A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Science in Epidemiology (Epidemiology and Biostatistics). 2016 / Background Reducing severe acute respiratory illness (SARI)-associated mortality in African children remains a public health priority and an immense challenge. The pneumococcal conjugate vaccine (PCV) was introduced into the South African routine immunization programme in 2009. The objectives of this study were: I. To describe the demographic characteristics, clinical presentation, respiratory pathogens of children aged <5 years hospitalized with SARI in an urban (Chris Hani-Baragwanath Hospital, Soweto) and a rural (Matikwana and Mapuleng Hospitals, Mpumalanga) setting in South Africa from 2009-2013 and II. To compare the factors associated with mortality among children aged <5 years hospitalized with SARI in these two sites separately. Methods Hospitalized children with SARI were enrolled into an active, prospective sentinel surveillance program. Clinical and epidemiologic data were collected until discharge. Nasopharyngeal aspirates were tested for influenza (A and B) and eight other respiratory viruses. In-hospital case-fatality proportion (CFP) and risk factors for mortality were determined for each hospital site separately using unconditional logistic regression. Results The in-hospital CFP was significantly higher in the rural (6.9%, 103/1486) than the urban (1.3%, 51/3811) site (p<0.001). This was observed among both HIV-infected (urban: 6.6%, 17/257) vs. (rural: 12.9%, 30/233) (p=0.019) and HIV-uninfected children (urban: 0.6%, 13/2236) vs. (rural: 4.2% 36/857) (p<0.001). In the urban site the only factor that is independently associated with death on multivariate analysis was HIV infection (odds ratio (OR) 12.1, 95% confidence interval (CI) 5.8-25.2). In the rural site HIV infection (OR 3.5, 95% CI 1.7-6.9), age <1 year (OR 3.5, 95% CI 2.0-6.1) vs. 1-4 years, any respiratory virus detected (OR 0.4, 95% CI 0.2-0.6), pneumococcal infection(OR 4.5, 95% CI 1.8-10.8) and malnutrition (OR 12.8, 95%CI 1.2-134.6) were independently associated with mortality. Conclusion SARI mortality was higher in the rural setting. Even in the era of PCV availability pneumococcus is still associated with mortality in rural areas. Efforts to prevent and treat HIV infections in children and reduce malnutrition may reduce SARI deaths. / MT2017

Severe acute respiratory syndrome (SARS) is an emerging infectious disease that first manifested in humans in China in November 2002 and has subsequently spread worldwide. According to the World Health Organization, 8098 cases occurred during the outbreak, and healthcare workers accounted for 1707 (21%) of the cases. To determine the prevalence of SARS infection of healthcare workers in Taiwan, we performed a serosurvey by the recombinant protein-based enzyme-linked immunosorbent assay (ELISA) to test for immunoglobulin (Ig) G antibodies to the SARS coronavirus (SARS-CoV) among 1525 healthcare workers in 26 hospitals that admitted SARS patients in mid-May, 2003. Then, a case-control study was carried out to evaluate the risk factors of SARS infection among the healthcare workers. A total of 52 infected staffs and 78 hospital and age matched non-infected controls were recruited. The seroprevalence rate was 3.68% (58/1525) for healthcare workers. Univariate analysis showed that with the habit of drinking coffee or tea, taking care of fever patients more than 8 days, ever practice of CPR, suction of sputum, taking patient¡¦s temperature, use of P100 mask, use of N95 mask, use of face cover, use of goggles, use of gown, removing gloves after work, working in isolation area or fever screen station were significantly protective factors. In addition, eating jujube was a risk factor for SARS infection. Then, the multivariate analysis showed that use of P100 ¡]OR: 0.056, 95%CI: 0.019-0.162, p value: <0.001¡^and working in isolation area ¡]OR: 0.153, 95%CI: 0.029-0.810, p value: 0.027¡^or fever screen station¡]OR: 0.103, 95%CI: 0.011-0.963, p value: 0.046¡^were the most important protective factors for SARS infection. These findings suggest that nosocomial infection of SARS can be prevented effectively by use of P100 and the triage screening in emergency departments.

