Rotavirus infection is the most common cause of severe dehydrating gastroenteritis in infants and young children and is responsible for about 453,000 deaths annually among children <5 years of age, primarily in developing countries.1 The virus is primarily transmitted by fecal-oral route and good sanitation and access to clean water does not reduce the rate of rotavirus infection,2 and thus vaccination is considered as an effective strategy to prevent rotavirus disease. In response to the substantial global disease burden due to rotavirus, the World Health Organization (WHO) recommends the inclusion of rotavirus vaccine in the routine immunizations programs of all countries. Preliminary impact studies of rotavirus vaccination have shown significant reduction of rotavirus hospitalizations since 2006.3,4 To formulate effective policies to control rotavirus disease through immunization, reliable epidemiological data is needed to assess the burden of rotavirus disease, to examine trends in seasonality and age patterns of incidence and to determine the serotypes of strains currently in circulation.5
In Mauritius, limited data are available regarding molecular characterization of rotavirus strains responsible for acute gastroenteritis. One previous study in 2000 found that 65% of circulating strains were G9P, 12.5% were G9P and 7.5% were G9P/P showing an enormous prevalence of G9 strains.6 The G2 rotavirus strains and 4 mixed genotypes infections were also detected.
The aim of this study is to describe the epidemiology and genetic diversity of rotavirus strains circulating in Mauritian children <5 years of age who are admitted with acute gastroenteritis to the paediatric wards of 2 regional hospitals from June 2008 to December 2010. Specifically, this study seeks to determine age-specific and season-specific incidence rates of diarrhea caused by rotaviruses and to determine the P and G types of rotavirus strains in circulation. This information can be used to direct policy decision on the introduction of rotavirus vaccine.
MATERIALS AND METHODS
Surveillance and Stools Collection
In Mauritius, rotavirus surveillance is conducted by the Ministry of Health and Quality of Life (MoH&QL) with support from WHO/AFRO according to WHO Generic protocol7 at 2 sentinel sites, the pediatric wards of Victoria and Jawaharlal Nehru regional hospitals, with bed capacity of 15 per site. Briefly, a sample of children, <5 years of age who were admitted for treatment of acute gastroenteritis, with at least 3 looser than normal stools and/or two or more episodes of vomiting in a 24-hour period were enrolled and a stool specimen was collected within 48 hours of admission.
Detection of Group A Rotavirus
The 10% fecal suspensions were screened for the presence of group A antigen at the Virology Department, Central Health Laboratory, Victoria Hospital, using a commercially available enzyme immunoassay (EIA; ProSpecT rotavirus kit, Oxoid Ltd, Basingstoke, UK). The test was performed according to the manufacturer’s instructions. Rotavirus-positive stool samples and 10% negative samples were stored at −70°C. A subset of rotavirus-positive stools selected at random was subjected to polyacrylamide gel electrophoresis (PAGE) and VP4 and VP7 genotyping at MRC/Diarrhoeal Pathogens Research Unit, Department of Virology, University of Limpopo MEDUNSA Campus, Pretoria, South Africa.
Polyacrylamide Gel Electrophoresis
PAGE analysis was used to differentiate between rotavirus short and long electrophoretypes by comparing the migration pattern of the eleven dsRNA segments. The viral RNA was extracted using 1M NaAc containing 1% sodium dodecyl sulfate and (1:1) phenol and chloroform and the RNA was precipitated by ice-cold ethanol. The extracted RNA was resolved on 10% polyacrylamide gels with 3% stacking gels with use of a discontinuous buffer system at 100 V for 18 hours at room temperature.8 The RNA segments were then stained by silver nitrate as described by Herring et al.9
Viral RNA Extraction and RT-PCR
Viral RNA was extracted from 10% rotavirus-positive fecal suspensions, using QIAamp Viral RNA Mini Extraction Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The principle of this extraction method utilizes lysis of the viral particle to liberate the dsRNA which are then captured on a spin column and washed several times to remove inhibitors. The eluted RNA extracts are then used as templates for RT-PCR whereby copies of cDNA are produced. RT-PCR involves three steps: denaturation of dsRNA, reverse transcription of dsRNA and PCR amplification of cDNA.
The consensus primer pairs sBeg/End9 and Con2/Con3 were used to generate full-length copies of the VP7 gene (1062 bp) and to amplify a fragment of the VP4 gene (876 bp), respectively.10,11 The purified RNA was reverse transcribed with avian myeloblastosis virus reverse transcriptase at 42°C for 30 min, followed by 30 cycles of PCR (denaturing at 94°C for 1 min, annealing at 42°C for 1 min and extension at 72°C for 1 min) and a final extension cycle (72°C for 7 min).
