Comparative Study of the Epidemiology of Rotavirus in Children from a Community-Based Birth Cohort and a Hospital in South India
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《微生物临床杂志》
Department of Gastrointestinal Sciences, Community Health, Child Health Unit III, Christian Medical College, Vellore, India
Virus Reference Department, Centre for Infection, Health Protection Agency, London, United Kingdom
London School of Hygiene and Tropical Medicine, London, United Kingdom
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas
ABSTRACT
Rotavirus gastroenteritis is the major cause of severe dehydrating diarrhea in children worldwide. This study compares rotavirus diarrhea in 351 children in a community-based cohort and 343 children admitted to a hospital during the same period. Clinical information and fecal specimens were obtained during diarrheal episodes. Fecal samples were screened for VP6 antigen, and the positive samples were G and P typed by reverse transcription-PCR. Rotavirus was detected in 82/1,152 (7.1%) episodes of diarrhea in the community and 94/343 (27.4%) cases in the hospital. The median age of affected children (7.5 versus 10.5 months) and the mean severity of symptoms (Vesikari score, 7.6 ± 3.4 versus 11 ± 2.5) were lower in the community. A larger proportion of children in the community were breast-fed than were children admitted to the hospital (73% versus 34.8%). In the community, the genotypes identified in symptomatic patients, in order of frequency, were G1 (36.5%), G10 (17.1%), G2 (15.9%), and G9 (7.3%) and mixed infections (7.3%). The most common G-P combinations were G1P[8], G2P[4], G1P[4], and G10P[11]. The distribution of G types from hospitalized children was G1 (46.8%), G9 (19.1%), G2 (8.5%), G10 (1.1%), and 4.3% mixed infections. The most common G-P combinations were G1P[8] and G9P[8]. This study documents significant genetic heterogeneity of rotaviruses in the community and the hospital. G10P[11] strains resembling a vaccine candidate strain caused disease in the community, indicating the need for careful epidemiological studies as well as safety studies for the vaccine candidates.
INTRODUCTION
Rotaviruses are the major etiological agents causing diarrhea in infants and young children. A recent review estimates that more than 600,000 deaths occur annually in children under the age of 5 years due to rotavirus infection, with a majority of these deaths occurring in developing countries (36).
Rotaviruses belong to the family Reoviridae and possess a genome of 11 segments of double-stranded RNA that are located inside triple-layered viral particles. Rotaviruses are classified into seven different serogroups (A to G), based on the antigenic specificities of the capsid proteins. For group A viruses, typing schemes were introduced based on antigenic epitopes on the proteins that form the inner capsid (VP6; subgroups I and II) and on proteins of the outer capsid, the glycoprotein VP7 (G serotypes) and the spike protein VP4 (P serotypes). In recent years, reverse transcription-PCR was used in molecular epidemiological studies. All known G serotypes have been correlated with genotypes; however, there are more P genotypes than serotypes identified, leading to a serotype/genotype dual nomenclature for P types. At least 15 different G types and 26 different P types have been found in human and animal infections (11, 24, 29, 46). The incidence and distribution of group A rotavirus serotypes and genotypes vary between geographical areas during a rotavirus season and from one season to the next. Globally, different surveys indicate that G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8] are the most common G and P types (1, 2, 14, 18). However, in several regions, viruses of other G-P combinations have been found: G5P[8] in Brazil, G8P[6] in Malawi, G9P[6] in India and Bangladesh, and G10P[11] in India (6, 20, 28, 39, 44).
The first vaccine to be licensed, RotaShield, included reassortants of the most frequently encountered viral strains G1 to G4 (38). The vaccine was withdrawn due to a temporal association with intussusception (33). Promising results of large-scale clinical trials on two new vaccines, Rotateq and Rotarix, have recently been published (40, 47). The anticipated availability of an effective vaccine highlights the need to better define the epidemiology and disease burden associated with rotavirus. It is important to study local distribution of rotavirus strains and conduct active surveillance programs for emerging reassortant strains prior to and after the introduction of vaccines.
Most of the body of knowledge on rotavirus infections in children is based on hospital-centered studies. There is an inherent referral bias if these studies alone are considered. A clear understanding of the spectrum of disease can result only from a combination of community- and hospital-based studies which investigate both mild and severe disease. A study based in the community provides geographically representative information on the disease burden, strain prevalence, and incidence rates of rotavirus infection in the community. In contrast, the hospital-based surveillance system provides detailed information on severity and strain prevalence in children presenting to a hospital with diarrhea. In this report, we highlight the differences between rotavirus diarrhea in a community birth cohort and in a hospital in southern India over a 2-year period.
MATERIALS AND METHODS
Patients from the community. A cohort of 452 children was recruited from an urban slum area in Vellore, South India, over a period of 18 months extending from March 2002 to August 2003. The population of this area was 33,390, of which 2,895 (8.7%) were under the age of 5 years. Sixty-seven children (14.6%) dropped out of the study. Thirty-eight children moved out of the study area, 25 refused to continue in the cohort, and 4 children died after recruitment. The remaining 385 children were monitored between May 2002 and May 2004. Fecal samples were collected from these children in the neonatal period, and a baseline assessment of the domestic environment was conducted by interview. Thereafter, each child was visited at home twice a week by a field worker in order to collect information on any illness of the child and the family. Any child who developed diarrhea was assessed clinically and treated appropriately. Samples of feces were collected from the child during the diarrheal episode. The child was followed up daily until cessation of diarrhea.
In this cohort, 351 of 385 children had a total of 1,299 episodes of diarrhea between May 2002 and May 2004. Samples collected from these children during this period were analyzed for the comparative study.
Patients from the hospital. The study was carried out at the Christian Medical College, a 1,800-bed hospital in Vellore, India. Vellore is an urban area with a population of 400,000 and is estimated to have approximately 42,000 children less than 5 years of age. All children under 5 years of age presenting to the hospital with acute gastroenteritis and requiring hospitalization for rehydration for at least 6 h were enrolled in the study. A total of 343 children less than 5 years of age, hospitalized for acute diarrhea between January 2002 and December 2003, were screened for rotavirus.
