Class of 2009 Abstract / OBJECTIVES: To determine patient preference for the type of visit for the receipt of the influenza vaccine from the pharmacist and to determine patient preference for contact method for annual notification of the influenza vaccine program. METHODS: This was a descriptive study using a short telephone survey. The first dependent variable was the preferred type of visit comparing appointment-based and predetermined walk-in clinics. The second dependent variable was the preferred method of contact for annual notification of a pharmacist administered influenza vaccination program (telephone, US post mail, email). RESULTS: The telephone survey was completed by 206 patients. Overall, study participants preferred appointment-based visits ( 81.2 %; p < 0.05) compared to a predetermined walk-in clinic (18.8%). Overall, study participants significantly preferred to be contacted for annual notification of a pharmacist administered influenza vaccination program via telephone (75.7%; p< 0.05) compared with US post mail and email. Based on the percentages observed, the second preferred method of contact was email (12.6%) and US post mail was the third preferred method of contact (11.7%). CONCLUSIONS: Patient preference for type of visit for pharmacist-administered influenza vaccine was appointment-based as opposed to predetermined walk-in clinic based. Patient preference for contact method for annual notification was telephone as opposed to email or postal mail.

Class of 2009 Abstract / OBJECTIVES: To evaluate patients’ perceptions of receiving a pharmacist-administered influenza vaccine in the community pharmacy setting. METHODS: All patients receiving a pharmacist-administered influenza vaccine at a Safeway Pharmacy in Tucson, Arizona were invited to participate in the survey. Participants completed the survey in a waiting area outside the pharmacy. At the completion of the study time frame, surveys were collected, and each response was entered into an Excel spreadsheet for data analysis. RESULTS: Seventy-five patients completed the Flu Shot Survey. One hundred percent of patients reported that getting the influenza vaccine at a grocery store pharmacy is convenient. Respondents reported being either very confident (97.3%) or somewhat confident (2.7%) in pharmacists as immunizers. Only 18.7% reported having never received an influenza vaccine from a pharmacist, and 13.3% reported having no prior knowledge that Arizona pharmacists could administer the influenza vaccine. CONCLUSIONS: All patients responded that receiving the influenza vaccine from a community pharmacist was convenient. Patients wanted to receive the vaccine next year from a pharmacist, and the majority of respondents were confident in the pharmacist as an immunizer. Few patients reported never receiving the influenza vaccine from a community pharmacist, and even fewer patients were unaware that pharmacists in Arizona can immunize.

Influenza is suspected to be endemic in tropical climates, with peaks during and/or following cold or rainy seasons. To date, only one study has been conducted examining the epidemiology and seasonality of influenza in Uganda. The focus of this analysis is to determine whether a change in the seasonality of influenza can been seen between three distinct regions of Uganda: Central, Northwest, and Western. Secondary data analysis was conducted on surveillance data collected by the Uganda Virus Research Institute (UVRI) between April 2007 to September 2010 from 10 surveillance sites. Surveillance sites were grouped for this analysis into three regions: Central, Northwest, and Western. A total of 3,944 samples were collected and tested for any strain of influenza. The prevalence of influenza over the 4 years of surveillance was 10.1%. The majority of cases came from the Central region (81.7%) and the highest prevalence of influenza-positive samples were collected in the Central region (88.7 cases/1,000 persons). A clear difference in influenza activity was observed during the 2009 H1N1 pandemic. Uganda reported its first case of H1N1 in July 2009 (Relief Web). The Central region experienced its initial flux of influenza activity in July and August 2009 (Figure 1). However, the Northwest region did not experience a flux in activity until October 2009 (Figure 2). Influenza activity in the Central and Northwest regions appear to coincide with colder temperatures and both rainy seasons. The Northwest region was the only region to experience a peak corresponding with warmer weather. Results showed a slight change in the seasonality of influenza between the Central and Northwest regions of Uganda from surveillance data collected between April 2007 and September 2010.

