|Year : 2019 | Volume
| Issue : 3 | Page : 187-192
Molecular characterization of rotavirus genotype-A in children with acute diarrhea attending a tertiary hospital in Ilorin, Nigeria
Dele Ohinoyi Amadu1, Idris Nasir Abdullahi2, Anthony Uchenna Emeribe3, Peter Omale Musa4, Lawal Olayemi5, Thairu Yunusa6, Chisom Emmanuel Okechukwu7, Matthew Oluwafemi Salami8
1 Department of Medical Microbiology and Parasitology, University of Ilorin Teaching Hospital, Ilorin, Nigeria
2 Department of Medical Laboratory Science, College of Medical Sciences, Ahmadu Bello University, Zaria, Kaduna, Nigeria
3 Department of Medical Laboratory Science, University of Calabar, Calabar, Nigeria
4 Department of Medicine, Ahmadu Bello University, Zaria, Kaduna, Nigeria
5 Department of Medicine, Faculty of Health Sciences, National University of Samoa, Samoa
6 Department of Medical Microbiology and Parasitology, University of Abuja, Abuja, Nigeria
7 Department of Medical Microbiology, School of Medical Laboratory Science, Usmanu Danfodiyo University, Sokoto, Nigeria
8 Department of Medical Microbiology, Federal School of Medical Laboratory Technology, Jos, Nigeria
|Date of Submission||21-Nov-2018|
|Date of Acceptance||06-May-2019|
|Date of Web Publication||05-Aug-2019|
Dr. Idris Nasir Abdullahi
Department of Medical Laboratory Science, College of Medical Sciences, Ahmadu Bello University, PMB 06 Shika, Zaria
Source of Support: None, Conflict of Interest: None
BACKGROUND: Despite indications that severe rotavirus diarrhea in children under <5 years of age is a major public health problem, only limited specific data on rotavirus burden are available in Sub-Saharan Africa. This study aimed to carry out molecular characterization of circulating rotavirus strains causing acute diarrhea among under-five children attending University of Ilorin Teaching Hospital (UITH), Ilorin, Nigeria.
MATERIALS AND METHODS: Ninety-three under-five children who are hospitalized with severe diarrhea were enrolled and their stool samples were collected for the detection and subsequent characterization for G and P rotavirus serotypes.
RESULTS: Of a total of 93 samples comprising 54 (58.1%) males and 39 (41.9%) females, 25 (26.9%) samples were positive for rotavirus by ELISA. Genotyping by RT-PCR was done on 25 samples. The most prevalent type-able VP7 G types were G9 (28%), G1 (24%), followed by G12 (20%), G2 (12%), and G10 (4%); on the other hand, the most prevalent type-able VP4 P types are P8 (48%), P4 (24%), and P6 (16%). The most common G-P combination was G9P (4) (20%) followed by G12P (8) and G1P (4) (16%, respectively); then GNTP (8) and G1P (6) (8%, respectively); and G9P (8), G12P (4), G10P (6), G2P (8), and G2P (6) (4%, respectively). The rotaviruses isolated from these children were from those <24 months of age (100%). There is statistical relationship between the prevalence of rotavirus infection with age (P = 0.033) but not with gender (P = 0.765).
CONCLUSION: This study highlights the rotavirus disease burden and diversity of rotavirus strains circulating in UITH. Continued sentinel surveillance will provide useful information to policy-makers with regard to rotavirus vaccine introduction.
Keywords: Diarrhea, molecular characterization, pediatric infection, rotavirus
|How to cite this article:|
Amadu DO, Abdullahi IN, Emeribe AU, Musa PO, Olayemi L, Yunusa T, Okechukwu CE, Salami MO. Molecular characterization of rotavirus genotype-A in children with acute diarrhea attending a tertiary hospital in Ilorin, Nigeria. Int J Health Allied Sci 2019;8:187-92
|How to cite this URL:|
Amadu DO, Abdullahi IN, Emeribe AU, Musa PO, Olayemi L, Yunusa T, Okechukwu CE, Salami MO. Molecular characterization of rotavirus genotype-A in children with acute diarrhea attending a tertiary hospital in Ilorin, Nigeria. Int J Health Allied Sci [serial online] 2019 [cited 2019 Aug 20];8:187-92. Available from: http://www.ijhas.in/text.asp?2019/8/3/187/263954
| Introduction|| |
Rotavirus is classified as a genus within the Reoviridae family and contains a genome of 11 segments of double-stranded RNA that encode six structural (VP1–VP4, VP6, and VP7) and six nonstructural (NSP1–NSP6) proteins. The two outer capsid proteins, VP4 and VP7, define serotype P (protease-sensitive proteins VP4), encoded by gene segment 4 and G (glycoprotein VP7) encoded by the gene segment 7, 8, or 9, depending on the strain. Both proteins elicit neutralizing antibody responses and form the basis for classifying Group-A rotavirus intoPand G serotypes and genotypes.