Informed consent was obtained from the parents of all children enrolled in the community- and hospital-based studies. The studies were approved by the guidelines of the Research Committee of the Christian Medical College, Vellore, India.
All samples from the hospital and the community were transported on ice chests to the laboratory and were processed immediately for the detection of rotavirus. Four 2-g aliquots were stored at –70°C for strain characterization and future testing.
Rotavirus screening. Samples were screened for rotavirus VP6 antigen by a latex agglutination assay (Meridian Diagnostics, Inc.) until September 2002 (2% of samples from the community and 39% from the hospital). Thereafter, all samples were examined for group A rotavirus by an enzyme-linked immunosorbent assay (Rota IDEIA; DakoCytomation Ltd., United Kingdom) to detect VP6 antigen following the manufacturer's instructions.
RNA extraction and cDNA synthesis. Viral RNA was extracted from 200 μl of the 10% of rotavirus-positive fecal suspension in balanced salt solution, using guanidine isothiocyanate-silica according to the method described by Boom et al. (4). It was then eluted in 50 μl of RNase-free distilled water containing 40 units of RNase inhibitor (RNasin; Ambion, United Kingdom).
cDNA was generated from 40 μl of the extracted RNA by reverse transcription in the presence of random primers (hexamers) [Pd(N)6; Pharmacia Biotech, United Kingdom], using 400 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen, Life Technologies, United Kingdom).
Genotyping PCR. The resulting cDNA was the template for both VP7- and VP4-specific typing PCRs, using the oligonucleotide primers described earlier (13, 15, 17, 19). Primers complementary to conserved regions of VP7 were used for the first-round VP7 PCR to amplify an 881-bp region of the VP7 gene (19). The second-round typing PCR was a multiplex PCR and incorporated the 3'-end-labeled primer (VP7 Reverse) and the G-type-specific primers G1, G2, G3, G4, G8, G9, and G10 (19).
Primers Con2 and Con3 were used for the first-round PCRs to amplify an 876-bp fragment of the VP4 gene (17). The second-round PCR included primers specific for genotypes P[4], P[6], P[8], P[9], P[10], and P[11] and the consensus primer Con3 (19).
Assessment of severity. Diarrhea was defined as the passage of three watery stools in a 24-h period. In children less than 6 months of age, a change in number or consistency of stools reported by the mother was considered indicative of diarrhea. An episode was defined as at least 1 day of diarrhea, preceded and followed by 2 or more days without diarrhea (32). The episode was considered to have ended on the day bowel movements returned to normal.
The severity of diarrhea was assessed using the Vesikari scoring system (41). Information was collected on duration of diarrhea, maximum number of stools passed per day, duration and peak frequency of vomiting, degree of fever, presence and severity of dehydration, and treatment. To permit calculation of this score, the episode was considered mild for a score of 5, moderately severe for a score of 6 to 10, and severe for a score of >10.
For calculating the severity scores for children in the community, a modified Vesikari scoring system was followed. Since accurate temperature measurements were not possible in the field, temperatures were recorded as normal, low-grade fever, and high-grade fever as reported by the caregivers.
Statistical analysis. Data were entered in Microsoft Access and analyzed using SPSS v. 9.0. Chi square and Student's t tests were performed to determine the significance of differences observed between the two different groups of patients.
RESULTS
Rotavirus diarrhea. A total of 351 children in the community cohort presented with 1,299 episodes of diarrhea between May 2002 and May 2004. The mean age of the group at the end of this monitoring period was 17 ± 4.3 months. There were 175 males (49.9%) and 176 females (50.1%). Diarrheal samples were collected during 88.7% of the episodes of diarrhea. The age range of children presenting with diarrhea was 0 to 24 months (interquartile range [IQR], 3 to 10 months, indicating that 50% of the cases occurred between 3 and 10 months). Rotavirus was detected in 82/1,152 (7.1%) episodes for which samples were obtained.
In the hospital, 343 children were admitted with diarrhea from January 2002 to December 2003. Rotavirus was detected in 94 children, accounting for 27.4% of all diarrhea cases. The age range of children presenting with diarrhea to the hospital was 0 to 60 months (IQR, 5 to 12 months). In seven cases, none of whom were rotavirus positive, age was not recorded on the patient charts. Demographic, virological, and clinical data of children in the community and the hospital with rotavirus diarrhea are shown in Table 1.
The median age of detection of rotavirus in children from the community was 7.5 months (IQR, 4.7 to 10.2) compared to 10.0 months (IQR, 7.5 to 12.5) in the hospital (P < 0.007). A larger proportion of those admitted in the hospital were male children (63.8%) than were those in the community (53.7%). However, this difference did not reach statistical significance (P = 0.15). A greater proportion of children in the community were breast-fed than were those admitted to the hospital (73% versus 34.8%) (P < 0.001) at the time they presented with gastroenteritis.
In the community, 32.9% of the episodes of rotavirus diarrhea were mild, 45.1% were moderate, and 22.0% were severe. During these episodes, 13.4% of children were treated at home, 79.3% were taken to the study clinic, and 7.3% required hospitalization. Not all severe cases required admission, because they received supervised oral rehydration at the clinic. In the community, six young children with severe dehydration were admitted (mean age, 4.3 months; range, 0 to 10 months; four of six breast-fed). In contrast, only 3.3% of rotavirus diarrheal cases admitted in the hospital were mild, 40.0% were moderate, and 56.7% were severe (P < 0.001). The mild cases were admitted to the hospital to alleviate parental anxiety. The mean Vesikari score was also higher in the hospital than in the community (11.0 ± 2.5 versus 7.6 ± 3.4) (P < 0.001). There was one rotavirus-associated death in the community (1.2%) and no documented mortality in the hospital.
The distribution of rotavirus infection in the community and hospital by age is given in Table 2. The largest proportion of rotavirus diarrhea was noted in the 12- to 17-month age group in the community and in the 6- to 11-month age group in the hospital (11.5% and 42.3%, respectively). The smallest proportion of rotavirus diarrhea was noted in the 0- to 6-month age group in both settings. The difference in severity across age groups was not found to be significant in both the community (P = 0.3) and the hospital (P = 0.6) (Table 3). Severity scores could not be determined for four children admitted to the hospital because they were discharged before the visit of the study medical officer.
