PEDIATRICS Vol. 117 No. 5 May 2006, pp. e827-e832 (doi:10.1542/peds.2005-1556)
Intussusception After Administration of the Rhesus Tetravalent Rotavirus Vaccine (Rotashield): The Search for a Pathogenic Mechanism
a Viral Gastroenteritis Section, Division of Viral and Rickettsial Diseases
b Infectious Disease Pathology Activity, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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OBJECTIVES. The rhesus tetravalent rotavirus vaccine (RRV) was withdrawn from the routine program for childhood immunization in the United States because of the rare and unexpected occurrence of intussusception in the 2-week period after administration of the first dose.
METHODS. To search for the pathogenesis of this association, we compared the pathology of surgical specimens from infants who had surgical reduction of their intussusceptions within 2 weeks of receiving the vaccine (case patients; n = 8) with the pathology of specimens from children who had surgery >2 weeks after immunization (n = 6) or who had never been immunized (n = 26). Tissue was examined for evidence of the vaccine strain of rotavirus by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemical staining.
RESULTS. RRV was identified by RT-PCR in tissue samples from 7 of the 8 case patients and in 2 of the 6 children who received the vaccine at a more distant time (29 and 58 days before surgery), but it was not identified in samples from any of the nonvaccinated children. No evidence of rotavirus tissue involvement was detected in any of the children by in situ hybridization or immunohistochemical staining. Pathologic evidence (for example, inclusion bodies, smudge cells) of adenovirus infection was present in 35% of the 37 specimens examined by routine staining and immunohistochemistry.
CONCLUSIONS. The fact that RRV was detected by RT-PCR but not by either of the other assays could be explained by RRV being present in the lumen of the gut but not in the tissues of appendix, ileum, or Peyer's patches. The Peyer's patches were not hyperplastic, and we could not establish the pathogenic mechanism for this association.
Key Words: rotavirus • intussusception • vaccines • adverse events
Abbreviations: RRV—rhesus tetravalent rotavirus • RT-PCR—reverse transcriptase-polymerase chain reaction • IHC—immunohistochemistry • ISH—in situ hybridization • CDC—Centers for Disease Control and Prevention • H&E—hematoxylin and eosin • HRV—human rotavirus
In August 1998, the US Food and Drug Administration licensed the first rotavirus vaccine, Rotashield, a live, orally administrated, tetravalent rhesus-human reassortant (RRV-TV) vaccine. The vaccine was subsequently recommended by the Advisory Committee on Immunization Practices and the American Academy of Pediatrics for the routine immunization of children on a 2-, 4-, and 6-month schedule.1,2 Between November 1998 and July 1999, an estimated 600000 children received 
1 doses of the vaccine. In July 1999, reports of intussusception after administration of RRV-TV3 led to a suspension of the recommendation. An investigation was conducted to determine if the vaccine was causally associated with intussusception.4 When results from a series of epidemiologic studies confirmed the association,5–9 The Advisory Committee on Immunization Practices and American Academy of Pediatrics withdrew their recommendations for use of the vaccine in November 1999,10 and the manufacturer, Wyeth Lederle Vaccines and Pediatrics (Pearl River, NY), withdrew the vaccine from the market.
