Immunopathogenesis and diagnosis of cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS) in persons living with HIV

Cryptococcal meningitis (CM) has become the most common cause of adult meningitis in HIVinfected patients in southern and central Africa, with more than one million cases a year estimated to occur globally. Morbidity and mortality is significant, with two-thirds of patients estimated to die within 3 months of CM diagnosis. Untreated, HIV-CM co-infection is uniformly lethal. HIV-infected patients with CM who receive antifungal therapy and combination antiretroviral therapy (cART) remain at risk of further neurological deterioration (ND). This is mostly due to cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS). While consensus clinical definitions for C-IRIS have been published, without a clear diagnostic marker, C-IRIS remains a diagnosis of exclusion. Recognising clinical, immunological and fungal determinants of C-IRIS could enable identification of those at greatest risk of C-IRIS. Understanding the immunopathogenesis of C-IRIS is important for the development of prevention, diagnosis and treatment strategies. We established a new prospective cohort study in Durban, South Africa where we enrolled 130 HIV-infected, cART-naïve patients experiencing their first episode of CM. Therapeutic lumbar punctures were performed and patients were administered antifungal therapy and cART. Patients were followed for a further 24 weeks for episodes of ND – these were classified into C-IRIS, not-C-IRIS and indeterminate. Quantitative and qualitative cerebrospinal fluid (CSF) cryptococcal cultures were performed to ascertain fungal burden. Immunological markers were explored using flow cytometry and bead array assays both in fresh whole blood and CSF. We hypothesised that C-IRIS is associated with alterations of chemokine receptor expression on T-cells and chemokine concentrations in CSF which enhance recruitment of Th1 T-cells and/or myeloid cells to the central nervous system (CNS). We also explored the utility of a novel whole blood assay of cryptococcal mannoprotein (CMP)-reactive T cells (CD25+/CD134+) as a potential field-based diagnostic test for C-IRIS. Of 106 patients commencing cART, 27 (25.5%) developed C-IRIS, 16 (15.1%) ND-not C-IRIS and 63 (59.4%) no-ND. On multivariable analysis, C-IRIS was associated with persistent CSF cryptococcal growth (p=0.026) and lower CSF protein (p=0.059) prior to cART initiation and lower CD4+ T-cell increases while on cART (p=0.026). Importantly, using survival analysis, patients with a negative cryptococcal culture pre-cART commencement (n=51; 48.1%) compared to patients with culture positivity (n=55; 51.9%) at the time of initiation of cART experienced fewer episodes of ND, C-IRIS and cryptococcal relapse/persistence (p=0.0003, 0.0042 and, 0.0004, respectively). Together, our data suggest that persistent CSF cryptococcal growth at cART initiation and poor CD4+ T-cell increases on cART are strong clinical predictors of C-IRIS and approaches aimed at achieving CSF culture negativity prior to cART should be evaluated as a strategy to reduce rates of C-IRIS. C-IRIS patients compared to patients with no-ND had higher CSF ratios of CCL2/CXCL10 and CCL3/CXCL10 and higher proportions of CXCR3+CCR5+CD8+T-cells in CSF compared to blood (p=0.0250, 0.053, 0.0177 respectively); suggesting a role for increased CD8+ T-cell and myeloid cell trafficking to the CNS in C-IRIS. C-IRIS patients compared to patients with no-ND had lower 24-hour-CMP-induced IFN-gamma production at the time of initiation of cART (p=0.0437), giving further evidence to the importance of IFN-gamma in cryptococcosis control. We suggest that low-IFN-gamma responders may potentially benefit from adjunctive exogenous IFN therapy. Early diagnosis and treatment is necessary in tackling HIV and CM co-infection. Further translational studies should include risk stratification treatment strategies, best therapy to achieve CSF cryptococcal sterility and adjunctive exogenous IFN-gamma therapy for low IFN-gamma responders to CMP. Clinical studies incorporating basic science research is important in our quest towards understanding C-IRIS immunopathogenesis. Ongoing advocacy for improved drug access and health care access is necessary