From the *Department of Medical Microbiology, Virology Section, University of Zimbabwe; †Zimbabwe Ministry of Health and Child Welfare; ‡WHO country office, Harare, Zimbabwe; §WHO Regional Office for Africa, (WHO/AFRO), Brazzaville, Congo Republic; ¶Medical Research Council/UL Diarrhoeal Pathogens Research Unit and WHO Rotavirus Regional Reference Laboratory for Africa, University of Limpopo Medunsa Campus and National Health Laboratory Service, Pretoria, South Africa; ‖Chitungwiza Central Hospital, Chitungwiza, Zimbabwe; **Parirenyatwa Group of Hospitals; and ††Harare Central Hospital, Harare, Zimbabwe.
Accepted for publication July 31, 2013.
The authors have no other funding or conflicts of interest to disclose.
Address for correspondence: Arnold Mukaratirwa, The Department of Medical Microbiology, College of Health Sciences, University of Zimbabwe, P.O.Box A178, Avondale, Harare, Zimbabwe. E-mail: email@example.com.
Rotavirus diarrhea affects almost all children whether rich or poor by the age of 5 years, kills nearly 453,000 children worldwide a year. More than 80% of rotavirus deaths occur in the developing countries where resources are limited. Now that rotavirus vaccine is a reality, knowing the baseline trends in rotavirus diarrhea and circulating genotypes before vaccine introduction are important for effective monitoring of the benefits. Two live-attenuated, oral rotavirus vaccines have been developed, which have the potential to decrease the global burden of rotavirus disease1–3: Rotarix (GlaxoSmithKline Biologicals, Rixenstart, Belgium)4 which is a monovalent G1P attenuated human rotavirus strain and RotaTeq (Merck, Whitehouse Station, NJ)5 which is a pentavalent vaccine consisting of a mixture of 5 human-bovine reassortants expressing G1–G4 and P.
To address the problem of diarrheal disease caused by rotavirus in children and contribute towards achieving Millennium Development Goal 4 to reduce child mortality, the Zimbabwean Ministry of Health plans to introduce the rotavirus vaccine in January 2014. The introduction plan is aligned with the comprehensive multi-year plan 2012–2016. The country has chosen to introduce Rotarix and all children <1 year of age will be eligible to receive 2 doses at 6 and 10 weeks of age. This vaccine will be integrated into the current Expanded Programme on Immunization schedule.
In anticipation of rotavirus vaccine introduction, the Zimbabwe Ministry of Health initiated rotavirus surveillance in 2008 as there are limited published data describing rotavirus infection in Zimbabwe. In a small study in 1988, rotavirus was detected in almost a quarter of acute diarrhea cases in young children and occurred most commonly in the cool, dry months.6 In the early 2000s, a small number of rotavirus-positive samples were characterized from Parirenyatawa Hospital, Zimbabwe, showing the high prevalence of G1P and G3P strains circulating.7 The objective of this article is to describe epidemiological trends of rotavirus diarrhea and circulating genotypes among children <5 years of age before rotavirus vaccine introduction.
Active hospital-based rotavirus surveillance for diarrhea was conducted in 3 sentinel sites, Parirenyatwa Group Hospitals, Harare Central Hospital and Chitungwiza Central Hospital (CCH). The Parirenyatwa Group Hospital has almost 200 paediatric beds and the catchment population is about 1.7 million, which comprise mainly the urban and elite group. The Harare Central Hospital has about 150 paediatric beds and serves an urban population of about 1 million, which comprise both the elite and poor groups. The CCH has close to 150 paediatric beds and serves the population of about 1.2 million which comprise both urban and rural residents.
Children <5 years who presented with acute gastroenteritis, the occurrence of ≥3 episodes of diarrhea (stools of a less formed character than usual) within a 24-hour period of ≤7 days duration as a primary illness and were admitted to a hospital ward or treated at the emergency unit at 1 of the 3 sentinel sites during the period January 2008 to December 2011, were enrolled in the surveillance program. Stool specimens were collected.8 Children ≥5 years of age, with bloody diarrhea, with symptoms ≥7 days or who acquired gastroenteritis during hospitalization for treatment of other diseases, were excluded. Faecal specimens were collected within 48 hours of admission and not >7 days after onset of acute diarrhea.
All information regarding inclusion criteria and basic demographic and clinical information for each case was captured on the World Health Organization rotavirus surveillance form.
Faecal specimens were transported at 4°C to the Department of Medical Microbiology, Virology Laboratory for detection of group A rotavirus antigen using enzyme immunoassay (EIA) according to instructions of the manufacturer, ProSpect Rotavirus microplate assay (Oxoid Ltd, Cambridge, UK). Every faecal specimen received in the Zimbabwe National Virology Laboratory was accompanied by the rotavirus surveillance form.
Faecal specimens stored at −20°C, which tested positive for EIA rotavirus antigen detection, were transported to the University of Limpopo, South Africa for Polyacrylamide gel electrophoresis (PAGE) and reverse transcription polymerase chain reaction (RT-PCR).