Group-A rotaviruses are the most important etiological agents of severe diarrhea in infants and young children. Globally, rotavirus-associated mortality has significantly declined from 528,000–215,000 death in 2000 and 2013, respectively, of which 75% occur in Africa and Asia. Nigeria accounted for 14%. It has been estimated that one of six children born in Sub-Saharan African will die before 5 years of age and one-fifth of these deaths will be from diarrhea.
In Sub-Saharan Africa, gastroenteritis remains a major cause of childhood morbidity and mortality and a leading cause of childhood illness as a result of poor economy, infrastructure, and political instability. Improvements in sanitation and availability of clean water have not decreased the rate of rotavirus diarrhea in developed countries, illustrating the need to develop vaccines as the first strategy of prevention. Knowledge of rotavirus strain distribution has become of increased importance because new rotavirus vaccines have just been licensed, and it will be critical to assess how effective these are against the diversity of strains in circulation. A safe and effective rotavirus vaccine is required to reduce this high disease burden.,
Two effective rotavirus vaccines, a single-strain attenuated human rotavirus vaccine (Rotarix, GlaxoSmithKline Biological) and a multi-strain bovine-human reassortant vaccine (RotaTeq, Merck and Company), are now available. The World Health Organization (WHO) recommends the inclusion of rotavirus vaccines in all national routine immunization schedules.
In countries where diarrheal deaths account for ≥10% of mortality among children <5 years of age, the introduction of rotavirus vaccine is strongly recommended. Efficacy of these vaccines has ranged from 80% to 98% in industrialized countries, including Latin America, and 39% to 77% in developing countries such as Africa and Asia.,
It was speculated that an effective and properly administered rotavirus vaccine in Africa could potentially prevent 170,000–210,000 deaths (about 1 in 20) annually based on the assumptions that 20%–25% of all childhood diarrhea deaths are due to rotavirus., In the last 5 years, there is a paucity of information on the epidemiology and genetic characteristics of the circulating human rotavirus in most of the developing nations. This will be crucial to guide control and prevention strategies so as to ensure that rotavirus infection is reduced. In view of the aforementioned, this study sought to present findings from a year of hospital-based rotavirus sentinel surveillance at the University of Ilorin Teaching Hospital (UITH), Ilorin, Kwara State, Nigeria, using serological, multiplex reverse transcription-polymerase chain reaction (RT-PCR) assays and molecular characterization of rotavirus strains circulating among under-five children.
| Materials and Methods|| |
A total of 93 stool samples were collected within the period of January 2013 to August 2014 from children <5 years old. All the children were either out patients consulted for gastroenteritis or hospitalized (in patients) at UITH. Prior to Transportation to the regional laboratory at Noguchi memorial institute for medical research in Accra, Ghana, the stool specimens were stored in cryovials at − 70°C in glycerol.
Subjects recruitment and case definition
Diarrhea was defined by the occurrence of three or more liquid stools in a 24-hour period and presence of diarrhea at the time of clinical presentation with or without admission to the inpatient diarrheal treatment unit. The exclusion criteria were as follows: Hospital-acquired diarrhea, which was defined as onset of diarrhea >48 h after hospitalization; bloody diarrhea; and chronic and/or persistent diarrhea, which was defined as diarrhea that lasted for >2 weeks. Inclusion and exclusion criteria for diarrhea cases were applied as specified in the WHO AFRO standard operating procedures, which were based on WHO Generic Protocol.
Children presenting to the pediatric and child's care unit of UITH were screened for diarrhea by medical officers or nurses of UITH. Children aged <60 months with acute diarrhea and from whom stool sample could be recovered within 48 h of hospitalization were enrolled.
Sample size calculation
The sample size was determined using the prevalence rate of 6.0%. Therefore, the minimum sample size at 95% confidence level was 88. A total of 93 children were enrolled.
Ethical approval and informed consent
This study was conducted in accordance with the Declaration of Helsinki, and the ethical research committee of the UITH approved the protocol. The study was appropriately explained to all participants' parents or guardian. More so, they individually gave verbal and/or written consent for inclusion before recruited for the study. All data were analyzed anonymously throughout the study.
