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

真菌隐球菌是撒哈拉以南非洲成年人脑膜炎的主要原因，结果非常糟糕，大多数患者死亡或出现脑损伤。它主要影响艾滋病毒感染的患者。尽管儿童感染被认为比成人罕见，但由此对发育中的大脑造成的损害是严重的，儿童感染的数量在很大程度上是未知的。世界卫生组织建议对患有晚期艾滋病毒的10岁以上儿童进行检测，以寻找隐球菌感染的证据，因为预防性治疗挽救了生命。该项目的目标是：a)确定SA儿童中隐球菌抗原血症和CM的患病率和临床管理。将对SA的所有公共医疗机构进行回溯性队列研究，包括2017至2022年间国家传染病研究所(NICD)监测数据仓库确定的所有CRAG阳性结果。流行率将使用南非统计局的年中人口分母和Thembisa模型计算。一项前瞻性研究将使用Germs-SA，一种活跃的NICD监测系统来建立。临床分离株和脑脊液将被储存，用于AIM 2和AIM 3的宿主-真菌相互作用研究。已经收集和储存的儿科隐球菌分离株也可用于AIM 2)比较成人和儿科临床分离株的新生隐球菌细胞壁组成。5个来自成人的临床分离株和5个来自儿科感染和野生型新生隐球菌的临床分离株将在模拟宿主的条件下培养。将使用储存的分离物(NICD)，并在可用时添加来自前瞻性研究的新鲜样本。通过甲壳素、壳聚糖、壳寡聚体、葡聚糖和甘露聚糖的染色以及在流式细胞仪上测量荧光强度来比较细胞壁和胶囊的成分。形态将通过对细胞表面成分的染色以及共聚焦和电子显微镜的可视化来比较。这项工作将告知AIM 3.c)使用体内和体外模型描述儿童新生芽孢杆菌分离株的神经炎症效应。儿童脑脊液样本(AIM 1)将通过ELISA分析其细胞因子谱(例如，肿瘤坏死因子、IL-6、干扰素-α)，并与成人样本进行比较。我们将使用已建立的小鼠模型来表征儿童对隐球菌感染的神经免疫反应。为了在细胞水平上描述儿科神经隐球菌病的分子机制，体外，器官型小鼠脑片培养将被使用。取6天龄小鼠的器官型脑片，在人工脑脊液中的半透膜上培养，然后用临床分离的隐球菌进行刺激。如上所述，将使用免疫组织化学和细胞因子分析来测量神经炎性激活。Dangarembizi博士的实验室可以从癫痫手术中获得儿科脑组织，因此有可能发展到人类器官切片培养。该项目将提供有关南非儿科隐球菌抗原血症和隐球菌病的重要流行病学信息。这将有助于填补关于隐球菌性脑膜炎发病机制的现有知识空白，以及在成人和儿童人群中观察到的差异。

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.