PEDIATRICS Vol. 122 No. 2 August 2008, pp. 392-397 (doi:10.1542/peds.2007-2290)
ARTICLE |
Analysis of Rotavirus Antigenemia and Extraintestinal Manifestations in Children With Rotavirus Gastroenteritis
a Departments of Pediatrics
b Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
c Department of Pediatrics, Kariya Toyota General Hospital, Kariya, Aichi, Japan
d Department of Pediatrics, Toyokawa Municipal Hospital, Toyokawa, Aichi, Japan
e Department of Pediatrics, Showa Hospital, Konan, Aichi, Japan
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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OBJECTIVE. This study was conducted to examine the association between rotavirus antigenemia and clinical features, particularly extraintestinal manifestations, and the association between serum cytokine levels and rotavirus antigen quantity.
METHODS. Sixty hospitalized children who received a diagnosis of acute rotavirus gastroenteritis were enrolled in this study. Paired serum samples were collected from the 60 children when admitted to and discharged from the hospital. Associations among viral antigen levels and fever, elevated transaminase levels, and seizures were evaluated to determine whether antigenemia correlated with disease severity. Viral antigen was measured by using an in-house enzyme-linked immunosorbent assay that detected VP6 antigen. A flow-cytometric bead array was used to measure serum cytokine levels.
RESULTS. Rotavirus antigen levels were significantly higher in serum collected at the time of hospital admission than at the time of discharge. Serum rotavirus antigen levels peaked on day 2 of the illness (2.02 ± 0.73), followed by a gradual decrease in antigen levels to nearly undetectable levels by day 6. The quantity of rotavirus antigen was significantly higher in serum collected from patients with fever than those without fever. The presence or absence of elevated transaminase levels and seizures was not associated with serum rotavirus antigen levels. A weak but significantly positive association was observed between interleukin 8 levels and antigenemia. A weak but significantly negative association was observed between interleukin 10 levels and antigenemia.
CONCLUSIONS. Rotavirus antigenemia is frequently observed in a patient's serum during the acute phase, and viral antigen levels change dramatically during the acute phase of the illness. Because patients with fever had higher rotavirus antigen levels, antigenemia severity might contribute to fever. The host immune response plays an important role in controlling antigenemia levels.
Key Words: rotavirus • antigenemia • cytokine
Abbreviations: ELISA—enzyme-linked immunosorbent assay • PBST—phosphate-buffered saline that contains Tween 20 • CBA—cytometric bead array • OD—optical density • IL—interleukin
Rotavirus is the major cause of gastroenteritis in young children worldwide. Severe dehydration caused by rotavirus-induced diarrhea and vomiting can be fatal in developing countries. Meanwhile, gastroenteritis induced by rotavirus infection causes a large economic burden in developed countries. Initially, rotavirus replication was thought to be limited to the gastrointestinal tract in patients with gastroenteritis; however, it is widely known that rotavirus gastroenteritis is sometimes complicated by high fever, elevated transaminase levels,1,2 seizures,3–5 and encephalitis,6,7 which may be caused by systemic viral infection. Rotavirus RNA has been detected in the cerebrospinal fluid of patients with convulsions8; however, it is unclear whether the RNA was really there. Rotavirus antigens and RNA were detected in the serum of children with rotavirus infection.9–11 In addition, several investigators have demonstrated that rotavirus antigen is detected not only in serum but also in multiple organs, including the stomach, intestine, liver, lung, spleen, kidney, pancreas, thymus, and bladder, in rotavirus-infected animals.12 These findings suggest that rotavirus spreads beyond the intestine in children with rotavirus gastroenteritis, resulting in systemic viral infection.
