|Year : 2020 | Volume
| Issue : 4 | Page : 151-156
Changing serotypes of rotavirus in Western India and clinical severity of diarrhoea
Kshitija Rane1, Pankhuri Kothari2, Mansi Thakur1, Dattatraya Joshi1, Nitin Kadam2, Nimain Mohanty2
1 Department of Medical Genetics & Biotechnology, MGMIHS, Kamothe, Navi Mumbai, Maharashtra, India
2 Department of Pediatrics, MGM Medical College and Hospital, Navi Mumbai, Maharashtra, India
|Date of Submission||01-Nov-2021|
|Date of Decision||29-May-2021|
|Date of Acceptance||06-Jun-2021|
|Date of Web Publication||22-Dec-2021|
Department of Pediatrics, MGM Medical College, Navi Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Introduction: Rotavirus and Norovirus diarrhea pose highest threat to neonates and toddlers all over the world. Diarrhoea happens to be the second most common cause of under-5 mortality in India after Pneumonia. India has the highest incidence of rotavirus diarrhoea, contributing to about 1,50,000 inder-5 deaths annually. Virus serotypes do undergo constant changes, varying in different regions, thought of responding to vaccines differently. Objecive: A prospective observational study was undertaken in Kamothe, Navimumbai (India); enlisting 207 cases of acute watery diarrhoea among under-5 children to study incidence of Rotavirus and Norovirus as causal agents and their serotypes. Efforts were made to understand if virus positivity and their types were related to clinical severity of diarrhoea. Method: Stool samples were collected and tested for rotavirus and norovirus from cases of acte diarrhoea on clinical presentation and after excluding cases of dysentery on clinical presentation. 10.62% cases were found positive for rotavirus RNA by ELISA and 4.83% by Electropherotyping. Cases of acute watery diarrhoea having rotavirus positive and negative stool showed remarkable difference with diarrhoea severity assessed by Vesikary scores. Severe dehydration, stool frequency, vomiting and weight loss were associated with rotavirus infection in younger age group. Long electropherotypes were more prevalent (86.36%) and were associated with higher clinical severity of diarrhoea. P was predominant (77.27%) followed by P (13.63%) genotypes. Neither P nor P was detected. Those found positive for rotavirus in their stool samples, were negative in their serum by eletropherotyping. Only 0.48% of diarrhoea stool samples were found positive for Norovirus by ELISA. Conclusion: Rotavirus diarrhoes shown a decreasing trend in Navimumbai area as compared to previous years. Incidence of Norovirus infection was found very low. Rotavirus positive cases, particularly of long arm electropherotypes, showed clinical severity of diarrhoea as compared to short-arm types and in those found negative for rotavirus or Norovirus.
Keywords: Acute watery diarrhoea, Rotavirus, Norovirus, Vesikary score
|How to cite this article:|
Rane K, Kothari P, Thakur M, Joshi D, Kadam N, Mohanty N. Changing serotypes of rotavirus in Western India and clinical severity of diarrhoea. J Pediatr Assoc India 2020;9:151-6
|How to cite this URL:|
Rane K, Kothari P, Thakur M, Joshi D, Kadam N, Mohanty N. Changing serotypes of rotavirus in Western India and clinical severity of diarrhoea. J Pediatr Assoc India [serial online] 2020 [cited 2023 Jun 3];9:151-6. Available from: http://www.jpai.in//text.asp?2020/9/4/151/333375
| Introduction|| |
Diarrhea has been described as the second leading cause of mortality worldwide among children under 5 years of age. Every year diarrhea kills over 760,000 children worldwide. Studies from various countries suggest that after Rotavirus, Norovirus and Astrovirus, followed by Adenovirus, have been implicated in childhood acute viral diarrhea. National burden of Rotavirus for India has been given by John et al. which postulates that out of 686,277 outpatient visits, 291,756 were hospitalized and 26,985 were died. The ratio of severe diarrhea due to Rotavirus increased from an average of 25% (ISV: 21%–28%) prior to 2000–38% (ISV: 19%–50%) after 2005.
