Comparison of cytokine and immunoglobulin levels in blood between asymptomatic serum sryptococcal antigen positive and negative HIV-positive individuals with CD4 counts <100 cells/ μl. A nested case-control study in Harare.

Abstract

Background: Cryptococcosis is still a leading cause of mortality among patients with advanced HIV infection. Although CD4 T-cell deficiency is necessary for HIV-associated cryptococcosis, not all severely immunocompromised individuals develop the disease despite serological evidence of prior exposure to the cryptococcus. At present, there are no host immune biomarkers in blood that can predict reactivation of latent cryptococcal infection among those with severe immunosuppression. Therefore, a nested case-control study was conducted in Harare to compare levels of different cytokines and immunoglobulins between asymptomatic individuals with reactivated disease, as defined by the presence of cryptococcal antigen (CrAg) in serum, and those without. Methodology: Cytokine and immunoglobulin levels were determined in sera and plasma samples, respectively, from serum CrAg positive (sCrAg+ve) and negative (sCrAg-ve) HIV positive individuals with CD4 counts <100 cells/μl who had no signs and symptoms suggestive of a meningitis. Experiments were performed in two sets with different sample sizes due to differences in the amount of reagents available. In the first set of experiments, levels of 26 cytokines were measured using Luminex in 66 serum samples. 46 were from sCrAg+ve participants and 20 were from sCrAg-ve participants. The cytokine panel comprised of IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17α, IP-10, sCD40L, TNF-α, TNF-β, Flt-3L, G-CSF, GM-CSF, IFN-α2, IFN-γ, MCP-1, MIP-1α and MIP-1β. The second set of experiments determined levels of total, laminarin (Lam)- and glucuronoxylomannan (GXM)-binding antibodies by Luminex and ELISA, respectively, in 237 plasma samples.112 were from sCrAg+ve participants and 125 were from sCrAg-ve participants. All the included participants had provided written informed consent for their samples to be used for this study. Results: Cytokine analysis was performed on 66 serum samples from 66 participants, 66.7% (n=44) were males and 69.7% (n=46) were sCrAg+ve. Median (IQR) age and CD4 count were 38(32.8-42.3) years and 17(10-32.3) cells/µl, respectively. Median (IQR) serum IL-17α [0.64 (0.64-1.8) vs 1.18 (0.64-3.14)pg/ml, p=0.017] and MIP-1β [35.51 (25.45-124.8) vs 17.01(2.48-47.77)pg/ml, p=0.011] levels were lower in sCrAg+ve compared with sCrAg-ve participants, whereas IP-10 levels were higher in sCrAg+ve compared with sCrAg-ve participants, [2074(1601-2770) vs 1304(664.4-2898)pg/ml, p=0.031]. Antibody analysis was conducted on 237 plasma samples, 112(47.3%) were sCrAg+ve with a median (IQR) serum CrAg titre of 1:20 (1:5-1:80) and the remaining 125 samples were from sCrAg-ve participants. Majority (59.5%, n=141) were males. Median (IQR) age and CD4 count were 38(33-42) years and 26(14-50) cells/µl, respectively. Levels of total IgM and IgG2, and GXM-IgG were significantly higher, whereas GXM-IgM, Lam-IgM, and Lam-IgG were lower in sCrAg+ve participants compared to sCrAg-ve participants. Conclusion: Reactivation of latent cryptococcal infection (serum CrAg positivity) is associated with decreased serum levels of MIP-1β and IL-17α and increased levels of IP-10. Reactivation is also heralded by an increase in plasma total IgM and IgG2, GXM-binding IgG and a decrease in IgM and IgG antibody levels that react with β-glucans. These results provide some insights into the possibility of employing cytokine and antibody measurements as adjunctive screening tools in identification of individuals at risk of reactivating latent cryptococcal infection.