In part ONE of this thesis, including the most up to date information on SARS virology, disease transmission, pathogenesis and laboratory diagnosis will be summarized and presented, including the results of many studies in which I have participated (these references will be underlined as they appear in text). This of course summarizes knowledge that is now known in 2006 but was largely unknown during the initial outbreak. In part TWO, six original clinical studies performed at PWH will be presented: study (1) describes the clinical manifestations and severity of SARS, and its potential to cause major hospital outbreaks; (2) demonstrates the importance of epidemiological linkage in diagnosing SARS; (3) reports the clinical outcomes of a stepwise treatment protocol, which includes the use of corticosteroid therapy as an immunomodulant; (4) demonstrates that corticosteroid therapy can retard viral clearance, and should be used judiciously; (5) demonstrates that a more robust humoral response is associated with severe SARS, thus indicating that passive immunity treatment strategies seem only suitable either during early illness or as prophylaxis; and (6) shows that SARS has few early discriminating laboratory features compared to other causes of community-acquired pneumonia, thus a high index of suspicion is needed to recognize this infection in the absence of worldwide transmission. A thorough review of the relevant published material will be included in the discussion section of each study. / Severe Acute Respiratory Syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. It caused a global outbreak in 2003, resulting in more than 8000 infections, 700 deaths, and major social and economic disruption. In the initial phase of the SARS outbreak, the medical profession had no knowledge regarding the responsible pathogen, nor the clinical manifestations of SARS and the course of illness. There was no reliable diagnostic tool and no known effective therapy. But for the first time in medical history, we witnessed the rapid accumulation of knowledge on a disease as it evolved, which in turn assisted its management and control. / Since conducting randomized-controlled trials during the 2003 crisis was almost impossible, most of the presented studies are either descriptive or case-controlled in design. However, these studies have laid foundations for recent and future research into the clinical diagnosis and management of SARS. Moreover, the construction of the SARS clinical database has contributed to the work of other investigators, which has resulted in over thirty-six publications. It is my hope that these research endeavors can contribute to the understanding of this emerging, deadly disease. / Lee Lai Shun, Nelson. / "April 2006." / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0205. / Thesis (M.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 264-292). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The thesis examines a mediated crisis and how The Straits Times and The Australian approach the reporting of Severe Acute Respiratory Syndrome (SARS). It looks at how this mediated crisis exemplifies the culture of the national newspaper and in turn how the national newspaper has an historical influence on the national psyche. A total of 649 reports and headlines and 141 letters about SARS in The Straits Times (including The Straits Time Interactive) were examined from April 2003 to November 2003 as were 125 headlines from The Australian. The early sections of the thesis discuss how a crisis makes news; examine how the media report a crisis and what emphasis is given to aspects such as: actors, primary definers, vocabulary, lexical choices, subjects, themes, issues and value dimension or stance. The first chapter defines crisis, journalism and crisis journalism and discusses where the latter sits within the continuing expansion and development of major theoretical frameworks, including living in a risk society. The implication here is that crisis and risk have a symbiotic relationship. Historical perspectives of news are discussed in Chapter 2, and the newspaper is placed within the context of contemporary media. The chapter discusses how newspapers are aligned with the concept of the national mind and demonstrates the roles and formations of the two newspapers in relation to the SARS crisis. Chapter 3 codes the headlines, article titles and subtitles of The Straits Times and The Australian and using content analysis of the headlines, analyses the reporting of a serious health crisis SARS that lasted from March to November, 2003. The quantification within content analysis enables a researcher to read and interpret questions that relate to the intensity of meaning in texts, their social impact, the relationships between media texts and the realities and representations they reflect (Hansen et al, 1998). The theory and method of content analysis is used in this chapter to consider differences between The Straits Times and The Australian and to exemplify the media’s representation of the narratives of SARS as it happened in the countries of Singapore and Australia. Aspects of crisis and risk, the newspaper and the national mind, narratives, presentations, and post SARS events are discussed in the last chapter. It is concluded from these discussions there is a world narrative that tells the story of how the human condition likes to live and rely on a safe social environment always being available. The relationship between a mediated crisis and risk are also discussed. In addition, it is maintained that reporting in 2003 was not just about SARS but a way of reporting that allowed one to view journalism as an aid to good governance, particularly with regard to living in a risk and crisis-ridden society.