The prevalence of serotypes and genotypes in the region will help to understand whether the lower efficacy of vaccine. About 50% of seroconversion occurred in developing countries such as Africa and Asia, as compared to over 85% in Europe and North America 10 years back could be due to the possible genetic drifts of viruses or the factors associated with variation in host gene polymorphisms in the Asian population, or both. The debate over the introduction of rotavirus vaccines' Universal Immunization Program at a colossal cost in India continues. It is desirable to monitor rotavirus diarrhea, its prevalence pattern, new emerging strains in any, and their genetic and regional variance in India.
| Materials and Methods|| |
Two hundred and seven children from 6 months to 5 years of age having acute viral diarrhea on the basis of clinical presentation were included in this study during 2017–2019.
(1) Acute diarrhea as defined by the WHO, (2) age: under 5 years (U5C), (3) parents willing to participate in the study after confirming their informed consent, and (4) not received rotavirus vaccine.
(1) Cases of dysentery evidenced by visible blood in stool, (2) high-grade fever and/or leukocytosis, (2) stools frequency >15/day, (3) cases of severe shock, and (4) altered sensorium.
A structured pro forma was filled in while interviewing/counseling parent or guardian of the patient at reporting to the hospital. It included demographic parameters such as age, sex, season, and details of clinical symptoms as per the Vesikari scoring system. In 1990, Ruska and Vesikari described a numerical scale to assess the severity of gastroenteritis on the basis of duration and frequency of diarrhea, vomiting, fever, extent of dehydration, and type of treatment required. These have been used to grade the severity of rotaviral diarrhea in epidemiological studies in the form of Vesikari scores from 0 to 20. Scores of all the seven parameters were added and the final value predicts severity of the disease. Less than 7 score shows mild disease, score between 7 and 10 shows moderate disease, while Vesikari scoring above 11 shows severe disease.
Stool samples were collected in a sterile screw-cap container. Clinical follow-up continued until symptoms subside. Wet mount analysis of the 5% stool suspension was performed to check the shedding of blood cells, parasitic infection, etc., Detection of rotavirus VP6 antigen in diarrheal stool samples was carried out using 'IVD Research Inc, USA' rotavirus antigen detection assay micro well enzyme immunoassay. For detection of Norovirus, “Ridascreen Norolisa” kit was used. Rotavirus double-stranded RNA was extracted from stool of infected clinical samples using Trizol-LS reagent (Life Technologies, Rockville, Md.). All fecal specimens were analyzed by polyacrylamide gel electrophoresis (PAGE) to identify the potential presence of rotavirus double-stranded ribonucleic acid (dsRNA). Electropherotyping permits monitoring of rotavirus infection as well as identification of electropherotypic strain involved. Three different types of strains specific to human hosts have been considered for this study, namely., long (4-2-3-2), short (3-2-2-2), and mixed (distinguished). Lowermost two bands specifically indicate the difference between long and short patterns.
Eight P serotypes of human rotaviruses have been characterized. Additional VP4 gene variants have been identified, so ultimately, the number of P serotypes may exceed 20. Theoretically, eighty different strains of rotavirus could result from various combinations of the known 10G and 8P serotypes of human rotaviruses. For vaccine development purposes, it is fortunate that only four common strains – PG1; PG3; PG4; and PG2 types predominate globally. Such prevalence varies from one geographic area to another. Few unusual strains are common in developing countries, for example., P (6) RV strains to extent of 9.5% of all rotaviruses from a multicenter collection in India. The frequency of P and P genotypes was higher compared to P genotypes for population studied in this region. P RV strain was a neonatal strain with less virulence detected incidentally at AIIMS, New Delhi, which forms the basis of our indigenous 116E rotavirus vaccine. Samples positive for Rotavirus either by electropherotyping or ELISA were processed for genotyping of the VP4 gene using semi-nested polymerase chain reaction (PCR). Primers flanking the VP8 gene were used in the first step of amplification. The second step was performed using genotype-specific reverse primers and same forward primer. Products of the final step were electrophoresed on 7% DNA PAGE gel and stained using silver nitrate (Sigma) and sodium carbonate (Himedia).
