Rotavirus detection and typing in the rotavirus vaccine era

Abstract

Diarrhoeal disease is a leading cause of morbidity and mortality worldwide. Rotaviruses are the most common cause of severe childhood diarrhoea globally. In Australia, before rotavirus vaccination, rotaviruses caused approximately 10,000 hospitalisations, 22,000 emergency department visits, and 115,000 general practice consultations annually in children aged less than 5 years. On 1 July 2007 Australia became one of the first countries to include rotavirus vaccine into their national immunisation programme. Rotarix was initially used in the Australian Capital Territory, New South Wales, Northern Territory, Tasmania, and Western Australia (Western Australia changed to using RotaTeq in 2009). RotaTeq was initially used in Queensland, South Australia, and Victoria. As of 1 July 2017, all states and territories now use Rotarix.While active hospital surveillance demonstrated the vaccine’s success against severe rotavirus infections, an intensive community-based cohort is required to further understand the full impact upon post-vaccine rotavirus epidemiology. Furthermore, rotavirus gastroenteritis requires laboratory confirmation, and rotavirus detection data can be biased by changes in testing methods. My PhD thesis comprised four sub-studies focusing on both rotavirus detection and rotavirus-related disease in the vaccine era.In Queensland, rotavirus infections are notifiable and notification data are used to examine the effect of rotavirus vaccine programmes. The accuracy of notification data is dependent upon the accuracy of laboratory methods used to confirm infection. In 2012, concerns were raised in Queensland about the specificity of the VIKIA Rota-Adeno assay (BioMerieux, France), an immunochromatographic (ICT) assay, following an unexplained increase in positive results and feedback from clinicians. By re-examining samples initially testing positive in the VIKIA Rota-Adeno assay with other commercially available ELISA rotavirus assays and, for a subset of specimens, by reverse transcription polymerase chain reaction (RT-PCR) assays, I discovered the specificity of this commercially available rotavirus ICT assay was seriously compromised. These studies translated into changes in routine rotavirus testing in Queensland and the reformulation of the VIKIA commercial method. My next study was nested in a broader community-based project, the Observational Research in Childhood Infectious Diseases (ORChID) study, and aimed to document the community-based epidemiology of rotavirus infections in the first 2 years of childhood. To gather initial information about the most prevalent enteric virus infections in the community in the rotavirus vaccine era, quantitative PCR (qPCR) assays were used to assess enteric viruses in the weekly stool samples collected from 5 healthy, fully rotavirus-vaccinated infants from the ORChID study over a 2 year period. Overall, 511 samples from the 5 patients were tested by 6 qPCR methods and the results compared. Rotavirus was not the most prevalent pathogen amongst these infants (only 7 positive samples from 3 subjects identified), and most (5/7) were vaccine strains. Other viruses, particularly adenovirus (131/511 samples, 25.6%, including types 1, 2, 5, 12, 31 and 41), were more commonly found. Frequent, silent shedding of up to 3 months by one or more of the other viruses was observed. These data highlight the complexity of gastroenteritis diagnosis, and show that a positive PCR result for enteric viruses may not always indicate the cause of gastroenteritis.The main component of my thesis involved comprehensively investigating rotavirus genotypes and shedding in an unselected community-based birth cohort of Australian infants. This involved all available samples from infants (n = 158) enrolled in the ORChID cohort. Newborn infants were progressively enrolled between 2010 and 2012, and were followed until their second birthday. Parents recorded symptoms daily and collected weekly nappy swabs from their children and mailed these to the laboratory. The samples were tested for rotavirus by RT-qPCR, and rotavirus-positive samples were subjected to P and G-genotyping. Viral shedding, genotype, load and associations with symptoms were investigated. Rotavirus was frequently detected in the stool samples of infants from the cohort (1068/11,139 samples; 9.6%); but when genotyped these were mainly vaccine viruses (95.7%), which across each of the 3 doses were shed for a median (interquartile range) 2 (1-3) weeks. Symptomatic wild-type rotavirus detections, but not vaccine virus, were associated with higher viral loads. However, the predictive value of these load data were insufficient to be useful clinically. In conclusion, a problem related to an unexplained increase in rotavirus notifications was addressed and led to changes in pathology testing practices. Post-implementation of the rotavirus vaccine programme, my community-based cohort studies found the virus was no longer a common gastrointestinal pathogen. In contrast, rotavirus vaccine strain shedding occurred frequently and was more prolonged than previously documented in clinical trials and post-licensure studies. Prolonged shedding of vaccine virus and increases in asymptomatic detections may be a potential problem for RT-qPCR diagnostics, requiring assays that specifically distinguish vaccine from wild-type infection. Future work should focus on addressing changes in laboratory methods to improve specificity in detecting wild-type disease, and expanding the use of routine genotyping, an important tool to understand rotavirus prevalence and epidemiology in the vaccine era.