Epidemiology and risk factors for candidaemia at Chris Hani Baragwanath hospital (2009-2010)

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment for the requirements for the degree of Master of Medicine in Microbiology. Johannesburg, 2017Background Invasive Candida infections (ICI) have emerged as an important cause of increased morbidity and mortality in specific patient populations in recent years. Multiple risk factors coupled with changes in epidemiology have made clinical management of these patients challenging. A laboratory-based surveillance project, Tracking Resistance to Antifungal drugs for Candida species in South Africa (TRAC-SA) was conducted at Chris Hani Baragwanath Hospital (CHBH) from 2009 to 2010 and allowed for collection of laboratory information related to episodes of candidaemia, delineation of the situation at the hospital and distribution of information to relevant stakeholders to help make informed clinical decisions. Objective Determine the clinical epidemiology and risk factors for bloodstream Candida infection at CHBH over an 18-month period Methods A retrospective, cross-sectional analysis was carried out on cases of blood culture-confirmed candidaemia from inpatients from 1 February 2009 until 31 August 2010. These cases were identified from the TRAC-SA database, inpatient files were traced and clinical data recorded on a standard case report form. Additional laboratory data of selected tests done within 72 hours of the initial blood culture were obtained from the National Laboratory Health Service Corporate Data Warehouse (CDW). Results A total of 167 episodes of candidaemia were identified during the study period with an incidence of 2.09 per 1000 admissions. The distribution of episodes occurred among 55 children (33%), 41 adults (25%) and 71 neonates (43%). The overall species distribution was Candida species other than C. albicans (98/167, 58.7%) and C. albicans (69/167, 41.3%). Candida species other than C. albicans comprised mainly of C. parapsilosis (73/167, 43.7%), C. glabrata (10.2 %, 17/167) and other species combined including C. tropicalis and C. krusei (8/167, 4.7%). Factors associated with C. albicans (versus Candida species other than C. albicans) infection included older age, use of 2 or more antibiotics, use of broad spectrum antibiotics specifically meropenem, aminoglycosides, vancomycin, co-trimoxazole and mechanical ventilation (p < 0.001). The overall case-fatality was 59/163 (35.3%). The highest case fatality was noted among adults with C. albicans infection, i.e. 15/22 (68.18%). Significant risk factors associated with in-hospital mortality were use of central lines, urinary catheters, total parenteral nutrition, 2 or more antibiotics, beta lactam - beta lactamase inhibitors, proton pump inhibitors, aminoglycoside and abdominal surgery (p < 0.01). Of the C. parapsilosis isolates tested, 40 (57. 9%) tested non-susceptible to fluconazole. Risk factors associated with fluconazole resistance included neonatal age, involvement of the respiratory system, mechanical ventilation, chemotherapy, use of a prior antifungal agent and use of 2 or broader spectrum antibiotics (p<0.01). Of 71 neonates, 16 (22.5%) received empiric antifungals, in comparison to children (5/55, 9.0%) and adults (4/41, 9.7%) (p = 0.272). Conclusion CHBH had a high incidence of candidaemia with a predominance of Candida species other than C. albicans especially in the neonate age group. Risk stratification of in-patients is of paramount importance in choice of empiric antifungal drug due to the differing azole resistance patterns observed.MT201

Biological characteristics of polysccharide based contrast agents for cancer diagnostics

Despite all the progress made in the treatment of cancer in recent years, it is still necessary to continue with the research of more effective and specific drugs. In recent years, there has been a growing interest in personalized medicine and its application through drug delivery systems, which could help increase the specificity of cancer treatment and subsequently its effectiveness. Drug delivery systems can use different platforms for their design, whether they are liposomes, micelles, nano crystals or others. A very interesting platform for the construction of drug delivery systems are polysaccharides, which were, as carriers of contrast agents in order to effectively display tumours, characterized in this doctoral thesis. But polysaccharides are interesting for more reasons. Both by its availability, and by its biocompatibility and non-toxic character. In this doctoral thesis we deal with two types of polysaccharides conjugates with linked contrast agents for magnetic resonance and fluorescent imaging. The first type of polysaccharide is glycogen, the second is mannan. Both constructs - glycogen and mannan based, were synthesized in a version with and without polymethyloxazolin, which should prolong their circulation in the organism. Both types of polysaccharide conjugates used passive...I přes veškerý pokrok v léčbě nádorových onemocnění, který proběhl v uplynulých letech, je stále nutné ve výzkumu účinnějších a specifičtějších léčiv pokračovat. V posledních letech je na vzestupu zájem o personalizovanou medicínu a její uplanění skrze nosiče léčiv (drug delivery systémy), které by mohly pomoci se zvýšením specificity léčby nádorových onemocnění a následně i účinnosti. Drug delivery systémy mohou využívat různých platforem pro svoji konstrukci, ať už jde o liposómy, micely, nanokrystaly či jiné. Velmi zajímavou platformu pro konstrukci drug delivery systémů představují polysacharidy, které byly, jakožto nosiče kontrastních látek s cílem efektivního zobrazení nádorů, charakterizovány v této disertační práci. Polysacharidy jsou zajímavé z více důvodů. Jednak svojí dostupností, dále také svojí biokompatibilitou a netoxickým charakterem. V této disertační práci se zabýváme dvěma typy polysacharidových konjugátů s navázanými kontrastními látkami pro magnetickou rezonanci a fluorescenční zobrazování. Prvním typem polysacharidu je glykogen, druhým pak manan. Oba konstrukty, jak glykogenový, tak i mananový, byly nasyntetizovány ve verzi s i bez polymetyloxazolinu, který by měl prodloužit jejich cirkulaci v organismu. Oba typy polysacharidových konjugátů využívaly pasivního cílení do nádoru...Department of PhysiologyKatedra fyziologieFaculty of SciencePřírodovědecká fakult

