Douglas K. Mitchell

The changing epidemiology of astrovirus-associated gastroenteritis: a review

Our understanding of the epidemiology of astrovirus-associated gastroenteritis has changed markedly with each improvement in detection method. In early surveys based on electronmicroscopy (EM), astroviruses appeared to be a rare cause of gastroenteritis, being found in fewer than 1% of children with diarrhea, usually in small outbreaks of disease and primarily during the winter season. The development and use of monoclonal antibodies and enzyme immunoassays (EIA) to detect astroviruses led to reports of a higher prevalence (2.5%-9%) of astrovirus infection among patients hospitalized with diarrhea. Astroviruses appeared second only to rotaviruses as a cause of hospitalization for childhood viral gastroenteritis. Studies based on EIA detection of astroviruses indicate that astroviruses are common causes of diarrhea in children worldwide, and that most children are infected during their first two years of life. The elderly and the immunocompromised represent high-risk groups as well. The observations that newborns monitored prospectively rarely have repeat disease and that the rate of detection decreases with increasing age suggest that immunity to astroviruses, as immunity to rotaviruses, may develop early in life. The cloning and sequencing of astroviruses have led to more sensitive assays to detect the viruses by reverse transcription, polymerase chain reaction (RT-PCR). Application of RT-PCR for detection of astroviruses in children in day-care centers showed a marked increase in the detected prevalence of astrovirus-associated diarrhea, the rate of asymptomatic infection, and the duration of shedding of virus among those infected, when compared with studies that used other methods. As with rotaviruses, neither the mode of transmission nor the reservoir of astrovirus infection has been identified. Both immune and molecular-based assays to detect astrovirus serotypes indicate that serotype 1 is most common worldwide, although the predominant serotypes may vary by region and time. In the absence of obvious strategies to prevent astrovirus-associated diarrhea, vaccines might be considered if further studies establish that the disease burden would render such a vaccine cost-effective.

Prebiotic Effect Of Fructo-Oligosaccharide Supplemented Term Infant Formula at Two Concentrations Compared with Unsupplemented Formula and Human Milk

Background: Human milk components, including oligosaccharides, affect the gastrointestinal flora of infants. Previous studies in adults have demonstrated that fructo-oligosaccharides increase potentially beneficial fecal bacteria, including bifidobacteria. The purpose of this study was to determine the prebiotic effect of infant formula supplemented with fructo-oligosaccharides. Methods: Healthy term infants 2 to 6 weeks of age were enrolled in a 5-week, prospective, randomized, crossover, single-site study with a nonrandomized human milk comparator group. Washout weeks preceded and followed a week of feeding with fructo-oligosaccharide-supplemented formula (1.5 or 3.0 g/L). Stool specimens were quantitatively cultured weekly for bacteroides, lactobacilli, bifidobacteria, clostridia and enterococci and were tested for Clostridium difficile toxin. Results: Seventy-two of 87 infants completed the trial; 58 were formula fed and 14 were human milk fed. Mean counts of bifidobacteria and lactobacilli were similar in all groups at entry and no group experienced a significant change in counts with fructo-oligosaccharide supplementation. After 7 days of fructo-oligosaccharide supplementation the bifidobacteria counts were greater in the 1.5 g/L fructo-oligosaccharide formula group than in the human milk fed or 3.0 g/L fructo-oligosaccharide formula groups. Formula-fed infants had higher counts of enterococci and bacteroides before fructo-oligosaccharide supplementation, and these counts did not change after supplementation. Clostridium counts increased 7 days after supplementation in the 1.5 g/L fructo-oligosaccharide formula group (P = 0.0356). No human milk fed infants had C. difficile toxin in stools. Fructo-oligosaccharide (3.0 g/L) supplementation resulted in more frequent and significantly softer stools. Conclusions: Infant formula supplemented with 1.5 or 3.0 g/L fructo-oligosaccharides was safe but had minimal effect on fecal flora and C. difficile toxin.

Immunogenicity of a recombinant human cytomegalovirus gB vaccine in seronegative toddlers

BACKGROUND Immunization of young children against cytomegalovirus (CMV) might decrease child-to-child and child-to-adult transmission of CMV and thereby reduce maternal infection during pregnancy. We conducted a Phase I trial in CMV-seronegative toddlers to evaluate the reactogenicity and immunogenicity of a CMV gB vaccine administered with MF59, an oil and water adjuvant. METHODS Eighteen children between 12 and 35 months of age received either 20 microg of CMV gB/MF59 (n = 15) or a control hepatitis A vaccine (n = 3) at 0, 1 and 6 months. The study was open-label for the first six children and then observer-blinded and randomized. Children were monitored for local and systemic reactions and for the development of antibodies to the envelope protein gB and CMV-neutralizing antibodies. RESULTS Adverse reactions were uncommon and mild. Two children were excluded from the immunogenicity analysis because they had serologic evidence of CMV infection. Reciprocal geometric mean neutralizing titers were: 0 preimmunization (n = 18); 90 (range, 53 to 188) after Dose 2 (n = 6); and 638 (range, 210 to 1645) 1 month after Dose 3 (n = 13). The reciprocal geometric mean neutralizing titers of antibody to gB by EIA were: 0 preimmunization (n = 18); 857 (range, 307 to 2073) after Dose 1 (n = 12); 27 457 (range, 9312 to 55,080) after Dose 2 (n = 6); and 98,264 (range, 35,480 to 228,780) 1 month after Dose 3 (n = 5). After Dose 3 antibody responses of toddlers were greater than those of naturally infected adults and were notably higher than among 149 adults given 3 doses of the same vaccine in other trials. CONCLUSION The CMV gB vaccine is well-tolerated and highly immunogenic in toddlers.