Patógenos associados a aves migratórias podem causar doenças em aves domésticas. As aves de subsistência são possíveis fontes de disseminação destes patógenos pela ausência de biossegurança na criação e pelo contato com aves silvestres. A região do Parque Nacional da Lagoa do Peixe (PNLP), no Rio Grande do Sul, se caracteriza como um importante sítio de aves migratórias. Devido a isto, é possível uma interface entre aves migratórias e aves de subsistência criadas na região. A influenza aviária (AI) é a principal doença associada a aves migratórias que afeta o sistema respiratório, sendo estas os principais disseminadores do vírus. Estudos prévios indicam que há risco de introdução de um vírus de AI de alta patogenicidade (HPAIV) na região. Porém, o padrão e impacto do espalhamento deste patógeno na região é desconhecido, visto a ausência de relatos. Cabe ressaltar que outras enfermidades respiratórias, como a Doença de Newcasle (ND) e a micoplasmose por Mycoplasma gallisepticum (MG), também são associadas a aves migratórias e podem causar doenças em aves domésticas. Neste contexto, o objetivo deste trabalho é realizar um inquérito epidemiológico de doenças respiratórias associadas a aves migratórias em aves de subsistência e propor um modelo de espalhamento de HPAIV na região do PNLP. Com esta finalidade, realizamos uma avaliação (i) da presença de anticorpos contra AI, ND e MG nas aves de subsistência por ELISA, (ii) da presença dos agentes de AI e ND por RT-PCR em tempo real nas aves de subsistência, (iii) dos possíveis fatores de risco associados aos patógenos e (iv) das consequências da introdução de HPAIV na região através de um modelo matemático de espalhamento. Nas propriedades de aves de subsistência amostradas da região, foi detectada a presença de anticorpos contra AI, ND e MG na frequência de 4,2%, 87,5% e 58,3%, respectivamente. Todas as amostras foram negativas no RT-PCR em tempo real realizado para AI e ND. A avaliação de fatores de risco foi possível nas propriedades soropositivas para ND e MG, possibilitando realizar-se a primeira análise de fatores de risco para estes agentes em aves de subsistência no Brasil. Para ND, o risco foi maior nas propriedades nas quais os criadores adotam a prática de reposição própria para manter a criação (PR=1,64; 95% IC: 1,11 – 2,42). Além disso, o aumento na distância das granjas em relação ao estuário da Laguna do Peixe diminui o risco para ND (PR=0,94; 95% IC: 0,90 – 0,99). Já para MG, foram considerados fatores de risco a prática de confinar as aves (PR = 3,40, 95% IC: 1,93 – 5,99) e a interação entre a “troca de aves ou ovos com outros produtores e vizinhos possuírem aves” (PR = 2,16, 95% IC: 1,24 – 3,76). Nas simulações da modelagem matemática de espalhamento de HPAIV na região, a maioria das propriedades se tornaria infectada até o 30º dia de infecção. Além disso, na média das simulações, a infecção atingiria em torno de 80 a 90% das propriedades, alcançando até 30 km de distância do caso índice. Os resultados indicam a circulação de patógenos associados a aves migratórias na região do PNLP. Devido à detecção destes nas aves de subsistência, estas podem ser consideradas sentinelas destes agentes na região. Além disso, os dados obtidos incrementam o conhecimento sobre o espalhamento e a dinâmica dessas doenças em propriedades com aves de subsistência, bem como sobre os indicadores de risco para ocorrência. Estes dados podem ser utilizados para o estabelecimento de medidas de biossegurança e de manejo adequados para este tipo de criação e de programas de monitoria visando à contenção da disseminação de patógenos. / Pathogens associated with migratory birds can cause disease in domestic birds. Backyard poultry are possible source of pathogens dissemination due to the lack of biosecurity measures and the contact with wild birds. The region of Lagoa do Peixe National Park (PNLP), in Rio Grande do Sul state, Brazil, is characterized as an important site for migratory birds. Thus, it is a possible interface place for migratory and backyard birds. Avian influenza (AI) is the most important respiratory disease associated with migratory birds, the main virus disseminating agents. Previous studies have indicated a risk for introduction of higly pathogenic avian influenza virus (HPAIV) in PNLP region. However, the impact and spreading pattern of AI virus in this region is unknown due to the lack of disease reports. It is noteworthy that other respiratory diseases, such Newcastle Disease (ND) and micoplasmosis due to Mycoplasma gallisepticum (MG), are also associated with migratory birds and can cause disturbances in domestic poultry. In this context, the aims of this work are to perform an epidemiological survey of respiratory diseases associated with migratory birds in backyard poultry and to propose a model of HPAIV spreading in PNLP region. For this, it was evaluated (i) the presence of antibodies against AI, ND and MG in backyard poultry by ELISA, (ii) the presence of AI and ND virus in backyard poultry by real time RT-PCR, (iii) the potential risk factors associated with these pathogens, and (iv) the consequences of HPAIV introduction in PNLP region using a mathematical model of virus spreading. In the sampled properties, frequency of antibodies against AI, ND and MG were 4.2%, 87.5% and 58.3%, respectively. All samples were negative for AI and ND in real time RT-PCR analysis. The first evaluation of risk factors associated to ND or MG in backyard poultry from Brazil was performed in this work. For ND, the risk was increased in the properties in which farmers used their own replacement poultry to restock their flock (PR=1.64; 95% CI: 1.11–2.42). Furthermore, the increasing distance of the household flock to the “Laguna do Peixe” estuary is associated with decreasing NDV seropositivity (PR=0.94; 95% CI: 0.90 – 0.99). For MG, seropositivity was significantly associated with bird confinement (PR = 3.40, 95% CI: 1.93 – 5.99) and with interaction between “poultry/egg exchange and neighbors have poultry” (PR = 2.16, 95% CI: 1.24 – 3.76). In the simulations from the mathematical model of HPAIV spreading in PNLP region, most of the properties of this region would become infected up to 30 days outbreak beginning. Moreover, the infection would affect about 80 to 90% of the properties from the region, reaching up to 30 km distance from the index case. The results showed the circulation of pathogens associated with migratory birds in PNLP region. Since these pathogens were detected in backyard poultry, these birds can be considered sentinels for these agents in this region. Additionally, the data obtained improve the knowledge on spreading and dynamics of these diseases in properties with backyard poultry, as well as on potential risk factors. The set of these results can be useful to development of adequate biosecurity procedures for backyard flocks and surveillance programs to avoid pathogen dissemination.

