|Year : 2004 | Volume
| Issue : 4 | Page : 212-221
Rotavirus infection among infants and young children in Chennai, South India
P Saravanan , S Ananthan , M Ananthasubramanian
Department of Microbiology, Dr. ALM PGIBMS, University of Madras, Taramani Campus, Chennai - 600 113, India
|Date of Submission||19-Jul-2003|
|Date of Acceptance||13-Mar-2004|
Department of Microbiology, Hindustan College of Arts and Science, Padur, Chennai - 603 103, India
BACKGROUND: Group A rotavirus has been recognized as the major etiologic agent of childhood gastroenteritis in infants and young children worldwide. Rapid progress towards the development of an efficacious rotavirus vaccine has warranted extensive epidemiological studies on rotavirus serotypes that cause severe disease in developing and developed countries and to monitor the emergence of newer and unusual strains in different geographical settings that could represent variants not covered by existing vaccines. METHODS: In this study, we determined the prevalence of rotavirus infection and characterised group A rotavirus in stool samples by using monoclonal antibody (MAb) based ELISA and polyacrylamide gel electrophoresis (PAGE). Stool samples were collected from 745 children of 0-3 years of age presenting to the hospital with acute diarrhea between March 1995 and August 1999. These were assayed for antigenic (group, subgroup, serotype) and genomic (viral RNA profile and VP7 and VP4 genotype) characterization by ELISA and PAGE. RESULTS: Out of 745 stool samples analysed 168 were found to be positive for rotavirus. Among these 118 could be assigned a subgroup (SG), serotype and electropherotype (E-type). The study has evidenced the predominant occurrence of strains with short E-type, SGI and serotype G2 in 66.1% of the samples. The presence of strains representing 10 different E-types and mixed genotype specificities with G2 P[4,8] and G1-G2 P[4,8] has documented the prevailing high genomic diversity of rotaviruses in this geographical area. CONCLUSION: This study has described the predominant strains of rotavirus in south India. There is a need for further detailed studies on the molecular characterization of rotaviruses which would have important implications in vaccine evaluation programmes.
|How to cite this article:|
Saravanan P, Ananthan S, Ananthasubramanian M. Rotavirus infection among infants and young children in Chennai, South India. Indian J Med Microbiol 2004;22:212-21
|How to cite this URL:|
Saravanan P, Ananthan S, Ananthasubramanian M. Rotavirus infection among infants and young children in Chennai, South India. Indian J Med Microbiol [serial online] 2004 [cited 2019 Feb 23];22:212-21. Available from: http://www.ijmm.org/text.asp?2004/22/4/212/12810
Group A rotaviruses are the single most important cause of severe acute diarrhoea in young children throughout the world. It is a cause of significant morbidity and mortality among children younger than 5 years of age in India. Of the approximately 600,000 annual deaths due to rotavirus (RV) worldwide, more than 150,000 occur in India., Also, 20 to 30 percent hospitalized cases of diarrhea are due to rotaviruses,, making the development and testing of an effective RV vaccine a particular priority in India. The licensure of the recent tetravalent vaccine has been withdrawn following continued reports from vaccine adverse event reporting system (VAERS) on increased risk of intussusception in vaccine recipients.
Epidemiological and molecular studies in many countries show complex patterns of change from year to year in the serotypes and electropherotypes that cause diarrhoea in hospitalized children from the same geographical areas. Recent epidemiological studies in India, Bangladesh and the United States show that other G and P types (G5, G6, G8, G9, G10, P2A, P8 can be common and may be of emerging importance in some communities.,,,,
Thus, extensive epidemiological studies on rotavirus serotypes are required to map annual changes in strains in different geographical areas. The data can be useful to select areas for vaccine trials and to serve as baselines for identification of new strains, should they emerge. In the present study, the pattern of rotavirus infection in Chennai, southern India, and their antigenic and genomic characterisation was done.
| ~ Materials and Methods|| |
Patients and samples
A total of 745 children aged less than 3 years suffering from acute diarrhea, attending the Gastroenterology department of the Institute of Child Health and Hospital for Children (ICH&HC), and Communicable Diseases Hospital (CDH), Chennai during March 1995 and August 1999, were included in the study. Single faecal specimen was collected from each patient within first to fourth day of illness.
