Ruth F. Bishop

Clinical immunity after neonatal rotavirus infection: a prospective longitudinal study in young children.

To determine whether rotavirus infection in newborn babies conferred immunity to postneonatal rotavirus infection, we studied 81 babies at birth and kept them under clinical and serologic study for three years. During the first 14 days of life, 44 of the infants excreted rotavirus, and 37 did not. Fifty-five per cent of those with neonatal infection and 54 per cent of those without it had rotavirus infection during the next three years. Symptoms associated with postneonatal rotavirus infection were significantly less frequent and less severe in the infants who had had neonatal infection (P = 0.003) than in those who had not. Thirty-eight per cent of the former group (9 of 24 infants) had symptoms of mild (3 infants) or moderate (6) severity during the first postneonatal infection. In contrast, 85 per cent of the latter group (17 of 20 infants) had mild (3), moderate (6), or severe (8) symptoms. We conclude that neonatal rotavirus infection does not confer immunity against reinfection but does protect against the development of clinically severe disease during reinfection.

Rotavirus antigenaemia and viraemia: a common event?

BACKGROUND Rotavirus infection is thought to be confined to the intestine. Reports of rotavirus RNA in the cerebral spinal fluid and serum of children infected with rotavirus suggest the possibility that rotavirus escapes the intestine into the circulatory system. We assessed whether rotavirus antigen, RNA, or both, were present in serum samples from immunocompetent rotavirus-infected children and animals. METHODS We obtained sera from immunocompetent mice, rats, rabbits, and calves 1-10 days after inoculation with rotavirus or matched vehicle. We obtained sera retrospectively from immunocompetent children diagnosed with rotavirus diarrhoea (n=33), healthy children (n=6) and adults (n=12), children convalescing from rotavirus (n=6), and children with non-rotavirus diarrhoea (n=11). Samples were analysed for the presence of rotavirus antigen or RNA by EIA or RT-PCR, respectively. FINDINGS Rotavirus antigen was present in sera from rotavirus-infected animals, but not in sera from control animals. Infectious rotavirus or rotavirus RNA was detected in sera of mice and calves, respectively. Antigen was present in 22 of 33 serum samples from children with confirmed rotavirus infection but in none of 35 samples from controls. Detection of serum antigen was inversely related to the number of days between symptom onset and sample collection, and directly related to stool antigen concentration. Rotavirus RNA was detected by RT-PCR in three of six rotavirus-positive sera. INTERPRETATION Rotavirus can escape the gastrointestinal tract in children, resulting in antigenaemia and possible viraemia. This finding is important for the understanding of the pathogenesis, immunology, and clinical manifestations of rotavirus infection.

Extended excretion of rotavirus after severe diarrhoea in young children

BACKGROUND Rotaviruses are the major cause of severe childhood diarrhoea. Knowledge of the natural history of infection, including duration of intestinal virus shedding, is important in the understanding of transmission, sources of infection, and immune responses. METHODS We carried out a study of rotavirus excretion in 37 children admitted to hospital with severe rotavirus diarrhoea. Sequential faecal specimens were collected from each child during 100 days of surveillance, and screened for rotavirus by EIA and by amplification of genome double-stranded RNA by reverse-transcription PCR. IgA coproantibody was estimated by EIA. FINDINGS Duration of rotavirus excretion ranged from 4 to 57 days after onset of diarrhoea. Excretion ceased within 10 days in 16 (43%) children, and within 20 days in 26 (70%) children. Extended excretion was detected for 25-57 days in the remaining 11 (30%) children owing mainly to continued excretion of the primary infecting strain. Extended excretion was significantly associated with antirotavirus IgA coproantibody boosts during 100 days of surveillance (p=0.001, log-rank test), and with recurrence of mild diarrhoea symptoms during convalescence (p=0.006, Fishers exact test). INTERPRETATION Severe rotavirus disease in young children may be followed by extended excretion of rotavirus. The risk of transmission to others may be greater than previously believed. Extended excretion could also explain some cases of the postgastroenteritis syndrome.

Distribution of rotavirus genotypes after introduction of rotavirus vaccines, Rotarix® and RotaTeq®, into the National Immunization Program of Australia.