We have demonstrated that viremia generally correlates with illness severity in children with varicella-zoster virus infection13 and human herpesvirus 6 infection.14 Although several lines of evidence suggest that systemic rotavirus infection occurs in infected children,9–11 it is not well understood whether rotavirus antigenemia levels correlate with disease severity. Only 1 report has postulated that rotavirus RNAemia is associated with high fever, but this study did not present statistical analyses on these findings.15 Moreover, it has been suggested that cytokines may play an important role in rotavirus gastroenteritis pathogenesis16–21; however, no clinical studies have investigated the correlation between levels of rotavirus antigenemia and serum cytokines to date. We hypothesized that the grade of systemic rotavirus replication and cytokine levels induced by viral infection play an important role in extraintestinal manifestations such as fever, elevated transaminase levels, and seizures; therefore, in this study, we examined the association between rotavirus antigenemia and clinical features, particularly extraintestinal manifestations. Moreover, because it has been suggested that cytokines are involved in the pathogenesis of rotavirus gastroenteritis, we also examined the association between serum cytokine levels and rotavirus antigen levels.
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METHODS |
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Patient Characteristics and Sample Collection
Sixty hospitalized children with a diagnosis of acute rotavirus gastroenteritis were enrolled in this study. All patients were admitted to 1 of 4 pediatric departments (Fujita Health University, Kariya Toyota General Hospital, Toyokawa City Hospital, or Showa Hospital) between December 2004 and March 2006. The patients' guardians consented to their participation in this study. This study was approved by the review boards of all 4 institutes. Diagnosis of rotavirus gastroenteritis was confirmed by detection of rotavirus antigen in stool samples by using an immunochromato assay (Dipstick [Eiken, Tokyo, Japan]). Paired serum samples were collected from the 60 children (age: 1.4 ± 1.4 years; gender: 40 boys and 20 girls) at the time of hospital admission (days 1–5; day 1 was defined as the date of onset of symptoms [eg, fever, vomiting, diarrhea]) and discharge (days 4–13). In addition, 20 serum samples were collected from age-matched control children whose stool samples were negative for rotavirus antigen by the immunochromato assay.
Clinical features of the children were examined retrospectively by using medical charts. To assess whether serum rotavirus antigen levels correlated with disease severity, we examined the association between viral antigen levels and fever (>37.5°C), elevated transaminase levels (alanine aminotransferase: >50 IU/L), and seizures, symptoms that are caused by systemic viral infection. The presence or absence of these clinical manifestations was evaluated from the data collected at the time of hospital admission.
Rotavirus Antigen Detection
Rotavirus antigen was measured using an in-house enzyme-linked immunosorbent assay (ELISA) that detects VP6 antigen of the virus. Fifty microliters of diluted (1:16) serum was used to detect rotavirus antigen. The dilution ratio was determined from preliminary studies by using several different serum samples that were collected from patients with rotavirus gastroenteritis. As shown in Fig 1, a 1:16 dilution was appropriate to measure quantitatively viral antigen in patients' serum. Ninety-six–well plates (Nalgen Nunc International, Rochester, NY) coated with a monoclonal antibody against the VP6 antigen of rotavirus (YO-156)22 were used for the ELISA.23 The antibody YO-156 (immunoglobulin G2a subclass) is highly reactive with a common epitope of group A rotaviruses, and the antibody can detect all of the group A rotavirus strains that have been examined. Specificity of the antibody was confirmed by immunoprecipitation assay, immune electron microscopy, and Western blotting analysis.22 After blocking with 1% bovine serum albumin in phosphate-buffered saline that contains Tween 20 (PBST), the plate was washed with PBST. The plate was then incubated with 50 µL of diluted patients' serum at 4°C overnight. After washing the plate with PBST, 50 µL of antihuman rotavirus hyperimmune rabbit serum diluted 1:5000 with PBST that contained 2.5% skim milk was added to each well. The plate was incubated at 37°C for 1.5 hours. After the plate was washed with PBST, it was incubated with a 1:5000 dilution of peroxidase-conjugated donkey antirabbit immunoglobulin G (Jackson ImmunoResearch Laboratory, Inc, West Grove, PA) at 37°C for 1.5 hours. The quantity of monoclonal antibody bound to rotavirus VP6 antigen was assessed after addition of the substrate. The optical density (OD) was read by spectrophotometry at a 492-nm wavelength. To establish an appropriate cutoff value to distinguish between rotavirus-positive and -negative samples, we tested 20 serum samples that were collected from control subjects. Because the mean OD of the control samples was 0.084 ± 0.014, we defined 0.13 (mean ± 3 SD) as the baseline value in this study.
