Medicine - Doctoral Theses
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Item Evaluation of biomarkers for the diagnosis, management and follow-up of women with gestational trophoblastic disease(University College Cork, 2024) Joyce, Caroline Martha; O'Donoghue, Keelin; McCarthy, Tommie V.; Coulter, John; Irish Research CouncilGestational Trophoblastic Disease (GTD) describes a spectrum of disorders arising from the abnormal proliferation of trophoblastic tissue. Human chorionic gonadotrophin (hCG) is an excellent biomarker for most forms of this disease as its concentration accurately reflects trophoblastic activity. Hydatidiform mole (HM) or molar pregnancy is the most common form of GTD which may be suspected on ultrasound but requires pathological confirmation for diagnosis. Prognosis is generally excellent however close surveillance with hCG monitoring is imperative to detect cases progressing to malignant disease, referred to as gestational trophoblastic neoplasia (GTN). Clinical management and treatment of women with GTN in specialist centres achieves cure rates approaching 100%, emphasising the importance of accurate hCG monitoring for early detection of disease persistence to ensure optimal outcome. This thesis aims to evaluate pathological, genetic, and biochemical biomarkers used in the diagnosis, monitoring, and follow-up of women with GTD. In this thesis, a scoping review was performed to generate evidence and identify gaps in the GTD knowledge base to inform areas for future research. Key findings were the need to standardise hCG immunoassays for effective GTD surveillance, to improve diagnostic reporting pathways and to establish more accurate incidence rates. In order to identify patient priorities and inform initiatives to enhance the quality of care, a service evaluation was performed with women enrolled in the National GTD Registry invited to participate. Feedback revealed a knowledge gap regarding GTD amongst healthcare professionals outside GTD centres. This study also revealed shortcomings in psychological support and bereavement counselling offered to women after a molar pregnancy. To enhance the diagnostic accuracy of HM reporting, an in-situ hybridisation ploidy assay was developed with a customised scoring system to aid partial hydatidiform mole (PHM) diagnosis. This innovative technique provides a reliable adjunct to morphological assessment for PHM classification. The accuracy of this ploidy technique was confirmed by evaluation with molecular short tandem repeat genotyping. A national pathology questionnaire was performed to gather information on GTD diagnostic rates. Data collected was cross-referenced with cases documented in the National GTD Registry. This revealed a concerning under-enrolment of women with GTD (42%) onto the National GTD Registry by their clinicians. An audit of HM diagnostic rates over a 3-year period was performed following the Implementation of the new ploidy technique to aid diagnosis . This revealed a higher local PHM incidence rate (1 in 296 births) than reported nationally in the pathology audit (1 in 484 births). An electronic questionnaire was distributed to all European laboratories offering hCG for GTD management to gather insights on 5 key areas: hCG methodology, quality parameters, reporting and operational procedures, and use of hCG for non-GTD testing. This revealed considerable inter- and intra-laboratory variability in practice, emphasising the need to promote standardisation and harmonisation of immunoassays for GTD-derived hCG. A review of five challenging cases managed at the Irish GTD centre was conducted to evaluate the role of serum hCG and molecular genotyping in guiding management decisions, treatment strategies, and risk stratification for women with molar pregnancy, GTN and GTD mimics. These complex cases confirmed the pivotal role of serum hCG and molecular genotyping in monitoring disease persistence, prognostic stratification, and clinical management Novel strategies to measure hCG were pursued to address the lack of centralised hCG testing for GTD in Ireland. A proof-of-concept study for remote capillary blood collection was performed to evaluate the clinical efficacy and user acceptability of this method of collection for hCG monitoring in early pregnancy. The study confirmed the equivalence of capillary and venous blood hCG testing, demonstrated complete clinical concordance, thereby offering an alternative to venepuncture for hCG measurement. To explore the accessibility of hCG testing in the community, a point-of-care testing (POCT) device was evaluated for its suitability to triage women with pregnancy of unknown location (PUL) in the early pregnancy unit. The Abbott iSTAT®1 POCT instrument was chosen for this purpose and was clinically concordant with central laboratory hCG results facilitating the use of clinical decision thresholds for PUL. The research presented in this thesis provides valuable clinical