Acute Respiratory Distress Syndrome (ARDS) is an inflammatory disease characterized by pulmonary edema, stiff lungs and hypoxemia. Patients with ARDS are more susceptible to VILI (ventilator induced lung injury). Under mechanical ventilation, lung stress and strain are the main determinants of VILI and in patients with muscle effort patient-ventilator asynchrony may enhance this phenomenon. Ventilation modes PCV and VCV with auto-flow can minimize patient-ventilator asynchrony, but then can liberate the offer of flow and tidal volume, compromising the protective ventilatory strategy in ARDS. This study aimed to evaluate the influence of muscle effort and patient-ventilator asynchrony on pulmonary stress and strain in a mechanic lung model of acute respiratory distress syndrome. An experimental bench study was performed, using a lung simulator, ASL 5000TM, in which was configured a lung model with restrictive respiratory mechanics with complacency of 25ml/cmH2O and resistance of 10 cmH2O/L/sec. Muscle effort was adjusted in three situations: no muscular effort (Pmus = 0), with inspiratory muscle effort (Pmus = -5 cmH2O) and inspiratory and expiratory effort (Pmus = -5/+5 cmH2O), all with breathe rate (b) of 20 bpm. Five ventilators were connected to the simulator through and endotracheal tube No 8.0 mm and adjusted on VCV, VCV with Auto-flowTM (in the ventilator in which it was available) and PCV modes, all with tidal volume (VT): 420 ml, PEEP: 10 cmH2O and breath rate set in two situations: b = 15 bpm (lower than b of the respiratory muscle effort) and b = 25 bpm (higher than b of the respiratory muscle effort). Variables analyzed were: maximum VT, alveolar pressure at the end of inspiration, effective PEEP, driving pressure, transpulmonary pressure at the end of inspiration and expiration, average transpulmonary pressure, inspiratory peak flow and analysis of mechanic curves. In the studied lung model the b of the ventilator adjusted higher of the b of the patient and not the muscle effort was the main determinant for the development of patient-ventilator asynchrony, causing large variations of the VT and pulmonary pressures, intensifying the lung stress and strain. The ventilatory modes had similar behavior, although VCV Auto-flowTM and PCV have presented slightly higher values of VT and pulmonary pressures. Thus it is concluded that the proper adjustment of the programed breath rate in the assisted/controlled modes can minimize patient-ventilator asynchrony, reducing lung stress and strain. / A SÃndrome da AngÃstia RespiratÃria Aguda (SARA) à uma doenÃa inflamatÃria caracterizada por edema pulmonar, pulmÃes rÃgidos e hipoxemia. Pacientes com SARA estÃo mais suscetÃveis à VILI (ventilator induced lung injury). Sob ventilaÃÃo mecÃnica, o stress e o strain pulmonares sÃo os principais determinantes da VILI e nos pacientes com esforÃo muscular a assincronia paciente-ventilador pode potencializar este fenÃmeno. Os modos ventilatÃrios PCV e VCV com AutoFlow podem minimizar a assincronia paciente-ventilador, mas por outro lado podem liberar a oferta de fluxo e volume corrente, comprometendo a estratÃgia ventilatÃria protetora na SARA. Objetivou-se avaliar as influÃncias do esforÃo muscular e da assincronia paciente-ventilador sobre o âstrainâ e o âstressâ pulmonares em modelo pulmonar mecÃnico de sÃndrome da angÃstia respiratÃria aguda. Foi realizado um estudo experimental de bancada, utilizando um simulador de pulmÃo, ASL 5000 no qual foi configurado um modelo pulmonar com mecÃnica respiratÃria restritiva, com complacÃncia de 25ml/cmH2O e resistÃncia de 10 cmH2O/L/sec. O esforÃo muscular foi ajustado em trÃs situaÃÃes: sem esforÃo muscular (Pmus=0), com esforÃo muscular inspiratÃrio (Pmus= -5cmH2O) e esforÃo inspiratÃrio e expiratÃrio (Pmus= -5/+5 cmH2O), todos com frequÃncia respiratÃria (f) de 20rpm. Ao simulador foram conectados cinco ventiladores atravÃs de um tubo orotraqueal n 8,0 mm e ajustados nos modos VCV, VCV com sistema AutoFlow (no ventilador que tinha o sistema disponÃvel) e PCV, todos com volume corrente (VC): 420 ml, PEEP: 10 cmH2O e frequÃncia respiratÃria programada em duas situaÃÃes: f=15rpm (< que a f de esforÃo muscular respiratÃrio) e f=25rpm (> que a f de esforÃo muscular respiratÃrio). As variÃveis analisadas foram: VC mÃximo, a pressÃo alveolar no final da inspiraÃÃo, PEEP