Thursday 2 February 2023

Rotaviral Diarrhoea in Children - A Detail Analysis

The present study entitled, “A study of Molecular Epidemiology of Rotavirus Gastroenteritis among children under Five Years attending NEIGRIHMS, Shillong” was conducted in the Department of Microbiology, NEIGRIHMS from January 2017 to December 2018 with the following objectives: -
1. Understanding the incidence of Rotavirus Gastroenteritis among the children below five years with acute diarrhea attending NEIGRIHMS
2. Molecular characterization of Rotaviruses associated with acute gastroenteritis in children under
five years
3. Study of the clinico-epidemiological profile of Rotaviral Gastroenteritis in children under five years
4. Understanding the correlation between clinical and epidemiological conditions with circulating genotypes of rotaviruses
 In this study, a total of 55 cases of diarrhea fulfilling the case definition were included. Stool samples were collected and each sample was immediately aliquoted up to 3 collection tubes with
proper levelling. 1st set was kept at 2-4 degree Celsius for Rotavirus Group A antigen-based ELISA and others were for kept at -80 degree Celsius for studying the molecular epidemiological pattern
by SDS-PAGE analysis and nested RT-PCR. Childhood diarrhea due to rotavirus can cause significant mortality and morbidity among under five children but unfortunately most of the time
it remains undiagnosed. Moreover, enormous genomic diversity of rotavirus strains is seen across different geographical regions which can challenge the efficacy of currently available rotavirus

The summary of the results and observations made in our study are as follows-
•Amongst a total of 55 under five children, the most common age group that presented to the hospital was 7-9 months which included 13 (23.63%) children. •Rotavirus infection was found to be present in 22(40%) out of 55 total cases. •The highest 9(40.9%) positivity was
again seen in the age group of 7-9 months followed by 4(18.18%) children in the age group of 4-6 months.
•Statistically significant gender specific preponderance was not seen in rotavirus infected children.
•Amongst 22 Rotavirus positive cases, majority 15 (68.18%) was from Meghalaya and Assam 4 (18.18%). Urban and rural distribution of rotavirus positive cases was not found to be significant. Therefore, the present study reaffirms the current concept that Rotavirus is a ‘Democratic Virus’ which can infect children irrespective of the socioeconomic
background of their parents and families.
•In our study, Rotavirus gastroenteritis was found to be most common during the winter months with low temperature and relative humidity. 19(86.36%) rotavirus gastroenteritis cases presented during the months of January to March followed by 3(13.63%) cases who presented between October to December.
•Diarrhea (90%), vomiting (95.45%), fever (90.9%), dehydration’ (90.9%), irritability
(100%), lethargy (100%), were seen as constant clinical features in all the rotavirus gastroenteritis children and found to be statistically significant. 1(4.5%) rotavirus infected child had two episodes of convulsions and loss of consciousness. Dyselectrolytemia in the
form of Hypernatremia (Na+ >145 meq/L) was seen in 16 (72.72%) children while
hypokalemia (K+ <3.5 meq/L) was present in 6(27.27%) children out of 22 Rotavirus
positive gastroenteritis cases. Both these findings were statistically significant.
•This study showed that exclusive breastfeeding per se till the age of 6 months does not confer any overall protection against rotavirus infection.
• A total of 55 stool samples were subjected to sandwich ELISA for Rotavirus Antigen.Rotavirus antigen was positive in 22 (40%) samples. All the ELISA positive samples were subjected to SDS-PAGE electrophoresis in which 11(50%) samples were electropherotyped and characteristic 4:2:3:2 pattern of Rotavirus dsRNA was
demonstrated. All these 11 samples which were electrophoresed showed ‘long
electropherotype’ of Rotaviral dsRNA. ‘Short electropherotype’ of rotavirus was not detected in our study based on the relative mobility pattern of the 11th segment of dsRNA.
• There was no statistically significant age, gender and clinical feature specific difference in the distribution of ‘long electropherotypes’ in our study. No concrete comment could be made in our
study regarding the seasonal distribution of ‘long electropherotype’ of rotavirus.
•All 22 Rotavirus ELISA positive stool samples were subjected to nested RT-PCR for G and P genotyping irrespective of the results obtained in SDS-PAGE electrophoresis.
•Amongst a total of 22 Rotavirus ELISA positive samples, first round of nested RT-PCR for G genotyping gave VP7 amplicon(1062bp) in 11(50%) samples. G genotype was successfully assigned to all these 11 samples (50%) in second round amplification of nested RT-PCR using G- genotype specific primers for G1, G2, G3, G4, G9. All these 11 samples (100%) revealed the presence of G1 genotype. Rest of the 11 samples remained negative in both first and second rounds of nested RT- PCR.

