Rotavirus antigenemia in children is associated with viremia

Abstract

Background: Antigenemia is commonly detected in rotavirus-infected children. Although rotavirus RNA has been detected in serum, definitive proof of rotavirus viremia has not been shown. We aimed to analyze a defined patient population to determine if infectious virus could be detected in sera from children with rotavirus antigenemia.

Methods and findings: Serum samples obtained upon hospitalization from children with gastroenteritis (57 stool rotavirus-positive and 41 rotavirus-negative), children with diagnosed bronchiolitis of known (n = 58) or unknown (n = 17) viral etiology, children with noninfectious, nonchronic conditions (n = 17), and healthy adults (n = 28) were tested for rotavirus antigen by enzyme immunoassay (EIA). Results of serum antigen testing were assessed for association with clinical and immunological attributes of the children. Rotavirus antigenemia was detected in 90% (51/57) of children with rotavirus-positive stools, in 89% (8/9) of children without diarrhea but with rotavirus-positive stools, in 12% (2/17) of children with bronchiolitis of unknown etiology without gastroenteritis, and in 12% (5/41) of children with gastroenteritis but with rotavirus-negative stools. Antigenemia was not detected in sera from children with noninfectious nonchronic conditions, children with bronchiolitis of known etiology and no gastroenteritis, or healthy adults. Neither age nor timing of serum collection within eight days after onset of gastroenteritis significantly affected levels of antigenemia, and there was no correlation between antigenemia and viral genotype. However, there was a negative correlation between serum rotavirus antigen and acute rotavirus-specific serum IgA (r = -0.44, p = 0.025) and IgG (r = -0.40, p = 0.01) titers. We examined 11 antigen-positive and nine antigen-negative sera for infectious virus after three blind serial passages in HT-29 cells using immunofluorescence staining for rotavirus structural and nonstructural proteins. Infectious virus was detected in 11/11 (100%) sera from serum antigen-positive children and in two out of nine (22%) sera samples from antigen-negative children (p = 0.002).

Conclusions: Most children infected with rotavirus are viremic. The presence of viremia is directly related to the detection of antigenemia and is independent of the presence of diarrhea. Antigenemia load is inversely related to the titer of antirotavirus antibody in the serum. The finding of infectious rotavirus in the blood suggests extraintestinal involvement in rotavirus pathogenesis; however, the impact of rotavirus viremia on clinical manifestations of infection is unknown.

Figure 1. Rotavirus Antigenemia in Children Does Not Depend on the Presence of Diarrhea

(A) Sera obtained from children with noninfectious, nonchronic conditions (n = 17), healthy adults (n = 28), children with gastroenteritis but rotavirus stool negative (n = 41), and children with gastroenteritis that were rotavirus stool positive (n = 57) were diluted 1:10 and tested for rotavirus antigen by EIA. Values for each sample were recorded as OD × 1,000. The cut-off value for positive samples (line) was established by calculating two standard deviations above the mean OD value of the children with noninfectious, nonchronic conditions and healthy adults. Numbers indicate mean OD values ± standard deviation. *p = 0.001 compared to children with noninfectious, nonchronic conditions. (B) Children with gastroenteritis (n = 98) with or without rotavirus present in the stool were further stratified on the basis of criteria of the presence or absence of diarrhea. Numbers indicate mean OD values ± standard deviation. Boxes indicate children from whom serum samples were analyzed for increases in antibody titers between acute and convalescent samples. ^# p = 0.001 compared to rotavirus (RV) Neg No Diarrhea group. ⁺ p = 0.88 compared to rotavirus Pos No Diarrhea group.

Figure 2. Detection of Rotavirus Antigenemia within Eight Days after Onset of Symptoms Does Not Depend on the Number of Days after Onset of Illness or the Age of Child

The ODs of serum samples obtained from children with rotavirus-positive stools were plotted against days after onset of illness (A) or age of child in months (B). Correlation analysis was performed (line) and R ² values calculated.

Figure 3. Infectious Rotavirus Is Present in Children with Rotavirus Antigenemia

A total of 11 antigen-positive and nine antigen-negative sera were examined for infectious virus by focus-forming assay after three blind serial passes in HT-29 cells. Infected HT-29 cells were identified using a rabbit antiserum against the ALA strain of rotavirus (A–F) or against NSP4 peptide 114–135 (G, H) followed by a FITC-labeled secondary antibody. Shown are: (A) Rhesus rotavirus (RRV) as a positive control; (B, G) Antigen negative serum; and (C–F, H) antigen positive serum.