Although the statistical association between RRV and intussusception was documented in epidemiologic studies, the mechanism of this relationship remains unclear. Lack of understanding of the mechanism could impede development of other live oral rotavirus vaccines11,12 and raise doubts about licensed oral vaccines against typhoid, cholera, and polio.13 For instance, concerns were immediately raised that oral poliovirus vaccine might also be associated with intussusception, but this was not supported by an international consultation on the issue.13 The pathogenesis of intussusception is not well understood, but the presence of mesenteric lymphadenopathy or inflamed Peyer's patches supports an infectious or inflammatory process, and viruses such as adenovirus, human herpesvirus 6 and 7, Epstein-Barr virus, cytomegalovirus, and wild-type human rotavirus (HRV) have been implicated.14
We examined the putative role of RRV-TV as a specific etiologic agent of intussusception and wanted to examine tissue to determine if we could identify a pathologic basis for this association. We developed assays to detect and type rotaviruses in formalin-fixed tissues by immunohistochemistry (IHC), in situ hybridization (ISH), and reverse transcriptase-polymerase chain reaction (RT-PCR).15 Tissues from children who underwent surgery for intussusception in the 2 weeks after receipt of RRV-TV (case patients) were compared with surgical specimens from children who had received RRV-TV at a time more distant from the intussusception and with specimens from children who had never been vaccinated with RRV-TV (controls).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Tissue Sources
We obtained surgical specimens of intestinal or lymph node tissues from patients with intussusception after RRV-TV and from children with intussusception identified in the multistate case-control study who did not receive the vaccine.5 We obtained tissue from intussusception patients who were reported to Vaccine Adverse Events Reporting System (11 vaccinees) and from children investigated as part of the Centers for Disease Control and Prevention (CDC) case-control study (4 vaccinees, 25 controls). The study was approved by the Institutional Review Boards of the CDC and of 14 participating states (California, Florida, Georgia, Illinois, Indiana, Minnesota, New Jersey, New York, Ohio, Pennsylvania, South Carolina, Tennessee, Texas, and Washington). For cases identified through Vaccine Adverse Events Reporting System, the CDC used the contact information on the report form to contact the child's physician or parents. The CDC then contacted the appropriate department of pathology to request the specimen. Informed consent was obtained from parents or guardians of infants in the case-control study to release to the CDC the name of their child, to obtain all information and data collected from the original study, and to secure tissues specimens from the pathologists involved for additional analysis.
Acceptable Tissues
Any available surgical specimens were acceptable for this study (for example, tissue from resected bowel, appendix, and mesenteric lymph nodes). Preferred specimens were tissues that were formalin-fixed, paraffin-embedded, or fresh-frozen. Unstained slides were also accepted for limited examination. Tissue specimens were collected on the basis of 2 criteria: (1) the child had a radiologically or surgically confirmed case of intussusception and underwent surgery to reduce the intussusception and (2) the child was 2 to 11 months of age at the onset of intussusception.
Histopathologic Evaluation
Pathologists were blinded to the vaccination status of infants whose tissues were tested, and the clinical and vaccine data were combined with the pathology results only after examination. Routine histopathologic evaluation was performed on hematoxylin and eosin (H&E)-stained slides, and specimens were tested by using 3 diagnostic techniques: IHC, ISH, and RT-PCR.15 The antibody used in the IHC assays was a polyclonal rabbit antibody directed against HRV Wa strain, which crossreacts with RRV. The positive controls used in the IHC assays included cells infected with HRV and RRV, respectively, and small intestinal tissues from experimentally infected piglets (provided by Linda Saif, MD, Ohio State University, Worster, OH). The negative control antibody was preimmune serum from the same rabbit from which the polyclonal antibody was generated. Uninfected cells were used as negative controls. A monoclonal antiadenovirus antibody was used in the IHC assay for adenovirus.16 For ISH, the RNA probes were generated from PCR products amplified from the rotaviral genes encoding VP4, NSP4, and NSP1 and tailored with the T7 promoter.17 For RT-PCR, RNA was isolated from formalin-fixed tissue using a commercial RNaid Plus kit (Bio101, Vista, CA) as described previously.15 RNA extracts were analyzed with a 1-step procedure followed by confirmation of the PCR products by probe hybridization using type-specific primers and an oligonucleotide probe to detect the RRV VP4 gene. The HRV VP4 gene was detected with degenerate primer pair con1/con2 (spanning nucleotides 676–887) and digoxigenin-labeled oligonucleotide probe A VP4-c1(nucleotides 743–763), which is homologous to common HRV P[8] and P[4] strains (J.R.G., unpublished data). Negative controls included extracts from rotavirus-negative tissues. Positive controls were RNAs extracted from Wa (P1A[8], G1) or DS-1 (P1B [4], G2) rotavirus-infected cell lysates.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Study Population
Infants had to be <7 months old to receive the first dose of vaccine, and most had received only 1 dose; consequently, case patients (ie, those who developed intussusception in the 2-week period after vaccination with RRV-TV) were younger than controls (P = .007) and developed intussusception only after the first vaccine dose (Table 1). No other significant differences were noted between cases and controls.
