|Year : 2016 | Volume
| Issue : 1 | Page : 1-5
Rota virus genotypes and the clinical severity of Diarrhoea among children under 5 years of age
Ezeonwu Bertilla Uzoma1, Chinedu Chukwubuikem2, Efe Omoyibo1, Oguonu Tagbo3
1 From the Department of Paediatrics, Federal Medical Centre, Asaba, Delta State, Nigeria
2 Department of Microbiology, University of Nigeria Teaching Hospital, Enugu, Nigeria
3 Department of Paediatrics, University of Nigeria Teaching Hospital, Enugu, Nigeria
|Date of Web Publication||13-Apr-2016|
Ezeonwu Bertilla Uzoma
From the Department of Paediatrics, Federal Medical Centre, Asaba, Delta State
Source of Support: None, Conflict of Interest: None
Background: Diarrhoeal disease still remains one of the common causes of morbidity and mortality in children under 5 years of age. It is caused notably by the different serotypes of rotavirus.
Objectives: To ascertain the prevalence and risk factors for rotavirus diarrhoea in children under 5 years of age seen at Federal Medical Centre, Asaba, and to determine the different serotypes and their relationship with diarrhoea severity.
Subjects and Methods: A hospital-based cross-sectional study in which all children under 5 years of age, with diarrhoea had their stool samples tested for rotavirus antigen with enzyme immunoassay. Significant values of variables were determined using t-tests and Chi-square tests as appropriate.
Results: One hundred and thirty-two children were studied: 52.3% were males and 46.7% were females. Children 1-11 months of age constitute 49.2%, whereas those 12-59 months were 50.8%. Only 49 (37.1%) children were ELISA positive for rotavirus and of this number, 31 (63.3%) were within 1-11 months of age, P = 0.013. The three most common rotavirus genotypes were G3P in 24.5%, G1P in 12.2% and G12P in 10.2% of the samples, respectively. Duration of vomiting was more prolonged with G3P infection, P = 0.029, whereas dehydration and the overall severity of the diarrhoea were more with G12P infection, P = 0.026 and 0.010, respectively.
Conclusion: The emerging G12 rotavirus genotype was isolated in Asaba.
Keywords: Children, diarrhoea, genotype, rotavirus
|How to cite this article:|
Uzoma EB, Chukwubuikem C, Omoyibo E, Tagbo O. Rota virus genotypes and the clinical severity of Diarrhoea among children under 5 years of age. Niger Postgrad Med J 2016;23:1-5
|How to cite this URL:|
Uzoma EB, Chukwubuikem C, Omoyibo E, Tagbo O. Rota virus genotypes and the clinical severity of Diarrhoea among children under 5 years of age. Niger Postgrad Med J [serial online] 2016 [cited 2019 Jan 24];23:1-5. Available from: http://www.npmj.org/text.asp?2016/23/1/1/180108
| Introduction|| |
Rotavirus is among the most common cause of diarrhoea among children worldwide.  In Nigeria, rotavirus was reported in 10-56% of cases of acute diarrhoeal diseases. ,,, Different genotypes of rotavirus are seen in different parts of the world and it can differ from season to season and within the same geographical region.  The G-genotypes G1-G4 were reported globally as the most common with G1 being the predominant type followed by G2. , In Africa, all the serotypes have been implicated in acute diarrhoea diseases in children with G1 genotype also predominating. In Nigeria, despite the emergence of G12 variant of the rotavirus genotypes among children with diarrhoea, the G1 and G2 still maintained their predominance. ,
It appears that the distribution of the genotypes varies with time and change with the introduction of vaccines.  As a requisite for the introduction of such vaccines, it is pertinent that the spectrum of the prevailing species of the virus in any environment is determined. Therefore, an update on the behaviour and predominant genotype in a given population is needed for the development of effective vaccine. 
This study was, therefore, performed to determine the predominant genotype at our tertiary institution and to also relate such to the clinical characteristics of diarrhoea.
| Subjects and Methods|| |
This study was carried out at Federal Medical Centre, Asaba, which lies in the tropical rain forest region of Nigeria, with two major climatic seasons; dry season, which spans from November to March, and rainy season from April to October of each year. Children under 5 years of age who have diarrhoea constitute 25.0% of admission in Children Emergency Room annually (with infant: preschool-aged ratio of 1:1). Ethical approval was sought and obtained from the Health Research and Ethics on 24 th December 2012, before commencing the study.
This was a cross-sectional, descriptive study in which all consenting caregivers of children under 5 years of age, with diarrhoea had their wards enrolled using a consecutive sampling method from December 2012 to November 2013 (12-month period). Basic demographic characteristics such as age, sex, child's institution, source of drinking water, method of waste disposal and feeding practice were obtained.
The children were stratified into two groups: 0-11 months and 12-59 months. The severity of rotavirus diarrhoeal disease was categorised using the Vesikari clinical severity scoring system manual which scores 7 parameters.  Five of the parameters are scored from 1 to 3 points: Maximum number of stools per day, duration of diarrhoea in days, maximum number of vomiting episodes per day, duration of vomiting in days and temperature. Other two parameters scored are dehydration (scored 2 and 3 points) and treatment (scored 1 and 2 points). The severity score is between 0 and 20 points where the maximum score is 20 points: Mild category is <7 points, moderate is 7-10 points while severe is >11 points. 