insights through a series of nine research studies and has broadened the knowledge base in GTD. The findings offer an opportunity to positively impact diagnostic practices. The knowledge gap regarding GTD amongst healthcare professionals revealed in the patient survey points to a need for targeted educational initiatives in this area. Widespread integration of the HER2 ploidy technique to support morphology and standardise HM reporting could improve diagnostic accuracy, addressing a suspected underdiagnosis of PHM nationally and possibly internationally. It also suggests the need to re-evaluate incidence rates following the introduction of advanced diagnostic techniques such as ploidy assessment and molecular genotyping. This thesis identified an under-registration of women with GTD in Ireland supporting the need for initiatives such as mandatory registration or advocacy by professional organisations to increase registration rates. The inter and intra-laboratory variation in hCG immunoassays revealed in the European laboratory survey merits renewed harmonisation efforts through pilot external quality assurance and sample exchange programmes. The use of novel methods to enhance the accessibility of hCG monitoring for GTD in the community, through capillary blood collection or use of point-of-care analysis, warrants further study. The positive patient feedback indicated a preference for this approach, suggesting better compliance with serial test monitoring, a priority for GTD management.Item Mapping the microbiota in hidradenitis suppurativa(University College Cork, 0022) McCarthy, Siobhán; O'Toole, Paul W.; Shanahan, Fergus; Murphy, MichelleHidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by the formation of nodules, abscesses, and fistula at intertriginous sites. Pain, pruritus, malodour, and suppuration have a significant impact on quality of life for HS patients. The skin-gut axis is an area of emerging research in inflammatory skin disease and is a potential contributory factor to the pathogenesis of HS. Skin microbiome alterations in HS have become an area of expanding research with its role in the pathogenesis of HS becoming clearer. Gut microbiome alterations have been described in many inflammatory diseases including inflammatory bowel disease, rheumatoid arthritis, and cancer, but little is known about the gut microbiome in HS. Our hypothesis is that gut, as well as skin, microbiota alterations and their metabolites, may be responsible for skin inflammation in HS. The level of pain experienced by 150 patients with HS was measured using the visual analogue scale and EQ-5D-5L questionnaires. 59 patients with HS provided faecal samples, nasal and skin swabs of affected sites for analysis. These 59 patients also provided serum samples and completed questionnaires including the Dermatology Life Quality Index (DLQI), Morisky Medication Adherence Scale, International Physical Activity Questionnaire, and Food Frequency Questionnaire. 30 healthy controls provided fecal samples and 20 healthy controls provided nasal and skin swabs. We performed bacterial 16S rRNA gene amplicon sequencing on total DNA derived from the samples. Calprotectin was determined by enzyme-linked immunoassay (CALPROLAB™ Calprotectin ELISA (HRP). Complement C5a levels were assessed using enzyme-linked immunosorbent assay (Abcam ab193695 Complement C5a Human ELISA Kit). Pain was widely reported in 134 responders to the visual analog scale questionnaire. 82.1% reported some level of pain and 35.8% reported high levels of pain. In the EQ-5D-5L questionnaire, 81.4% of responders indicated that they felt pain or discomfort, and 31.0% expressed “severe” or “extreme” pain/discomfort. Weak correlation was seen between levels of pain and one marker of gut microbiota alpha diversity (Chao1), but this failed to be repeated for all indices of microbiota species richness. A significant impact on quality of life with a mean DLQI score of 12.15 (SD 7.67) was observed in patients with HS, which is in line with other severe dermatoses. 59% of patients reported a large or extremely large impact on their quality of life. The DLQI score did not correlate with gut microbial diversity. In this study, over 40% of patients with HS also reported some difficulty remembering to take their medications with 20% reporting that they forgot to take their medication in the previous 2 weeks. Patients with HS also reported moderate-to-high levels of physical activity; however this may be overestimated. Alterations in gut microbiota diversity did not correlate with levels of physical activity. Microbiome alpha diversity was significantly lower in the faecal, skin and nasal samples of individuals with HS which may be secondary to disease biology or related to antibiotic usage. Ruminococcus gnavus was more abundant in the faecal microbiome of individuals with HS, which is also reported in Crohn’s disease (CD), suggesting comorbidity due to shared gut microbiota alterations. No significant difference in faecal microbiota composition or overall habitual diet was seen in the patients with HS and diet was not associated with disease severity. Diet in HS was significantly different to controls, and similar to patients with CD. Finegoldia magna was over-abundant in HS skin samples relative to healthy controls. It is possible local inflammation is driven by F. magna through promoting the formation of neutrophil extracellular traps (NETs). 