Detection of rotavirus in clinical specimens was done by an indirect sandwich ELISA as reported earlier. The ELISA positive samples were subjected to monoclonal antibody based ELISA that incorporates monoclonal antibodies specific for group A, subgroup I and subgroup II antigens and for serotype G1, G2, G3, and G4 antigens for their antigenic characterization according to the procedures described, with minor modifications as reported earlier. Analysis of rotavirus RNA was done by polyacrylamide gel electrophoresis (PAGE) and silver nitrate staining of the gels as described with some modification as reported earlier. VP7 and VP4 characterization of the representative rotavirus specimens was done by using reverse transcription and polymerase chain reaction (RT-PCR) technique at Viral Gastroenteritis Section, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, as per the procedure described. Data on various weather parameters were collected from the Indian Meteorological Department, Meenambakkam, Chennai, for the study period to analyse the seasonal pattern of rotavirus infection in Chennai.
| ~ Results|| |
The study showed an overall infection rate of 22.55% among children with acute diarrhea. The age group analysis of rotavirus positive children with acute diarrhoea manifested a significant increase in the infection rate among the age group of 7-12 months with 29.95% ( 2 = 9.79, P < 0.005; [Table - 1]). Major proportion of the rotavirus positive cases fell under the age group between 7-18 months with a rate of 62.5%. No association of rotavirus infection could be discerned between male (23.85%) and female (21.13%) children analysed [Table - 1]. Analysis of seasonal variation pertaining to rotavirus [Table - 2] revealed that the hotter months (March - August) in all the three years (1996, 1997 and 1998) had decreased rate of rotavirus associated diarrhoea (17.6, 17.0 and 14.3% respectively) than the cooler months (September - February) (21.8, 31.8 and 22.5% respectively). The same pattern could be appreciated when the summation of seasons were viewed (16.4% in hot months and 25.9% in cool months).
Even though no statistical significance was manifested in the rate of rotavirus infection between hot and cool months of 1996 ( 2 = 0.778, P > 0.25), a significant variation in having peak periods in cooler months of 1997 ( 2 = 12.029, P < 0.001) and 1998 ( 2 = 4.146, P < 0.05) were observed [Table - 2].
Analytical data of climatological factors such as rainfall, temperature (mean minimum and maximum) and mean relative humidity (RH) percentage in relation to rotavirus infections were grouped for convenience to analyse the significance of relationship of such factors with rotavirus diarrhoea between 1995 and 1999 [Table - 3]. Mean rainfall and mean RH percentage were greater during October through January than during the remaining months of the year, and mean minimum temperature was relatively low during October through February. These factors well coincided with the higher prevalence of rotavirus during these months. A significant relationship was observed with mean minimum temperature of 24 C wherein, a higher occurrence of rotavirus associated diarrhoea (27.7%) was recorded ( 2 = 5.676, P < 0.025).
Characterization of strains
A total of 118 of the 168 (70.2%) rotavirus positive specimens, which contained adequate material for repetitive analysis and for co-electrophoretic runs of viral RNA, were assigned a serotype, subgroup (SG) and electropherotype (E-type). The study revealed predominant occurrence of G2 type strains with 66.1%, followed by G4 (13.6%), G1 (9.3%) and G3 (1.7%). Dual infection was observed in 11 (9.3%) specimens, of which G1-G2 constituted 63.6%.
Examination of RNA migration pattern in polyacrylamide gels showed the occurrence of 10 different E-types, majority of them were of short RNA pattern seen in 89 (75.4%) and those with long RNA pattern in 29 (24.6%) specimens studied [Figure - 1].
Short pattern strains were found to predominate significantly during the years 1995 (P<0.05), 1996 (P<0.001), 1997 (P<0.001) and 1998 (P<0.001). Seven different short E-types (Lanes 3,4,5,7,8, 10,11) and three different long E-types (Lanes 6,9,13) were revealed based on their respective migration characteristics of segments presented in [Table - 4].