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Specimen Evaluation
We received tissue specimens or slides from 41 infants with intussusception. Of these, 37 were suitable for testing by IHC and ISH, whereas no additional testing could be performed on 4 samples submitted with H&E slides only. Of the 37 patients with suitable specimens, 19 had intestinal tissue, 17 had appendices (4 with lymph nodes), and 1 had a lymph node only. Of these, 8 tissues were obtained from case patients immunized in the 2 weeks preceding the onset of intussusception, the period of highest estimated relative risk for intussusception associated with receipt of RRV-TV.5
Virus Detection
The presence of a virus (rotavirus or adenovirus) was detected in 23 (62%) of 37 specimens. Adenovirus was the most commonly detected (13 of 37 [35%] samples), and it was associated with distinct histopathologic findings, including Cowdry type A inclusion bodies and smudge cells, on H&E staining of the mucosal epithelial cells. RRV-TV was detected in 9 (36%) of 25 specimens tested, and a wild-type HRV was detected in 1 (4%) of 25 specimens.
Rotavirus
A total of 25 specimens were tested by RT-PCR for rotavirus. Of these, 8 were from case patients vaccinated in the 2 weeks preceding onset of intussusception, 7 were from children vaccinated >2 weeks before onset and whose intussusception was believed to be unrelated to the vaccine, and 10 were from controls chosen from the entire group on the basis of the quality of the specimens available. Of the 25 specimens, 10 were positive for rotavirus; a natural rotavirus strain was detected in a sample from a nonvaccinated control, and RRV-TV strains were detected in samples from 9 vaccinees. Of the latter, 7 were detected in the 8 case patients who had received the vaccine in the previous 2 weeks, and the remaining 2 were detected in children who had received the vaccine 29 and 58 days before the onset of intussusception. For these 2 late cases, virus was present in small quantity because specimens were positive only on probe hybridization of the RT-PCR product. None of the tissues, including those positive for RV by RT-PCR, was positive by ISH or IHC. By H&E staining, none of the lymph nodes or Peyer's patches from infants who had been recently vaccinated showed definitive evidence of lymphoid hyperplasia.
Adenovirus
Adenovirus was the most common virus detected by histopathologic evaluation and IHC testing in 13 (35%) of the 37 specimens examined (Fig 1). These specimens had distinct histopathologic features, including smudge cells and Cowdry type A inclusion bodies. The frequency of detection did not differ significantly between controls (9 of 22 [41%]) and vaccinees (4 of 15 [27%]).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This study was done to gain greater understanding of the possible role of RRV-TV in the pathogenesis of RRV-TV-associated intussusception. We identified RRV-TV RNA by RT-PCR in tissue from 7 of 8 vaccinees who received RRV-TV in the 2 weeks before surgery but found no evidence of RRV in the same tissues by IHC or ISH. These findings can be explained in several ways. First, the sensitivity of RT-PCR allows for detection of as few as 10 viral particles,15 and in a child recently vaccinated with a live oral rotavirus vaccine, a positive test result is not unexpected. This finding confirms the high sensitivity of the assay to detect a small quantity of virus when present in pathology specimens. In fact, in 2 vaccinees, RRV-TV RNA was detected at 29 and 58 days after vaccination by probe testing of the RT-PCR product, a finding that indicates how long an attenuated rotavirus can remain in the gastrointestinal tract even at a low level of detection and that because of this extra probing of the PCR product is required. Second, tissues available for testing in this study were mostly from the ileocecal junction, appendiceal or lymph node tissues. The primary site of natural rotavirus infection is in the small intestine,18 but tissues from these sites were not available for testing. Last, as PCR testing does not localize RRV RNA to a particular tissue or cell type, the positive PCR results could only represent virus in the intestinal lumen, whereas the negative IHC and ISH results suggest no direct tissue invasion. In addition, by direct staining, no evidence of hyperplasia was noted in the Peyer's patches, a finding that in the past has been associated with intussusception.