Temporal distribution. No significant seasonal trends were observed in the community as well as in the hospital. However, there appeared to be a peak of rotavirus diarrhea in the months of July to September in 2003, corresponding to the rainy season.
Genotyping. All of the 82 positive episode samples collected from the community were available in sufficient quantity for VP7 genotyping. G and P typing results could be obtained for 84.2% and 58.5% of the samples, respectively. The G types identified in children with diarrhea from the community, in order of frequency, were G1 (36.5%), G10 (17.1%), G2 (15.9%), and G9 (7.3%). The P types identified were P[4] (28.0%), P[8] (15.9%), P[11] (8.5%), and P[6] (1.2%) (Table 4). In children requiring admission from the community, the G types were G10 (50%), mixed infections with G1 and G9 and with G1 and G10, and untypeable strains.
For the hospital samples, G and P typing results could be obtained on 79.7% and 65.9% of the samples, respectively. The most common G type was G1 (46.8%), followed by G9 (19.1%) and G2 (8.5%) (see Table 6). The most common P type was P[8] (55.2%). Other P types included P[4] (5.3%), P[6] and P[11] (2.2%), and P[10] (1.1%) (Table 5).
The most frequent G-P combination in the community was G1P[8] (13.4%; 95% confidence interval [CI], 6.0 to 20.8%) and G2P[4] (13.4%; 95% CI, 6.0 to 20.8%), followed by G1P[4] (12.2%; 95% CI, 5.1 to 19.3%), G10P[11] (8.5%; 95% CI, 2.5 to 14.5%), and G9P[8] (1.2%; 95% CI, –1.2 to 3.6%). Mixed infections with more than one G type accounted for 7.3% of rotavirus infections. Twelve samples (14.6%) remained untypeable for both VP7 and VP4 (Table 4) despite being positive in a VP6 PCR.
The most common G-P combinations seen in the hospital were G1P[8] (38.3%; 95% CI, 28.5 to 48.1%), G9P[8] (18%; 95% CI, 10.2 to 25.8%), and G2P[4] (5.3%; 95% CI, 0.8 to 9.8%) (Table 5). One case each of infection by G9P[6] and G10P[11] was also seen. Mixed infection was seen in four samples, accounting for 4.3% of all rotavirus infections. Seventeen samples remained untypeable (18.1%) for both VP7 and VP4 (Table 5) despite being positive in a VP6 PCR. The dual infections in the community and hospital were G1-G2, G1-G9, G1-G10, and G2-G10 combinations. One instance of triple infection was seen in the hospital setting with G1-G2-G9.
Eight children in the community-based cohort had a repeat episode of rotavirus diarrhea during this period of follow-up. Three children were reinfected with the same G type of rotavirus during the second episode, two with G1, and one with a G10 strain. The durations between infections in these children were 16, 24, and 60 weeks. In the remaining five children, the durations between infections were 3, 6, 16, 24, and 28 weeks. Mixed infections were seen in three children during the second episode, and two were severe.
DISCUSSION
This study is a comparison of rotavirus diarrhea in two different settings within the same geographical location during overlapping 2-year periods. There were 82 rotavirus-associated diarrheal episodes in the community and 94 rotavirus-associated diarrheal admissions to the hospital which were subjected to comparative analysis.
Rotavirus-infected children admitted to the hospital were older than their counterparts in the community (P = 0.007). This difference may have been due to the closer monitoring of the community children, resulting in the early identification of symptomatic children and prompt treatment. It is also possible that the severity of rotavirus is greater in older children, and this accounted for the older age of the children admitted to the hospital.
A significantly smaller proportion of children who presented with rotavirus diarrhea in the hospital were breast-fed than were children from the community (P < 0.001). A relationship between breast-feeding and rotavirus diarrhea has been demonstrated, with a twofold-greater risk of rotavirus diarrhea in non-breast-fed children than in those who were exclusively breast-fed (27). Lactadherin in human milk is believed to interfere with rotavirus replication (34). The increased replication in non-breast-fed children may partially explain the greater severity of rotavirus diarrhea in the children admitted to the hospital. However, in the absence of information on the socioeconomic status of hospitalized children and cultural beliefs related to breast-feeding, the lower levels of breast-feeding in children requiring hospitalization cannot be explained, except possibly by their older age.
A recent review of 24 community-based studies and 72 hospital-based studies indicated that rotavirus accounted for a median of 8.1% and 21.3% of diarrheal episodes in the two settings, respectively (37). The percentage of diarrheal episodes attributable to rotavirus in our study conformed to this trend, with rotavirus accounting for 7.1% of diarrheal episodes in the community and 27.4% in the hospital (P < 0.0001). The diarrhea seen in the hospital was more severe (56.7% versus 22.0%), and the mean Vesikari score was also significantly higher for children in the hospital than for children in the community (P < 0.001). This difference may be due to an inherent referral bias, as more-severe cases need admission in the hospital. Economic considerations may also be responsible for this referral bias. While it is possible in other settings that severe cases in the community have greater mortality rates and do not reach the caregiver because of an economic disadvantage, we did not find any significant difference in rotavirus-associated mortality between the two groups (P = 0.28). The pooled mortality rate of all rotavirus diarrheal episodes was 0.57% in our study and is consistent with the published estimates of rotavirus-associated mortality of 0.34% (37). Despite being a large referral hospital, free treatment is given to children and economic reasons may not play a significant role in determining health-seeking behavior of patients in this setting.
In developed countries, the most prevalent rotavirus strains causing childhood diarrhea are G1 to G4 and G9 (21). In contrast, there is significant diversity of circulating rotavirus strains in India, though G1 and G2 are the most commonly isolated strains (21, 25). We found that the G1 strain accounted for 36.5% of episodes of rotavirus diarrhea in the community and 46.8% in the hospital, showing that it still continues to be the dominant strain. The most common G-P combinations in the community were G1P[8] and G2P[4], each accounting for 13.4% of all rotavirus diarrheal episodes. In comparison, G1P[8] accounted for 38.3% of all episodes of diarrhea in the hospital setting. A recent review of global rotavirus genotypes states that G1P[8] accounts for 70% of rotavirus infections in North America, Australia, and Europe, whereas it represents about 30% of infections in South America and Asia and about 23% in Africa (42).