Pattern of appearance and prevalence of different E-types manifested the predominance of strains with a particular E-type each year with a constant change in the predominant types. Strains with pattern of lane 4 dominated in 1996 with 57.7%, followed by those of lane 5 in 1997 with 33.3% [Table - 5]. Co-circulation of more than one type of strain was observed annually.
Analysis of relationship between E-types, subgroup and serotype antigenic specificities revealed a significant association of short type, G2 serotype and SGI in 78 of the 89 (87.6%) strains studied ( 2 = 63.87, P < 0.001). Two long E-types with dual SGI+II antigenic specificities belonged to serotype G3, and other long E-types were SGII with either G1 or G4 specificity [Table - 6].
Comparison of clinical characteristics of patients infected with SGI and SGII rotaviruses revealed a significant presentation of severe dehydration ( 2 = 7.931, P < 0.005) and severe diarrhoea with more than 10 stools a day ( 2 = 12.509, P < 0.001) in children with SGI rotavirus infection (more common). The duration of diarrhoea was significantly higher among children infected with SGI rotavirus (t = 2.935, at 107 DF; P < 0.01) [Table - 7].
VP7 and VP4 genotypic characterization of representative short electropherotype strains (predominant type encountered during the study period) evidenced the predominant presence of strains with G2, P(4) genotype specificity, which represented lanes 3, 4, 5, 7 and 11. Those strains of lane 10 pattern showed G1-G2, P(4,8) genotype specificity. Strains representing lane 8 pattern exhibited G2, P(4,8)genotype, which demonstrate the presence of dual infection among rotavirus genogroups, a pre-requisite for the emergence of reassortant rotaviruses.
| ~ Discussion|| |
An overall prevalence rate of 22.6 percent was associated with rotavirus diarrhea in children in this study ranging from 33.3% in 1995 to 19.0% in 1998. Earlier short and long term studies from other parts of India has revealed varying rates of prevalence that ranged from as low as 4 to 62.6%.,,,,,, These wide ranges obviously reflect differences in age group studied, detection methods employed, geographical location, time of onset and duration of the investigation.
In the present study, a peak infection was observed in 29.95% of 7 - 12 months old children. More than 60% of the positive cases occurred among 7 - 18 months age group children, which is concordant with recent and earlier reports from India that ranged from 22 to 74 percent.,,, It appeared that infants below 4 months of age were initially protected to some extent by maternal antibodies against severe diarrhoea due to rotavirus, and they seem to have acquired adequate immunity between 12 and 16 months of age. The greater risks of infants and young children in the interim period of 6 to 12 months with declined levels of maternal antibodies to rotavirus infection have been documented.- No predilection of infection was seen among sex of the patients analysed in our study, which is in contrast to other studies wherein a preponderance of infection observed in male children ranged from 60.1 to 76.6 percent.,,,
Analysis of meteorological data in relation to rotavirus infection revealed a significant association with cooler months of 1997 (P< 0.001) and 1998 (P< 0.05), which is commensurate with previous reports from India and other countries.,,,, It has been observed that temperature influences the stability of human and animal rotaviruses that contributes to the efficient transmission of the human rotaviruses. Moreover the influence of low relative humidity in the home has been suggested as a facilitating factor for the survival of rotaviruses on surfaces.,,, A significant negative correlation with mean relative humidity and low mean minimum temperature was observed in all studies. This is suggestive of the indirect but important influence of meteorological factors on the complex epidemiology of human rotavirus infection.
Antigenic characterization studies showed predominance of subgroup (SG) I virus with 66.1% similar to that observed in Delhi.,, On the contrary, SGII virus was found to predominate in regions of Bangladesh and Japan. Moreover, all these studies have recorded low incidence rate of strains with dual SG specificity ranging from 1.8 to 3 percent as against a higher proportion of 14.5 percent observed in the present study. In contrast to previous reports, a significant association of clinical presentation of SGI virus infected patients with severe dehydration, higher mean duration of diarrhoea and severe diarrhea of more than 10 stools per day was found compared to SGII virus.