Molecular characterization of a novel recombinant strain of human astrovirus associated with gastroenteritis in children.

Summary. We report a naturally occurring human astrovirus (HAstV) strain detected in two different geographic locations. We identified two isolates of this strain in a diarrhea outbreak at a child care center in Houston, Texas; and two isolates in diarrhea stool samples from two children in Mexico City. All four isolates were detected in stool samples by enzyme immunoassay (EIA). One of the Mexican isolates was typed by EIA and all four isolates were HAstV-5 by typing RT-PCR. The four isolates were >97% nucleotide-identical in two different genomic regions: ORF1a (246nt), and the 3′ end of the genome (471nt). One isolate from each geographic location was further sequenced in the transition region from ORF1b to ORF2 (1255nt) and this region of the two isolates showed ≥ 99% nt identity. Phylogenetic analyses of sequences of eight HAstV antigenic types and the novel strain in the transition region demonstrated the new strain being closely related to HAstV-3 in ORF1b, but closest to HAstV-5 in ORF2. These results and high sequence identity among all HAstV antigenic types in the transition region and RNA structural predictions supported a potential recombination site at the ORF1b/ORF2 junction. This is the first evidence that recombination occurs among human astroviruses.

A prospective study of astrovirus diarrhea of infancy in Mexico City

AIM To describe the epidemiologic and clinical characteristics of astrovirus-associated diarrhea in a cohort of young children from a periurban community in Mexico City. METHODS From November, 1988, through December, 1991, a total of 214 children were enrolled in a longitudinal study of diarrhea and monitored from birth to 18 months of age. A stool specimen was collected during each episode of diarrhea. Specimens from a total of 510 diarrhea episodes were tested for astrovirus by enzyme immunoassay and examined for other enteric pathogens. The antigenic types of astrovirus were determined by a typing enzyme immunoassay. RESULTS Astrovirus was detected in 26 (5%) of 510 diarrhea episodes, with an incidence rate of 0.1 episode/child year; the highest rate was in children 13 to 18 months of age. Astrovirus-associated diarrhea was characterized by a median of 4 stools (range, 2 to 10) during the first 24 h, a median duration of 3 days (range, 1 to 21), vomiting (20%), and fever (7%). No cases of dehydration or repeat symptomatic infections were observed. Coinfection with another pathogen was detected in 11 of the 26 episodes (42%). Serotype 2 (35%) was most common, followed by serotypes 4 (15%), 3 (11%), and 1 and 5 (4% each); 31% were nontypable. Astrovirus-associated diarrhea was less severe, as measured by the number of stools (4.3 +/- 1.9), than diarrhea caused by rotavirus (7.1 +/- 2.8) or when coinfections occurred (5.5 +/- 1.6; P = 0.008). CONCLUSIONS Astrovirus was associated with 5% of the episodes of diarrhea in this cohort of young Mexican children and presented as a mild secretory diarrhea. Five predominant antigenic types were detected with type 2 being the most common.

Application of electronmicroscopy, enzyme immunoassay, and RT‐PCR to monitor an outbreak of astrovirus type 1 in a paediatric bone marrow transplant unit

During 1997, an extensive outbreak of astrovirus occurred in a unit where paediatric patients were being treated for leukaemias and inherited immune deficiency disorders. Prolonged shedding of virus for many months following infection was demonstrated in three patients who had undergone bone marrow transplantation. Comparison of reverse transcription‐polymerase chain reaction (RT‐PCR), enzyme immunoassay (EIA), and electronmicroscopy (EM) to monitor the outbreak showed that many subclinical infections, mainly in children aged > 3 years could only be detected by RT‐PCR. Use of RT‐PCR revealed that several patients were infected earlier and shed virus for longer than by using EM or EIA. The virus responsible for the outbreak was identified as HAstV‐1 and was shown to have a sequence that differed from a strain obtained in 1988. J. Med. Virol. 57:313–321, 1999.

