Norovirus and adenovirus have long been recognized as causes of self-limited gastroenteritis. Because the commercial fecal enzyme immunoassays are limited in sensitivity and norovirus cannot be cultured or visualized on electron microscopy, it was only with the advent of nucleic acid amplification testing that it has become evident that norovirus is a frequent cause of sporadic childhood and traveler's diarrhea and the predominant cause of outbreaks of gastroenteritis in developed countries.1 We report an immunocompromised patient with prolonged diarrhea and shedding of both norovirus and adenovirus.
An 8-month-old boy with intestinal failure syndrome secondary to gastroschisis developed cholestasis from parenteral nutrition. He had an uneventful combined liver, pancreas, and small-bowel transplant. Induction immunosuppression was methylprednisolone and daclizumab and maintenance immunosuppression was tacrolimus and sirolimus. Donor and recipient were both seronegative for cytomegalovirus (CMV). Increased ileostomy output on day 23 posttransplant resolved spontaneously within 3 days, and small bowel biopsies done on this and 8 other occasions before discharge on day 57 were normal.
On day 61, the child was readmitted with dehydration from vomiting and increased ileostomy losses. Small bowel biopsy showed changes that were most marked in the ileum and consisted of surface epithelial damage with immature cells lining the villi, suggesting increased cell turnover with relatively immature cells migrating up the villi. Rare isolated apoptoses were identified with no mucosal necrosis and no evidence of rejection. Specific immunohistochemistry of the native bowel and proximal transplant was negative, but the transplanted ileum showed many cells staining for CMV early antigen confined to the nuclei, yet no CMV inclusions were evident. Biopsy was most compatible with viral infection and ganciclovir was started. Repeat biopsies on days 64 and 68 showed mild mucosal lesions with loss of surface enterocyte polarity, migration of immature cells into the villi, and a few smudgy intranuclear inclusions which was thought to be most suggestive of adenovirus infection but adenovirus specific immunochemistry staining was not performed. Multiple stool samples were negative for bacteria, Clostridum difficile toxin, ova and parasites, and CMV culture.
Despite negative CMV immunohistochemistry on the repeat biopsies, ganciclovir was continued because of the previous positive results. High ileostomy losses continued until day 75 when output transiently decreased and the child developed retching. Repeat small bowel biopsy on day 76 showed focal mucosal hemorrhage but resolution of the changes consistent with viral infection. On day 82, retching had resolved but he had fever and increased ileostomy losses with ileostomy prolapse. Small bowel biopsy now showed mucosal edema and a persisting mild lesion characterized by intraepithelial and crypt lymphocytosis and occasional neutrophilic leukocytosis with no confluent crypt apoptosis, inactivated lymphoid infiltrate, or viral inclusions, which was again thought to be most compatible with viral infection.
On day 84, a volvulus was diagnosed and he had resection of 15 cm of necrotic small bowel with small bowel biopsy now showing hemorrhagic necrosis, acute and chronic inflammatory cells and glassy appearance of enterocytes suggestive of viral infection. CMV early antigen staining was again positive with no intranuclear inclusions and the possibility of artifactual staining from a cross-reacting antigen was raised. He completed a 36-day course of ganciclovir on day 100, but large ileostomy losses continued (up to 3 L/d). Immunosuppression was not reduced and tacrolimus and sirolimus were maintained within therapeutic levels throughout the illness.
He was discharged home to receive total parenteral nutrition (TPN) on day 107 despite continuing large ileostomy losses. Repeat small bowel biopsy on day 117 still showed diffusely positive CMV early antigen, especially in the surface epithelium of the villi. He finally achieved full oral feeding with discontinuation of TPN on day 168. The ileostomy losses slowly decreased from more than 3 L/d to 500 mL/d by day 171. On day 196, his ileostomy was closed and small bowel biopsy was normal. Buffy coat for CMV antigenemia and shell vial cultures were the standard diagnostic methods used at the time in the laboratory and were negative for CMV on days 25, 33, 47, 54, 66, 98, and 111 and he remains CMV seronegative 3 years posttransplant.
