The epidemiology and host-fungal neuroimmune interaction of paediatric cryptococcal infection in South Africa.

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The fungus Cryptococcus is the leading cause of meningitis in adults in Sub-Saharan Africa and has very poor outcomes with most patients dying or developing brain damage. It predominantly affects patients with HIV infection. Although childhood infection is recognised to be rarer than in adults, the resulting damage to the developing brain is significant and the number of infections in children is largely unknown. The World Health Organization recommends testing every child over 10 years old with advanced HIV disease for evidence of cryptococcal infection as pre-emptive treatment saves lives.The aims of the project are:a) Define the prevalence and clinical management of cryptococcal antigenaemia and incidence of CM in children in SA.A retrospective cohort study will be performed across all public healthcare facilities in SA including all CrAg positive results from children <18 yrs between 2017 and 2022 identified by the Surveillance Data Warehouse of the National Institute for Communicable Diseases (NICD). Prevalence will be calculated using mid-year population denominators from Statistics South Africa and the Thembisa model. A prospective study will be set-up using GERMS-SA, an active NICD surveillance system. Clinical isolates and CSF will be stored for host-fungus interaction studies for aim 2 and 3. Already collected and stored paediatric cryptococcal isolates are also available for use in aim 2.b) Compare the Cryptococcal neoformans cell wall composition of adult and paediatric clinical isolates.Five clinical isolates from adult and five from paediatric infections and wild-type C. neoformans will be grown in host-mimicking conditions. Stored isolates (NICD) will be used, with fresh samples from the prospective study added when available. Cell wall and capsule components will be compared by staining for chitin, chitosan, chito-oligomers, glucan and mannans and measuring fluorescence intensity on flow cytometry. Morphology will be compared by staining cell surface components and visualisation with confocal and electron microscopy. This work will inform the isolates used in aim 3.c) Describe the neuroinflammatory effects of paediatric C. neoformans isolates using in vivo and ex vivo models.The paediatric CSF samples (aim 1) will be analysed by ELISA for their cytokine profile (e.g. TNF, IL-6, INF-a) and compared to adult samplesWe will characterize the paediatric neuroimmune response to Cryptococcus infection using an established mouse model. To delineate molecular mechanisms underlying paediatric neurocryptococcosis at the cellular level, ex vivo, organotypic murine brain slice cultures will be used. Organotypic brain slices obtained from six-day-old mouse pups will be cultured on a semi-permeable membrane in artificial CSF then stimulated with clinical Cryptococcal isolates. Neuroinflammatory activation will be measured using immunohistochemistry and cytokine assays as described above. Dr Dangarembizi's laboratory has access to paediatric brain tissue from epilepsy surgery so there is a possibility of advancing to human organotypic slice cultures.This project will provide important epidemiological information about paediatric cryptococcal antigenaemia and cryptococcosis in South Africa. It will help fill the existing gap in knowledge of the pathogenesis of cryptococcal meningitis and the differences observed in the adult and paediatric population.