Molecular Epidemiology of Childhood Astrovirus Infection in Child Care Centers

This study assessed the role of human astrovirus (HAstV) in outbreaks and sporadic cases of diarrhea among children attending child care centers (CCCs) and determined the infecting astrovirus antigenic types by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis. Eight astrovirus outbreaks occurred in 6 CCCs. Of 179 children with diarrhea, 36 (20%) had astrovirus-associated diarrhea. Diarrhea stools obtained during diarrhea outbreaks were more likely to contain astrovirus (40/476) than were samples not associated with a diarrhea outbreak (14/452) (P<.001). Type-specific RT-PCR and DNA sequencing identified 5 outbreaks associated with HAstV-1 and 3 outbreaks with HAstV-2. Sequential outbreaks in 2 CCCs occurred with a different type in the same year. Phylogenetic analysis identified 6 clades of HAstV-1 and 2 clades of HAstV-2 during this 1-year surveillance. Astrovirus was a significant cause of diarrhea outbreaks, and 2 antigenic types were present in the community during 1 diarrhea season.

ROLE OF ASTROVIRUSES IN CHILDHOOD DIARRHEA

Human astrovirus (HAstV) is a significant cause of acute diarrhea among children, resulting in outbreaks of diarrhea and occasionally in hospitalization. Improved detection methods for eight antigenic types of HAstV and studies assessing the frequency and severity of HAstV diarrhea have further defined the impact of HAstV infections in children. These studies have shown that HAstV infections are clinically milder (diarrhea, vomiting, fever) than rotavirus infections. However, frequent coinfection of HAstV with rotavirus and caliciviruses in childhood diarrhea complicates the epidemiology. Seroprevalence studies have provided evidence that the majority of children are infected by HAstV by 6 years of age. The route of transmission is probably fecal-oral from food or water sources. Recent and planned studies will help to define the epidemiology and in the future lead to prevention strategies, which could include vaccination.

Molecular Characterization of Astroviruses by Reverse Transcriptase PCR and Sequence Analysis: Comparison of Clinical and Environmental Isolates from South Africa

ABSTRACT A comparative analysis was performed with 25 isolates of astroviruses (AstVs) detected in sewage sources and 22 concurrently identified clinical AstV isolates from the Tshwane (Pretoria) Metropolitan Area in South Africa. The samples and specimens were screened for AstVs by using an enzyme immunoassay and/or a reverse transcriptase PCR (RT-PCR) for the highly conserved untranslated region (3′ end) of the genome. The RT-PCR results were confirmed by oligonucleotide probe dot blot hybridization. Viable viruses were propagated in cell cultures for amplification when a minimal specimen was available or indeterminate sequences were obtained. AstV strains were characterized by RT-PCR and partial sequence analysis of the capsid region. The presence of multiple human AstV (HAstV) types in a single sewage sample complicated identification of individual strains, and additional type-specific RT-PCR and sequence analyses of the capsid region were required for characterization. Amplification and characterization of one genotype from a sample, therefore, did not preclude the possibility that a sample harbored additional different genotypes. Genotype and sequence information obtained from AstVs in wastewater samples were compared to information obtained from AstV strains from human stools. HAstV type 1 (HAstV-1), as well as HAstV-3, -5, -6, and -8, were identified among the clinical isolates, and HAstV-1, -2, -3, -4, -5, -7, and -8 were identified among the environmental samples. Phylogenetic analysis demonstrated that HAstV-1, -3, -5, and -8, which were present in human stool and sewage samples, clustered together, indicating that these viruses are closely related. The concurrent presence of identical HAstV strains in wastewater samples and in hospitalized patients suggests that AstVs present in the environment pose a potential risk to communities in which fecally contaminated water is used for recreational and domestic purposes.

Prevalence of antibodies to astrovirus types 1 and 3 in children and adolescents in Norfolk, Virginia.

OBJECTIVE To determine the prevalence of antibody to human astrovirus types 1 (HAstV-1) and 3 (HAstV-3) in children. METHODS Sera from children hospitalized in Norfolk, VA, for noninfectious conditions were collected for a 1-month period every 6 months from 1993 to 1996 and tested by enzyme immunoassay for antibody to HAstV-1 and HAstV-3 with the use of baculovirus-expressed recombinant capsid proteins as antigens. RESULTS The seroprevalence of 393 infants and children to HAstV-1 decreased from 67% in infants <3 months of age to 7% by 6 to 8 months of age, consistent with loss of transplacental antibodies. Children acquired HAstV-1 antibody with a peak prevalence of 94% at 6 to 9 years of age (P < 0.001). Antibodies to HAstV-3 exhibited a lower prevalence, with 26% positive at <3 months, 0% at 6 to 11 months and 42% by 6 to 9 years of age. HAstV-1 seroprevalence in children O to 2 months of age decreased from 89% in November, 1993, to 40% in November, 1996 (P = 0.009). CONCLUSIONS Astrovirus type-specific antibody prevalence can be measured by baculovirus-expressed capsid antigens in an enzyme immunoassay. Children developed antibody to HAstV-1 (94%) and to HAstV-3 (42%) by 6 to 9 years of age indicating frequent exposure to these enteric viruses in infancy and early childhood.