Background: Rotavirus vaccines, RotaTeq and Rotarix, were introduced into the Australian National Immunization Program on July 1, 2007. The simultaneous introduction in different Australian states and territories provides a unique opportunity to compare the affect of each vaccine on the types of circulating rotavirus strains. This report describes the rotavirus genotypes responsible for the hospitalization of children during the first 2-year period after vaccine introduction. Methods: A total of 764 rotavirus-associated diarrheal cases were collected from children presenting to hospital in 10 Australian centers. Rotavirus genotype was determined using reverse transcription polymerase chain reaction assays. Results: G1P[8] was the dominant genotype nationally (52%), followed by G2P[4] (19.8%), G9P[8] (12.2%), and G3P[8] (11%). Differences in the prevalence rates of G2P[4] and G3P[8] were seen in the various states. G2P[4] strains were more prevalent in states using Rotarix, whereas G3P[8] strains were more prevalent in states using RotaTeq. Conclusions: Differences in rotavirus genotypes were observed across Australia, which suggest that different immune pressures are exerted by the different vaccines, but do not necessarily imply lack of protection by either vaccine. These differences may simply be related to the variation that can occur because of natural annual fluctuation in rotavirus strain prevalence.

Mycobacterium avium subspecies paratuberculosis in children with early-onset Crohn's disease.

Background: Mycobacterium avium subspecies paratuberculosis (MAP) is the most enduring infectious candidate that may be associated with inflammatory bowel disease (IBD). It is possible that the inconsistencies in the prevalence studies of MAP in adults reflect clinical differences in adult patients studied, including duration of disease and treatment regimens, and also in lack of specificity of some of the assays used. The aim was to determine the presence of MAP in children with symptoms of Crohns disease (CD) and ulcerative colitis (UC), using gut biopsy tissue and peripheral blood mononuclear cells (PBMC) collected at initial endoscopic examination prior to clinical treatment. Methods: Mucosal biopsies and/or PBMC specimens were collected from a total of 142 children, comprising 62 with CD, 26 with UC, and 54 with non‐IBD. MAP‐specific IS900 polymerase chain reaction (PCR) analysis was performed on all biopsies and PBMC specimens. Conventional MAP culture technique was performed on a subset of 10 CD, 2 UC, and 4 non‐IBD patients to isolate MAP. Results: MAP was identified by IS900 PCR significantly more often in mucosal biopsies from CD 39% (22/56) than from non‐IBD 15% (6/39) patients (P < 0.05), and in PBMC from CD 16% (8/50) than from non‐IBD 0% (0/31) patients (P < 0.05). Viable MAP were cultured from mucosal biopsies from 4/10 CD, 0/2 UC, and 0/4 non‐IBD patients, but were not cultured from PBMC specimens. Conclusions: This unique study on the occurrence of MAP in gut tissue and blood from pediatric IBD patients suggests the possible involvement of MAP in the early stages of development of CD in children. Inflamm Bowel Dis 2009

Early phase II trial of human rotavirus vaccine candidate RV3

A naturally attenuated, human neonatal strain, rotavirus vaccine candidate RV3, was tested in a limited phase II randomized double-blind controlled trial. Doses of 1 ml, containing placebo or 6.5 x 10(5) fluorescent cell forming units (fcfu) of virus in AGMK cells, were given at 3, 5 and 7 months of age. Limited replication in the small intestine is implied by the lack of virus excretion, and by the occurrence of an immune response in only 46% of the infants. However, those who developed an immune response were partially protected against rotavirus disease during the subsequent winter epidemic (protective efficacy 54%), supporting observations of protection induced by natural infection by this strain. Protection appeared to be heterotypic. Further trials are warranted, employing strategies to increase immunogenicity of this human rotavirus candidate vaccine.

Molecular Detection of Human Calicivirus in Young Children Hospitalized with Acute Gastroenteritis in Melbourne, Australia, during 1999

ABSTRACT Reverse transcription-PCR and sequence analysis identified calciviruses in 32 of 60 stool specimens (negative for other enteric pathogens) obtained from children admitted to our hospital with acute gastroenteritis. The overall annual incidence rate for calcivirus was 9% (32 of 354 children). Molecular analysis identified 30 “Norwalk-like virus” genogroup II (predominantly Lordsdale cluster) and 2 “Sapporo-like virus” strains.