  First round of nested RT-PCR on 22 Rotavirus ELISA positive samples for P genotyping showed VP4 amplicon(663bp) in 10(45.45%) samples. Rest of the 12 samples failed to yield VP4 amplicon (663bp) in our study. Second round of nested RT-PCR was done on all 22 samples using P-genotype specific primers for P[4],P[6],P[8],P[9],P[10]. Amongst 10 samples which gave VP4 amplicons in first round amplification, P [8] genotype was detected in 7 samples (70%) and P [4] was detected in 3 samples (30%). Remaining 12 samples negative in first round, also failed to demonstrate any P-genotype specific amplicon in second round of nested RT-PCR.
•Amongst different combination of G and P genotypes, the most common genotype found in our study was G1P [8] (38.46%) followed by G1P [4] (23.07%).
•Three samples (23.07%) with G1 genotype remained untypeable for P genotypes (VP4 gene).Two samples (15.38%) with P [8] genotype were untypeable for G-Genotype.
    Amongst 22 ELISA positive samples, 11 remained untypeable for both VP7 and VP4 genes.
•Definite remarks could not be made regarding the correlation of demographic and clinical features with different G and P genotypes of rotavirus found in our study.
•This study highlighted the different genotypes of rotavirus presently circulating amongst the under five years age children from different north eastern states attending NEIGRIHMS, Shillong.
      In our study we observed that amongst 55 under five years age children suffering from diarrhea ,22 (40%) came positive for Rotavirus Group A Antigen by sandwich ELISA. The most common clinical features in these 22 rotavirus infected cases were diarrhea (90.9%), vomiting(95.45%), ‘some dehydration’ (90.09%), fever (90.9%).
       Molecular analysis of these 22 ELISA positive samples was done by SDS-PAGE
electrophoresis in which 11 samples (50%) were electropherotyped and all of them revealed ‘long electropherotype’ pattern of rotavirus dsRNA. Nested RT-PCR carried out on 22 rotavirus ELISA positive samples successfully assigned G -genotype to 11 samples and P- genotype to 10 samples.
All these 11 samples revealed G1 genotype while amongst 10 samples for P-genotyping P[8] was seen in 7 (70%) samples and P[4] in rest 3 (30%) samples. Analysis of different G and P genotype
combinations was also done in our study. The most common genotype combination found was G1P[8] ( 38.46% ) followed by G1 P[4] (23.07%). Rotavirus with genotypes G1P[8] and G1P[4] were found to be consistently associated with diarrhea (100%), vomiting (100%), ‘some
dehydration’(100%), lethargy (100%) and irritability (100%).
     Our study successfully revealed one type of G -Genotype (G1) and two types of P-Genotype (P[4], P[8] ) with the combination of G1P [8], G1P[4] circulating amongst the under five children with rotaviral gastroenteritis attending NEIGRIHMS Shillong .The outcome of our study will be helpful for better understanding of the detail molecular epidemiology of the circulating genotypes of rotaviruses in this region with inclusion of larger sample size for designing the appropriate strategy in control and prevention of Rotavirus gastroenteritis among under five years age children. Further in-depth study in this regard will also help in identifying the appropriate vaccine candidate for development of safe and effective vaccine against rotavirus associated childhood diarrhea.

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