A significant finding in this study was the high percentage of G1P[4] strains in the community, accounting for 12.2% of rotavirus diarrhea. This strain was not detected in hospitalized children. In a previous hospital-based study from Vellore, this strain comprised 11% of the total rotavirus strains (22). It has been reported in 14% of isolates from Argentina (1, 16) and may be an emerging natural reassortant strain.
Rotavirus serotype G9 is recognized as the most widespread of the emerging genotypes, representing 4.1% of global rotavirus infections and accounting for up to 70% of rotavirus infections in recent reports (26, 30, 42, 43, 48). In our study, this strain was identified in 7.3% of rotavirus diarrhea cases from the community and 19.1% of cases from the hospital, where it was the second most common strain.
The G10P[11] strain is a bovine strain which has been identified as a cause of asymptomatic neonatal infection in India (10). Recent studies from India identified this strain in infants and young children as a cause of diarrhea (20). In our study, the G10P[11] strain was detected in 8.5% of rotavirus diarrhea cases in the community and in 1.1% of those in the hospital. However, the G10 genotype was the second most common G type in children from the community, accounting for 17.1% of all rotavirus diarrhea cases. The G10 strains are suspected to have arisen in the human population through interspecies transmission or animal-human rotavirus reassortment (7, 45). In Indian communities, there is close proximity of humans and animals, facilitating the potential for zoonotic transmission. The cohort is based in an urban slum setting, and the difference of G10 prevalence between the community and the hospital children may have been due to differences in socioeconomic status between the two populations. It is possible that the children from the community cohort belong to a lower socioeconomic status, making them more prone to acquire animal strains.
The rates of untypeable strains were 14.6% in the community children and 18.1% in the hospital children. Genotyping of G types has been more successful than that of P types in all Indian studies published so far, and the results presented here are comparable with previously published data (23). Due to the constant accumulation of point mutations through genetic drift and to the emergence of novel genotypes, possibly zoonotic transmission and subsequent reassortment, the reagents and methods used for genotyping require close monitoring and updating. We have developed methods and oligonucleotide primers which were used to overcome failures to type G9, G10, and P rotavirus strains (11, 23). In other studies, the detection rates of nontypeable strains have ranged from 6.5% to 16% (12, 31).
There have been two previous epidemiological studies of rotavirus diarrhea from the years 1983 to 1985 and 1995 to 1998 in Vellore (5, 22). A comparison of the data for the various rotavirus strains isolated from previous studies and the combined community and hospital data from the current study shows an interesting pattern emerging. The contributions of various strains as causative agents of diarrhea in these three time periods (1983 to 1985, 1995 to 1998, and 2002 to 2004) are as follows: G1, 32.6%, 39.6%, and 42%; G2, 6.5%, 19%, and 11.9%; G3, 7%, 0.8%, and 0%; G4, 30%, 23.8%, and 0%; G9, 0%, 3.9%, and 13.6%; and G10, 0%, 0%, and 8.5%. Based on these data, there appears to have been a gradual disappearance of G3 and G4 strains and an emergence of G9 and G10 strains.
The impact of the type of circulating rotavirus strains in the community and the hospital on vaccine development cannot be overemphasized. Current vaccine development efforts are targeted at the six most common types of rotavirus, G1 to G4, G9, and P[8]. A live attenuated vaccine based on a human strain, Rotarix, has been on trial in four countries and has shown efficacy against severe rotavirus gastroenteritis caused by G1 and non-G1 types, including the emerging G9 type (B. De Vos, A. C. Linhares, G. Ruiz-Palacios, L. Guerrero, B. Salinas, I. Perez-Schael, Abstr. 8th Intl. Symp. Double-Stranded RNA Viruses, Lucca, Italy, abstr. RT-4, 2003), although protection against the G9 type may have been due to all G9 strains being combined with P[8]. It has currently been licensed for use in Mexico (9). In our study, the G1 strain constituted 37% of all rotavirus diarrhea cases in the community and 47% of cases in the hospital. Unless there is a significant heterotypic response to this strain, it appears that this vaccine will fail to protect a majority of the children in this population. Another candidate vaccine, Rotateq, is a reassortant pentavalent vaccine with a backbone of bovine rotavirus and surface proteins of human serotypes G1, G2, G3, G4, and P[8] (35). Preliminary data from a Finnish study suggest that there was about 75% protection for overall rotavirus infection (35). This vaccine has been licensed for use in the United States. The presence of a significant proportion of G9 and G10 rotavirus strains in the community would theoretically make this vaccine effective in only 55% of affected children.
The observation that rotavirus strains infecting neonates are usually asymptomatic and that neonatal infection protects against subsequent rotavirus infection has led to the interest in these strains as putative vaccine candidates (3). In this context, it is important to evaluate the safety of the neonatal strain I 321, a G10P[11] strain (7). We have previously noted that this strain causes symptomatic diarrhea in neonates and older children, and sequencing of VP7, VP6, VP4, and NSP4 has shown significant homology between circulating G10P[11] strains and I 321 strains (20). In this study, G10P[11] was responsible for 8.5% of rotavirus diarrhea cases in the community and 1.1% in the hospital. In addition, the median severity score for G10 infection was in the moderately severe range (community, 6; and hospital, 9 [as shown in Table 6]). In light of these findings, it is imperative that stringent safety trials are conducted prior to the use of this strain as a vaccine.
This comparative study of community and hospitalized children with rotavirus diarrhea has several important implications. It has clearly shown that there are distinct differences in the ages of infection and severities of symptoms in these two settings. Variations in genotypes suggest a possible difference in genotype-specific severity, possible zoonotic spread, and the emergence of reassortant viruses. The broad diversity of rotavirus strains also indicates that unless a vaccine induces a broad heterotypic response, there may be a limited protective response and the composition of the vaccine may need to be changed frequently to reflect the changes in circulating viruses.