Nucleic Acid Detection
was performed on 100 EIA rotavirus-positive specimens. The reagent preparation for rotavirus dsRNA extraction, PAGE and silver-staining methods was carried out according to standard operating procedure used in the MRC Diarrhoeal Pathogens Research Unit and WHO Rotavirus detection Manual.9,10 The migration pattern of the rotavirus RNA segments on the gel identified the rotavirus strain as either “short” or “long” electropherotype.
The rotavirus RNA extraction from faecal specimens for detection of rotavirus using RT-PCR was carried out using a commercially available kit, the QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany). The RT-PCR was performed using several primer pairs designed to target highly conserved regions of the RNA genome. Con2 and Con3 primers were used to amplify the fragment of VP4 gene (876 bp),11 and the End9 and sBeg9 primers were used to amplify the VP7 gene (1062 bp).12
The amplicons for RT-PCR products were used for genotyping both VP7 and VP4 genes. The VP7 genotyping was carried out using genotype-specific primers for G1, G2, G3, G4, G8, G9 and G12. Genotype-specific primers which were used to genotype VP4 were: Con3, dP,7 2T-1, 3T-1, 4T-1, 5T-1, mP and P (SE-1).
The statistical analysis of the data was done using STATA 10.1 software program. Descriptive analysis were performed to determine the prevalence of EIA rotavirus antigen-positive specimens collected from acute diarrhea cases and compare the characteristics of rotavirus-positive and rotavirus-negative cases. The prevalence of electropherotypes in rotavirus diarrhea cases was determined and the prevalence of the separate G genotypes, P genotypes and the combination genotypes was calculated.
A total of 3728 faecal samples collected from hospitalized children were analyzed by rotavirus antigen detection EIA and of which, 1804 (48.5%) tested positive for rotavirus. The sentinel site positivity rate varied from 40.9% to 55.6%. The highest rate of rotavirus infection was observed mainly in CCH which serves mainly the rural population, while Parirenyatwa Group of Hospitals with a catchment area of mostly the elite urban population had lowest rate. The overall annual positivity rate varied from 29% to 59.7% (Table 1). The highest prevalence of rotavirus diarrhea was found during the dry, cool season (Fig. 1) in children ≤ 59 months of age. Male children comprised 59% of rotavirus-positive cases and 56% of rotavirus-negative cases. Although the male cases had higher chances of being infected by rotavirus than female cases, this was not statistically significant (P = 0.06). Rotavirus positivity peaked in children 3–17 months of age with almost 80% of cases occurring in this age range (P ≤ 0.001). Over 90% of rotavirus-positive cases occurred in children <2 years of age (Table 2). Compared with rotavirus-negative cases, rotavirus-positive cases were more likely to be dehydrated (26% vs. 14%, P ≤ 0.001), have vomiting (77% vs. 57%, P ≤ 0.001) and less likely to have fever (17% vs. 24%, P = 0.03). Similar proportions of rotavirus-positive and rotavirus-negative cases received oral rehydration (43% and 44%, respectively), but rotavirus cases were less likely to receive intravenous rehydration (20% and 26%, respectively).
Of the 193 specimens that were genotyped, combination of G and P genotype specificities was successful for 127 rotavirus-positive strains. The predominant G and P type combinations found were: G9P accounting for 43.3% (55/127), followed by G1P (n = 15), G2P (n = 11), G2P (n = 11) and G12P (n = 11) each of which accounted for 11.8%, 8.7%, 8.7% and 8.7% of the characterized strains, respectively (Table 3). Other G and P combinations observed were G9P 6(4.7%), G8P 8(6.3%) and G1P 5(3.9%). Some strains were detected at low level: G9P G8P, G12P and G12P. All of these G and P combination strains were found in children ≤25 months of age. Mixed infections were found in 10 samples (7.9%) and they were associated with G1/G8/G9, G8/G12, P/P/P, P/P, P/P and P/P. P was only detected in 2010 and G3 and G4 were not detected.
The typical 4-2–3-2 grouping of the rotavirus group A RNA segments was seen in 66 of 100 EIA rotavirus-positive isolates that were tested using PAGE and 34 (34%) produced negative results. A total of 51 (77.3%) of the isolates that tested positive were long electropherotype and 15 (22.7%) were short RNA electropherotype and no specific genotype could be associated with a particular electropherotype. Many of the circulating rotavirus that caused acute gastroenteritis had long electropherotype, 51/66 (77.3%), while the short RNA electropherotypes were displayed in 15/66 (22.7%) rotavirus-positive samples.
This study found that 48% of children with severe diarrhea who were hospitalized at the 3 referral hospitals situated in the urban areas of Harare and Chitungwiza from January 2008 to December 2011 were positive for rotavirus with the annual rotavirus positivity ranging from 29% to 60%. The 48% rotavirus detection rate determined in this study was less than that reported (66% incidence) in a study carried out in Harare urban community (Nziramasanga P, Berejena C, Chibukira P, et al., unpublished data, 2010). The detection rate of rotavirus in children with acute gastroenteritis was higher than those reported in other African countries which were about 36%. Although no data was gathered about the place of residence for the hospitalized children, most of the severe diarrhea cases were probably coming from the urban locations.