efetiva, driving pressure, pressÃo transpulmonar no final da inspiraÃÃo e expiraÃÃo, pressÃo transpulmonar mÃdia, pico de fluxo inspiratÃrio e anÃlise das curvas de mecÃnica. No modelo pulmonar estudado a f do ventilador pulmonar ajustada acima da f do paciente e nÃo o esforÃo muscular o principal determinante para o desenvolvimento de assincronia paciente ventilador, causando grandes variaÃÃes de VC e pressÃes pulmonares, o que intensificou o stress e strain pulmonares. Os modos ventilatÃrios tiveram comportamento semelhante, embora os modos VCV AutoFlow e PCV tenham apresentado valores discretamente maiores de VC e pressÃes pulmonares. Desta forma conclui-se que o ajuste adequado da frequÃncia programada nos modos assistido/controlado podem pode minimizar a assincronia paciente ventilador reduzindo o stress e strain pulmonares. Palavras-

Masters of Science / The Coronaviridae family consists of RNA viruses within the order Nidovirales. The family is classified into two genera, namely the corona- and toroviruses. Coronaviruses are enveloped, single stranded, positive sense RNA viruses with genomes ranging between 27-32kb in size. The 5’ two-thirds of the genome encodes for the 1a/b polyprotein, while the 3’ one-third of the genome encodes for the structural proteins that mediate viral entry into the host cell. These structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The nucleocapsid protein is expressed at high levels within an infected cell. Studies have shown that this protein plays a key regulatory role in different cellular pathways, including the inhibition of interferon production and the up-regulation of the AP1 signal transduction pathway, amongst others. Also, the N protein is vital in the formation of the ribonucleocapsid core by binding to the viral RNA during virion assembly. The focus of this study is the immune response in whole blood cultures to the presence of human coronavirus (HCoV) NL63 N protein. To characterise the stimulation of the immune activity against HCoV-NL63 N in blood cultures, the HCoV-NL63 N gene was expressed in a bacterial system. In this pilot study, GSTtagged N constructs were then purified and used to treat whole blood cultures from three volunteers. ELISAs were used to measure the cytokine response in these treated whole blood cultures. Results showed that the nucleocapsid protein has an inflammatory response on whole blood cultures. These results have generated vital information in the potential function of the HCoV-NL63 N protein on the immune system. It is suffice to say that the HCoV-NL63 N protein is able to elicit an effective inflammatory response within the host cell. Future studies into the cellular pathways affected by the HCoV-NL63 N protein will clarify its exact role in stimulating the host immune system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the coronavirus disease of 2019 (COVID-19) pandemic, which has infected millions of people worldwide. To date, several vaccines and antivirals have been developed against SARS-CoV-2; however, its tendency to mutate rapidly poses a continued threat to human health. As such, the development of better pan-coronavirus therapeutics is still required. Recently, the SARS-CoV-2 non-structural protein 13 (Nsp13) helicase has been shown to be an attractive therapeutic target given its high conservation rate among coronaviruses and indispensable role in viral replication. Based on this, we sought to further study the biochemical mechanisms behind Nsp13's binding and unwinding activities, along with its interactions with host cells, to provide further insight for future therapeutic development. To study the binding and unwinding activity of Nsp13, we site-specifically incorporated the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into Nsp13 to act as a bioorthogonal handle for fluorescent labelling. We identified five potential sites for AzF incorporation in Nsp13 and assessed their reactivities towards a conjugated Cy5 fluorophore through strain-promoted alkyne-azide cycloaddition (SPAAC). Further experiments were also performed to ensure that the unwinding activity was not adversely affected before these Nsp13-AzF constructs were utilized in fluorescence resonance energy transfer (FRET)-based binding assays. Ultimately, the F81AzF construct was identified to be the most suitable for monitoring