To the Editor:
Cryptococcal meningitis is the most important worldwide cause of meningitis in the immunocompromised (HIV-infected) patient.1 Moreover, Cryptococcus neoformans is emerging as an important pathogen in organ transplant recipients and is currently already the third most common fungal infection in these patients,2 with death rates between 20% and 100%.3 As is true for other systemic mycoses, treatment of disease caused by C. neoformans has improved dramatically over the last 2 decades. Before 1950, disseminated cryptococcal disease was uniformly fatal. With the advent of polyene antifungal agents (particularly amphotericin B) and azoles like fluconazole, successful outcomes are achieved in as many as 60% to 70% of patients with C. neoformans meningitis, depending on the status of the host at the time of presentation.4,5 Development of elevated intracranial pressure is an important contributor to the morbidity and mortality of C. neoformans meningitis. Almost three quarters of HIV-infected patients in a recent National Institute of Allergy and Infectious Diseases (NIAID)–sponsored Mycoses Study Group trial had elevated intracranial pressure.5 Benefit from management of intracranial pressure is inferred from reduced mortality in this population. Other than repeated lumbar puncture, no medical management has been shown to be effective for treatment of this complication, however.5 The elevated intracranial pressure in this setting is partly thought to be caused by interference with cerebrospinal fluid (CSF) reabsorption in the arachnoid villi as a result of high levels of fungal polysaccharide or excessive growth of the organism per se. There is little evidence to support or refute this hypothesis, however.
Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability and angiogenesis. VEGF has been implicated in the pathogenesis of brain edema related to ischemia, trauma, and tumors.6 Previously, we have reported elevated CSF VEGF levels in bacterial meningitis patients.7 Recently, elevated VEGF values in the CSF of adult patients with tuberculosis meningitis (TBM) were reported, suggesting that VEGF might have a role in the pathophysiology of TBM.8 There is considerable experimental evidence and a modest but growing base of sound clinical data to indicate that adjunctive corticosteroid therapy can significantly reduce the incidence of vascular and other neurologic complications and improve mortality in TBM.9 Corticosteroids decrease vascular permeability and limit cerebral edema in clinical TBM, as evidenced by a more rapid resolution of edema associated with tuberculomas and tuberculous encephalitis as well as a greater decrease in serial CSF protein levels.10 Studies in a rat model of brain tumor–associated edema suggest that inhibition of VEGF by corticosteroids may explain the effect of corticosteroids on cerebral edema.11
Previously, it has been shown that the cryptococcal capsular polysaccharides can induce cerebral edema.12 Recently, we have shown that the C. neoformans capsular components glucuronoxylomannan (GXM) and mannoprotein-4 (MP-4) can induce VEGF production in neutrophils, monocytes, and peripheral blood mononuclear cells (PBMCs). Furthermore, 30% of (HIV-infected) patients with C. neoformans meningitis showed elevated levels of VEGF in CSF.13 We therefore studied the effect of corticosteroids on GXM-induced VEGF production in PBMCs. We isolated GXM from the heavily encapsulated strain NCPF3168 and PBMCs from peripheral blood obtained from healthy volunteers after informed consent, both as described by us previously.13 For VEGF induction, PBMCs (1.25 × 106 cells) were seeded into 48-well tissue culture plates, supplemented with the various stimuli, and incubated at 37°C in a water-jacketed CO2-supplemented incubator for 72 hours, which is optimal for VEGF production. Optimal VEGF induction was evoked by GXM at a concentration of 0.5 mg/mL. To inhibit VEGF release, dexamethasone was added in a clinically relevant concentration series ranging between 10 nM and 10 μM. As a positive control, cells were incubated with 10 μg/mL of tumor necrosis factor-α (TNFα). No stimulus was added to negative control wells. After incubation, samples were centrifuged at 350g for 10 minutes. Supernatants were collected and stored at −70°C until assayed for VEGF content. Preparations of GXM were negative for endotoxin contamination using a Limulus assay (Kabi Diagnostica, Mölndal, Sweden) with a sensitivity of 100 pg/mL of Escherichia coli lipopolysaccharide (LPS). Nevertheless, all experiments were carried out under sterile conditions and in the presence of 10 μg/mL of polymyxin B sulfate to neutralize any undetected LPS contamination. A commercially available human VEGF enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Europe, Abingdon, UK) was used to measure VEGF in culture supernatants and CSF according to the manufacturer’s guidelines. This assay is sensitive to 20 pg/mL of VEGF and recognizes the soluble isoforms VEGF121 and VEGF165. The assay does not cross-react with platelet-derived growth factor (PDGF) or other homologous cytokines. As can be seen in the Figure 1, dexamethasone could inhibit VEGF secretion completely (n = 6; P < 0.001 for all comparisons between GXM-treated group vs. GXM/dexamethasone groups; differences between groups were analyzed using a 2-tailed Student t test).
Finally, in a recent small retrospective study, it was shown that corticosteroids may have a role in preventing blindness (5% vs. 50%) and halting of visual loss (13% vs. 70%), both thought to be induced by increased intracranial pressure in C. neoformans var. gattii meningitis in immunocompetent patients.14 We are currently analyzing the relation between VEGF levels and clinical parameters such as increased intracranial pressure in a cohort of patients with C. neoformans meningitis from Thailand. A correlation between increased VEGF levels and increased intracranial pressure may warrant a clinical trial of corticosteroids in HIV-infected patients with C. neoformans meningitis.
Andy I. M. Hoepelman, Prof MD, PhD
Michiel Van der Flier, MD, PhD
Frank E. J. Coenjaerts, PhD
*Department of Acute Medicine and Infectious Diseases,, University Medical Center Utrecht (UMCU),, Utrecht, The Netherlands;, †Eijkman-Winkler Center for Microbiology and Infectious Diseases,, UMCU, Utrecht, The Netherlands; and, ‡Wilhelmina Children’s Hospital, UMCU,, Utrecht, The Netherlands
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