ACKNOWLEDGMENTS
This work was supported by the Wellcome Trust under the Trilateral Cooperative Initiative for Research in Infectious Diseases in the Developing World (grant number 063144).
FOOTNOTES
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Virus Reference Department, Centre for Infection, Health Protection Agency, London, United Kingdom
London School of Hygiene and Tropical Medicine, London, United Kingdom
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas
ABSTRACT
Rotavirus gastroenteritis is the major cause of severe dehydrating diarrhea in children worldwide. This study compares rotavirus diarrhea in 351 children in a community-based cohort and 343 children admitted to a hospital during the same period. Clinical information and fecal specimens were obtained during diarrheal episodes. Fecal samples were screened for VP6 antigen, and the positive samples were G and P typed by reverse transcription-PCR. Rotavirus was detected in 82/1,152 (7.1%) episodes of diarrhea in the community and 94/343 (27.4%) cases in the hospital. The median age of affected children (7.5 versus 10.5 months) and the mean severity of symptoms (Vesikari score, 7.6 ± 3.4 versus 11 ± 2.5) were lower in the community. A larger proportion of children in the community were breast-fed than were children admitted to the hospital (73% versus 34.8%). In the community, the genotypes identified in symptomatic patients, in order of frequency, were G1 (36.5%), G10 (17.1%), G2 (15.9%), and G9 (7.3%) and mixed infections (7.3%). The most common G-P combinations were G1P[8], G2P[4], G1P[4], and G10P[11]. The distribution of G types from hospitalized children was G1 (46.8%), G9 (19.1%), G2 (8.5%), G10 (1.1%), and 4.3% mixed infections. The most common G-P combinations were G1P[8] and G9P[8]. This study documents significant genetic heterogeneity of rotaviruses in the community and the hospital. G10P[11] strains resembling a vaccine candidate strain caused disease in the community, indicating the need for careful epidemiological studies as well as safety studies for the vaccine candidates.
INTRODUCTION
Rotaviruses are the major etiological agents causing diarrhea in infants and young children. A recent review estimates that more than 600,000 deaths occur annually in children under the age of 5 years due to rotavirus infection, with a majority of these deaths occurring in developing countries (36).
Rotaviruses belong to the family Reoviridae and possess a genome of 11 segments of double-stranded RNA that are located inside triple-layered viral particles. Rotaviruses are classified into seven different serogroups (A to G), based on the antigenic specificities of the capsid proteins. For group A viruses, typing schemes were introduced based on antigenic epitopes on the proteins that form the inner capsid (VP6; subgroups I and II) and on proteins of the outer capsid, the glycoprotein VP7 (G serotypes) and the spike protein VP4 (P serotypes). In recent years, reverse transcription-PCR was used in molecular epidemiological studies. All known G serotypes have been correlated with genotypes; however, there are more P genotypes than serotypes identified, leading to a serotype/genotype dual nomenclature for P types. At least 15 different G types and 26 different P types have been found in human and animal infections (11, 24, 29, 46). The incidence and distribution of group A rotavirus serotypes and genotypes vary between geographical areas during a rotavirus season and from one season to the next. Globally, different surveys indicate that G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8] are the most common G and P types (1, 2, 14, 18). However, in several regions, viruses of other G-P combinations have been found: G5P[8] in Brazil, G8P[6] in Malawi, G9P[6] in India and Bangladesh, and G10P[11] in India (6, 20, 28, 39, 44).
The first vaccine to be licensed, RotaShield, included reassortants of the most frequently encountered viral strains G1 to G4 (38). The vaccine was withdrawn due to a temporal association with intussusception (33). Promising results of large-scale clinical trials on two new vaccines, Rotateq and Rotarix, have recently been published (40, 47). The anticipated availability of an effective vaccine highlights the need to better define the epidemiology and disease burden associated with rotavirus. It is important to study local distribution of rotavirus strains and conduct active surveillance programs for emerging reassortant strains prior to and after the introduction of vaccines.
Most of the body of knowledge on rotavirus infections in children is based on hospital-centered studies. There is an inherent referral bias if these studies alone are considered. A clear understanding of the spectrum of disease can result only from a combination of community- and hospital-based studies which investigate both mild and severe disease. A study based in the community provides geographically representative information on the disease burden, strain prevalence, and incidence rates of rotavirus infection in the community. In contrast, the hospital-based surveillance system provides detailed information on severity and strain prevalence in children presenting to a hospital with diarrhea. In this report, we highlight the differences between rotavirus diarrhea in a community birth cohort and in a hospital in southern India over a 2-year period.
MATERIALS AND METHODS
Patients from the community. A cohort of 452 children was recruited from an urban slum area in Vellore, South India, over a period of 18 months extending from March 2002 to August 2003. The population of this area was 33,390, of which 2,895 (8.7%) were under the age of 5 years. Sixty-seven children (14.6%) dropped out of the study. Thirty-eight children moved out of the study area, 25 refused to continue in the cohort, and 4 children died after recruitment. The remaining 385 children were monitored between May 2002 and May 2004. Fecal samples were collected from these children in the neonatal period, and a baseline assessment of the domestic environment was conducted by interview. Thereafter, each child was visited at home twice a week by a field worker in order to collect information on any illness of the child and the family. Any child who developed diarrhea was assessed clinically and treated appropriately. Samples of feces were collected from the child during the diarrheal episode. The child was followed up daily until cessation of diarrhea.
In this cohort, 351 of 385 children had a total of 1,299 episodes of diarrhea between May 2002 and May 2004. Samples collected from these children during this period were analyzed for the comparative study.
Patients from the hospital. The study was carried out at the Christian Medical College, a 1,800-bed hospital in Vellore, India. Vellore is an urban area with a population of 400,000 and is estimated to have approximately 42,000 children less than 5 years of age. All children under 5 years of age presenting to the hospital with acute gastroenteritis and requiring hospitalization for rehydration for at least 6 h were enrolled in the study. A total of 343 children less than 5 years of age, hospitalized for acute diarrhea between January 2002 and December 2003, were screened for rotavirus.
Informed consent was obtained from the parents of all children enrolled in the community- and hospital-based studies. The studies were approved by the guidelines of the Research Committee of the Christian Medical College, Vellore, India.