The clinical data were analyzed using SPSS version 17 software. Independent sample t-test was used for comparison. Significance was taken at P < 0.05.
| Results|| |
Frequency of stool passage and vomiting
The mean stool frequency and duration of rotavirus diarrhea were higher compared to nonrotavirus diarrhea cases. While there was no much difference in frequency, vomiting was significantly higher in 14 of 22 (63.6%) cases of rotaviral diarrhea as compared from 43 of 185 (23.2%) nonrotaviral diarrhea (P < 0.001).
Degree of dehydration
Dehydration is divided into categories as per the WHO criteria: Very mild or no dehydration, some or mild-to-moderate dehydration, and severe dehydration. Among several symptoms and signs, abnormal skin turgor, dryness in mucosal surfaces, and prolonged capillary refill time are the ones most reliable. Among 207 children of acute viral diarrhea diagnosed on clinical ground, 82.10% had no dehydration. About 14.40% had some dehydration and 3.10% had severe dehydration. However in rotavirus-positive cases, 45.4% had no dehydration, 36.4% had some dehydration, and 18.2% had severe dehydration showing higher disease severity [Figure 1]. Among nonrotaviral diarrhea cases, 87.8% had no dehydration, 10.8% cases had mild dehydration, and only 1.6% had severe dehydration. The difference is statistically very significant (P = 0.002). Shariff et al. and Nafi reported 76% and 72.3% dehydration, respectively, of diarrhea cases at admission. About 7.4% had severe dehydration in Sheriff 's study.
|Figure 1: Degree of dehydration compared. Between rotavirus stool positive and negative cases|
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Dehydration results in weight loss. Dehydration and weight loss were observed more in children with Rotavirus proven. [Figure 2] clearly shows this difference in average weight loss higher in rotavirus diarrhea (5.87%) than among children having nonrotavirus diarrhea (3.87%) [Table 1]. The difference is statistically significant, particularly in a country like India, where the prevalence of undernutrition is to the tune of 48% (NFHS-4) and an acute diarrhea could push the child to the higher grade of malnutrition [Table 1].
|Figure 2: Electropherotyping of rotavirus from stool samples. Lane 1 and lane 5 showing long arm type, lane 2 and 3 short arm types, Lane 4 mixed-type|
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|Table 1: Primers used for molecular detection of rotavirus P genotype using Semi nested PCR|
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Vesikari score was used to assess the disease severity on a 0–20 scale. It ranged from 4 to 17 with a mean of 8.6. On the whole, Of 207 cases, 66 (31.9%) had mild, 90 (43.5%) had moderately severe disease, and 51 (24.6%) had severe disease. The mean Vesikari score was 12.5 (±4.03) for rotaviral diarrhea and 8.12 (±2.65) for nonrotaviral diarrhea. The difference was highly significant.
Detection of rotavirus RNA by electropherotyping and enzyme immunoassay
Out of 207 stool samples screened by ELISA for rotavirus-A antigen, Ten came positive (4.83%). All 207 stool samples were also screened for rotavirus by electropherotyping [Figure 3]. Twenty-three samples were found positive by this method (11.11% positivity) as compared to 10 by ELISA.
|Figure 3: Weight loss in diarrhoea cases. Rotavirus positive cases showed higher weight loss on an average where as rotavirus negative cases (NRV) showed lower weight loss|
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Frequency of norovirus infection
All 207 stool samples were screened for the presence of norovirus antigen by ELISA. Only three samples were found positive for norovirus (0.64%). No other molecular studies on Norovirus were available locally. Thus, the incidence of norovirus infection in this hospital-based study over period of 3 years in Navi Mumbai area was found to be negligibly low.