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Cytokine Measurements
The flow-cytometric bead array (CBA) was used according to the manufacturer's protocol (Becton Dickinson, San Diego, CA).24 CBA measured the following cytokines: interleukin (IL)-8, IL-1β, IL-6, IL-10, tumor necrosis factor
, and IL-12. Fifty microliters of sample (standards or test) were added to 50 µL of a cocktail of capture beads and detector antibodies, and the mixture was incubated for 1.5 hours at room temperature in the dark. Excess unbound detector antibody was removed by washing, and 50 µL of reagent was added before data acquisition. Two-color flow-cytometric analysis was performed using a flow cytometer (FACScan [Becton Dickinson, Franklin Lakes, NJ]). A total of 1800 events were acquired by following the protocol supplied. Analysis was performed using CBA dedicated analysis software (CellQuest [Becton Dickinson]). All samples for which the calculated cytokine concentration was below the given sensitivity were treated as undetectable.
Statistical Analysis
Statistical analyses were performed by using Stat View 5.0 (SAS Institute, Cary, NC). Patient gender was compared between the 2 groups by using a 
2 test. Unpaired comparisons between the patient's age and days of sampling were performed by using a Student's t test. Mean peak absorption levels were compared between the 2 groups by using either a Wilcoxon signed-ranks test or Mann-Whitney U test. Spearman's rank correlation coefficients were used to measure the strength of the association between cytokine levels and rotavirus antigen quantity.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Kinetics of Rotavirus Antigenemia
First, we compared rotavirus antigen levels at the time of hospital admission with levels at the time of discharge. Serum samples that were collected at the time of admission were between days 1 and 5 of the illness, and those that were collected at the time of discharge were between days 4 and 13 of the illness. Rotavirus antigen was significantly higher in serum that was collected at the time of admission (days 1–5) than in those collected at the time of discharge (days 4–13; P < .0001; Fig 2). In addition to the antigen levels, detection rate of serum rotavirus antigen was also high in serum that was collected at the time of admission (54 of 60 [90%]) in comparison with the samples that were collected at the time of discharge (19 of 60 [31.7%]). To determine rotavirus antigen kinetics after the onset of illness, we monitored the optical densities in all 120 serum samples. As shown in Fig 3, serum rotavirus antigen levels peaked on day 2 (2.02 ± 0.73), followed by a gradual decrease to nearly undetectable levels by day 6. High detection rates (ranging between 89% and 94%) were also observed in serum samples that were collected until day 5.
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Association Between Rotavirus Antigenemia Levels and Clinical Symptoms
OD values in serum samples that were collected at the time of admission were compared between patients with and without each clinical manifestation (Fig 4). Although patients' backgrounds were not different between the groups with (gender: 28 boys and 16 girls; age: 1.4 ± 1.3 years; sampling time: 2.7 ± 1.2 days) and without fever (gender: 11 boys and 5 girls; age: 1.4 ± 1.5 years; sampling time: 3.2 ± 1.3 days), the quantity of rotavirus antigen was significantly higher in the serum that was collected from the patients with fever (1.70 ± 0.87) than those without fever (1.07 ± 0.98; P = .0273). No febrile episode was observed during admission period in patients without fever at the time of admission to the hospital. Rotavirus antigen levels in patients with (alanine aminotransferase: 84.8 ± 61.8 IU/L) and without elevated transaminase levels were 1.16 ± 0.96 and 1.60 ± 0.92, respectively, with no statistical difference in the antigenemia levels between the 2 groups (P = .2351). Finally, we tested whether the level of rotavirus antigenemia was correlated with seizures. The number of patients who experienced seizures was small (n = 7). Moreover, 4 of the 7 patients had febrile seizures, whereas the remaining 3 patients had afebrile seizures, which were diagnosed as benign convulsions associated with mild gastroenteritis. No statistical difference was observed in rotavirus antigenemia levels between patients with (1.38 ± 1.04) and without seizures (1.56 ± 0.93; P = .6451).