the binding of Nsp13 to a series of nucleic acid substrates in a distance-dependent manner by FRET. The next steps of this project would be to implement F81AzF in single-molecule FRET (smFRET) experiments to directly monitor the positioning and dynamics of this helicase on its substrate. In addition, interactions between Nsp13 and host cellular and biological processes were investigated to provide further insight into potential mechanisms that can be exploited for novel therapeutic development. From transcriptomic profiling analyses of A549 cells, we uncovered that Nsp13 influences microRNA (miRNA) expression and signalling pathways; in particular, miR-146a, a potent mediator of inflammation and immune responses, was found to be induced upon Nsp13-overexpression. Further experiments revealed that this may lead to the inhibition of NF-κB signalling, through the repression of the upstream targets TRAF6 and IRAK1, to suppress the production of proinflammatory cytokines and facilitate viral infection. Collectively, from this work, we propose that further exploration of these miR-146a-mediated signalling pathways may present alternative strategies for antiviral investigations.

Background. Medical imaging has played an important role in the diagnosis, progress monitoring and follow-up of most disease entities, in particular chest infections. The emergence of a novel chest disease poses an immediate challenge to the pillars of imaging, namely chest radiography and computed tomography. The characteristic imaging appearances, differential diagnoses and diagnostic pitfalls need to be established for correct diagnosis and appropriate management. The sensitivity and utility of the different imaging modalities will also need to be addressed. / In the event of an outbreak or epidemic, these challenges are made more difficult by an overwhelming number of patients and limited resources. In March 2003, we were faced with such a situation in our institution and the disease was later termed Severe Acute Respiratory Syndrome (SARS). / Patients in Hong Kong were treated with a combination of an antiviral agent and corticosteroids in addition to respiratory support. The majority of patients improved with treatment, although between 20--36% required treatment in an intensive care unit. / Problems and importance. This novel disease of high infectivity, morbidity and mortality posed a major threat to public health and a challenge to health authorities both locally and internationally. With regard to medical imaging, the following research questions were identified: (1) What are the imaging signs of this new disease? (2) Does chest imaging provide a high degree of sensitivity for diagnosing the infection? (3) Are the imaging signs disease-specific or are they similar to other pathology? (4) Does the progressive evolution of the imaging appearance correlate with the clinical status of the patient? (5) Could the imaging appearance be useful for predicting the final outcome? (6) Are there complications that require detection by imaging? / The lung parenchyma is the main site of infection and the resultant microscopic pathology included: pulmonary exudate, sequestration of macrophages, diffuse alveolar damage, proliferation of epithelial cells and hyaline membrane formation. Macroscopic features include alveolar consolidation in the early stages and later, organizing pneumonia or bronchiolitis obliterans organizing pneumonia. / These answers to these questions are essential to our understanding of the disease and to increase our diagnostic ability. (Abstract shortened by UMI.) / This newly emerged disease is a respiratory infection with a high morbidity/mortality and was found to be caused by a coronavirus (SARS CoV). By the end of the outbreak a total of 8098 probable cases of infection were reported worldwide, with a mortality rate of 9.6% (774 deaths). Hong Kong was one of the hardest hit regions, totaling 1755 probable cases of infection and 299 deaths by the end of the outbreak. / Antonio Gregory Ernest. / "September 2005." / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3745. / Thesis (M.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 245-258). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