Biological characteristics of polysccharide based contrast agents for cancer diagnostics

Despite all the progress made in the treatment of cancer in recent years, it is still necessary to continue with the research of more effective and specific drugs. In recent years, there has been a growing interest in personalized medicine and its application through drug delivery systems, which could help increase the specificity of cancer treatment and subsequently its effectiveness. Drug delivery systems can use different platforms for their design, whether they are liposomes, micelles, nano crystals or others. A very interesting platform for the construction of drug delivery systems are polysaccharides, which were, as carriers of contrast agents in order to effectively display tumours, characterized in this doctoral thesis. But polysaccharides are interesting for more reasons. Both by its availability, and by its biocompatibility and non-toxic character. In this doctoral thesis we deal with two types of polysaccharides conjugates with linked contrast agents for magnetic resonance and fluorescent imaging. The first type of polysaccharide is glycogen, the second is mannan. Both constructs - glycogen and mannan based, were synthesized in a version with and without polymethyloxazolin, which should prolong their circulation in the organism. Both types of polysaccharide conjugates used passive...I přes veškerý pokrok v léčbě nádorových onemocnění, který proběhl v uplynulých letech, je stále nutné ve výzkumu účinnějších a specifičtějších léčiv pokračovat. V posledních letech je na vzestupu zájem o personalizovanou medicínu a její uplanění skrze nosiče léčiv (drug delivery systémy), které by mohly pomoci se zvýšením specificity léčby nádorových onemocnění a následně i účinnosti. Drug delivery systémy mohou využívat různých platforem pro svoji konstrukci, ať už jde o liposómy, micely, nanokrystaly či jiné. Velmi zajímavou platformu pro konstrukci drug delivery systémů představují polysacharidy, které byly, jakožto nosiče kontrastních látek s cílem efektivního zobrazení nádorů, charakterizovány v této disertační práci. Polysacharidy jsou zajímavé z více důvodů. Jednak svojí dostupností, dále také svojí biokompatibilitou a netoxickým charakterem. V této disertační práci se zabýváme dvěma typy polysacharidových konjugátů s navázanými kontrastními látkami pro magnetickou rezonanci a fluorescenční zobrazování. Prvním typem polysacharidu je glykogen, druhým pak manan. Oba konstrukty, jak glykogenový, tak i mananový, byly nasyntetizovány ve verzi s i bez polymetyloxazolinu, který by měl prodloužit jejich cirkulaci v organismu. Oba typy polysacharidových konjugátů využívaly pasivního cílení do nádoru...Department of PhysiologyKatedra fyziologiePřírodovědecká fakultaFaculty of Scienc

Synthesis and characterization of caffeic acid and dihydrocaffeic acid derivatives as antifungal agents

Invasive fungal infection, a major cause of morbidity and mortality in immuno-compromised hosts, is of importance and is significantly increasing in incidence in recent years. Although there are a number of antifungal agents currently available, they are associated with various limitations. Being inhibitors of fungal cell wall biosynthesis, the class of echinocandins is characterized by outstanding safety profiles and great potential in combination antifungal therapy, but they fall short in oral bioavailability. A discovery of peptidomimetic analogues of echinocandin B, with potential inhibitory activity against 1,3-β-D-glucan synthase (an enzyme essential for the biosynthesis of fungal cell walls), was initiated by our research group. A series of structurally related derivatives (chlorogenic, quinic, caffeic and dihydrocaffeic acid derivatives) were later designed to mimic the backbone of echinocandins. Further studies have been carried out on optimization of synthesis and modifications of a caffeic acid derivative. Due to the instability problems of previously reported compounds, a new molecule, a dihydrocaffeic acid derivative, has been designed and synthesized. It was found that adopting a few optimized procedures can not only improve the yield and purity of the caffeic acid derivatives, but also save a lot of time in synthesis