Virologic Methods and Results
Norovirus and rotavirus real time-polymerase chain reaction (RT-PCR) was performed as previously described. All stool samples tested negative for rotavirus.2,3 Adenovirus RT-PCR was performed using the ABI PRISM 7000 Sequence Detection System with primers and probes selected from the hexon gene of human adenovirus. Ten stool samples from days 68, 82, 88, 93, 102,112, 117, 131, 138, and 145, tested positive for adenovirus by electron microscopy and 15 stool samples from days 68, 82, 88, 93, 102,112, 117, 131, 138, 145, 154, 159, 171, 175, and 182 posttransplant tested positive for norovirus and enteric adenovirus by real-time RT-PCR and PCR assay, whereas stool from day 10 (before the onset of the increased ileostomy losses) tested negative for both viruses. No further stool samples were obtained as the ileostomy losses had normalized. Peak norovirus load (2.78E08 copies/g stool) was detected on day 88 when tacrolimus level was 10.9 ug/L (Fig. 1 online only). Adenovirus loads were not measured.
Plasma on day 67, 117, 122, 134, and 138 tested positive for adenovirus with the samples from days 42, 145, 176, and 183 testing negative. All plasma samples were negative for norovirus.
Purified PCR product was directly sequenced using Thermo Sequenase Primer Cycle Sequencing Kit (Amersham, USA) on OpenGeneTM DNA Sequencing System (Visible Genetics, Ontario, Canada). Sequence analysis of norovirus from C region (Capsid gene) from day 88 and day 172 stool samples showed a 94% homology to Snow Mountain strain.
Norovirus Serology Assays.
Available sera (collected pretransplant and on posttransplant day 42, 117, 134, 176, 225, and 264) were tested for antibodies to GII/2 and GII/4. The assay was modified from a previously described method.4 Virus-like particles representing genetic cluster GII/2 (Chesterfield virus) and GII/4 (Burwash Landing virus) were used as the antigens. Individual IgG units were calculated using a standard curve generated from serial titrations of reference sera. Seropositivity for the patient was defined as optical density (OD) values ≥0.15. Seroconversion was defined as a 4-fold or greater increase in antibody units between compared sera pairs. Seroconversion for norovirus antibodies GII/2 and GII/4 was documented on the day 176 sample.
Using PCR and RT-PCR, this solid organ transplant recipient had norovirus and adenovirus detected in multiple stool specimens during a 114 day period and adenovirus detected by PCR in multiple plasma specimens during a 72 day period, with increased ileostomy losses occurring during the time the viruses could be detected in stool. All but 1 of the 5 small bowel biopsies taken during the time of increased ileostomy losses was reported as being most compatible with a viral infection. Therefore, it seems likely that 1 or both viruses were the cause of the increased ileostomy losses. The role of CMV is less clear as early antigen was detected on several occasions resulting in a long course of ganciclovir, yet donor and recipient were both seronegative pretransplant, viral inclusions were never identified, viremia was never detected and seroconversion did not occur. This suggests that the staining observed may have represented a false positive result because of nonspecific staining or cross-reacting antigen. Cross-reaction between CMV and adenovirus by immunofluorescence staining has been reported in the early development of monoclonocal antibodies against CMV,5 but the amount of cross-reactivity of the CMV immunofluorescence diagnostic assay with adenovirus and norovirus is not known.
More prolonged shedding of norovirus has been described in immunocompromised hosts. Shedding in stool was detected by nucleic acid amplification testing for a median of 23 days and a maximum of 140 days after an outbreak in a pediatric oncology unit with the child with the most prolonged shedding having diarrhea for at least 4 months.11 Shedding and persistent diarrhea was documented for 156 days in a child with a bone marrow transplant for a T-cell immunodeficiency.12 A previous report describes 5 children aged 9–17 months at the time of combined liver and intestinal transplantion with prolonged diarrhea and shedding of a single strain of norovirus (type Miami Beach) for up to 138 days.13 Adenovirus was detected before the norovirus in one of these cases and concurrently with calicivirus in a second case. Nucleic acid amplification testing is not widely available for testing plasma or stool for norovirus so it is likely that other cases of chronic diarrhea in solid organ transplant recipients are attributable to norovirus.
The role that adenovirus played in the prolonged diarrhea in our case is not clear. Duration of shedding of enteric adenovirus as detected by monoclonal antibody-based enzyme immunoassay ranged from 1 to 14 days in day care outbreaks.14 Latent infection of respiratory adenovirus with prolonged respiratory excretion is well recognized and 1 study suggested lymphocytes from tonsillar or adenoid tissue as the site of latency,15 but prolonged shedding of enteric adenoviruses has rarely been documented. Presence of adenovirus in multiple plasma samples in the current case suggests ongoing infection rather than just stool carriage. Disseminated adenoviral infection and adenoviral enteritis are particular problems in intestinal transplant recipients, presumably as gut-associated lymphoid tissue can harbor latent adenovirus, both donor and recipient are likely to be young and to have an incomplete immune response to adenovirus, and more immunosuppression is required than for other transplants.15 Treatment of adenovirus with cidofovir has been described in transplant recipients,15 but was not attempted in the current case as the efficacy is not clear and the child was well apart from chronic diarrhea. The role of decreasing immunosuppression in hastening recovery is not clear, and was not used in the current case.