27.1% of patients with HS had a raised faecal calprotectin level of >50mg/kg with markedly elevated levels of >150mg/kg in 8.5% suggesting occult gastrointestinal inflammation. Median complement C5a level in patients with HS was elevated at 47 ng/ml (IQR 30.55 ng/ml) which is in keeping with prior studies. These alterations in both the gut and skin microbiome in HS warrant further exploration, and therapeutic strategies including faecal microbiota transplant or bacteriotherapy could be of benefit. Faecal calprotectin is a simple test which should be considered in patients with HS, with further investigations warranted in patients with elevated levels. C5a may provide a therapeutic target in patients with HS and serum C5a may act a biomarker of the disease.Item Characterisation of the effect of electroporation and electrochemotherapy on cancer cells and immune cells in the tumour microenvironment(University College Cork, 2022-10) Bendix, Maura; Brint, Elizabeth K.; Houston, Aileen M.; Amu, Sylvie; Forde, Patrick; Health Research Board; Breakthrough Cancer ResearchLung cancer is the leading cause of cancer-related death worldwide, with the lung cancer incidence rate expected to rise further. Despite recently developed novel therapy options, 5-year survival rates for lung cancer patients remains below 20% generally and below 5% for late-stage diagnosis, thus additional therapy options are still needed. Electrochemotherapy (ECT), the application of an electric pulse to deliver chemotherapy drugs into cells, could be a new treatment option for lung cancer patients. ECT is a locally very effective treatment, with local tumour reduction of up to 85%, while the systemic effects are more varied. For clinical application ECT treatment modalities have been standardized since 2006, after the ESOPE study, which optimized ECT parameters to 8 pulses at 1000V/cm with 100µs pulse length at 1Hz frequency and either bleomycin or cisplatin as the drug of choice. To evaluate whether ECT could be a potential treatment option for lung cancer patients’ ECT parameters, the needed electric field strength and the needed drug and drug concentration, were optimized for in vitro lung cancer research. In our study, we initially developed a standard operating protocol (SOP) to determine the optimal electric field strength for a given cancer cell line in vitro, while keeping the other ESOPE parameters constant. The developed SOP combined short-, medium-, and long-term assays to fully visualize the impact treatment, at a given field strength, has on the tested cancer cell line. This evaluation showed that human lung cancer cell lines (A549, H460 and SK-MES 1) and the human pancreatic Pan02 cell line have an optimal electric field strength of 800V/cm, the melanoma A375 (human) and B16F10 (murine) cell lines as well as the murine pancreatic Mia-PACA2 cell line have an optimal electric field strength of 700V/cm, while the murine Lewis Lung carcinoma (LLC) cell line has an optimal electric field strength of 1300V/cm. In addition, our study findings demonstrate that cisplatin at 11µM would be the drug of choice when using ECT for lung cancer treatments. In recent years while the importance of the immune system in lung cancer development and treatment results has become increasingly clear, little is known about how ECT treatments impact immune cells. Therefore, the impact of ECT on murine T cells, dendritic cells (DCs) and macrophages was evaluated in vitro. Our data indicates that while T cells are able to tolerate electric field strengths of up to 1400V/cm, DCs and macrophages are significantly negatively impacted by electric field strengths exceeding 800V/cm. Further investigation on the impact of ECT on dendritic cells demonstrated that DCs die via necrosis following ECT treatment, while ECT at electric field strength exceeding 1000V/cm leads to DC maturation and activation of the surviving cells. In addition, DCs remain partially functional following ECT treatment in a stimuli and treatment dependent manner with distinctively different sets of genes upregulated 4-hours post treatment at 800V/cm compared to treatment at 1000V/cm and 1300V/cm. Taken together, our data indicates that it is worthwhile to further investigate ECT as a potential therapy option for lung cancer patients, while more attention needs to be paid to the impact ECT has on immune cells in order to maximize treatment results.Item Phenotyping for a personalised approach to CVD prevention(University