VP4 and VP7 Genotyping
The genotyping of VP4 and VP7 were performed by nested multiplex PCR from the complementary DNA to examine rotavirus strains diversity using type-specific primers. The VP4 genotypes were performed using VP4-specific primers namely Con3, 1T-1d, 2T-1, 3T-1, 4T-1 and 5T-1, which are specific for the human rotavirus P, P, P, P and P strains as described by Gentsch et al9 and Iturriza-Gómara et al.12 VP7-specific primers, such as RVG9, aBT1, aCT2, aDT4, mG3, mG9, mG10 and G12 to determine G1 to G4 and G8, G9, G10 and G12 genotypes were used for G typing specified by Gouvea et al11 and Iturriza-Gómara et al.13 PCR was performed at 94°C × 2 min followed by 30 cycles of 94°C × 1 min, 42°C × 1 min, 72° × 1 min and a final extension 72°C × 7 min. PCR products were resolved by electrophoresis on a 2% agarose gel, stained with ethidium bromide and visualized under UV.
During the 30-month period of the rotavirus surveillance study (June 2008 through December 2010), 787 diarrheal (40.7%) stool samples were collected from 1932 children <5 years of age admitted with acute diarrhea in the paediatric wards of the 2 regional hospitals in Mauritius.
Overall, the median rotavirus detection rate during the study period was 327/787 (41.6%) ranging from 33.5% in 2008, 37.8% in 2009 and 53% in 2010 (Fig. 1). Rotavirus infections were detected in all age groups in this study with the highest prevalence of rotavirus infection occurring in children aged 12–23 months (125/327; 38.2%) followed by children <12 months (70/327; 21.4%). Lowest rotavirus infections were observed from children 49–59 months of age (19/327; 5.8%; Fig. 2). Rotavirus affected more males (60%) than females (40%).
Seasonal distribution of rotavirus infections in Mauritius from June 2008 through December 2010 showed 3 peak periods. The rotavirus positivity rate peaked in winter months in 2008 and 2010 and in summer months in 2009 (Fig. 3). During these rotavirus peak seasons, the prevalence of rotavirus infections was 54.2%. In July through October 2008, the positivity rate for rotavirus was 37.4% with a peak of 47% in July 2008. In October through December 2009, the positivity rate for rotavirus was 58.9% with a peak of 66.7% in October 2009. Furthermore, 76.2% of fecal samples were positive from July through October 2010 with a peak of 77.0% in September. The greatest number of admissions due to gastroenteritis occurred during the rotavirus seasons (Fig. 3). From April to August 2009 and March to April 2010 when rotavirus was infrequently detected, the number of diarrheal admissions was lowest.
In this study, all the 116 fecal specimens subjected to molecular characterization showed only long electrophoretypes by PAGE. Genotyping results indicates that 89% of rotavirus strains circulating from June 2008 to December 2008 were G3P and the remaining 11% were G3P. In 2009, the predominant strain responsible for rotavirus diarrhea was G4P (76%) and the other rotavirus genotypes were G3P (8%), G9P (3%), G3/G4/G9P (3%), G4P/P (5%) and G[NT]P(5%). The rotavirus strains circulating in 2010 were G1P (90%), G9P (2%), G4P (4%), G8P (2%) and G[NT]P (2%; Fig. 4).
A different rotavirus genotype was responsible for each rotavirus epidemic during the 30 months of study and practically no particular rotavirus strain were continuously present throughout the study. Rotavirus strains G3P, G4P and G1P were responsible for many admissions due to rotavirus diarrhea in 2008, 2009 and 2010, respectively. The prevalence rate of G3P, G4P and G1P during the overall study period was 23.1%, 26.5% and 41%, respectively. The predominant rotavirus strain circulating during a particular rotavirus season gradually tailed off and disappeared during the next rotavirus season with the emergence of another predominant strain. Genotype P (96%) was predominant throughout the study period.
The rotavirus genotype G1P was responsible for the greatest number of admissions among children <5 years old and accounted for the highest rotavirus positivity rate during the study period and accounting for 90% of the rotavirus strains circulating in 2010 (Fig. 4).
This is the first standardized surveillance study conducted in Mauritius to describe the epidemiology and document the diversity of rotavirus strains among children of <5 years of age suffering from acute gastroenteritis.
In this study, rotavirus was detected in 41.6% of children with acute diarrhea with a peak positivity rate of 53% in 2010, thereby demonstrating rotavirus as the most common cause of diarrhea among hospitalized children. In addition, the greatest number of admissions due to gastroenteritis occurred during the rotavirus seasons. This observation was also noted in other countries which showed that rotavirus was the most common etiologic agent causing diarrhea.14,15 During the rotavirus peak seasons in Mauritius, the prevalence of rotavirus infections was 54.2% with highest positivity rate of 76.2% in 2010. The high proportion of rotavirus detected in diarrheal stools in this study was also documented in other studies which collected stools during rotavirus seasons.16,17
During the surveillance period, rotavirus gastroenteritis that required hospitalization occurred mostly in children <2 years of age (59.6%), predominantly in children in the range of 12–23 months (38.2%). However, studies in South Africa,18 India,19 Zimbabwe20 and Egypt21 have reported that most of rotavirus infections were detected in children <2 years old of age but occurred mostly in children <1 year of age. In developed countries, rotavirus infections were found to be more common in children 9–15 months of age.4 This difference in findings in this study could be attributed to the fact that younger children tend to seek treatment in private, in non-surveillance hospitals than older children.