Rotavirus detection using enzyme-linked immunosorbent assay
Ten percent stool suspension in phosphate-buffered solution (PBS) was prepared and stored at − 20°C prior to use. Each Stool sample was tested for the detection of Group A Rotavirus antigen using an enzyme-linked immunosorbent assay (ELISA) with a commercial available rotavirus ELISA kit (ProSpectOxiod) as per the manufacturer's instructions. Positive and negative control samples were included during the ELISA run. Similarly, external quality assurance of results was conducted to ensure reliability and reproducibility.
Polyacrylamide gel electrophoresis
After dsRNA extractions by bender technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), all ELISA-positive samples were subjected to SDS-PAGE according to the technique described by Kuta et al.
Reverse transcription-polymerase chain reaction
For the RT-PCR, RNA was extracted using the QIAGEN technique from previously prepared 10% stool sample suspensions in PBS. The purified RNA was reverse transcribed to cDNA and genotyped using a RT-PCR.
Detection of rotaviruses in clinical specimens and determination of the G-type and P-type was accomplished by extraction of the viral RNA from fecal specimens and analysis by RT-PCR with primers specific for the VP7 genes of G serotypes 1, 2, 3, 4, 8, 9, 10, and 12 and VP4 genes of P serotypes 4, 6, 8, 9, 10, and 11. Rotavirus genotyping was done in collaboration with Noguchi Memorial Institute of Medical Research, University of Ghana, and Rotavirus Regional Reference Laboratory, Ghana.
Data generated from the investigation were systematically analyzed. Data were presented as percentages, 2 × 2 contingency table; Chi-square test for independence was used to determine relationship of gender/age with rotavirus positivity. Statistical Package for the Social Sciences (SPSS) software version 20 (California Inc., Chicago, USA) was used for all the analyses. A two-sided P ≤ 0.05 at 95% confidence interval was considered statistically significant.
| Results|| |
Of a total of 93 samples comprising 54 (58.1%) males and 39 (41.9%), 25 (26.9%) samples were positive for rotavirus by ELISA. All the rotaviruses isolated from these children were from those <24 months of age (100%) [Table 1]. Rotavirus was isolated more from males 17 (68.0) than the female counterparts [Table 2]. There is statistical relationship between the prevalence of rotavirus infection with age (P = 0.033) but not with gender (P = 0.765) [Table 2].
|Table 1: Age-stratified prevalence of rotavirusassociated with acute diarrhea among subjects|
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|Table 2: Gender-stratified prevalence of rotavirusassociated with acute diarrhea among subjects|
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June 2013 recorded the highest incidence of rotavirus infection and none during August–December 2013, as well as February–March and May–August 2014. October–December is the peak of rainy and dry seasons in Ilorin metropolis, Nigeria [Figure 1].
In this study, analyses using PAGE showed that of the 25 human ELISA-positive samples tested by PAGE, 17/25 (68%) yielded electrophoretic patterns typical of rotavirus while 8/25 (32%) showed no profile. Of the 17-rotavirus strains that yielded RNA profile, 11/17 (64.7%) had classical long rotavirus RNA electrophoretic patterns and 6/17 (35.3%) exhibited classical short RNA profile. Genotyping by RT-PCR was done on the 25 samples. The most prevalent type-able VP7 G types are G9 (28%), G1 (24%), followed by G12 (20%), G2 (12%), and G10 (4%). On the other hand, the most prevalent type-able VP4 P types are P8 (48%), P4 (24%), and P6 (16%), The most common G-P combination was G9P (4) (20%) followed by G12P (8) and G1P (4) (16%, respectively); then GNTP (8) and G1P (6) (8%, respectively); and G9P (8), G12P (4), G10P (6), G2P (8), and G2P (6) (4%, respectively) [Table 3].
| Discussion|| |
Rotavirus has been identified to be the single most important pathogen associated with diarrhea cases in both hospital patients and outpatients.,
Rotavirus was most common during January and April. However, no case was recorded during August–December (peak rainy season). The possible explanations for months with no cases are peak rainy season, non-recruitment of study participants, and hospital staff industrial action (which will lead to hospital services closure).
