Serum levels of inflammatory cytokines in Rift Valley fever patients are indicative of severe disease

Abstract

Background: Rift Valley fever (RVF) is a mosquito-borne viral zoonosis affecting domestic and wild ruminants, camels and humans. Outbreaks of RVF are characterized by a sudden onset of abortions and high mortality amongst domestic ruminants. Humans develop disease ranging from a mild flu-like illness to more severe complications including hemorrhagic syndrome, ocular and neurological lesions and death. During the RVF outbreak in South Africa in 2010/11, a total of 278 human cases were laboratory confirmed, including 25 deaths. The role of the host inflammatory response to RVF pathogenesis is not completely understood.

Methods: Virus load in serum from human fatal and non-fatal cases was determined by standard tissue culture infective dose 50 (TCID50) titration on Vero cells. Patient serum concentration of chemokines and cytokines involved in inflammatory responses (IL-8, RANTES, CXCL9, MCP-1, IP-10, IL-1β, IL-6, IL-10, TNF and IL-12p70) was determined using cytometric bead assays and flow cytometry.

Results: Fatal cases had a 1-log10 higher TCID50/ml serum concentration of RVF virus (RVFV) than survivors (p < 0.05). There were no significant sequence differences between isolates recovered from fatal and non-fatal cases. Chemokines and pro- and anti-inflammatory cytokines were detected at significantly increased (IL-8, CXCL9, MCP-1, IP-10, IL-10) or decreased (RANTES) levels when comparing fatal cases to infected survivors and uninfected controls, or when comparing combined infected patients to uninfected controls.

Conclusions: The results suggest that regulation of the host inflammatory responses plays an important role in the outcome of RVFV infection in humans. Dysregulation of the inflammatory response contributes to a fatal outcome. The cytokines and chemokines identified in this study that correlate with fatal outcomes warrant further investigation as markers for disease severity.

Fig. 1

Individual and average RVFV viral loads in serum over time. Individual viral loads in serum are indicated per each time point post onset of disease: blue squares indicate individual non-fatal cases and red dots indicate individual fatal cases. Median viral loads at each time point are indicated by open blue squares (non-fatal) and red circles (fatal). Certain days are represented by only one data point per data set, in which case no medians are shown (days 5, 6, 10). Maximum two samples for respective days were available from fatal cases, thus averages are shown and not medians

Fig. 2

Individual and average cytokine concentrations in serum over time. Individual cytokine concentrations in serum are indicated per each time point post onset of disease: blue squares indicate individual non-fatal cases and red dots indicate individual fatal cases. Average concentrations at each time points are indicated by open blue squares (non-fatal) and red circles (fatal). The continuous green line indicates the average concentration in negative control samples (RVF naïve individuals) and thus has no bearing on the days indicated on the x-axis. Certain days are represented by only one data point per data set, in which case no medians are shown (days 1, 4, 6, 10 and 13). For days where less than three samples were available per dataset, averages are shown instead of medians. Note: because of the 1:4 dilution of serum for the cytokine analysis, a “0 pg/ml” value represents a limit of detection rather than absolute absence of the detected protein

Fig. 3

Heatmap showing relative chemokine and cytokine levels between fatal cases, non-fatal cases and negative controls. Relative levels are indicated by color. Red indicates a high level, orange indicates an intermediate level, blue indicates a low level and absence of color indicates that no protein was detected. Brackets indicate differences which are statistically significant (p < 0.01)

Fig. 4

Correlations between viral load and chemokine/cytokine levels. The relationship between serum levels of chemokines/cytokines and viral load (log) was evaluated using the Spearman correlation coefficient. Obvious outliers were excluded from analyses. Only significant correlations (P <0.05) are shown and were observed in non-hospitalized non-fatal cases (a) and in fatal cases (b). The P values are indicated