Incidencia y factores pronósticos para el desarrollo de infección fúngica invasiva en pacientes hematológicos de alto riesgo

La infección fúngica invasiva (IFI) es una causa cada vez más frecuente de morbi-mortalidad en pacientes con enfermedades hematológicas malignas en tratamiento con quimioterapia intensiva y los receptores de un trasplante alogénico de progenitores hematopoyético (alo-TPH). El objetivo de esta tesis por compendio de publicaciones fue realizar un estudio epidemiológico de la IFI en nuestro entorno y, recoger la práctica clínica habitual en la profilaxis y el tratamiento de la IFI. Para ello se recogen los resultados de tres estudios publicados en revistas de alto impacto especializadas en hematología y trasplante hematopoyético. Los estudios comparten un nexo común, el análisis de las infecciones fúngicas invasivas en dos grupos de pacientes con muy alto riesgo de desarrollar estas infecciones graves y en los que se usan diferentes estrategias de profilaxis antifúngica, como son los receptores de un alo-TPH y los pacientes con leucemia mieloblástica aguda (LMA) sometidos a quimioterapia intensiva. Estos estudios prestan especialmente atención al impacto de la profilaxis antifúngica administrada (eficacia, toxicidad, desarrollo de IFI de brecha), a la epidemiología de la IFI en nuestro entorno, a la incidencia y evolución de esta complicación, analizando también los factores de riesgo que permitan una mejor adaptación, si es necesario, de las estrategias profilácticas y terapéuticas disponibles. Sus conclusiones fueron: 1. La incidencia de IFI en pacientes hematológicos de alto riesgo en nuestro entorno continúa siendo elevada. Entre el 8 y el 11% de los receptores de alo-TPH desarrollaron una IFI y hasta el 10% en pacientes con LMA en tratamiento intensivo. 2. El pulmón fue el órgano más frecuentemente afectado y los hongos aislados con más frecuencia fueron del género Aspergillus. 3. Se identificaron cinco variables con valor pronóstico independiente en pacientes sometidos a alo-TPH tras el injerto: edad mayor a 40 años, TPH previo, neutropenia antes del injerto de más de 15 días, reactivación de CMV antes del día 180 y EICH crónico extenso. Estas variables sirvieron para construir un modelo de predicción multivariable que fue validado y logra segregar a los pacientes en riesgo bajo, intermedio o alto de desarrollar IFI. 4. La profilaxis con itraconazol y voriconazol a dosis bajas (100 mg/12h) resultó ineficaz para prevenir episodios de IFI en receptores de alo-TPH. La profilaxis con voriconazol a dosis plenas (200 mg/12h) redujo el riesgo de IFI tras el injerto mieloide en los pacientes de riesgo bajo o intermedio, pero no tuvo un impacto en los pacientes de alto riesgo. El voriconazol a dosis plenas (200 mg/12h) mostró una toxicidad similar al voriconazol a dosis bajas. 5. La profilaxis con voriconazol a dosis plena (200 mg/12h) en pacientes con LMA tratados con quimioterapia intensiva de primera línea demostró reducir la incidencia de IFI, el tiempo de hospitalización y el uso de terapia antifúngica empírica, cuando se comparó con la profilaxis con fluconazol o itraconazol. Otro factor protector de IFI encontrado en el análisis multivariante fue recibir un ciclo de quimioterapia sin antraciclinas (citarabina a altas dosis). 6. La revisión periódica de la incidencia de IFI, sus factores pronósticos, y el impacto de las profilaxis antifúngica utilizadas en cada centro resulta fundamental para optimizar las estrategias profilácticas, diagnósticas, y terapéuticas