Recent studies on rotavirus VP7 in India and other countries have shown, G1 type as the most frequently reported virus.,,,,,, On the other hand in our study, we found G2 virus to predominate throughout the study period with 66.1 percent , which is in concordance with another Indian study. Predominance of G4 type virus has been reported in France and Malaysia., Moreover recent study reports have also documented the emergence of newer and common prevalence of unusual strains in developing countries: G6,G8,G9,G10 in India,, G5 and G10 in Brazil and G9 in Bangladesh.
Electropherotyping have been used as a potential tool for studying temporal variation in rotavirus infection in children. In the present study, rotaviruses with short RNA pattern was preponderant throughout the study in contrast to another study from India which has recorded higher incidence of rotavirus with long RNA pattern. The incidence pattern of different E-types revealed sequential appearance of E-types and predominance of a single E-type strain at a time along with co-circulation of less common types (as seen with strains representing lane pattern 3 in 1995, lane pattern 4 in 1996, lane pattern 5 in 1997), which is in concordance with previous molecular epidemiological studies on rotaviruses.,,, However, rotaviruses with super-short RNA pattern and atypical viruses have been recorded in some studies.,,,
The occurrence of strains representing 10 different electropherotypes (E-types) including seven short E-types and three long E-types, and those with mixed VP7 and VP4 genotype specificities (G2 P(4,8), G1-G2 P(4,8) in our study is imperative of the prevailing genomic diversity of rotaviruses and a prerequisite for the emergence of reassortant viruses in the course of natural infection. Strains with mixed genotype specificities and emergence of newer strains with unusual genotypes (G1 P(4), G2 P(8), P(6), G4 P(4), P(8), G10 P(11), G9 P(11), G9 P(6) have been evidenced from recent studies in India and Bangladesh.,,,,
Comparative analysis of VP7 serotype, subgroup and E-type showed a significant association of short E-type with SGI and G2 specificity (P< 0.001). On the contrary, other studies from India and Philippines have documented the prevalence of unusual strains with long E-type with SGI and short E-type with SGII antigenic specificities.,, A high antigenic polymorphism was observed among G2 viruses in the present study with seven different E-types (lane pattern 3, 4, 5, 7, 8, 10, 11) and G1 with 3 different E-types (lane pattern 6, 9, 13).
Although, in the present study correlation of immunological markers and rotavirus infection was not done, prior studies have documented that, the immune response following homologous and heterologous rotavirus infection correlated to quantitative differences in the ability of the two types of infection to elicit local IgA response to rotavirus and depends to a greater extent on the dose of immunizing virus observed with heterologous immunization. Analysis of antibody response to VP6, VP4 and VP7 has been correlated with protection. The recent rhesus rotavirus based quadrivalent vaccine has been found to achieve significant reduction of severe diarrhoea due to rotavirus with protection rates of 80 to 91 percent. On the whole, the earlier study results substantiate the hypothesis that heterologous infection is a less effective strategy to stimulate local immunity than is homologous infection.,,
Overall, the present study results have discerned the prevailing high antigenic and genomic diversity of rotaviruses in this part of southern India. Since co-infection with multiple rotavirus strains can lead to the emergence of newer reassortants, a constant monitoring of antigenic diversity of rotaviruses is warranted from an epidemiological standpoint which would have implications for future vaccine strategies in India.
| ~ Acknowledgement|| |
The authors are grateful to Dr.Graham Beards, Public Health Laboratory Services, Birmingham, UK, and Dr.Shozo Urasawa, Sapporo Medical University, Sapporo, Japan, for providing standard rotavirus strains for the first four serotypes (G1 to G4) and human rotavirus Group-, Subgroup-, and Serotype - specific monoclonal antibodies respectively. We acknowledge the help of Dr. Jon R. Gentsch, Viral Gastroenteritis section, CDC, Atlanta, Georgia, U.S.A. for VP7 and VP4 genomic characterization of the rotavirus strains. The authors thank the Indian Meteorological Department, Meenambakkam, Chennai, for providing the data on various climatic factors of Chennai.
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