All samples from the hospital and the community were transported on ice chests to the laboratory and were processed immediately for the detection of rotavirus. Four 2-g aliquots were stored at –70°C for strain characterization and future testing.
Rotavirus screening. Samples were screened for rotavirus VP6 antigen by a latex agglutination assay (Meridian Diagnostics, Inc.) until September 2002 (2% of samples from the community and 39% from the hospital). Thereafter, all samples were examined for group A rotavirus by an enzyme-linked immunosorbent assay (Rota IDEIA; DakoCytomation Ltd., United Kingdom) to detect VP6 antigen following the manufacturer's instructions.
RNA extraction and cDNA synthesis. Viral RNA was extracted from 200 μl of the 10% of rotavirus-positive fecal suspension in balanced salt solution, using guanidine isothiocyanate-silica according to the method described by Boom et al. (4). It was then eluted in 50 μl of RNase-free distilled water containing 40 units of RNase inhibitor (RNasin; Ambion, United Kingdom).
cDNA was generated from 40 μl of the extracted RNA by reverse transcription in the presence of random primers (hexamers) [Pd(N)6; Pharmacia Biotech, United Kingdom], using 400 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen, Life Technologies, United Kingdom).
Genotyping PCR. The resulting cDNA was the template for both VP7- and VP4-specific typing PCRs, using the oligonucleotide primers described earlier (13, 15, 17, 19). Primers complementary to conserved regions of VP7 were used for the first-round VP7 PCR to amplify an 881-bp region of the VP7 gene (19). The second-round typing PCR was a multiplex PCR and incorporated the 3'-end-labeled primer (VP7 Reverse) and the G-type-specific primers G1, G2, G3, G4, G8, G9, and G10 (19).
Primers Con2 and Con3 were used for the first-round PCRs to amplify an 876-bp fragment of the VP4 gene (17). The second-round PCR included primers specific for genotypes P[4], P[6], P[8], P[9], P[10], and P[11] and the consensus primer Con3 (19).
Assessment of severity. Diarrhea was defined as the passage of three watery stools in a 24-h period. In children less than 6 months of age, a change in number or consistency of stools reported by the mother was considered indicative of diarrhea. An episode was defined as at least 1 day of diarrhea, preceded and followed by 2 or more days without diarrhea (32). The episode was considered to have ended on the day bowel movements returned to normal.
The severity of diarrhea was assessed using the Vesikari scoring system (41). Information was collected on duration of diarrhea, maximum number of stools passed per day, duration and peak frequency of vomiting, degree of fever, presence and severity of dehydration, and treatment. To permit calculation of this score, the episode was considered mild for a score of 5, moderately severe for a score of 6 to 10, and severe for a score of >10.
For calculating the severity scores for children in the community, a modified Vesikari scoring system was followed. Since accurate temperature measurements were not possible in the field, temperatures were recorded as normal, low-grade fever, and high-grade fever as reported by the caregivers.
Statistical analysis. Data were entered in Microsoft Access and analyzed using SPSS v. 9.0. Chi square and Student's t tests were performed to determine the significance of differences observed between the two different groups of patients.
RESULTS
Rotavirus diarrhea. A total of 351 children in the community cohort presented with 1,299 episodes of diarrhea between May 2002 and May 2004. The mean age of the group at the end of this monitoring period was 17 ± 4.3 months. There were 175 males (49.9%) and 176 females (50.1%). Diarrheal samples were collected during 88.7% of the episodes of diarrhea. The age range of children presenting with diarrhea was 0 to 24 months (interquartile range [IQR], 3 to 10 months, indicating that 50% of the cases occurred between 3 and 10 months). Rotavirus was detected in 82/1,152 (7.1%) episodes for which samples were obtained.
In the hospital, 343 children were admitted with diarrhea from January 2002 to December 2003. Rotavirus was detected in 94 children, accounting for 27.4% of all diarrhea cases. The age range of children presenting with diarrhea to the hospital was 0 to 60 months (IQR, 5 to 12 months). In seven cases, none of whom were rotavirus positive, age was not recorded on the patient charts. Demographic, virological, and clinical data of children in the community and the hospital with rotavirus diarrhea are shown in Table 1.
The median age of detection of rotavirus in children from the community was 7.5 months (IQR, 4.7 to 10.2) compared to 10.0 months (IQR, 7.5 to 12.5) in the hospital (P < 0.007). A larger proportion of those admitted in the hospital were male children (63.8%) than were those in the community (53.7%). However, this difference did not reach statistical significance (P = 0.15). A greater proportion of children in the community were breast-fed than were those admitted to the hospital (73% versus 34.8%) (P < 0.001) at the time they presented with gastroenteritis.
In the community, 32.9% of the episodes of rotavirus diarrhea were mild, 45.1% were moderate, and 22.0% were severe. During these episodes, 13.4% of children were treated at home, 79.3% were taken to the study clinic, and 7.3% required hospitalization. Not all severe cases required admission, because they received supervised oral rehydration at the clinic. In the community, six young children with severe dehydration were admitted (mean age, 4.3 months; range, 0 to 10 months; four of six breast-fed). In contrast, only 3.3% of rotavirus diarrheal cases admitted in the hospital were mild, 40.0% were moderate, and 56.7% were severe (P < 0.001). The mild cases were admitted to the hospital to alleviate parental anxiety. The mean Vesikari score was also higher in the hospital than in the community (11.0 ± 2.5 versus 7.6 ± 3.4) (P < 0.001). There was one rotavirus-associated death in the community (1.2%) and no documented mortality in the hospital.
The distribution of rotavirus infection in the community and hospital by age is given in Table 2. The largest proportion of rotavirus diarrhea was noted in the 12- to 17-month age group in the community and in the 6- to 11-month age group in the hospital (11.5% and 42.3%, respectively). The smallest proportion of rotavirus diarrhea was noted in the 0- to 6-month age group in both settings. The difference in severity across age groups was not found to be significant in both the community (P = 0.3) and the hospital (P = 0.6) (Table 3). Severity scores could not be determined for four children admitted to the hospital because they were discharged before the visit of the study medical officer.