College Cork, 2022-03-01) McEvoy, John William; Eustace, Joe; Fanning, Liam J.Cardiovascular disease (CVD) prevention requires interventions at the level of the population (public health) and at the level of individuals (medical care). Therapeutic options to reduce the risk of CVD among at risk individuals are increasingly available, but access to and implementation of these evidence-based therapies is suboptimal and costs to health systems are often a deterrent to payers. Therefore, advanced phenotyping of individuals, for example using biomarkers of subclinical CVD, in order to better personalize their preventive care is an attractive approach. In the corpus of work presented for this PhD by prior publication, a series of eleven published papers is included that examines the role of biomarkers of subclinical CVD (specifically high-sensitivity Troponin measured in blood and coronary artery calcium imaging) in the management of persons with hypertension and abnormalities in diastolic blood pressure.Item Experiences of pregnancy with major fetal anomalies(University College Cork, 2020-09-24) Power Walsh, Stacey; O'Donoghue, Keelin; Meaney, SarahBackground: Two to three percent of pregnancies will receive a diagnosis of a congenital anomaly, of which a proportion are fatal. While the prevalence of congenital anomalies is low it is the leading cause of fetal death and child mortality. More women are receiving a diagnosis of a fetal anomaly during their pregnancy as a result of advancements in technology. While there is no universally agreed definition, the term fatal fetal anomaly (FFA) is widely used to describe a condition likely to lead to death of the fetus in utero or within 28 days of birth. Delivering a diagnosis of a FFA is usually unexpected and is an unwanted and traumatic event for parents. Little is known about what conditions are most responsible for perinatal mortality. Following a FFA diagnosis, parents face multiple challenges, primarily whether to continue and avail of PPC or terminate the affected pregnancy. Regardless of the choice, the outcome is ultimately the same, ending in a fetal, neonatal or infant death. The need for the decision to be an informed one is paramount. Information is an essential factor to develop patient’s knowledge and empowers them in their decision-making. Media offers an insight into health-related information widely available to the public. FFA has generated international media attention as termination of pregnancy (TOP) for FFA was legislated for, for the first time in Ireland. However, it is identified that what the people voted for has not materialised in the legislation relating to practical terms of delivering abortion care. Following pregnancy loss or perinatal death, parents require various levels of support during their bereavement. There is an over reliance on voluntary organisations to provide peer support to families to meet their needs that often surpass that of standard maternity hospital provision. Education is essential in healthcare in order to keep up-to-date with best practice. This is of particular importance as TOP for FFA, without gestational limits, was provided for within legislation for the first time in Ireland from 2019. This change in legislation warrants an exploration into Volunteers experiences of supporting families, and Fetal Medicine Specialists’ (FMS) experiences of caring for parents following a FFA diagnosis. The overall aim of this thesis was to explore various aspects of pregnancies diagnosed with FFA, during this change in service provision. Methodology: To address the thesis’s aims, both qualitative and quantitative methods were employed. Employing a mixed methods approach to study a phenomenon allows for flexibility to explore different aspects in each of the studies. A secondary analysis was undertaken on anonymised data obtained from the National Perinatal Epidemiology Centre, pertaining to perinatal deaths from January 2011 to December 2016 in Ireland. A national cross-sectional telephone survey was undertaken to assess the public knowledge on FFA. For these quantitative studies, descriptive and inferential statistics were utilised to analyse data. A critical discourse analysis was undertaken to examine the relations between discourse and social and cultural phenomena. Habermasian’s framework facilitated an objective analysis of text in the Irish media, to facilitate interpretation and understanding of socially produced meanings. A modified Delphi study, involving two rounds and inclusive of free text, was undertaken to identify educational needs of Volunteers. Lastly, for two of the qualitative studies an interpretive descriptive approach facilitated the researcher to delve into Volunteers’ and FMS experiences of supporting and caring for parents who receive a diagnosis of a FFA at a time where TOP for FFA is being provided for, for the first time. This inductive approach represents a co-constructed truth, it