Enteric colonization in sporadic neonatal necrotizing enterocolitis.

Summary Microflora of the gut was studied close to the onset of sporadic necrotizing enterocolitis (NEC) in neonates. Enteric flora in 25 babies with NEC differed from that in 23 matched controls. Bacteroides spp and lactobacilli were less common in babies with NEC compared with controls: 32 versus 61% (p = 0.03) and 12 versus 48% (p = 0.006), respectively. Clostridium perfringens was isolated from 40% of babies with NEC compared with 13% of controls (p = 0.03). It was present in 33% of babies with NEC aged less than 14 days but was not detected in control babies of the same age. The incidences of C. butyr icum and C. difficile were similar in patients and controls. Colonization with C. perfringens in the absence of a protective barrier flora may be crucial in the pathogenesis of severe sudden onset NEC. Two potentially valuable marker factors to predict onset of this form of sporadic NEC are the use of fecal smears and estimation of fecal tryptic activity.

Rotavirus detection and characterisation in outbreaks of gastroenteritis in aged-care facilities.

BACKGROUND Although rotavirus is a major cause of gastroenteritis in children, its role in adult gastroenteritis and the sensitivity of different methods for its detection in specimens collected from adults are less well understood. OBJECTIVES (1) To examine the frequency and seasonality of rotavirus-associated gastroenteritis outbreaks in aged-care facilities in Victoria, Australia. (2) To determine rotavirus type in these outbreaks. (3) To determine whether other enteropathogenic agents are present in specimens from these outbreaks. (4) To examine the sensitivity of different methods (electron microscopy (EM), reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (EIA) and latex agglutination (LA)) for the detection of rotavirus in specimens from adults. STUDY DESIGN Specimens from gastroenteritis outbreaks in aged-care facilities forwarded to this laboratory for the years 1997-2000 were tested for enteropathogenic agents by various methods. Epidemiological, clinical and seasonal data from the rotavirus-positive outbreaks were analysed. RESULTS Rotavirus was detected by EM in 18 out of 29 individuals associated with seven out of 53 (13%) gastroenteritis outbreaks in aged-care facilities; norovirus was detected in 22 outbreaks (42%) and astrovirus in one outbreak (2%). No mixed viral infection was found in any outbreak. All rotaviruses were typed as Group A by RT-PCR. The rotaviruses in the seven outbreaks were G-typed as follows: G2 (three outbreaks), G4 (two outbreaks), G1 (one outbreak) and G9 (one outbreak). The rotavirus-associated outbreaks were concentrated in mid-winter to mid-spring. The relative sensitivities of the Group A rotavirus detection methods (for the 29 specimens tested) were EM (18), first-round RT-PCR (11), second-round PCR (19), EIA-visual (19), EIA-photometric (19) and LA (13). CONCLUSIONS In Victoria, Australia, outbreaks of gastroenteritis associated with rotavirus are quite common in aged-care facilities. They involve Group A rotavirus and have a winter/spring seasonality. G-types G1, G2, G4 and G9 were all detected. EIA, second-round PCR and EM proved sensitive methods for rotavirus detection whereas first-round RT-PCR and LA did not.

Derivation of neutralizing monoclonal antibodies to human rotaviruses and evidence that an immunodominant neutralization site is shared between serotypes 1 and 3

Neutralizing monoclonal antibodies were derived to human rotaviruses RV-4 (serotype 1), RV-5 (serotype 2), and ST-3 (serotype 4). By enzyme immunoassay and fluorescent focus neutralization, eight of the antibodies appeared to be specific for the immunizing serotype, and so have potential as reagents for rotavirus serotyping by enzyme immunoassay. Seven of these were shown by Western blotting, enzyme immunoassay for antibody additivity, and reaction with rotavirus reassortants, to be directed against the major outer capsid glycoprotein. The remaining serotype-specific antibody immunoprecipitated the 84-kD outer capsid protein. One antibody reacted with all serotype 1 and 3 rotaviruses but not with serotypes 2 or 4. When tested with virus mutants, this antibody recognized an immunodominant determinant of neutralization shared between serotypes 1 and 3 on the major outer shell glycoprotein. Our results suggest that two outer capsid proteins possess determinants of neutralization, and that viruses of different serotypes may share immunodominant neutralization sites.