Rotavirus hospitalizations are often observed to have marked seasonality, particularly in developed countries.22 In temperate climates, most of the rotavirus gastroenteritis occurred in winter and was uncommon in summer.24 Other studies have found no clear relationship between the timing of the peak in rotavirus activity with either season.25 In some tropical regions, rotavirus infections have been detected year round, but the prevalence seems to increase during periods of low rainfall or low humidity or decrease during periods of high rainfall and/or high humidity.23 However, in Mauritius, during the surveillance study, 2 rotavirus peaks were detected during winter seasons from July to October in 2008 and 2010, and 1 rotavirus peak detected during the summer season from October to December 2009. This trend was also observed in the past in Mauritius, with high rotavirus positivity rate in winter season in 2002, 2003 and 2004, and in 2005 and 2006 rotavirus infection occurred mainly in summer (unpublished data).
Several different rotavirus strains circulated during each year of this study, as has been observed in other studies.16,25 In 2008, 2 rotavirus strains, namely G3P (89%) and G3P (11%), were detected. In 2009, the predominant rotavirus genotype was G4P (76%), but other 5 rotavirus genotypes, G3P (8%), G9P (3%), G3/G4/G9P (3%), G4P/P (5%) and G[NT]P (5%), were also detected. In 2010, 90% of rotavirus diarrhea was caused by G1P but G9P (2%), G4P (4%), G8P (2%) and G[NT]P (2%) were also detected. Only two strains were detected in 2008, possibly due to the fact that the study started in June 2008 at the beginning of the rotavirus season.
Genotypes G1P, G2P, G3P, G4P and G9P represented almost 72% of all strains detected worldwide and were thought to be an important cause of diarrhea in young children.26,27 In the present study, the predominant rotavirus strains responsible for rotavirus infections were G3P in 2008, G4P in 2009 and G1P in 2010, and these strains accounted for 90% of all strains typed. Highest proportion of rotavirus hospitalizations were due to strain G1P (41%). G1P is the most prevalent genotype worldwide (reviewed in (26,28)). However, in Mauritius, during the study period, G1P was detected only in 2010. In a previous study in Mauritius, 65% of 40 rotavirus-positive samples collected in 2000 were G9P. G9P was rarely detected in our study with only 1 case detected in each of 2009 and 2010.
A constant change in the predominant strain, with a new strain emerging as the most common has been reported in some studies.8,16 The succession of rotavirus strains occurred in a manner in which some strains disappeared as new strains emerged. Similarly, results generated by this study demonstrated that each year there is a variation in the predominant rotavirus strain whereby the genotype G3P in 2008 was replaced by G4P in 2009 and eventually G1P emerged in 2010.
This study has several limitations. First, 3 full years of surveillance data were not available. Data from 2008 was only available from June to December which coincided with a peak in rotavirus infection. Thus, the percentage positive of 33.5% may be an overestimate of an annual percentage positive and cannot be directly compared with the percentage positivity for 2009 and 2010. Second, we only collected stool specimens from 787 (41%) of the 1932 eligible children <5 years of age who were hospitalized with diarrhea. Since we do not have any information regarding those children who were not enrolled, we do not know if the enrolled children are representative of all eligible children. Third, if the treatment seeking patterns for children <1 year of age differs from that of older children (ie, younger children are more likely to receive care in private, non-surveillance hospitals than older children), then our estimate of rotavirus positivity may be an underestimate if rotavirus is common in children <1 year of age and our age distribution will be skewed toward older age groups. Finally, we did not genotype all rotavirus-positive specimens collected. However, since we randomly selected specimens for genotyping, we expect our results to be representative of all positive specimens.
The diversity of rotavirus strains detected in this study highlights the need for a continuous surveillance. Moreover, the diversity of rotavirus strains in circulation, reliable data on the epidemiology of rotavirus infections, burden of the disease and circulating strains will provide information necessary to advocate for the introduction of rotavirus vaccines.
The authors thank staff of MoH&QL for contributing to this study; K.D. Jokhun and K. Maureemootoo for collecting clinical data and stool samples; G. Bhugeloo for input of data and H. Caussy for his helpful comments. The authors acknowledge WHO/AFRO for the financial and technical support for surveillance activities, provision of ELISA kits and training at the Regional Reference laboratory to this study and staff of the MRC/MEDUNSA Diarrheal Pathogens Research Unit for technical assistance for rotavirus characterization.
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