Impact of Cerebrospinal Fluid Leukocyte Infiltration and Neuroimmmune Mediators on Survival with HIV-Associated Cryptococcal Meningitis

Abstract

Introduction: Cryptococcal meningitis remains a prominent cause of death in persons with advanced HIV disease. CSF leukocyte infiltration predicts survival at 18 weeks; however, how CSF immune response relates to CSF leukocyte infiltration is unknown.

Methods: We enrolled 401 adults with HIV-associated cryptococcal meningitis in Uganda who received amphotericin and fluconazole induction therapy. We assessed the association of CSF leukocytes, chemokine, and cytokine responses with 18-week survival.

Results: Participants with CSF leukocytes ≥50/μL, had higher probability 68% (52/77) of 18-week survival compared with 52% (151/292) 18-week survival in those with ≤50 cells/μL (Hazard Ratio=1.63, 95% confidence intervals 1.14-2.23; p=0.008). Survival was also associated with higher expression of T helper (Th)-1, Th17 cytokines, and immune regulatory elements. CSF levels of Programmed Death-1 Ligand, CXCL10, and Interleukin (IL)-2 independently predicted survival. In multivariate analysis, CSF leukocytes were inversely associated with CSF fungal burden and positively associated with CSF protein, interferon-gamma (IFN-γ), IL-17A, tumor necrosis factor (TNF)-α, and peripheral blood CD4⁺ and CD8⁺ T cells expression.

Conclusion: 18-week survival after diagnosis of cryptococcal meningitis was associated with higher CSF leukocytes at baseline with greater T helper 1 (IFN-γ, IL-2 and TNF-α cytokines), T helper 17 (IL-17A cytokine) and CXCR3⁺ T cell (CXCL10 chemokine) responses. These results highlight the interdependent contribution of soluble and cellular immune responses in predicting survival with HIV-associated cryptococcal meningitis.

Keywords: CSF white cells; HIV-associated cryptococcal meningitis; antifungal therapy; cerebrospinal fluid; chemokines; cytokines; fungal burden; immune response; survival.

Figure 1.. the correlation of CSF and blood clinical features.

A - correlation of CSF features with 18 weeks survival by levels of CSF white cells. A (i) – CSF white cells, A (ii) – CSF proteins and A (iii) CSF cryptococcal fungal burden. B – correlation of peripheral blood features with 18 weeks survival by levels of CSF white cells. B (i) – peripheral blood white cells, B (ii) - CD4⁺ T cells, and B (iii) - CD8⁺ T cells. Error bars – median and 95% confidence intervals. * - statistically significant variables reported at p-value <0.050, at 95% confidence intervals.

Figure 2.. Correlation of CSF cytokines and chemokine levels with CSF leukocyte counts

A- Th1 cytokines; A (i) - Interleukin 2, A (ii) - Interferon gamma, A (iii) - Tumor necrosis factor alpha. B - Th17 cytokine, IL-17A. C – Immune regulatory elements; (i) – Interleukin 10, and programmed death 1 ligand. D Chemokines; D (i) CXCL10/IP-10 and D (ii) – CCL11/Eotaxin. The CSF white cells; (≤50 cells/μL; n=318), (51–200 cells/μL; n=57) and (201–500 cells/μL; n=26) participants. Error bars – median and 95% confidence intervals. * - statistically significant variables reported at p-value <0.050, at 95% confidence intervals.

Figure 3.. correlation of CSF white cells with 18-week survival.

A –survival by CSF white cell intervals (<5; n=245), (5–20; n=31), (21–50; n=42), (51–100; n=26), (101–200; n=31), and (201–500; n=26). B - 18 weeks survival by CSF white cells; (≤50 cells; n=318), (51–200 cells/μL; n=57) and (201–500 cells/μL; n=26) participants. C – 18-week survival with CSF ≤50 cells/μL. D (i-iii) –illustrates univariate immune responses associated with survival between survivors and those who died during 18-weeks of follow-up. Statistics - Mann-Whitney unpaired t-test. * - show statically significant variables. NS- not significant. Error bars – show 95% confidence intervals. p-values, p<0.050 were statistically significant.

Figure 4.. Principal Component Analyses showing Eigenvector projection (A) and cumulative effect size contributed by each covariate the PCA model (B) examined among all participants.

The three planes demonstrated by the Eigenvector projects are; (i-ii) - diagonal plane between CSF fungal burden and host survival and CSF white cells, CSF protein, peripheral white cells, CD4⁺ and CD8⁺ T cells. (i-iii) orthogonal plane between CSF fungal burden and host survival and the cytokine/chemokine profile. (ii-iii) orthogonal plane between CSF fungal burden and host survival and CSF white cells, CSF protein, peripheral white cells, CD4⁺ and CD8⁺ T cells. Eigenvectors >5 shows greater power and association with the outcome variable among interacting covariates.