Understanding the causes of second episode cryptococcal meningitis to improve diagnosis and treatment - Abstract Cryptococcus is the most common cause of HIV-related meningitis with mortality >25%. Symptoms recur in 5–10% of survivors, typically due to either paradoxical immune reconstitution inflammatory syndrome (IRIS) with sterile cultures or culture-positive relapse of infection. Cerebrospinal fluid (CSF) cryptococcal antigen (CrAg) testing is highly effective to diagnose primary cryptococcal meningitis but cannot distinguish relapse from IRIS where culture is the gold standard. Culture’s slow turnaround (7–10 days) hinders timely clinical action. Thus, clinicians must choose empiric corticosteroids (IRIS) or amphotericin (relapse). Incorrect treatment means exposing patients to unnecessary toxicities without addressing the underlying illness, highlighting a clear clinical need for a rapid, accurate test to differentiate relapse from IRIS to improve outcomes and minimize drug-related harm. The BioFire Meningitis/Encephalitis PCR panel correctly identified 18 of 19 patients with relapse or IRIS. Yet, utility in cryptococcosis is limited by high cost, proprietary equipment, and poor sensitivity (29%) at low fungal burdens. Our Cryptococcus neoformans quantitative PCR assay showed excellent correlation with baseline CSF quantitative culture and fungal clearance rates in first-episode meningitis. Whether the assay can differentiate IRIS from relapse or detect early treatment failure has not been studied. Similarly, the roles of inadequate immune response (including due to poor antiretroviral therapy adherence), rising fluconazole minimum inhibitory concentrations (MICs), and insufficient fluconazole levels in relapse remain underexplored. Our overall objective is to reduce second episode cryptococcal meningitis mortality and morbidity by understanding the cause(s) and through cost-effective, rapid and accurate diagnostic tests. Our aims are: 1) To validate the diagnostic performance of a real-time CSF-based Cryptococcus qPCR assay for diagnosing culture-positive relapse in participants presenting with recurrent cryptococcal meningitis; 2) To determine the causes of culture-positive relapse by testing drug levels for antiretroviral and antifungal therapies, testing in vitro MICs to fluconazole, characterizing the host immune response and distinguishing new infection from recurrence by genomic sequencing of Cryptococcus yeast; and 3) To prospectively evaluate the diagnostic accuracy of Cryptococcus qPCR to detect real-time mycologic treatment failure with increasing quantitative CSF cultures. This proposal aims to transform the diagnosis of relapse from slow and often inaccurate to rapid, precise, and cost-effective. It also provides a tool to quickly identify mycological treatment failure for clinical care and trials. Additionally, the proposed work seeks to uncover relapse causes to inform future prevention strategies, such as higher fluconazole doses during secondary prophylaxis.
