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Quantitative Evaluation of Rotaviral Antigenemia in Children with Acute Rotaviral Diarrhea

  1. Pratima Ray1,
  2. Martijn Fenaux2,
  3. Sumit Sharma1,
  4. Jyoti Malik1,
  5. Swati Subodh1,
  6. Shinjini Bhatnagar1,
  7. Harry Greenberg2,
  8. Roger I. Glass3,
  9. Jon Gentsch3 and
  10. M. K. Bhan1
  1. 1Center for Diarrheal Disease Research, Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India;
  2. 2Department of Medicine and Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, and Veterans Affairs Palo Alto Health Care System, Palo Alto, California;
  3. 3Viral Gastroenteritis Section, Centers for Disease Control and Prevention, Atlanta, Georgia
  1. Reprints or correspondence: Dr. Pratima Ray, Center for Diarrheal Disease Research, Dept. of Pediatrics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India (pratimaray{at}gmail.com)
 
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Abstract

BackgroundRotaviral antigen and RNA have recently been identified in the serum of patients with rotaviral gastroenteritis, but the roles they play in disease remains undetermined

MethodsRotaviral antigen and RNA were quantified by enzyme-linked immunosorbant assay and by quantitative reverse-transcription polymerase chain reaction in stool and serum specimens from children with rotaviral diarrhea (n=102), children with nonrotaviral diarrhea (n=40), and nondiarrheal control children (n=30)

ResultsRotaviral antigenemia was detected in 64%, 3%, and 0% of the children with rotaviral diarrhea, the children with nonrotaviral diarrhea, and the nondiarrheal control children, respectively. The level of rotaviral antigen in serum was ∼2×102–fold lower than that in stool, and a moderate correlation was observed between the 2 levels. Rotaviral RNA was detected in 93% of the antigen-positive serum specimens. The median number of RNA copies in serum was ∼1×105-fold lower than that in stool, and no correlation was observed between the 2 levels. Serum levels of both antigen and RNA were inversely associated with baseline titers of rotaviral serum immunoglobulin G (P<.01). Antigenemia was also associated with G1 serotype

ConclusionsRotaviral antigenemia and viremia were common in children with rotaviral diarrhea, but antigen and RNA levels in serum were substantially lower than those in stool. Antigenemia was associated with infection with G1 strains and with low baseline titers of rotaviral serum antibody

Rotavirus is the most common cause of severe gastroenteritis worldwide. In India, rotavirus is responsible for 20% of diarrhea-related hospitalizations and for >100,000 deaths among children <5 years of age [1–4]. Rotavirus targets the mature villus enterocytes of the small intestine [5–7], causing severe watery diarrhea; therefore, treatment has been directed exclusively at correcting dehydration and the loss of fluids and electrolytes. However, it is unclear whether dehydration is the only reason that rotaviral infections are more often fatal among children in the developing world than in the developed world. Recently, another possible explanation for this difference in disease severity and outcome has been suggested by findings from both animal and human studies demonstrating that rotavirus can also infect extraintestinal sites [8–25]. Rotaviral antigen and/or RNA has been reported in the cerebrospinal fluid of children with seizures and in the livers and kidneys of immunocompromised children examined postmortem [26–30]. Blutt et al. have reported the very frequent detection of rotaviral antigen and RNA in serum specimens from rotavirus-infected animals and children with rotaviral diarrhea [8]. If extraintestinal rotaviral infection is an additional cause of more-severe and more-lethal disease, alternative strategies for treatment might be required to improve outcome

To better understand the roles played by viremia (defined here as the presence of rotaviral RNA in serum) and antigenemia in rotaviral diarrhea, we assessed the levels of rotaviral antigen and RNA in serum specimens from immunocompetent, nonmalnourished children with acute rotaviral or nonrotaviral gastroenteritis and from age-matched healthy children. Furthermore, we quantified rotaviral load in stool and serum specimens from rotavirus-infected patients by quantitative reverse-transcription polymerase chain reaction (QRT-PCR) and ELISA and assessed the level of viral shedding as a correlate of antigenemia. We also examined the contribution of age, clinical disease severity, the G serotype of the infecting strain, and the level of preexisting antibody to rotavirus as possible correlates of antigenemia in children with acute rotaviral diarrhea

