos. Grupos de coelhos inoculados com o BoHV-5 foram tratados com o inibidor da iNOS aminoguanidina (AG); com PFA; com ambos os fármacos; ou não receberam tratamento. Os índices de morbidade e mortalidade foram de 100% (6/6) nos grupos AG e controle; 66,7% (4/6) no grupo PFA e 83,3% (5/6) no grupo AG+PFA. O período de incubação foi significativamente menor (P<0,05) e os sinais neurológicos foram mais precoces e severos nos animais do grupo AG. Portanto, o tratamento com PFA reduziu a morbidade e mortalidade associadas com a infecção pelo BoHV-5; o tratamento com AG resultou no agravamento e na antecipação do quadro neurológico e no grupo tratado com ambos os fármacos observou-se um desenvolvimento mais tardio dos sinais neurológicos. Esses resultados contribuem para o conhecimento da patogenia da doença neurológica pelo BoHV-5 e abrem perspectivas para estudos adicionais de patogenia e terapêutica anti-herpesvírus.