Stool samples were collected and three drops of 1-3% glycerol were added to each sample and subsequently stored frozen at −20°C until transferred (at 2 monthly intervals) to the virology laboratory at University of Nigeria Teaching Hospital, Enugu. The samples were transported in a cold chain box to maintain the requisite ambient temperature, these were subsequently analysed using ProSpecT® Rotavirus Kit, Oxoid Ltd., (United Kingdom, batch/LOT 1024895) following standard operating procedures. Part of the sample (1-1.5 ml) was stored at −80°C in a freezer and later genotyped using semi-nested polymerase chain reaction (PCR) methods.
Viral RNA was extracted from 10% rotavirus-positive faecal suspensions, using QIAamp Viral RNA Mini Extraction Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The eluted RNA extracts were then used as templates for Viral RNA extraction and reverse transcriptase-PCR whereby copies of DNA (cDNA) are produced. Genotyping of the cDNA was performed by nested multiplex PCR using type-specific VP7 and VP4 primers. ,, PCR products were resolved by electrophoresis on a 2% agarose gel, stained with ethidium bromide and visualised under ultraviolet light.
All data were analysed using SPSS statistical software for Windows® version 20, 2012 (IBM Inc., Chicago Illinois, USA) and EpiInfo (version 3.5.4, CDC, Atlanta, USA) software. Variables such as sex and age were expressed in frequency tables. Significant values of variables such as continuous and categorical were determined using t-tests and Chi-square tests (Fisher's exact if indicated) as appropriate. Logistic regression analyses were used to determine risk associations of the different possible determinants such as age, gender, feeding practices, waste disposal and source of drinking water to rotavirus diarrhoea. Significant values were set at P < 0.05, with confidence intervals (CIs) of 95% where applicable.
| Results|| |
There were 244 cases of acute gastroenteritis admitted in children emergency room during the 12 months study period. Of this number, 149 children met the inclusion criteria (children under 5 years of age who had diarrhoea and whose mother consented) and 132 had their data complete and were analysed. Male children constituted 52.3% (69/132) of those analysed with a male to female ratio of 1.1:1. Among the age groups, 49.2% (65/132) were 1-11 months and 50.8% (67/132) 12-59 months old. The respective means (standard deviation) for the age (months), duration of diarrhoea before admission (days), duration of admission (days) and total duration of the illness (days) for the study population were 13.7 (8.6), 3.9 (5.9), 3.3 (3.5) and 7.2 (7.1). The mean Vesikari severity score for diarrhoea episodes was 13.7 (2.5) for the entire population. More cases of diarrhoea 71.2% (94/132) were seen in the dry season and hence more stool samples were collected during this period. Children whose stool samples were ELISA positive were 49 (37.1%), whereas 83 (62.9%) were negative.
Rotavirus antigen was detected in the stool samples of 37.1% (49/132) of children enzyme immunoassay (EIA positive): Out of which 63.3% (31/49) were 1-11 months of age and 36.7% (18/49) were between 12 and 59 months, P = 0.013. In the rotavirus group, there were 26 females and 23 males: Ratio 1:1.1, [Table 1]. [Table 2] shows the mean of the different characteristics of all the children (rotavirus positive and negative) with diarrhoea. Those with rotavirus diarrhoea presented to the hospital early in the course of the diarrhoeal disease than those who are EIA negative, P = 0.024. A greater percentage of ELISA positive stool samples 98.0% (48/49) were collected within 7 days of the onset of diarrhoea and isolation of rotavirus is 6 times more likely to be isolated from the stool if diarrhoea has lasted for 7 days or less (odds ratio [OR]: 6.57, 95% CI: 0.81-53.04, P = 0.044). The risk for rotavirus infection was 2 times more common among infants (OR: 2.48, 95% CI: 1.20-5.14, P = 0.013) and 2 times more common in the dry season (OR: 1.99, 95% CI: 0.86-4.56, P = 0.102). Multiple regression analysis shows that with the exception of younger age no other factors such as source of water, type of complementary feeding, method of feeding, history of exclusive breastfeeding or waste disposal poses a risk for rotavirus diarrhoea (P = 0.035). Children with rotavirus diarrhoea presented to the hospital earlier, [Table 2]. Presence of rotavirus did not affect the degree of dehydration of patient (P = 0.284), the severity of the diarrhoeal illness (P = 0.711) nor the patient outcome, P = 0.504.
|Table 1: Sociodemographic characteristics of the children with diarrhoea|
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|Table 2: Comparison of clinical features between the enzyme immunoassay positive and negative groups|
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Forty-four EIA positive stool samples were sent for determination of rotavirus genotype. The following genotypes were encountered: Four G genotypes (G3, G12, G1 and G9) and three P-genotypes (P, P and P).One G- and one P-genotype each were untypeable, [Table 3]. There were 17 different rotavirus strains with various G-P genotype combinations isolated. Some of the rotavirus diarrhoeal diseases (24.5%; 12/49) were due to mixed infections with G- and/or P-genotypes, [Table 3]. For G mix strains, G12, 1 accounted for 66.7% (2/3) and for the P mix, Paccounted for 66.7% (6/9), [Table 3]. The three most common rotavirus genotypes were G3P in 24.5% (12/49) of the samples, G1P in 12.2% (6/49) and G12P in 10.2% (5/49).