Infecções fúngicas : diagnóstico e susceptibilidade genética humana

Tese de Doutoramento Ciências da Saúde - Ciências Biológicas e BiomédicasInvasive fungal infections represent nowadays a major public health problem with associated high mortality rates. The lack of adequate diagnostic methods, together with the fact that many emerging fungal species are resistant to the currently available antifungal agents, contributes to the profound impact of these diseases in the health care systems, especially when dealing with immunocompromised patients. Taking this into consideration, the development of novel diagnostic applications has been considered a critical issue in recent years. We described a multiplex PCR-based strategy allowing the identification of eight of the most clinically relevant Candida species. The strategy, based on the amplification of fragments from the internal transcribed spacer regions of the ribosomal RNA genes, was shown to present both high specificity and sensitivity, in addition to other attractive features, including the individual discrimination of species present in mixture and the direct identification from clinical specimens, characteristics reinforcing the potential clinical application of the method. In addition to the need of more satisfactory diagnostic methods, the understanding of the host-fungi interaction, namely at the level of host genetic susceptibility, is critical to advance the knowledge regarding these infections and, in particular, the individual risk factors predisposing to them. Differences in human susceptibility to infectious diseases have been widely described with recent examples focusing on genetic variations within genes of the innate immune system, such as Toll-like receptors (TLRs), which may alter host-pathogen defence mechanisms, thus affecting susceptibility to infectious diseases, and in particular, fungal infections. Taking this into account, we developed a simple and rapid method based in the bi-directional PCR amplification of specific alleles (Bi-PASA) for genotyping known sequence variants in the TLR genes to be used in the forthcoming association studies regarding genetic susceptibility to fungal infections. The development of this methodology also allowed us to perform a characterization of the general Portuguese population regarding these polymorphisms, that can be used in future association studies, besides providing valuable information regarding stratification of patients most at risk of infection. Following the demonstration of the usefulness of Bi-PASA, we investigated the potential association between polymorphisms in the TLR genes and susceptibility to non-invasive forms of pulmonary aspergillosis. A significant association was observed between the presence of Asp299Gly (TLR4) and chronic cavitary pulmonary aspergillosis. In the same way, this variation was also linked with fungal colonization in the haematopoietic stem cell transplantation (HSCT) setting, suggesting that an abnormal TLR4 extracellular domain may be impairing the recognition of the fungus, thus contributing to an increased predisposition to these diseases. However, the same polymorphism was previously shown to have a protective role against invasive aspergillosis in HSCT patients. Thus, as shown for hyper-inflammatory states, such as atherosclerosis, impairments in the production of inflammatory cytokines contributing to disease susceptibility may be compensating the effect of the defective TLR4. Furthermore, susceptibility to another form of pulmonary aspergillosis, allergic bronchopulmonary aspergillosis, as well as viral infection in the HSCT setting, were shown to be associated with T-1237C (TLR9), highlighting the divergent function of TLRs in the pathogenesis of these infections. A shared susceptibility mechanism involving the T-1237C polymorphism in the promoter region of TLR9 was also observed to predispose to the development of non-Hodgkin lymphoma (NHL). This disease includes a set of heterogeneous lymphoproliferative malignancies often associated with an altered immunological function of the host and chronic inflammatory type of infections, in which TLR9 was already shown to play a critical role. The T-1237C polymorphism introduces a regulatory site that is trans-activated by the IL-6-dependent transcription factor IL-6 response element binding protein (IL-6 RE-BP), thus resulting in increased expression of TLR9. TLR9 activation of B lymphocytes leads to yet increased gene expression levels and sequentially to augmented proliferation rates, as well as higher production of IL-6. This was shown to result in a TLR9 activation loop leading to B lymphocyte-specific uncontrolled proliferation, making these cells more prone to acquire transforming mutations associated with the development of NHL. Besides uncovering a major risk factor for the development of NHL, the presented information has important implications on the recent usage of CpG agonists on several therapeutic strategies in cancer and autoimmune diseases. In summary, we have contributed to show that management of fungal infections, both invasive and non-invasive, involves not only the development of more satisfactory diagnostic procedures, but also considerable attention that has to be given to individual genetic variants that, as we showed, are able to alter susceptibility to these infections. These findings have potential relevance for the stratification of patients most at risk, not only of fungal infections, but also to diseases of other aetiological natures, whose