Temporal distribution. No significant seasonal trends were observed in the community as well as in the hospital. However, there appeared to be a peak of rotavirus diarrhea in the months of July to September in 2003, corresponding to the rainy season.
Genotyping. All of the 82 positive episode samples collected from the community were available in sufficient quantity for VP7 genotyping. G and P typing results could be obtained for 84.2% and 58.5% of the samples, respectively. The G types identified in children with diarrhea from the community, in order of frequency, were G1 (36.5%), G10 (17.1%), G2 (15.9%), and G9 (7.3%). The P types identified were P[4] (28.0%), P[8] (15.9%), P[11] (8.5%), and P[6] (1.2%) (Table 4). In children requiring admission from the community, the G types were G10 (50%), mixed infections with G1 and G9 and with G1 and G10, and untypeable strains.
For the hospital samples, G and P typing results could be obtained on 79.7% and 65.9% of the samples, respectively. The most common G type was G1 (46.8%), followed by G9 (19.1%) and G2 (8.5%) (see Table 6). The most common P type was P[8] (55.2%). Other P types included P[4] (5.3%), P[6] and P[11] (2.2%), and P[10] (1.1%) (Table 5).
The most frequent G-P combination in the community was G1P[8] (13.4%; 95% confidence interval [CI], 6.0 to 20.8%) and G2P[4] (13.4%; 95% CI, 6.0 to 20.8%), followed by G1P[4] (12.2%; 95% CI, 5.1 to 19.3%), G10P[11] (8.5%; 95% CI, 2.5 to 14.5%), and G9P[8] (1.2%; 95% CI, –1.2 to 3.6%). Mixed infections with more than one G type accounted for 7.3% of rotavirus infections. Twelve samples (14.6%) remained untypeable for both VP7 and VP4 (Table 4) despite being positive in a VP6 PCR.
The most common G-P combinations seen in the hospital were G1P[8] (38.3%; 95% CI, 28.5 to 48.1%), G9P[8] (18%; 95% CI, 10.2 to 25.8%), and G2P[4] (5.3%; 95% CI, 0.8 to 9.8%) (Table 5). One case each of infection by G9P[6] and G10P[11] was also seen. Mixed infection was seen in four samples, accounting for 4.3% of all rotavirus infections. Seventeen samples remained untypeable (18.1%) for both VP7 and VP4 (Table 5) despite being positive in a VP6 PCR. The dual infections in the community and hospital were G1-G2, G1-G9, G1-G10, and G2-G10 combinations. One instance of triple infection was seen in the hospital setting with G1-G2-G9.
Eight children in the community-based cohort had a repeat episode of rotavirus diarrhea during this period of follow-up. Three children were reinfected with the same G type of rotavirus during the second episode, two with G1, and one with a G10 strain. The durations between infections in these children were 16, 24, and 60 weeks. In the remaining five children, the durations between infections were 3, 6, 16, 24, and 28 weeks. Mixed infections were seen in three children during the second episode, and two were severe.
DISCUSSION
This study is a comparison of rotavirus diarrhea in two different settings within the same geographical location during overlapping 2-year periods. There were 82 rotavirus-associated diarrheal episodes in the community and 94 rotavirus-associated diarrheal admissions to the hospital which were subjected to comparative analysis.
Rotavirus-infected children admitted to the hospital were older than their counterparts in the community (P = 0.007). This difference may have been due to the closer monitoring of the community children, resulting in the early identification of symptomatic children and prompt treatment. It is also possible that the severity of rotavirus is greater in older children, and this accounted for the older age of the children admitted to the hospital.
A significantly smaller proportion of children who presented with rotavirus diarrhea in the hospital were breast-fed than were children from the community (P < 0.001). A relationship between breast-feeding and rotavirus diarrhea has been demonstrated, with a twofold-greater risk of rotavirus diarrhea in non-breast-fed children than in those who were exclusively breast-fed (27). Lactadherin in human milk is believed to interfere with rotavirus replication (34). The increased replication in non-breast-fed children may partially explain the greater severity of rotavirus diarrhea in the children admitted to the hospital. However, in the absence of information on the socioeconomic status of hospitalized children and cultural beliefs related to breast-feeding, the lower levels of breast-feeding in children requiring hospitalization cannot be explained, except possibly by their older age.
A recent review of 24 community-based studies and 72 hospital-based studies indicated that rotavirus accounted for a median of 8.1% and 21.3% of diarrheal episodes in the two settings, respectively (37). The percentage of diarrheal episodes attributable to rotavirus in our study conformed to this trend, with rotavirus accounting for 7.1% of diarrheal episodes in the community and 27.4% in the hospital (P < 0.0001). The diarrhea seen in the hospital was more severe (56.7% versus 22.0%), and the mean Vesikari score was also significantly higher for children in the hospital than for children in the community (P < 0.001). This difference may be due to an inherent referral bias, as more-severe cases need admission in the hospital. Economic considerations may also be responsible for this referral bias. While it is possible in other settings that severe cases in the community have greater mortality rates and do not reach the caregiver because of an economic disadvantage, we did not find any significant difference in rotavirus-associated mortality between the two groups (P = 0.28). The pooled mortality rate of all rotavirus diarrheal episodes was 0.57% in our study and is consistent with the published estimates of rotavirus-associated mortality of 0.34% (37). Despite being a large referral hospital, free treatment is given to children and economic reasons may not play a significant role in determining health-seeking behavior of patients in this setting.
In developed countries, the most prevalent rotavirus strains causing childhood diarrhea are G1 to G4 and G9 (21). In contrast, there is significant diversity of circulating rotavirus strains in India, though G1 and G2 are the most commonly isolated strains (21, 25). We found that the G1 strain accounted for 36.5% of episodes of rotavirus diarrhea in the community and 46.8% in the hospital, showing that it still continues to be the dominant strain. The most common G-P combinations in the community were G1P[8] and G2P[4], each accounting for 13.4% of all rotavirus diarrheal episodes. In comparison, G1P[8] accounted for 38.3% of all episodes of diarrhea in the hospital setting. A recent review of global rotavirus genotypes states that G1P[8] accounts for 70% of rotavirus infections in North America, Australia, and Europe, whereas it represents about 30% of infections in South America and Asia and about 23% in Africa (42).