The objectives of this research study are: 1. To probe into the widespread period of severe acute respiratory syndrome. 2. To study on this highly contagious and rapid spread of this new kind of disease At the time when nearby emergency department of medical center breaks out a suspicion of nasocomial infection. First, our emergency department immediately formed a strict fever screening station. After comparing the outcome of the prevention and spreading of this disease by the set-up of the emergency fever station between the central and southern medical institution. The research period was from May 15, 2003to July 15, 2003. Our research targets made by the emergency fever station were around 3730 patients with a random selection of 300 cases. This research uses the structure of fever screening measurement questionnaires to gather information and then adapted the EP1-INFO 10.0 version of statistical analysis. The results of the research are as follows: 1. Chief complaint of sore throat (38.3%) and fever (17%) fitted to the clinical symptoms of SARS. In relation to SARS before and after the spread of the disease, there are still other complaints such as the gastrointestinal system (18.7%) and cardiovascular disease (16.7%) that showed no obvious difference. 2. During the period of emergency fever screening station, an additional 50% of manpower are being arranged to screen probable or non-probable affected cases. (from the 300 randomly selected cases) 3. There are no obvious difference showed after comparing the outcome of the prevention and spreading of this disease by the set-up of the emergency fever center between the central and southern medical institution. 4. After tallying the number of doctors and nurses participated in screening procedure and number of non-medical staff developed similar symptoms to SARS, we can see the result of total number of medical staff from the emergency fever screening station that can successfully control the spread and prevention of the disease, making it the standard and model in the prevention and control of other communicable disease in the future. Key words: Severe acute respiratory syndrome, fever screening station, emergency room, emergency task force

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Coronaviridae family is composed of a number of ribonucleic acid (RNA)-containing viruses currently classified into two genera, the coronavirus and torovirus. The family is classified together with the Arteviridae in the order Nidovirales. Coronaviruses are enveloped single stranded positive sense RNA viruses about 80-160 nm in diameter. The coronavirus is, as in the case of all positive sense RNA virus, a messenger, and the naked RNA is infectious. The 5′-two thirds of the genome encodes for a polyprotein that contains all the enzymes necessary for replication, whereas the 3′-one third encodes for all the structural proteins that mediate viral entry into the host cell. The structural proteins include spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins.Nucleocapsid protein is one of the most crucial structural components of coronaviruses;hence major attention has been focused on characterization of this protein. Some laboratories have demonstrated that this protein interferes with different cellular pathways, thus implying it to be a key regulatory component of the virus (Zakhartchouk, Viswanathan et al. 2005). Furthermore, it has been shown that severe acute respiratory syndrome (SARS)-N protein interacts with cellular proteins, including cyclophilin A (CypA), heterogenous nuclear ribonucleoprotein (hnRNP) A1, human ubiquitin-conjugating enzyme, cyclin dependent kinase (CDK)-cyclin complex protein, Ikappaßalpha (IkBα), cytochrome (Cyt) P450 etc. For the purpose of this study, the focus is based on CypA interaction with human coronavirus (HCoV) NL63-N protein. These interactions might play a role in the pathology of HCoV-NL63. Using glutathione-S-transferase (GST), the interaction of CypA with the nucleocapsid protein can be clearly demonstrated to be direct and specific. Since the N protein is involved in viral RNA packaging to form a helical core, it is suffice to say that both NL63-N and CypA are possibly within the HCoV-NL63 replication/transcription complex and NL63-N/human CypA interaction might function in the regulation of HCoV-NL63 RNA synthesis. In addition, the results will demonstrate that HCoV-NL63-N has only a specific domain for interacting with CypA.