moves beyond the level of description of the phenomenon and articulates a meaning of and explanation for these experiences, generating implications for practice and application of these. Both of these studies adopted a data analysis methodology based on the principles of thematic analysis. Results: A lack of accurate knowledge relating to FFA, its classification, diagnosis and supports available to parents experiencing a pregnancy affected by a FFA among the general public was illustrated within this thesis. Additionally, the data identified misrepresentations in the information relating to FFA delivered to the general public. Outlined by these findings, opportunistic politicians were found to utilise the media to highlight their party’s ideological perspective regarding FFA, to derive political advantage to gain popularity and power. This thesis identified that the uncertainty of what conditions were deemed fatal in accordance with the legislation was related to the ambiguity and restrictiveness of the Irish TOP legislation as long-term survivors are known to many of the conditions considered a FFA. The lack of a universal agreement of what constitutes as a FFA and a list of conditions that are associated with this term, adds additional challenges to diagnosing a congenital anomaly as fatal. On examination of perinatal deaths with a congenital anomaly as a main cause of death, reported between 2011 and 2016, only 42% could be deemed a FFA in accordance with the criteria implemented by the Irish legislation. Many of the conditions in isolation may not be a FFA however, when combined they have the potential to be fatal. The suitability of the Irish legislation for TOP for FFA generated further difficulties for FMS due to its rapid introduction into clinical practice. FMS reported the lack of organisational support, time to prepare and additional resources as challenges for the introduction of a new service. These challenges were exacerbated by FMS fear of criminalisation attached to the TOP legislation if a TOP was carried out for a condition not deemed to be fatal and the subsequent media scrutiny. The data from these studies also revealed the importance of education and keeping up to date in order to deliver the best evidence based peer support and healthcare. The need for information specific to TOP for FFA provision in the Republic of Ireland was highlighted. Both Volunteers and FMS emphasised the importance of collaborative working (Volunteers requiring the support of healthcare professionals and FMS requiring the support of their colleagues) to meet the needs of parents following a FFA diagnosis while also acknowledging the importance of teamwork in supporting them in their peer support or clinical role. Organisational and collegial support is essential to sustain the delivery of peer-to-peer support for Volunteers and to reduce the feeling of isolation and judgement for FMS providing TOP for FFA. Thus, assisting both Volunteers and FMS in their own self-care while working in an emotive environment involving pregnancy loss and perinatal death. Conclusion: These data suggest there is a need for better knowledge of FFA, particularly regarding the complexity relating to the presentation of infants with a fetal anomaly that leads to them being fatal. These studies acknowledge the need for a universal term and definition that represents fetuses/infants with conditions that cause perinatal death to ensure a standard of care for women and their partners following a FFA diagnosis. It promotes the need for a universal database designed to collect essential epidemiologic information on congenital anomalies within the Republic of Ireland to support the labelling of a condition as a FFA. Additionally, the importance of education and the need to provide support and care to parents following FFA that reflects best practice and responds to their needs. FMS providing TOP for FFA services require organisational and collegial support to effectively deliver this new service. Furthermore, this thesis promotes the need to support both Volunteers and healthcare professionals involved in the delivery of care for parents following a FFA diagnosis through a collaborative approach, one that values and respects all members. The findings of these data outline several recommendations for the Irish TOP legislation, healthcare policy and clinical practice. It argues the need to reform the TOP legislation by removing the retained criminalisation, amending Section 11 and remove the restrictive 28 days. Furthermore, it is recommended that the Act refers to clinical practice guidelines informed by FMS and those healthcare professionals working within the abortion services. Finally, it identifies areas that warrant further research, such as the exploration of parents’ experiences of care and support received following a FFA diagnosis, to inform future intervention and improve care delivered to parents following a FFA diagnosis.