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Association Between Levels of Rotavirus Antigenemia and Cytokines
The associations between the levels of 6 cytokines and the quantity of rotavirus antigen in acute serum samples were examined by Spearman's rank correlation coefficients (Fig 5). We identified a weak (r = 0.36) but significantly (P = .0041) positive association between IL-8 levels and the severity of rotavirus antigenemia. Although a similar correlation was demonstrated between IL-6 levels and quantity of viral antigen (r = 0.23), it was not statistically significant (P = .0697). Meanwhile, there was a weak (r = –0.258) but significantly (P = .0464) negative association between IL-10 levels and rotavirus antigenemia. No statistical correlation was observed between the other 3 cytokines levels and antigen levels.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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An in-house ELISA that used a monoclonal antibody YO-156 directed to group A common epitope on VP6 was used to measure serum rotavirus antigen in this study. VP6 is an inner capsid protein in rotavirus particle and is the most abundant protein detected in rotavirus particle and in the cells that are infected with rotavirus. The VP6 carries group-specific and subgroup-specific antigens. Our monoclonal antibody can detect both of the 2 subgroups (I or II). VP4 and VP7 are the outer capsid proteins, and they are associated with P-type and G-type specificities, respectively. In human rotavirus, the presence of at least 10 P types and 10 G types has been reported. Thus, to detect rotavirus antigen efficiently, it is reasonable to use monoclonal antibody that commonly is reactive to VP6 of any human rotavirus strain. To determine the appropriate serum dilution to clarify differences in viral antigen levels, we measured OD values in serially diluted serum samples and found that serum diluted 16-fold was the best to quantify viral antigen (Fig 1). As shown in Fig 3, the detection rate of rotavirus antigen in patients' serum ranged between 89% and 94% until 5 days after illness onset, suggesting a high frequency of rotavirus antigenemia during the acute phase of rotavirus gastroenteritis. This result supports previous studies that demonstrated frequent detection of rotavirus antigen in acute-phase serum; however, the detection rate of rotavirus antigen was higher in our study than in 2 previous studies.10,11 The previous studies used undiluted serum to measure viral antigen, whereas we used serum diluted 16-fold, suggesting that our ELISA system has greater sensitivity than previously published ELISA methods. Indeed, in our comparison of the sensitivity between our in-house ELISA and a commercial kit (Rotaclone [Meridian Bioscience, Inc, Cincinnati, OH]) by using same serum samples, our in-house ELISA exhibited clearly higher sensitivity than the commercial kit (data not shown).
Previous reports demonstrated that rotavirus antigen levels decreased to baseline levels during a 3- to 4-week interval by testing paired serum samples10; however, this study demonstrated that rotavirus antigen levels were significantly lower at the time of discharge from the hospital than at the time of hospital admission (P < .0001). In addition, the viral antigen kinetic analysis showed that viral antigen peaked on day 2 of the illness, and antigen levels quickly returned to almost undetectable levels by day 6. Fischer et al10 also examined viral antigen levels after disease onset but demonstrated unclear viral antigen kinetics. Thus, to our knowledge, this is the first study to demonstrate the kinetics of rotavirus antigenemia during the acute phase of rotavirus gastroenteritis. The high sensitivity of our ELISA system and sampling schedule allowed us to clarify such a dynamic change of viral antigen during acute illness. Because both innate and adaptive immunities play an important role in viral clearance, an analysis of the antirotavirus host immune response and rotavirus antigenemia would clarify the pathogenesis of rotavirus gastroenteritis.
