Management of fungal infections: diagnosis and human genetic susceptibility

Invasive 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.

Name thesis:degree_name

Doutoramento em Ciências da Saúde (especialidade em Ciências Biológicas e Biomédicas)

Year dc:date.issued

2008

Author dc:creator

• Carvalho, Agostinho Albérico Rodrigues de

Advisors dc:contributor.advisor

• Rodrigues, Fernando José dos Santos
• Maciel, P.

Statement dc:rights

• openAccess

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1822/8163

OAI record oai:identifier

http://repositorium.sdum.uminho.pt/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arepositorium.uminho.pt%3A1822%2F8163