A significant finding in this study was the high percentage of G1P[4] strains in the community, accounting for 12.2% of rotavirus diarrhea. This strain was not detected in hospitalized children. In a previous hospital-based study from Vellore, this strain comprised 11% of the total rotavirus strains (22). It has been reported in 14% of isolates from Argentina (1, 16) and may be an emerging natural reassortant strain.
Rotavirus serotype G9 is recognized as the most widespread of the emerging genotypes, representing 4.1% of global rotavirus infections and accounting for up to 70% of rotavirus infections in recent reports (26, 30, 42, 43, 48). In our study, this strain was identified in 7.3% of rotavirus diarrhea cases from the community and 19.1% of cases from the hospital, where it was the second most common strain.
The G10P[11] strain is a bovine strain which has been identified as a cause of asymptomatic neonatal infection in India (10). Recent studies from India identified this strain in infants and young children as a cause of diarrhea (20). In our study, the G10P[11] strain was detected in 8.5% of rotavirus diarrhea cases in the community and in 1.1% of those in the hospital. However, the G10 genotype was the second most common G type in children from the community, accounting for 17.1% of all rotavirus diarrhea cases. The G10 strains are suspected to have arisen in the human population through interspecies transmission or animal-human rotavirus reassortment (7, 45). In Indian communities, there is close proximity of humans and animals, facilitating the potential for zoonotic transmission. The cohort is based in an urban slum setting, and the difference of G10 prevalence between the community and the hospital children may have been due to differences in socioeconomic status between the two populations. It is possible that the children from the community cohort belong to a lower socioeconomic status, making them more prone to acquire animal strains.
The rates of untypeable strains were 14.6% in the community children and 18.1% in the hospital children. Genotyping of G types has been more successful than that of P types in all Indian studies published so far, and the results presented here are comparable with previously published data (23). Due to the constant accumulation of point mutations through genetic drift and to the emergence of novel genotypes, possibly zoonotic transmission and subsequent reassortment, the reagents and methods used for genotyping require close monitoring and updating. We have developed methods and oligonucleotide primers which were used to overcome failures to type G9, G10, and P rotavirus strains (11, 23). In other studies, the detection rates of nontypeable strains have ranged from 6.5% to 16% (12, 31).
There have been two previous epidemiological studies of rotavirus diarrhea from the years 1983 to 1985 and 1995 to 1998 in Vellore (5, 22). A comparison of the data for the various rotavirus strains isolated from previous studies and the combined community and hospital data from the current study shows an interesting pattern emerging. The contributions of various strains as causative agents of diarrhea in these three time periods (1983 to 1985, 1995 to 1998, and 2002 to 2004) are as follows: G1, 32.6%, 39.6%, and 42%; G2, 6.5%, 19%, and 11.9%; G3, 7%, 0.8%, and 0%; G4, 30%, 23.8%, and 0%; G9, 0%, 3.9%, and 13.6%; and G10, 0%, 0%, and 8.5%. Based on these data, there appears to have been a gradual disappearance of G3 and G4 strains and an emergence of G9 and G10 strains.
The impact of the type of circulating rotavirus strains in the community and the hospital on vaccine development cannot be overemphasized. Current vaccine development efforts are targeted at the six most common types of rotavirus, G1 to G4, G9, and P[8]. A live attenuated vaccine based on a human strain, Rotarix, has been on trial in four countries and has shown efficacy against severe rotavirus gastroenteritis caused by G1 and non-G1 types, including the emerging G9 type (B. De Vos, A. C. Linhares, G. Ruiz-Palacios, L. Guerrero, B. Salinas, I. Perez-Schael, Abstr. 8th Intl. Symp. Double-Stranded RNA Viruses, Lucca, Italy, abstr. RT-4, 2003), although protection against the G9 type may have been due to all G9 strains being combined with P[8]. It has currently been licensed for use in Mexico (9). In our study, the G1 strain constituted 37% of all rotavirus diarrhea cases in the community and 47% of cases in the hospital. Unless there is a significant heterotypic response to this strain, it appears that this vaccine will fail to protect a majority of the children in this population. Another candidate vaccine, Rotateq, is a reassortant pentavalent vaccine with a backbone of bovine rotavirus and surface proteins of human serotypes G1, G2, G3, G4, and P[8] (35). Preliminary data from a Finnish study suggest that there was about 75% protection for overall rotavirus infection (35). This vaccine has been licensed for use in the United States. The presence of a significant proportion of G9 and G10 rotavirus strains in the community would theoretically make this vaccine effective in only 55% of affected children.
The observation that rotavirus strains infecting neonates are usually asymptomatic and that neonatal infection protects against subsequent rotavirus infection has led to the interest in these strains as putative vaccine candidates (3). In this context, it is important to evaluate the safety of the neonatal strain I 321, a G10P[11] strain (7). We have previously noted that this strain causes symptomatic diarrhea in neonates and older children, and sequencing of VP7, VP6, VP4, and NSP4 has shown significant homology between circulating G10P[11] strains and I 321 strains (20). In this study, G10P[11] was responsible for 8.5% of rotavirus diarrhea cases in the community and 1.1% in the hospital. In addition, the median severity score for G10 infection was in the moderately severe range (community, 6; and hospital, 9 [as shown in Table 6]). In light of these findings, it is imperative that stringent safety trials are conducted prior to the use of this strain as a vaccine.
This comparative study of community and hospitalized children with rotavirus diarrhea has several important implications. It has clearly shown that there are distinct differences in the ages of infection and severities of symptoms in these two settings. Variations in genotypes suggest a possible difference in genotype-specific severity, possible zoonotic spread, and the emergence of reassortant viruses. The broad diversity of rotavirus strains also indicates that unless a vaccine induces a broad heterotypic response, there may be a limited protective response and the composition of the vaccine may need to be changed frequently to reflect the changes in circulating viruses.
ACKNOWLEDGMENTS
This work was supported by the Wellcome Trust under the Trilateral Cooperative Initiative for Research in Infectious Diseases in the Developing World (grant number 063144).
FOOTNOTES
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