The result of this study is consistent with a previous study conducted in Sokoto metropolis (25.5%) and sentinel-based rotavirus surveillance system and hospital-based study results within the African region., Surprisingly, earlier studies carried out in Ilorin and different parts of the Nigeria reported low prevalence. The report of Pennap and Umoh showed rotavirus infection prevalence of 15.6% among children aged 0–60 months who presented with diarrhea in Northeastern Nigeria. Aminu et al. similarly reported rotavirus prevalence of 18% among diarrheic children and 7.2% among nondiarrheic children in a hospital setting in Northern Nigeria and prevalence of 9% in children under 5 years of age in a community-based study in the same region. Furthermore, other investigators reported lower prevalence of the infection in the northern region. The low prevalence reported in the community-based study is expected as higher prevalence of rotavirus infection is more likely to be encountered in hospital-based studies since rotavirus-positive cases are often severe and likely to be admitted in hospitals.
However, studies from Southern Nigeria revealed higher rotavirus prevalence values than those from Northern Nigeria.,, The differences in the prevalence recorded by different investigators had been attributed to differences in geographical location of the study, or changing trends of the burden of the rotavirus disease over the years. The possibility of direct transmission of animal rotavirus to human host and the uncommon serotypes detected in this study may explain the high prevalence of the disease among children with frequent contact with animals, in Northern Nigeria compared to those of Southern Nigeria who may not be exposed to occupations such as cattle herding at an early stage of life. The analysis of genotypes of rotavirus isolates provides information on genetic diversity of the virus and heterogeneity of circulating strains and can be useful in tracing spread through a population. Indeed, viruses of the same serotype could exhibit different electropherotypes and those of the same electropherotypes different serotypes.,
The inconsistency between the ELISA and PAGE results might be as a result of RNA degradation in which case strains will not yield RNA profile on PAGE., In addition, this is probably due to the differences in specificities and sensitivities of both techniques. PAGE has been established to indicate a higher specificity (100%) but lower sensitivity (79%) compared to those of commercial ELISA which are both 84%. The absence of RNA bands had also been attributed to too little RNA or its degradation during the phenol/chloroform extraction stage. Strains with long RNA profiles were the most prevalent strains in circulation in the study area. This is in consonant with previous studies., Interestingly, however, there were no unusual electropherotypes even in the samples containing the G12 strains. This observation was in agreement with that of Adah et al.
The RT-PCR VP7 serotyping results revealed that 22 (88%) of the rotavirus isolates were successfully assigned a specificity of VP7 (G-type), while 3 (21.2%) of the isolates were nontype-able. It was likely that the nontype-able strains did not contain enough RNA to permit typing similar to the observation of a previous study. It may also be as a result of the existence of serotypes, which the serotype specific primers used in the study could not detect. Previous studies had shown that the most common global human G-serotypes were G1, G2, G3, G4, and G9, with G1 being the most prevalent and G9 being the fastest emerging genotype worldwide., In earlier studies,, no G2 or G4 serotypes were detected; however, Aminu et al. had recently reported the detection of G2 serotypes in Northern Nigeria. The presence of G9 serotype in this study could be a result of genetic stability since G9 serotype was previously reported in northern region of the country.
The results from this study showed that genotype is now becoming more associated with symptomatic diarrhea. In fact, rotavirus with VP4 P (6) genotype was the only genotype detected in the study conducted by Aminu et al. This had been attributed to natural re-assortment, which appears to be detected more frequently in developing countries than in developed world owing to low levels of hygiene and poor immunological defense in infants that facilitate mixed infections and hence more re-assortment. In addition, more close contact among humans, livestock, and other animals in developing countries makes the possibility of emergence of virulent rotavirus strains very high as a result of gene re-assortment. Apart from gene re-assortment, interspecies transmissions of rotaviruses involving a whole genome constellation evidenced by molecular characterization of rotaviruses isolated from different species have been suggested.
Indeed, studies had indicated that uncommon human rotavirus strains are emerging as global strains, which has important implications for effective vaccine development. The detection of unusual G/P combinations in the present study adds to this pool of information and further confirms the emergence of these unusual strains. This study reported a high prevalence of rotavirus infection among diarrheic children attending UITH, Nigeria. Both common, uncommon, and combinations of various genotypes were identified as revealed by their electrophoresis pattern based on the VP4 and VP7 gene. Overall, findings from this report will contribute to the global understanding of the molecular epidemiology of human rotavirus in Nigeria. This will be useful in guiding the choice and application of rotavirus vaccines for effective control and preventions.
This study is not without limitations. These include the small sample size nature and study period not up to the recommended 2 years for seasonal disease so as to cover at least two full seasons. Hence, it is recommended that national large-scale and periodic studies be conducted on rotavirus to ascertain its geographic distribution and genetic diversity in Nigeria.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]