Our finding of adenovirus in 35% of the patient tissue samples supports the results of previous studies that have found these viruses in 30% to 50% of tissue samples from children with intussusception.14,19–23
This study was limited by the availability of tissues in pathology departments and by the types and quality of tissue submitted. For infants who did not have surgical resection of intussusception, only appendices or lymph nodes were available for testing. Intestinal tissues were obtained from infants who had surgical resections; however, the quality and quantity of tissues varied, and in some cases, small quantities of tissues may not have been representative of the ongoing pathologic process. Rotavirus normally infects the small intestine, but intussusception occurs distant, at the ileal-cecal junction, so epithelial invasion is not anticipated. The sensitivity and specificity of the 3 test methods was also an issue in interpretation of results, because the most specific method, ISH, did not confirm the results of the most sensitive method, RT-PCR.15 Last, although we originally estimated that >30 specimens would be available, given the initially large risk of intussusception estimated as a consequence of RRV-TV administration, a reassessment of and lowering of this risk during our study6 led to a substantially smaller number of specimens being available. In fact, we were successful in obtaining specimens from all recently vaccinated children for whom intussusception and resection could be documented.3,5
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CONCLUSIONS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Despite pathologic studies, the mechanism of intussusception in infants who received RRV-TV still eludes us. The detection of RRV-TV RNA indicates the presence of virus in the gastrointestinal tract, but the absence of RRV-TV antigen and RNA in tissue and the lack of representative intestinal tissues for extensive histopathologic examination precluded us from reaching more definitive conclusions. The molecular and immunologic methods developed in this study can be used in future studies of formalin-fixed tissues to explore further the role of adenovirus and HRV in the etiology of intussusception and the pathogenesis of gastroenteritis and the investigation of deaths associated with these agents. Such methods could also be used in future vaccine evaluation studies.
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ACKNOWLEDGMENTS |
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We thank Paul Gargiullo, Trudy Murphy, and David Nelson for providing the information gathered in the case-control study and Claudia Chesley for editorial assistance in preparing the manuscript. We also acknowledge the initial consultation from the Rotavirus Pathology Study Group (Dennis Lang [National Institute of Allergy and Infectious Diseases]; Diane Griffin [Bloomberg School of Public Health, Johns Hopkins University]; Richard Ward [Cincinnati Children's Hospital]; Harry B. Greenberg [Stanford University Medical School]; Albert Z. Kapikian [National Institute of Allergy and Infectious Diseases]; Amir Kende, Lena A. Kombo, and Mary Estes [Baylor College of Medicine]; David O. Matson [Eastern Virginia Medical School]; Melinda Wharton [National Immunization Program, Centers for Disease Control and Prevention]; Scott Campbell, Mitchell Wolfe, and Paul Offit [Children's Hospital of Pennsylvania]; Penny Heaton and Alan Shaw [Merck Vaccines]; Robert Breiman [National Vaccine Program Office]; and Steve Udem, Frederick E. Varricchio, and William Gruber [Wyeth Lederle Vaccines and Pediatrics]) and state health department investigators (Jerry Narramore [Tennessee], Natalie Smith [California], John Talarico [New York], Eddy A. Bresnitz [New Jersey], Charles E. Jennings [Illinois], Wayne Staggs [Indiana], Dennis Perrotta and Maggie Kownaski [Texas], John Iskander [South Carolina], Peter Lurie [Pennsylvania], and Kris Erismin [Minnesota]).
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FOOTNOTES |
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Accepted Nov 17, 2005.
Address correspondence to Roger I. Glass, MD, PhD, Viral Gastroenteritis Section, MS G04, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30333. E-mail: rglass{at}cdc.gov
The authors have indicated they have no financial relationships relevant to this article to disclose.
The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the funding agency.
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PEDIATRICS (ISSN 1098-4275). ©2006 by the American Academy of Pediatrics
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