These common genotypes had no predilection for age or gender and the G3P was the most common strain found during the dry season, whereas during the wet season, the most common strain was the G1P, P ≤ 0.001.The G3P strain was the predominant type of rotavirus among the breastfed infants. Duration of vomiting was more prolonged with G3P infection, P = 0.029 while the dehydration and the overall severity of the rotavirus diarrhoea were more with G12P infection, P = 0.026 and 0.010, respectively.
| Discussion|| |
The high prevalence of rotavirus diarrhoea infection noted is similar to reports from some parts of Nigeria including Enugu (56%)  and Jos (33%).  It is in contrast with findings in Ile-Ife (15%)  and Zaria (9%).  The differences in prevalence may be epidemiological or methodological because studies with high prevalence , were hospital-based, whereas the low prevalence were community-based. , Relatively more cases of rotavirus diarrhoea were found among infants compared to older children possibly due to the protection conferred by previous infections on the older age group. 
Rotavirus is disseminated by faecal-oral route most commonly through close person-to-person contact and by infected fomites and less commonly through water and food. , The spores can remain for weeks and months in fomites unless disinfected, , this ensures that rotavirus diarrhoeal diseases are prevalent all the year round, although peak periods are observed as noted in this study and others. ,,, Ensuring good water quality and sanitary condition does not have maximal effect in the control of rotavirus diarrhoea; hence, the emphasis on immunisation.  The methods of care such as institutional or non-institutional, bottle-feeding and type of complementary feeding were not predictors of rotavirus diarrhoea in these children. However, Kazemi et al.  in Iran observed that being bottle fed and cared for in a childcare centre contributed to the rotavirus diarrhoea. Although contaminated food (which could be from the bottle) aids transmission of rotavirus, this disparity could also stem from the strain of rotavirus involved.
There was only one case of mortality due to rotavirus diarrhoea was recorded and the mean duration of diarrhoea before the presentation was 3 days. The high rate of survival from the disease could be attributed to the early presentation and prompt attention given to the children by the health care providers in the study centre.
The G3, G1, P and P were among the genotypes encountered just like in many other studies globally. The P-genotype prevalence did not vary from the P, P and P isolated from other studies, , but for the G-genotypes, the G12 and G9 isolated were emerging genotypes. As at 1998, the G-genotypes G1 to G4 were reported globally as the most common with G1 being the predominant type followed by G2. , Rahman et al. in Belgium reported G9 as an emerging genotype in 1997 and by 2000-2003, it predominated.  In a review done by Li et al. in China, G3 was the prevalent genotype since year 2000.  In later years (2014), G12 was isolated in some African countries, notably Tanzania and Nigeria. Despite the emergence of these new variants of the rotavirus genotypes among children with diarrhoea, the G1 and G2 still maintained their predominance. ,,
There is no particular pattern of the prevalence of rotavirus strains as there appears to be a wide dispersion of the prevalent strain over geographical areas and time. For instance, previously in Africa, G3 was the prevalent genotype with G3P virus strain in the early 2000  but in 2010, the prevalent strains were G1P, G2P and G8P.  This study isolated G3P, GIP and G12P which were entirely different from the predominant strain documented worldwide: (G1P, G2P, G3P and G4P). , In Ivory Coast, the most common strains were G1P and G8P;  in Tanzania, G1P, G8P and G12P;  and recently in South Africa, G1P and emerging G12P.  Other parts of the world reported various G-P combinations: In China, G3P, G1P and G2P.  Multiple infections by different strains of the virus were documented in Africa in 2010,  mixed G1/G8 was reported in Ghana in 2012 and index study found mixed G and P infections involving G1/G12 and P/P/P. One of the subjects had both of the G and P mixed genotypes.
The variation in genotype variance underscores the need for constant surveillance to determine new variants and strains for possible incorporation into the vaccines. The reported emergence of G12 in Nigeria  which has also been observed from this study becomes important in rotavirus vaccine production. It is pertinent to note that the three common strains isolated in this study have some peculiarities. Despite the high incidence of rotavirus infection during the dry season, infection with G1P seems to be more in the wet season. G3P was associated with prolonged vomiting although this did not affect the severity of the infection. Furthermore, G12P caused significant dehydration as well as severe degree of infection. This study has brought to the fore the need to urgently tackle this emerging G12 genotype since the G1/G3/P/P genotypes have been in circulation worldwide.
| Conclusion|| |
Rotavirus diarrhoea is a common disease among infants presenting at our tertiary institution in Asaba. The emerging new variants in Nigeria that differ from known types that cause diarrhoeal disease were also isolated in this study. These genotypes show more severe form of disease than the previous types.
We are grateful to all the children who participated, with their parents/caregivers.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]
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