Serum positivity for rotavirus RNA in stool positive cases
None of the serum samples were found positive for Rotavirus by electropherotyping whether with fever or otherwise. Stool samples found positive from stool RNA analysis from children were negative for the presence of nucleic acid in serum concluding that stool examination method is the best to screen for rotavirus antigen.
Long-arm electropherotype strains of Rotavirus were associated with severe rotavirus disease.
Out of 22 samples positive by PAGE electropherotyping in the present study, 19 (86.36%) were of the long arm and 1 (4.54%) was of short arm type. Two out of 22 (9.09%) showed mix type of pattern of electropherotype [Table 2], [Table 3], [Table 4]. This is in contrast with the data reported from Vellore and Hyderabad. Those studies have shown only long-arm patterns and none of the short arm or mixed type. We observed higher disease severity with long electropherotypes, similar to our own observation in past from western Maharashtra. In the present study, 82.3% of cases of long type had severe disease with moderate-to-severe dehydration as against 16.6% of short arm type having severe disease [Figure 4]. This difference was highly statistically significant (P = 0.009). No such association has been reported from elsewhere.
|Table 3: yearly average incidence of Rotavirus and Norovirus in various countries as compared to present study.|
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|Table 4: Frequency of electropherotypic strains found in present study, as compared with other studies|
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|Figure 4: Disease severity according to electropherotypic types: Long and short arm. Long type shows more severe symptoms than short types. (P = 0.009)|
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P-genotyping by polymerase chain reaction
RNA isolated from stool suspensions using viral RNA isolation kit (Qiagen Viral RNA mini kit, Qaigen, Germany) was amplified against target ORF (Open Reading Frame) of gene VP8. PCR products were electrophoresed on 1.5% agarose gel and were subsequently stained with EtBr. Very specific and bright bands above 800 bp [Figure 5] were obtained. Twenty samples were analyzed to monitor the prevalence of P-genotype in this region. The second step of semi-nested PCR was used to amplify the region of the VP8 gene, specific for each genotype. These amplified PCR products were electrophoresed on TBE polyacrylamide gel, followed by silver staining protocol [Figure 6]. The data showed higher prevalence of P genotype (17/22, 77.27%) followed by P genotype (3/22, 13.63%) [Table 2]. Two samples (2/22, 9.09%) were of unknown genotype, i.e., other than of P, P, P, or P genotype.
|Figure 5: VP4 amplified product. Lane 1 is ladder (100bp) Lane 1 is NTC and Lane 3 to lane 6 are VP8 gene amplified PCR product band at 887bp|
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|Figure 6: DNA PAGE:P and P genotypes. Lane 2 showing 484bp band specific for P genotype and lane 4 showing 346bp band specific for Pgenotype. Lane 3-50bp ladder|
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| Discussion|| |
Almost two decades ago, studies demonstrated that four globally common rotavirus serotypes (G1–G4) accounted for >90% of the rotavirus strains in circulation. Subsequently, these two serotypes were used in the development of reassortant vaccines for serotype-specific immunity, However, much greater strain diversity has been identified since then. We now recognize strains with at least 42 P-G combinations., During active vaccine postlicensure surveillance in the United States, a novel rotavirus genotype G14P (24) was detected in a stool sample from a child who had diarrhea. Emergence of G12 and G9 strain in India may not be effectively tackled by certain vaccines as was the case in South Africa or Malawi. Although crossprotection by viral antigens is claimed, it is desirable to continuously monitor viral strains, besides investigating other factors such as hygiene hypothesis, gut microbiota, innate immunity, blood group histocompatibility antigen, the prevalence of FUT2 gene in the ethnic community, and several other factors thought to be playing their roles region wise determining individual susceptibility. Ethnic population having FUT2 gene expression in enterocytes of secretor types contract rotavirus infection easily, whereas the nonsecretor types do not. Breastmilk of FUT2 nonsecretor mothers has been found protective against rotavirus diarrhea in the baby.