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Subjects, Materials, and Methods

Subjects and serum specimensThe study subjects were (1) children ⩽3 years old who had acute watery diarrhea and dehydration of a duration of <72 h (n=142) and (2) age-matched children without gastroenteritis (the nondiarrheal control subjects) (n=30); patients were treated at the All India Institute of Medical Sciences (AIIMS) hospital. Of the 142 children with gastroenteritis, 102 had rotavirus detected in their stool samples (the case patients with rotaviral diarrhea), and 40 did not (the control subjects with nonrotaviral diarrhea). None of the children participating in the study were malnourished, and all had a weight-for-height value ⩾80% of the median of the National Center for Health Statistics’s reference standard. The study was approved by the Ethics Committee for Human Studies at AIIMS. Informed consent was obtained from the parents of the children. On admission to the hospital, clinical information on the study children was recorded, and stool and serum specimens were collected. Stool specimens were tested for rotavirus by ELISA, as described elsewhere [31]. Convalescent-phase serum specimens were obtained from 59 of the 142 children with gastroenteritis 2–3 weeks after hospital admission. Stool and serum specimens were stored in aliquots at −70°C until analyzed

Quantification of rotavirus in stool and serum specimens by ELISA Rotaviral antigen in stool and serum specimens was quantified using a monoclonal antibody–based ELISA kit (Premier Rotaclone; Meridian Diagnostics), in accordance with the manufacturer’s protocol [31]. Antigen levels were expressed as units per milliliter of serum or units per gram of stool, determined on the basis of a standard-dilution plot of antigen levels in reference stool. Control antigen specimens, 1 positive and 1 negative, were included on each plate, and the assay was considered to be valid only when the negative control specimen was clearly negative and the positive control specimen was within a 2-fold range of its assigned value, as described by McNeal et al. [32]. The cutoff point for serum antigen levels was ⩾6 U/mL, which was 2 SDs above the mean serum antigen level of the 40 control subjects with nonrotaviral diarrhea

Quantification of rotavirus in stool and serum specimens by QRT-PCRRNA copy numbers for rotavirus in stool and serum specimens were determined by QRT-PCR in a subset of 28 case patients with rotaviral diarrhea and antigenemia from whom sufficient stool and serum specimens were available. Stool and serum specimens (10% wt/vol) were handled separately; each specimen was transferred to a sterile tube, frozen in liquid nitrogen, and stored in aliquots before RNA extraction. Total RNA was extracted using the RNAwiz method (Ambion), in accordance with the manufacturer’s protocol, and then resuspended in 100 μL of water. The QRT-PCR assay was conducted with a G1 rotavirus– and G9 rotavirus–specific VP6 primer pair (India.1.1+12.F [5′-CCTTTTCAACATCATGC-3′] and INDIA.2.1+12.R [5′-GATTCACAAACTGCAG-3′]) and a G2 rotavirus–specific primer pair (India.1.14.F [5′-CCATTTGAACATCATGC-3′] and India.2.14.R [5′-ATTCACAGACTGCAG-3′]). The G1/G9- and G2-specific primers were designed using the VP6 sequences obtained from selected G1, G2, and G9 human rotaviral strains used in this analysis (GenBank accession nos. DQ359151, DQ359152, and DQ359153) and were based on the most universally conserved VP6 sequence in the G1/G9 and G2 strains. Cloned fragments of the G1/G9 and G2 VP6 sequences were used in standard dilutions and were run in parallel with each reaction as the standard curve. QRT-PCR was performed in a 384-well ABI optical plate, using QuantiTect SYBR Green RT-PCR reagents (Qiagen). The RT step was done at 40°C for 10 min and then at 95°C for 15 min, followed by 38 cycles of PCR (95°C for 15 s, 42°C for 15 s, and 72°C for 25 s)

G serotyping assayDouble-stranded rotaviral RNA was extracted from 79 stool specimens and was used to determine the G serotype of the infecting rotaviral strains. G serotypes of the infecting strains were determined by RT-PCR with primers specific for G serotypes 1–4 and 9, as described elsewhere [2, 33, 34]

