Early ART After Cryptococcal Meningitis Is Associated With Cerebrospinal Fluid Pleocytosis and Macrophage Activation in a Multisite Randomized Trial.

INTRODUCTION: Earlier antiretroviral therapy (ART) initiation in cryptococcal meningitis resulted in higher mortality compared with deferred ART initiation (1-2 weeks vs 5 weeks postmeningitis diagnosis). We hypothesized this was due to ART-associated immune pathology, without clinically recognized immune reconstitution inflammatory syndrome. METHODS: Three macrophage activation markers and 19 cytokines/chemokines were measured from cryopreserved cerebrospinal fluid (CSF) and serum during the Cryptococcal Optimal ART Timing (COAT) trial. Comparisons were made between trial arms (early vs deferred) at 1, 8, 14, and 21 days following meningitis diagnosis. RESULTS: More participants with early ART initiation had CSF white cell count (WCC) ≥5/µL at day 14 (58% vs 40%; P = .047), after a median of 6-days ART. Differences were mainly driven by participants with CSF WCC <5/µL at meningitis diagnosis: 28% (10/36) of such persons in the early ART group had CSF WCC ≥5/µL by day 14, compared with 0% (0/27) in the deferred arm (P = .002). Furthermore, Kampala participants (the largest site) receiving early ART had higher day-14 CSF levels of interleukin-13 (P = .04), sCD14 (P = .04), sCD163 (P = .02), and CCL3/MIP-1α (P = .02), suggesting increased macrophage/microglial activation. CONCLUSIONS: Early ART initiation in cryptococcal meningitis increased CSF cellular infiltrate, macrophage/microglial activation, and T helper 2 responses within the central nervous system. This suggests that increased mortality from early ART in the COAT trial was immunologically mediated.

Cryptococcus neoformans ex vivo capsule size is associated with intracranial pressure and host immune response in HIV-associated cryptococcal meningitis.

BACKGROUND: The Cryptococcus neoformans polysaccharide capsule is a well-characterized virulence factor with immunomodulatory properties. The organism and/or shed capsule is postulated to raise intracranial pressure (ICP) in cryptococcal meningitis (CM) by mechanical obstruction of cerebrospinal fluid (CSF) outflow. Little is known regarding capsule phenotype in human cryptococcosis. We investigated the relationship of ex vivo CSF capsular phenotype with ICP and CSF immune response, as well as in vitro phenotype. METHODS: In total, 134 human immunodeficiency virus (HIV)-infected Ugandan adults with CM had serial lumbar punctures with measurement of CSF opening pressures, quantitative cultures, ex vivo capsule size and shedding, viscosity, and CSF cytokines; 108 had complete data. Induced capsular size and shedding were measured in vitro for 48 C. neoformans isolates. RESULTS: Cryptococcal strains producing larger ex vivo capsules in the baseline (pretreatment) CSF correlated with higher ICP (P = .02), slower rate of fungal clearance (P = .02), and paucity of CSF inflammation, including decreased CSF white blood cell (WBC) count (P < .001), interleukin (IL)-4 (P = .02), IL-6 (P = .01), IL-7 (P = .04), IL-8 (P = .03), and interferon γ (P = .03). CSF capsule shedding did not correlate with ICP. On multivariable analysis, capsule size remained independently associated with ICP. Ex vivo capsular size and shedding did not correlate with that of the same isolates grown in vitro. CONCLUSIONS: Cryptococcal capsule size ex vivo is an important contributor to virulence in human cryptococcal meningitis.

Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contrast.

Cryptococcal meningitis is common in sub-Saharan Africa. Given the need for data for a rapid, point-of-care cryptococcal antigen (CRAG) lateral flow immunochromatographic assay (LFA), we assessed diagnostic performance of cerebrospinal fluid (CSF) culture, CRAG latex agglutination, India ink microscopy, and CRAG LFA for 832 HIV-infected persons with suspected meningitis during 2006-2009 (n = 299) in Uganda and during 2010-2012 (n = 533) in Uganda and South Africa. CRAG LFA had the best performance (sensitivity 99.3%, specificity 99.1%). Culture sensitivity was dependent on CSF volume (82.4% for 10 µL, 94.2% for 100 µL). CRAG latex agglutination test sensitivity (97.0%-97.8%) and specificity (85.9%-100%) varied between manufacturers. India ink microscopy was 86% sensitive. Laser thermal contrast had 92% accuracy (R = 0.91, p<0.001) in quantifying CRAG titers from 1 LFA strip to within <1.5 dilutions of actual CRAG titers. CRAG LFA is a major advance for meningitis diagnostics in resource-limited settings.

