Results
Patient Characteristics
Patient characteristics for the final study population are shown in Table 3. CFRs for the 55 laboratory-confirmed pediatric cases varied by age: 76.9% for children ≤5 years of age (n = 13), 37.5% for children 6–15 years of age (n =16), and 41.6% for adolescents 16–21 years of age (n = 26). In contrast, the CFR for the 161 adults with laboratory-confirmed cases was 56.5%. For the 45 pediatric patients for whom serum samples were available for analysis, the CFR was 100% for children ≤5 years of age (n = 8), 28.6% for those 6–15 years (n = 14), and 39% for those 16–21 years (n =23). Because all samples were available only from fatal cases for children ≤5 years of age, we initially excluded these samples from biomarker statistical analyses. However, because this exclusion might have introduced bias, we subsequently repeated the analysis including these 9 additional specimens and found no differences in the statistical significance of the models. Because the CFRs for child and adolescent populations were lower than those for adults, the 3 pediatric age groups were combined to increase the power of the study.
Patients were considered to have hemorrhagic manifestations if they exhibited any of the following signs: vomiting blood; blood in the stool; or bleeding from the gums, skin, or eyes. We found that a higher percentage of pediatric than adult patients exhibited hemorrhage, but overall CFR remained lower for children than for adults.
Viremia
To determine whether pediatric patients were more likely to survive as a result of lower levels of viral replication, we measured viremia levels in each sample by using real-time reverse transcription PCR and compared the results with a standard curve generated from stock virus of known titer. No statistically significant differences were found between viral loads in adults and pediatric patients (Figure 3, panel A). Viral loads were higher for patients who died (Figure 3, panel B), as previously demonstrated; however, in the pediatric population, this difference did not reach statistical significance, likely because of the small sample size and the wide range of observed values in the pediatric patients with nonfatal cases.
(Enlarge Image)
Figure 3.
Viral loads for Ebola virus disease patients infected with Sudan virus during outbreak in Uganda, 2000–2001. A) Relative TCID50, of pediatric patients (1–21 years of age) compared with those of adult patients (22–60 years of age); B) fatal and nonfatal outcomes for pediatric versus adult patients. Viral load determination was performed on all samples and quantitated by a reverse transcription PCR curve generated from a known titer stock of Sudan virus. Error bars represent SE. TCID50, 50% tissue culture infective dose.
Serum Chemistry Testing
Serum chemistry tests were performed on all samples that had sufficient available volume after initial testing. Blood urea nitrogen, creatinine, and albumin levels varied by age, as expected, given the normal physiological differences between adults and children (data not shown). No age-specific associations were found between any analyte in the serum chemistry results and death or hemorrhage. More labile analytes, such as carbon dioxide and electrolytes, were excluded from analysis.
Biomarkers of Inflammation
Cytokines and chemokines are a diverse group of proteins that modulate the immune response and have been extensively studied in many different disease processes. We analyzed 25 cytokines and chemokines. Of the 10 analytes that had a statistically significant association with age (Table 2), 4 were cytokines or chemokines, and 3 of those—IL-10, IP-10, and RANTES—were associated with an age-dependent survival outcome (Figure 1, panels A–C). IL-10 and IP-10 levels were higher in pediatric patients who died than in those who survived; adult patients had similar levels of these biomarkers regardless of outcome (Figure 1, panels A, B). Serum samples from this outbreak have been analyzed in the past, and increased levels of IL-10 were reported in patients with fatal outcomes; however, age was not analyzed in the prior study. RANTES levels were higher in pediatric patients than in adult patients and were further elevated in pediatric patients with nonfatal outcomes (Figure 1, panel C). RANTES is the only biomarker we identified as associated with increased survival in pediatric patients. Macrophage colony-stimulating factor levels were higher in pediatric patients than in adult patients, but no age-specific associations with hemorrhage or survival outcomes were observed (Figure 4, panel A).
(Enlarge Image)
Figure 4.
Biomarkers associated with age but not survival outcome or hemorrhage for patients with Ebola virus disease: black, adult; white, pediatric. A) Macrophage colony-stimulating factor; B) total IgG; C) tissue factor. Mean levels are depicted in each patient group as a function of time after symptom onset. Error bars represent SE; * indicates p≤0.05. Numbers of specimens included in each group are as follows: adult at 0–5 days, 47; adult at 6–10 days, 46; adult at 11–15 days, 19; pediatric at 0–5 days, 25; pediatric at 6–10 days, 18; pediatric at 11–15 days, 6.
The acute-phase response refers to a constellation of host responses that occur during infection and other inflammatory processes. These responses are classically triggered by proinflammatory cytokines and lead to increased levels of acute-phase reactants. These markers of inflammation are often used clinically to assist in diagnosis and to track a patient's response to therapy in many infectious or inflammatory processes. Of the acute-phase reactants that we measured—SAA, C-reactive protein, ferritin, and IgG—only SAA and IgG levels demonstrated age-specific associations. SAA levels were higher for pediatric patients than for adult patients and were higher at later times of infection (6–15 days after symptom onset) for pediatric patients without hemorrhagic manifestations than for those with hemorrhagic manifestations (Figure 2, panel A). Total IgG levels were higher for samples from pediatric patients than for those from adult patients (Figure 4, panel B), but no age-specific associations with death or hemorrhage were observed for this biomarker.
Biomarkers of Endothelial Function
Given the role of the endothelium in maintaining vascular integrity and modulation of hemodynamic stability and the vascular leakage seen in EVD, we included several markers of endothelial function in our study (sICAM, sVCAM, and soluble E-selectin). sICAM and sVCAM demonstrated an age-specific association. ICAM and VCAM are expressed on endothelial cells and upregulated in response to proinflammatory cytokines. Both factors are shed from the surface of activated endothelial cells and can be measured in their soluble form in the serum. At 0–10 day after symptom onset, pediatric patients had higher levels of sICAM and sVCAM than did adults, and pediatric patients who died had higher levels of both factors than did those who survived (Figure 1, panels D, E).
Biomarkers of Coagulopathy
The frequent presence of hemorrhagic manifestations in EVD patients and the increased frequency seen in our pediatric population warranted an examination of the measureable factors that control coagulation and fibrinolysis. We measured PAI-1, fibrinogen, tissue plasminogen activator, D-dimer, thrombomodulin, and TF in all patient samples. PAI-1 levels were elevated in pediatric patients, and more so in those who died and those who had hemorrhagic manifestations (Figure 1, panel F; Figure 2, panel B). TF levels were slightly elevated in pediatric patients (Figure 4, panel C), but no age-specific associations with hemorrhage or death were shown.