The Quartz crystal microbalance (QCM) is a very sensitive mass-detecting device which is based on changes in to the vibrational frequency of quartz crystals after adsorption of substances to a modified crystal surface. In this study a QCM-based biosensor was developed for the rapid diagnosis of influenza viruses and its suitability and limitations were compared with currently available diagnostic methods on 67 clinical samples (nasal washes) received during the 2005 Australian winter. The type-specific and conserved viral M1 proteins of both A/PR/8/34 and B/Lee/40 viruses were used to prepare polyclonal antisera for the development of an ELISA. The limits of detection of ELISAs for the detection of purified A/PR/8/34 and B/Lee/40 ƒnviruses were 20ƒÝg/mL ƒnand 14 ƒÝg/mL using polyclonal antibodies, and 30 ƒÝg/mL and 20 ƒÝg/mL for monoclonal antibodies, respectively. The limit for detection of each virus was 104 pfu/mL, irrespective of whether antisera or monoclonal antibodies were used for capture. Non-purified cell culture-grown preparations of either virus could be detected at 103 pfu/mL The QCM utilised the same reagents used in ELISAs. However, a number of parameters were then further optimised to improve the sensitivity of the tests. These included blocking of non-specific binding, examination of the effects of flow-cell compression, the role of pH, flow rate, antibody concentration and the addition of protein A to the crystal surfaces of the biosensor. The lowest virus concentration that could be detected with the QCM was 104 pfu/mL for egg-grown preparations of both A/PR/8/34 and B/Lee/40, which could be detected within 30 min. However, conjugation of 13 nm gold nanoparticles to a second detector antibody resulted in a 10-fold increase in sensitivity and a detection limit of 103 pfu/mL that could be determined within 1 h. The direct detection of the influenza viruses in nasal samples was not possible by QCM because of the significant frequency fluctuation that was probably caused by the viscosity of the samples. Therefore, an additional culture step of 12 h was required, which increased the processing time to 2 days. The QCM/nanoparticle method was shown to be as sensitive as the standard cell culture method, and the QCM method as sensitive as the shell vial method. The QCM and QCM/nanoparticle methods were shown to be 81 and 87% as sensitive and both were 100% as specific as the real-time polymerase chain reaction. However, for use in rapid diagnosis, improvements are required to remove frequency fluctuation resulting from the direct use of nasal samples.