Acute Kidney Injury and Urinary Biomarkers in Human Immunodeficiency Virus-Associated Cryptococcal Meningitis.

BACKGROUND: Cryptococcus is the most common etiology of adult meningitis in Africa. Amphotericin B deoxycholate remains paramount to treatment, despite toxicities, including acute kidney injury (AKI). We assessed the ability of the following urine markers to predict AKI in patients who received amphotericin B: urine neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CysC), tissue inhibitor of metalloproteinases-2 (TIMP-2), and protein. METHODS: One hundred and thirty human immunodeficiency virus (HIV)-infected participants with cryptococcal meningitis were enrolled and received amphotericin and fluconazole for 2 weeks. We defined AKI as glomerular filtration rate (GFR) < 60 mL/min/1.73 m2; measured urine NGAL, CysC, TIMP-2, and protein; and explored AKI incidence, risk factors, and associations with mortality using Cox proportional hazards models. RESULTS: Participants were 48% female with a median age of 35 years, a median CD4 count of 21 cells/µL, and 44% died within 12 months. Incident AKI occurred in 42% and was associated with mortality (adjusted hazard ratio [aHR] = 2.8; P < .001). Development of AKI was associated with female sex (P = .04) and with higher CD4 count (49 vs 14 cells/µL; P < .01). Urine protein level in the highest quartile independently predicted AKI and mortality (aHR = 1.64, P = .04; aHR = 2.13, P = .02, respectively). Urine NGAL levels in the highest quartile independently predicted AKI (aHR = 1.65; P = .04). CONCLUSIONS: Acute kidney injury occurred in 42% of patients, and AKI was associated with mortality. Urine biomarkers, specifically urine protein, may be useful for antecedent prediction of amphotericin-associated AKI but need further evaluation.

Human Immune Response Varies by the Degree of Relative Cryptococcal Antigen Shedding.

Background. Cerebrospinal fluid (CSF) cryptococcal glucuronoxylomannan antigen (CrAg) titers generally correlate with quantitative fungal culture burden; however, correlation is not precise. Some patients have higher CrAg titers with lower fungal burdens and vice versa. We hypothesized that the relative discordancy between CrAg titer and quantitative culture burden reflects the relative degree of CrAg shedding by Cryptococcus neoformans and is associated with human immune responses. Methods. One hundred ninety human immunodeficiency virus-infected individuals with cryptococcal meningitis were enrolled in Uganda and South Africa. We compared initial CSF CrAg titers relative to their CSF quantitative cultures to determine low (n = 58), intermediate (n = 68), or high (n = 64) CrAg shedders. We compared cytokines measured by Luminex multiplex assay on cryopreserved CSF and 10-week mortality across shedding groups using linear and logistic regression and distribution of genotypes by multilocus sequence typing. Results. The relative degree of CrAg shedding was positively associated with increasing CSF levels of the following: interleukin (IL)-6, IL-7, IL-8, and tumor necrosis factor-α (each P < 0.01), which are all secreted by antigen-presenting cells and negatively associated with vascular endothelial growth factor (P = .01). In addition, IL-5, IL-13, granulocyte colony-stimulating factor, and macrophage chemotactic protein were decreased in low-CrAg shedders compared with intermediate shedders (each P ≤ .01). Type 1 T-helper cells (Th1) cytokine responses and 10-week mortality did not differ between the shedding groups. Cryptococcal genotypes were equally distributed across shedding groups. Conclusions. Discordancy between CrAg shedding and expected shedding based on quantitative fungal burden is associated with detectable immunologic differences in CSF, primarily among secreted cytokines and chemokines produced by antigen-presenting cells and Th2.

Analysis of CUGBP1 targets identifies GU-repeat sequences that mediate rapid mRNA decay.

CUG-repeat binding protein 1 (CUGBP1) mediates selective mRNA decay by binding to GU-rich elements (GREs) containing the sequence UGUUUGUUUGU found in the 3' untranslated region (UTR) of short-lived transcripts. We used an anti-CUGBP1 antibody to immunoprecipitate CUGBP1 from HeLa cytoplasmic extracts and analyzed the associated transcripts using oligonucleotide microarrays. We identified 613 putative mRNA targets of CUGBP1 and found that the UGUUUGUUUGU GRE sequence and a GU-repeat sequence were both highly enriched in the 3' UTRs of these targets. We showed that CUGBP1 bound specifically to the GU-repeat sequence and that insertion of this sequence into the 3' UTR of a beta-globin reporter transcript conferred instability to the transcript. Based on these results, we redefined the GRE to include this GU-repeat sequence. Our results suggest that CUGBP1 coordinately regulates the mRNA decay of a network of transcripts involved in cell growth, cell motility, and apoptosis.