IgG ELISASerum titers of IgG antibody to rotavirus were determined by ELISA, as described by McNeal et al. [32]. Tissue-culture lysate from SA11-infected or mock-infected cells was used as coating antigen. Antibody titers were expressed as units per milliliter, which were determined on the basis of a standard curve, as described elsewhere [31]

AnalysisA 20-point clinical disease severity score was calculated for each child: mild diarrhea was defined as a score of 1–8, moderate diarrhea was defined as a score of 9–14, and severe diarrhea was defined as a score of 15–20, as described elsewhere [2, 35]. Data analysis and parametric/nonparametric tests were done using SPSS for Windows (version 10.0; SPSS). Proportions were compared by the χ2 test. Odds ratios (ORs) for the strength of associations were determined using Epi Info (version 6; US Centers for Disease Control and Prevention)

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Results

Assessment of rotaviral loadOf the 142 children with acute gastroenteritis, 102 (72%) were case patients with rotaviral diarrhea (positive for rotavirus in stool specimens), and 40 (28%) were control subjects with nonrotaviral diarrhea (negative for rotavirus in stool specimens). Rotaviral antigen was detected by ELISA in acute-phase serum specimens from 65 (64%) of the 102 case patients with rotaviral diarrhea and from 1 (3%) of the 40 control subjects with nonrotaviral diarrhea. None of the 30 nondiarrheal control subjects had detectable rotaviral antigen in their serum specimens. Convalescent-phase serum specimens (collected 2–3 weeks after hospital admission) from 35 of the case patients with rotaviral diarrhea were also tested for serum rotaviral antigen, and only 1 (3%) was positive by ELISA (14 U/mL); this patient also had the highest level of serum antigen (360 U/mL) in his baseline serum specimen (collected at the time of hospital admission)

The relative amount of rotaviral antigen was measured in paired stool and serum specimens from the 102 case patients with rotaviral diarrhea. Among the children with antigenemia, the median level of rotaviral antigen in stool was 100-fold greater than that in serum (table 1). The median level of rotaviral antigen in stool was significantly greater among the 65 children whose serum specimens were positive for rotaviral antigen (2670 U/g [range, 40–53,270 U/g]) than among the 37 children whose serum specimens were negative (140 U/g [range, 30–8640 U/g]) (P<.01). Among the 65 children with rotaviral antigen in both their stool and serum specimens, we identified a statistically significant but modest correlation between stool and serum antigen levels (r=0.345; P<.01)

Figure 1
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Figure 1

A Correlation between serum rotaviral antigen level and baseline serum IgG titer among 65 children with acute rotaviral diarrhea and antigenemia. The correlation was evaluated by Spearman’s test (r=-0.532; P<.001). B Correlation between serum rotaviral RNA level and baseline serum IgG titer among 26 children with acute rotaviral diarrhea and antigenemia who were positive for rotaviral RNA. The correlation was evaluated by Spearman’s test (r=-0.567; P=.003)

Table 1
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Table 1

Quantification of rotaviral antigen and RNA in stool and serum specimens from children with acute rotaviral diarrhea and antigenemia

We estimated rotaviral RNA copy numbers by QRT-PCR in a subset of 28 children who had paired stool and serum specimens and whose serum specimens were positive for rotaviral antigen. Rotaviral RNA was detected in 26 (93%) of these children. Rotaviral RNA could not be detected in the serum specimens from 2 (7%) of the children despite the presence of a high level of rotaviral RNA in their stool specimens. QRT-PCR results revealed low levels of rotaviral RNA in serum—the median RNA copy number in serum specimens was ∼1.5×105–fold lower than that in stool specimens (table 1). There was no significant correlation between levels of RNA in serum and stool specimens (r=0.149; P=.466)