pathogenesis share common signalling/activation pathways such as those presented by the TLRs.Actualmente, as infecções fúngicas invasivas representam um importante problema de saúde pública, sendo responsáveis por elevadas taxas de mortalidade. A falta de métodos de diagnóstico adequados, associada à resistência que muitas das espécies fúngicas emergentes apresentam aos antifúngicos disponíveis, contribui para um profundo impacto destas doenças, especialmente no caso dos doentes imunocomprometidos. Tendo em consideração estes aspectos, o desenvolvimento de novas metodologias de diagnóstico tem sido encarado como uma necessidade prioritária. Neste sentido, desenvolveu-se uma nova estratégia, baseada em PCR multiplex, que permite a identificação de oito das espécies de Candida mais relevantes em termos clínicos. Este método, baseado na amplificação de fragmentos dos genes de RNA ribossomal, apresenta uma elevada especificidade e sensibilidade. A sua potencial aplicação clínica é ainda reforçada por outras características, nomeadamente a discriminação individual de espécies presentes em co-infecção e a identificação directa a partir de espécimes clínicos. Juntamente com a necessidade de desenvolvimento de métodos de diagnóstico mais satisfatórios, a compreensão da interacção hospedeiro-fungo, nomeadamente ao nível da susceptibilidade genética humana, é essencial para o avanço do conhecimento destas infecções. Diferenças na susceptibilidade humana a doenças infecciosas têm sido descritas, associadas nomeadamente a variações em genes do sistema imunológico inato, como os receptores “Tolllike” (TLRs). Recentemente, verificou-se que estas variações podem alterar os mecanismos de defesa antimicrobianos, afectando a susceptibilidade a doenças infecciosas. Assim, tendo em conta estes aspectos, desenvolvemos um método simples e rápido para genotipar polimorfismos nos genes TLR, de forma a aplicá-lo na análise da susceptibilidade a infecções fúngicas nos estudos de associação subsequentes. O desenvolvimento desta metodologia permitiu ainda a caracterização da população Portuguesa em relação a estes polimorfismos, dados que poderão ser usados em estudos futuros, para além de fornecer informação pertinente acerca da estratificação de pacientes com maior risco de desenvolverem infecções. Após a demonstração da utilidade da técnica de Bi-PASA, investigámos a potencial associação entre polimorfismos nos genes TLR e a susceptibilidade a formas não-invasivas de aspergilose pulmonar. De acordo com os resultsdos obtidos, observou-se uma associação entre a presença de Asp299Gly (TLR4) e a aspergilose pulmonar crónica cavitária. De forma idêntica, verificou-se uma associação entre o mesmo polimorfismo e a colonização fúngica em doentes sujeitos a transplante de células estaminais hematopoiéticas (HSCT), sugerindo que um domínio extracelular anómalo de TLR4 pode limitar o reconhecimento do fungo, contribuindo para uma maior predisposição para estas doenças. Contudo, a mesma variação apresentou um papel protector em relação a aspergilose invasiva, sugerindo que uma redução na produção de citocinas pró-inflamatórias poderá equilibrar o defeito anterior, tal como foi anteriormente demonstrado para estados patológicos de hiper-inflamação, como a aterosclerose. Adicionalmente, demonstrámos que o polimorfismo T-1237C (TLR9) estava associado a uma maior susceptibilidade a aspergilose broncopulmonar alérgica, uma outra forma de aspergilose pulmonar, assim como a infecções virais em pacientes HSCT, realçando a função divergente dos TLRs na patogénese destas doenças. Um mecanismo de susceptibilidade partilhado envolvendo o polimorfismo T-1237C surgiu também como predispondo para o desenvolvimento de linfoma não-Hodgkin (NHL). Este inclui um conjunto de doenças linfoproliferativas, frequentemente associadas a alterações imunológicas do hospedeiro e infecções associadas a respostas inflamatórias crónicas, nas quais já foi descrito um papel importante de TLR9. O polimorfismo T-1237C introduz um local de regulação que é trans-activado por um factor de transcrição dependente de IL-6, resultando numa elevada expressão de TLR9. A activação deste receptor em linfócitos B leva a um aumento da sua expressão genética e, consequentemente, a taxas de proliferação mais elevadas, assim como à produção excessiva de IL-6. Estas alterações, juntamente com uma activação persistente de TLR9, culminam numa proliferação descontrolada dos linfócitos B, tornando estas células mais susceptíveis à aquisição de mutações transformantes associadas com o desenvolvimento de NHL. Para além de termos evidenciado o polimorfismo T-1237C como um factor de risco para o desenvolvimento de NHL, a informação resultante apresenta ainda importantes implicações no uso, recentemente preconizado, de agonistas de TLR9 em diversas estratégias terapêuticas, nomeadamente cancro e doenças autoimunes. Em resumo, demonstrámos que a abordagem das infecções fúngicas deve abranger não só um interesse particular no fungo, através do desenvolvimento de métodos de diagnóstico mais eficazes, mas também uma atenção considerável às variações genéticas individuais que, como demonstrámos, podem modular a susceptibilidade a estas infecções. Os resultados aqui apresentados têm uma potencial relevância na estratificação de pacientes com maior risco para infecções fúngicas, bem como doenças de outras etiologias, cuja patogénese partilha vias comuns de sinalização/activação, como as apresentadas pelos TLRs.Fundação para a Ciência e a Tecnologia (FCT) bolsa SFRH/BD/11837/200