ELISA versus electropherotyping
ELISA is an efficient screening test for rotavirus antigen of A-type. However, it can miss B-type or mixed type rotavirus strains. Sensitivity and specificity calculation using medcalc are of better efficiency with electropherotyping for rotavirus screening or diagnosis. It is a nucleic acid-based test that was more sensitive for the detection of Rotavirus from stool samples. Earlier recommendations suggest that the sensitivity of both techniques is equivalent. There is a possibility that reassortment might have generated novel strains not yet serotyped as yet and hence remain undetectable by ELISA. It seems any kind of rotavirus strain can be detected by electophoresing dsRNA on polyacrylamide gel. This could be prime reason for higher sensitivity of electropherotyping over ELISA. Those found positive in any of the two tests were further analyzed for P-genotyping [Figure 5] and [Figure 6] of virus strain involved.
Clinical profile of rotavirus diarrhea
Duration of illness was significantly more in rotaviral diarrhea. Vomiting was a major symptom and associated more with rotaviral diarrhea. Stool frequency and degree of dehydration were found to be critical to differentiate between cases of rotavirus diarrhea and non-rotavirus diarrhea. Vesikari score was higher in rotaviral diarrhea, indicating more severe disease. Long electropherotypes were associated with severe diarrhea (P = 0.009). Symptoms such as coryza, cough, fever, bodyache, and rash. remained uncommon.
Electro-pherotypes of rotavirus and their relevance
Although rotavirus infection is generally considered to be confined to the gut, cases of the extra intestinal disease have been reported recently. In such cases, antigen levels in serum showed inverse association while antigen levels in stool were directly associated with progression of infection. More interestingly, strains found in serum were genetically different from the rotavirus strains found in stool samples. Underlying molecular mechanism behind this strain discordance so far remains unexplained. Liu et al., 2009 showed the presence of Group A rotavirus in cerebrospinal fluid, sera, and stool of six children with convulsions and gastroenteritis. The techniques used in this study were ELISA, reverse transcription PCR, and real-time PCR. In the present study, no viral antigen was found in the serum of any positive case by the electropherotyping technique. Human rotaviruses are generally found to be of short and long electropherotypes. There can be a mixed electropherotypic strains by reassortment of strains. There is no specific association of long and short electropherotypes with binomial typing of the virus. Higher 86.36% incidence of long electropherotype variant, 4.54% short electropherotypes, and 9.09% mixed types in Navi Mumbai area is contracted with other studies, as shown in [Table 2].
P-genotyping indicated that P was predominant, followed by P genotype in Navi Mumbai region. Studies undertaken in various parts of India showed similar trend. No case of P was reported in this region, unlike the African population. There was no specific association of long and short electropherotypes with P-genotype of the virus strain.
| Conclusion|| |
Rotavirus diarrhea is a major cause of morbidity and mortality among under-5 children worldwide. The viral serotypes vary from region to region, having relevance to clinical severity and extent of protection by vaccines. Higher incidence (10.62%) of rotavirus infection in children was found in this region as compared to norovirus infection (0.48%) being increasingly reported from the west. Long electropherotypes (86.36%) and P genotype (77.27%) are more prevalent in the region than other type of strains of Rotavirus. P strain was not found in our study which could be the result of the successful implementation of indigenous 116E strain vaccine. Disease severity of rotavirus diarrhea was found to be significantly high as compared to nonrotavirus acute viral diarrhea, objectively assessed by estimating Vesikari scores. Certain remarkable pointers were identified peculiar to cases where stool samples were positive for Rotavirus as compared to those found negative. Significant among those were the age group of 7–12 months, high stool frequency, severe dehydration, vomiting, and weight loss. ELISA screening test may miss few uncommon rotavirus strains of non-A or mixed antigen types.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]