Other factors associated with rotaviral antigenemia Several factors were significantly associated with rotaviral antigenemia. Among the 102 case patients with rotaviral diarrhea, the rates for detection of rotaviral antigen in serum varied by age (for 0–5 months, 14%; for 6–11 months, 78%; for 12–23 months, 58%; and for 24–36 months, 37%). Baseline titers of serum IgG antibody to rotavirus were also associated with antigenemia. Among the case patients with rotaviral diarrhea, antigenemia was less common in children with detectable baseline serum IgG titers (46%) than among those without them (83%) (OR, 0.17 [95% confidence interval {CI}, 0.06–0.47]). We plotted serum antigen and serum RNA levels against baseline serum IgG titers (figure 1A and 1B) and found a significant correlation between baseline IgG titers and the levels of serum antigen (r=-0.532; P<.001) and of serum RNA (r=-0.567; P=.003) (figure 1)

The G serotype of the infecting strain was determined in 52 of 65 specimens from serum antigen–positive case patients with rotaviral diarrhea and in 27 of 37 specimens from serum antigen–negative children (table 2). Children infected with G1 strains more often had antigenemia (85%) than did those infected with other G serotypes (48%) (OR, 6.08 [95% CI, 1.88–20.54]). However, the serum antigen levels of the children infected with G1 strains (mean±SD, 24.2±17.1) were not significantly different from those of the children infected with other G serotypes (mean±SD, 22.0±15.8)

Table 2
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Table 2

Relationship between G serotype of infecting strains and rotaviral antigenemia among children positive for rotavirus in stool

Among the case patients with rotaviral diarrhea, the mean clinical disease severity score for those who were positive for serum antigen was 14.0, compared with 13.7 for those who were negative for serum antigen (P=.45). However, the proportion with severe diarrhea (a severity score of >14) was significantly higher (P=.036) among those who were positive for serum antigen (49%) than among those who were negative (24%). We did not find any association between clinical symptoms or disease severity and the level of RNA in serum (r=0.022; P=.911)

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Discussion

The present study confirms and extends the findings of recent reports from the United States and Jamaica indicating that acute rotaviral gastroenteritis in otherwise healthy immunocompetent children is very frequently associated with antigenemia and viremia, as evidenced by the presence of viral RNA in serum [8, 15, 16]. This is the first study to quantify the relative load of rotavirus by QRT-PCR and ELISA in both stool and serum specimens from children and is the largest series of human stool and serum specimens analyzed to date. Of note, although the frequencies of antigenemia and viremia were high, the levels of viral antigen and RNA in serum were extremely low, compared with levels found in stool. This finding may explain why the observation of rotaviral infections at extraintestinal sites is rare and suggests that this viremia is usually benign and silent. It will be interesting to determine whether the level of viremia is significantly increased in those rare cases of infection in which central nervous system, hepatic, or other diseases manifestations occur. Whether rotavirus actually replicates at systemic sites—and, if so, how the level of replication would compare with that in the small bowel—remains to be determined. However, recent experiments in a mouse model of homologous rotaviral infection have shown that extraintestinal viral replication does routinely take place in a variety of organs, including the mesenteric lymph nodes, liver, and lungs of healthy infant mice [36]. In light of these observations, it certainly seems highly plausible that extraintestinal rotaviral replication also takes place in humans and that this may account for at least some of the antigenemia and viremia detected

In the present study, we examined factors that may contribute to antigenemia and viremia. The correlation between stool and serum levels was only moderate for rotaviral antigen and was absent for rotaviral RNA. This finding suggests that the ability of the virus to replicate to a high titer in the intestine may not be the critical determinant of antigenemia or viremia and that other factors, such as extraintestinal replication, may be important. This observation is consistent with the findings of recent experiments conducted in neonatal mice by Mossel and Ramig, who found no correlation between titers of rotavirus in the gut and detection of rotavirus in the liver [37]. Of note, products of the NSP3 gene of rotavirus were found to be the major determinant of extraintestinal spread. This relative lack of correlation between levels of intestinal replication and systemic viremia was also observed recently during a quantitative comparison of viremia in mice after murine versus simian rotaviral infection [36]. One potential explanation for the lack of correlation is that the major source of virus in serum is not the intestine but some other site. A recent animal study by Azevedo et al. has shown that viremia could occur from intranasal, oral, or gavage inoculation of Wa human rotavirus [38]