Although 3 extraintestinal manifestations were evaluated in this study, only fever was statistically associated with viral antigen levels. It has been demonstrated that younger patients generally have higher viral antigen levels than older patients, because viral antigen levels are higher in patients with primary rotavirus infections than those reinfected with the virus.11 Moreover, sampling time affects rotavirus antigen detection in serum, as shown in Fig 3. When we compared the mean age and sampling time between patients with and without fever, no significant difference was observed between these 2 groups. Thus, we believe that serum rotavirus antigen levels are associated with the occurrence of fever in patients with rotavirus gastroenteritis. There was no correlation between rotavirus antigen levels and 2 additional manifestations: elevated transaminase levels and seizures. The elevated transaminase levels that occurred in these children was mild, as demonstrated previously.2 It seems that systemic spread of rotavirus antigen is not involved in mild elevated transaminase levels in patients with rotavirus gastroenteritis. It is widely known that rotavirus can cause both febrile seizures5 and benign convulsions associated with mild gastroenteritis.3,4 Because the number of patients with seizures was small and the patients with seizures had 2 different types of convulsions, it is difficult to analyze conclusively the correlation between rotavirus antigenemia and seizures. A large number of patients with rotavirus gastroenteritis should be analyzed to determine an association between central nervous system complications, such as seizures and encephalopathy, and viral antigen levels. Moreover, rotavirus antigen should be measured in the cerebrospinal fluid to determine whether the virus can directly invade the central nervous system, as was previously suggested by reverse transcriptase–polymerase chain reaction analysis.8 Furthermore, because other variables, including genotype, primary or secondary infection, and coinfection with other pathogens, were not evaluated in this study, an association between these factors and antigenemia levels should be analyzed in future study.
It is widely known that cytokines play an important role in the pathogenesis of viral infections. Several in vivo and in vitro studies have suggested that cytokines are involved in the pathogenesis of rotavirus infection.16–21 Although Jiang et al20 demonstrated that several cytokines are associated with the severity of rotavirus gastroenteritis, a correlation between cytokine production and rotavirus antigen levels has not been studied. Six cytokines were measured in serum samples that were collected at the time of hospital admission by CBA, a method that can simultaneously measure multiple cytokine levels in a single reaction and therefore requires only small amounts of serum. Two (IL-8 and Il-10) of the 6 cytokines were statistically correlated with rotavirus antigen levels in this study. It has been demonstrated that rotavirus infection can induce the production of several chemokines, including IL-8, in intestinal cell lines and small animal models.18,21 IL-8 may play an important role in generating the mucosal immune response to rotavirus infection. This study showed that significantly higher IL-8 levels were observed in patients with higher levels of rotavirus antigenemia. The results of this study together with previous in vitro and in vivo studies18,21 suggest that increased systemic spread of the virus could trigger a strong immune response in the host.
Note that IL-10 levels were negatively correlated with rotavirus antigen levels. This cytokine has modulatory effects on both T-helper 1 and 2 cytokines and plays a regulatory role in inflammatory processes. It has been demonstrated that plasma IL-10 levels increase in the acute phase of rotavirus gastroenteritis17,20; however, the role of this cytokine in disease pathogenesis remains obscure.
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CONCLUSIONS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The results of this study demonstrate that IL-10 levels were significantly lower in patients with high rotavirus antigen levels, which suggests that this cytokine plays an important role in the immune response against systemic rotavirus infection.
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ACKNOWLEDGMENTS |
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This work was supported in part by a Grant-in-Aid for the 21st Century Center of Excellence Program of Medicine and the Open Research Center, both at Fujita Health University, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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FOOTNOTES |
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Accepted Dec 30, 2007.
Address correspondence to Tetsushi Yoshikawa, MD, Fujita Health University School of Medicine, Department of Pediatrics, Toyoake, Aichi 4701192 Japan. E-mail: tetsushi{at}fujita-hu.ac.jp
The authors have indicated they have no financial relationships relevant to this article to disclose.
| What's Known on This Subject Rotavirus antigen is detected in acute-phase serum collected from patients with rotavirus gastroenteritis.
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| What This Study Adds An association between rotavirus antigenemia and clinical features, particularly extraintestinal manifestations, and the association between serum cytokine levels and rotavirus antigen quantity are clarified.
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PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics
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