Swine influenza (SI) is an acute, highly contagious, respiratory disease of swine. The causative agent of SI infections is swine influenza virus (SIV). SIV is a type A influenza virus classified into the Orthomyxoviridae family and is an enveloped particle with a genome composed of eight negative-orientated RNA segments.<p> The mortality rate of influenza disease in pigs is generally low but morbidity can reach up to 100%. SI infections considerably contribute to respiratory disease in post-weaning pigs, causing significant economic losses due to an increase in the number of days pigs need to reach market weight. In addition, SI infections possess significant human public health concerns. Vaccination is the primary method for the prevention of SI. Currently available vaccines against SI are a combination of two inactivated antigenically distinct SIVs with oil adjuvant. The application of these vaccines induce mainly humoral immune responses. In contrast, application of live attenuated influenza vaccines (LAIV) mimics natural infection and induce strong, long-lived cell-mediated and humoral immunity. Furthermore, LAIV induces cross-protective immunity against different subtypes of influenza A viruses. LAIVs are developed for human and equine influenza viruses but at present no LAIV is available for SIVs.<p> The critical step in influenza virus infection is an initial interaction between virus and cell surface carbohydrates followed by receptor-mediated endocytosis and fusion of the viral and endosomal membranes. Influenza virus entry into cells is mediated by the viral surface glycoprotein hemagglutinin (HA). HA is primary synthesized as a polypeptide in HA0 form. In order to be infectious, HA0 must be cleaved by host proteases into HA1 and HA2 subunits. Therefore, this process is crucial determinant for virus pathogenicity.<p> Our objective was to generate a live attenuated SIVs, particularly a viruses with a modified HA cleavage site resistant to activation during natural infection but which can be activated in vitro by an exogenous protease. Using the reverse genetics technique, we generated two mutant SIVs of strain A/SW/SK/18789/02 (H1N1) containing a modified cleavage site within their HA. Mutant A/SW/SK-R345V (R345V) contained a mutation within HA segment at amino acid (AA) position 345 from Arginine (Arg) to Valine (Val) while the second mutant, A/SW/SK-R345A (R345A) encoded Alanine (Ala) instead of Arginine (Arg) at position AA345 on HA. We showed that HA cleavage in both mutants was strictly dependent on the presence of human neutrophil elastase in tissue culture. These tissue-culture grown mutant SIVs showed similar growth properties in terms of plaque size and growth kinetics, compared to the wild type virus. Both mutant SIVs were able to preserve introduced mutations after multiple passages in tissue culture suggesting that AA substitution within HA cleavage site did not alter genetic stability in the presence of appropriate protease. Furthermore, these mutant SIVs were highly attenuated in pigs but capable of inducing significant cell-mediated and humoral immune responses after two vaccinations via intratracheal (IT) and intranasal (IN) routes. Immune responses induced by vaccination with elastase dependent SIV were sufficient to confer full protection against parental homologous and antigenic variant of H1N1 SIVs and partial protection from heterologous subtypic H3N2 after the challenge. Therefore, elastase-dependent mutant SIV could serve as live vaccine against antigenically distinct swine influenza viruses in pigs.

Swine influenza (SI) is an acute, highly contagious, respiratory disease of swine. The causative agent of SI infections is swine influenza virus (SIV). SIV is a type A influenza virus classified into the Orthomyxoviridae family and is an enveloped particle with a genome composed of eight negative-orientated RNA segments.<p> The mortality rate of influenza disease in pigs is generally low but morbidity can reach up to 100%. SI infections considerably contribute to respiratory disease in post-weaning pigs, causing significant economic losses due to an increase in the number of days pigs need to reach market weight. In addition, SI infections possess significant human public health concerns. Vaccination is the primary method for the prevention of SI. Currently available vaccines against SI are a combination of two inactivated antigenically distinct SIVs with oil adjuvant. The application of these vaccines induce mainly humoral immune responses. In contrast, application of live attenuated influenza vaccines (LAIV) mimics natural infection and induce strong, long-lived cell-mediated and humoral immunity. Furthermore, LAIV induces cross-protective immunity against different subtypes of influenza A viruses. LAIVs are developed for human and equine influenza viruses but at present no LAIV is available for SIVs.<p> The critical step in influenza virus infection is an initial interaction between virus and cell surface carbohydrates followed by receptor-mediated endocytosis and fusion of the viral and endosomal membranes. Influenza virus entry into cells is mediated by the viral surface glycoprotein hemagglutinin (HA). HA is primary synthesized as a polypeptide in HA0 form. In order to be infectious, HA0 must be cleaved by host proteases into HA1 and HA2 subunits. Therefore, this process is crucial determinant for virus pathogenicity.<p> Our objective was to generate a live attenuated SIVs, particularly a viruses with a modified HA cleavage site resistant to activation during natural infection but which can be activated in vitro by an exogenous protease. Using the reverse genetics technique, we generated two mutant SIVs of strain A/SW/SK/18789/02 (H1N1) containing a modified cleavage site within their HA. Mutant A/SW/SK-R345V (R345V) contained a mutation within HA segment at amino acid (AA) position 345 from Arginine (Arg) to Valine (Val) while the second mutant, A/SW/SK-R345A (R345A) encoded Alanine (Ala) instead of Arginine (Arg) at position AA345 on HA. We showed that HA cleavage in both mutants was strictly dependent on the presence of human neutrophil elastase in tissue culture. These tissue-culture grown mutant SIVs showed similar growth properties in terms of plaque size and growth kinetics, compared to the wild type virus. Both mutant SIVs were able to preserve introduced mutations after multiple passages in tissue culture suggesting that AA substitution within HA cleavage site did not alter genetic stability in the presence of appropriate protease. Furthermore, these mutant SIVs were highly attenuated in pigs but capable of inducing significant cell-mediated and humoral immune responses after two vaccinations via intratracheal (IT) and intranasal (IN) routes. Immune responses induced by vaccination with elastase dependent SIV were sufficient to confer full protection against parental homologous and antigenic variant of H1N1 SIVs and partial protection from heterologous subtypic H3N2 after the challenge. Therefore, elastase-dependent mutant SIV could serve as live vaccine against antigenically distinct swine influenza viruses in pigs.

No description available.

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 63-70).

Thesis (M.P.H.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 63-67).