Prior exposure to rotavirus appears to protect against antigenemia and viremia. Here, children with preexisting antibodies, as evidenced by the presence of IgG to rotavirus in acute-phase serum, were less likely to develop detectable levels of antigen or RNA in serum. The rate of antigenemia was lower in both young infants (<5 month) and older children (24–36 months) than in those 6–23 months, an observation that might be explained by the presence of circulating antibody acquired either maternally or from natural exposure to rotavirus. Presumably, circulating antibody could scavenge and facilitate the removal of rotaviral antigen or RNA in the bloodstream. Alternatively, circulating antibody might inhibit viral replication at systemic, but not mucosal, sites

Finally, we noted a significant correlation between the G1 serotype of rotavirus and antigenemia. Although various viral strains can differ significantly in their tropism and other growth characteristics, a plausible biological basis of this association is currently lacking. An earlier study detected an association between infection with a G1 strain and severe illness [2]; however, in the present study, an association between antigenemia and disease severity was not confirmed. Furthermore, unlike Chiappini et al., we did not find an association between clinical symptoms or disease severity and the presence of RNA in serum, on the basis of 28 children assessed [15]

An issue of considerable relevance is to understand the basic mechanism of viral spread in immunocompetent children. Although rotavirus clearly spreads beyond the intestine in animal models [23–25, 38, 39], there is no direct proof of the presence of infectious viruses at extraintestinal sites in children, and evidence of rotaviral replication has been shown only rarely in immunodeficient children [9]. Therefore, in humans, it is not clear whether the virus transits from infected intestinal cells and then replicates in the blood or other tissues or is only passively present in the circulation, perhaps by transepithelial transport through the M cells of the intestinal epithelium. However, recent studies have indicated that extraintestinal replication does frequently occur during murine rotaviral infection in mice [36]. An investigation that might provide greater clarity on this issue would be the analysis of possible viral replication in peripheral-blood monocytes from human subjects by QRT-PCR

Our study failed to demonstrate an association between severe disease and viremia, although, in this case, severity was a measure of diarrhea and dehydration. It remains to be seen whether there is a correlation between viremia and other severity measures, such as death

In conclusion, our findings confirm previous observations that antigenemia and viremia occur frequently in children during rotaviral gastroenteritis and that factors other than the level of viral shedding, such as circulating antibody levels and the G1 serotype, appear to influence systemic spread. We also demonstrated that rotaviral RNA and antigen levels detected in serum were significantly lower than rotaviral RNA and antigen levels detected in stool. This indicates that rotaviral replication in children is primarily focused in the small intestine. However, the number (40,000 to 4 million) of rotaviral RNA copies per milliliter of serum is likely to represent a significant infectious viral load in the circulation, which could seed infection of other organs. Recent experiments in mice have shown that such organs as the mesenteric lymph nodes, liver, kidney, and lungs can support rotaviral replication [36]. Although rotaviral replication has not been detected in these organs in nonimmunocompromised children, rotaviral RNA has been detected in the spleen, heart, lungs, kidney, bladder, and pancreas of children who experienced rotavirus-associated deaths [30]. Whether the viral RNA detected in these children was the result of extraintestinal viral replication or simply contamination by blood is not clear. Future studies of tissue specimens obtained from children during acute rotaviral infection will be needed to directly answer these questions, but such studies will be very difficult to accomplish

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Acknowledgments

We thank Mary Wilson and Mariyamma Philip, for excellent technical support; Dr. Ekta Mutta and other doctors and nursing staff at the All India Institute of Medical Sciences hospital, for specimen collection; and the parents of the children, for their participation

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Footnotes

  • ↵Presented in part: XIII International Congress of Virology, San Francisco, 23–28 July 2005 (abstract 262-V)

    Potential conflicts of interest: none reported

    Financial support: Department of Biotechnology, Government of India, and the US Centers for Disease Control and Prevention, through the Indo-US Vaccine Action Program and the Program for Appropriate Technology in Health

  • Received January 13, 2006.
  • Accepted March 31, 2006.
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  1. J Infect Dis. (2006) 194 (5): 588-593. doi: 10.1086/505878
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