Serologic response with a 4-fold rise in titers to norovirus was detected by day 14 in infected healthy volunteers challenged with Snow Mountain virus.16 The delay in the antibody response in our case was likely caused by the child's immunosuppression. The observation of cross-reacting antibodies with GII/4 and GII/2 antigens would be related to homotypic and heterotypic immune response described with norovirus infections.17
In the absence of effective therapy for viral diarrhea, routine use of nucleic acid amplification testing for diagnosis is expensive. However, nucleic acid amplification testing for norovirus and adenovirus should be considered in cases of chronic diarrhea in immunocompromised hosts to identify the incidence and spectrum of chronic infection. Most importantly, identification of viral infection as the cause of posttransplant diarrhea should decrease the possibility of inappropriate and perhaps harmful treatment for presumed rejection or an alternate infectious organism.
1. Estes MK, Prasad BV, Atmar RL. Noroviruses everywhere: has something changed? Curr Opin Infect Dis
2. Pang X, Lee B, Chui L, Preiksaitis JK, Monroe SS. Evaluation and validation of real-time reverse transcription-pcr assay using the LightCycler system for detection and quantitation of norovirus
. J Clin Microbiol
3. Pang XL, Lee B, Boroumand N, Leblanc B, Preiksaitis JK, Yu Ip CC. Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea. J Med Virol.
4. Monroe SS, Stine SE, Jiang X, Estes MK, Glass RI. Detection of antibody to recombinant Norwalk virus antigen in specimens from outbreaks of gastroenteritis. J Clin Microbiol
5. Kari B, Lussenhop N, Goertz R, Wabuke-Bunoti M, Radeke R, Gehrz R. Characterization of monoclonal antibodies reactive to several biochemically distinct human cytomegalovirus glycoprotein complexes. J Virol
6. Lopman BA, Reacher MH, Vipond IB, Sarangi J, Brown DW. Clinical manifestation of norovirus
gastroenteritis in health care settings. Clin Infect Dis
7. Okhuysen PC, Jiang X, Ye L, Johnson PC, Estes MK. Viral shedding and fecal IgA response after Norwalk virus infection. J Infect Dis
8. Goller JL, Dimitriadis A, Tan A, Kelly H, Marshall JA. Long-term features of norovirus
gastroenteritis in the elderly. J Hosp Infect
9. Rockx B, De Wit M, Vennema H, et al. Natural history of human calicivirus infection: a prospective cohort study. Clin Infect Dis
10. Murata T, Katsushima N, Mizuta K, Muraki Y, Hongo S, Matsuzaki Y. Prolonged norovirus
shedding in infants < or =6 months of age with gastroenteritis. Pediatr Infect Dis J
11. Simon A, Schildgen O, Maria Eis-Hubinger A, et al. Norovirus
outbreak in a pediatric oncology unit. Scand J Gastroenterol.
12. Gallimore CI, Taylor C, Gennery AR, et al. Use of a heminested reverse transcriptase PCR assay for detection of astrovirus in environmental swabs from an outbreak of gastroenteritis in a pediatric primary immunodeficiency unit. J Clin Microbiol
13. Fewtrell L, Kaufmann RB, Kay D, Enanoria W, Haller L, Colford JM Jr. Water, sanitation, and hygiene interventions to reduce diarrhoea in less developed countries: a systematic review and meta-analysis. Lancet Infect Dis
14. Van R, Wun CC, O'Ryan ML, Matson DO, Jackson L, Pickering LK. Outbreaks of human enteric adenovirus
types 40 and 41 in Houston day care centers. J Pediatr
15. Hoffman JA. Adenoviral disease in pediatric solid organ transplant
recipients. Pediatr Transplant
16. Lindesmith L, Moe C, Lependu J, Frelinger JA, Treanor J, Baric RS. Cellular and humoral immunity following Snow Mountain virus challenge. J Virol
17. Rockx B, Baric RS, de Grijs I, Duizer E, Koopmans MP. Characterization of the homo- and heterotypic immune responses after natural norovirus
infection. J Med Virol