The rotavirus infection was higher in male children (59%) than in female children (41%), which is similar to findings reported previously.13 High detection rate during the dry, cool months of May to July, each year from 2008 to 2011, is also consistent with previous studies from elsewhere in Sub-Saharan Africa. Rotavirus detection rates are increased during the dry, cool months of May to August and wet months of November to December.14,15 This is probably due to ingestion of contaminated dry soil or inhalation of rotavirus in the dust and drinking contaminated water. In this 2008–2011 (4 years) study, children below the age of 24 months and predominantly children <1 year of age were the most severely affected by severe gastroenteritis. This finding was similar to findings from studies of urban and peri-urban children in other African countries.14,16
Young infants will be targeted to receive the available rotavirus vaccines, Rotarix and RotaTeq. All doses of the vaccines should be administered within the first 8 months of life.8 Now that vaccines are a reality, it is important to know the baselines trends in rotavirus diarrhea and rotavirus circulating strains before introducing vaccine so that the impact of the vaccines can be monitored. The identification of unusual P and G combinations as well as a large diversity of circulating strains in Zimbabwe will provide an opportunity to monitor the impact and effectiveness of currently available rotavirus vaccines against these strains. The long RNA electropherotype pattern was predominant in gastroenteritis cases.
Our study has several limitations. First, limited demographic information was collected on enrolled patients. Thus, we are unable to compare rotavirus positivity rates and circulating strains from children from urban and rural areas. Most of the children in our study were likely from urban and peri-urban communities based on the catchment population of the surveillance hospitals. Second, the number of children enrolled during 2008 and 2009 was substantially smaller than those enrolled in 2010 and 2011. This difference is likely due to a difference in case ascertainment rather than a true increase in diarrheal disease. Finally, only a subset of specimens from the 4-year study was genotyped making it difficult to monitor changes in genotypes over time. However, our study does capture the diversity of strains circulating in Zimbabwe. It was also interesting to note that, whereas in 2002 when the predominant strain from Parirenyatwa Hospital was G3P,7 in this 4-year period, no G3 strains were identified showing the diversity of strains over time in 1 setting.
In addition, the identification of a predominance of G9 strains confirms that this strain is widely spread in Africa and is common in many regions.17 The appearance of G12 strains highlights the emergence of this “new” rotavirus strain before any rotavirus vaccine introduction17–19 and supports the need for continual strain surveillance to monitor the strains.
In conclusion, rotavirus diarrhea results in a significant disease burden among young children in Zimbabwe. In response to this significant disease burden, the Zimbabwe Ministry of Health plans to introduce rotavirus vaccine in 2014. The data collected as part of this study will provide valuable baseline introduction regarding rotavirus disease burden that can be used to monitor the impact of rotavirus vaccine in Zimbabwe. The detection of some uncommon rotavirus strains and yearly variation in rotavirus positivity rates dictates the need to continue surveillance of rotavirus strains before and after rotavirus vaccine introduction.
The authors thank the Zimbabwe Ministry of Health and Child Welfare, World Health Organization AFRO and partners for their unwavering support. The authors also appreciate the support from MRC Diarrhoeal Pathogens Research Unit University of Limpopo (Medunsa) for genotyping the samples.
1. Adiku TK, Dove W, Grosjean P, et al. Molecular characterization of rotavirus strains circulating among children with acute gastroenteritis in Madagascar during 2004-2005. J Infect Dis. 2010; 202:(suppl)S175–S179
2. Steele AD, Neuzil KM, Cunliffe NA, et al. Human rotavirus vaccine Rotarix™ provides protection against diverse circulating rotavirus strains in African infants: a randomized controlled trial. BMC Infect Dis. 2012; 12:213
3. Patel M, Pedreira C, De Oliveira LH, et al. Association between pentavalent rotavirus vaccine and severe rotavirus diarrhea among children in Nicaragua. JAMA. 2009; 301:2243–2251
4. De Vos B, Vesikari T, Linhares AC, et al. A rotavirus vaccine for prophylaxis of infants against rotavirus gastroenteritis. Pediatr Infect Dis J. 2004; 23:(10 suppl)S179–S182
5. Heaton PM, Goveia MG, Miller JM, et al. Development of a pentavalent rotavirus vaccine against prevalent serotypes of rotavirus gastroenteritis. J Infect Dis. 2005; 192:(suppl 1)S17–S21
6. Tswana SA, Jorgensen PH, Halliwell RW, et al. The incidence of rotavirus infection in children from two selected study areas in Zimbabwe. Cent Afr J Med. 1990; 36:241–246
7. Steele AD, Ivanoff B. Rotavirus strains circulating in Africa during 1996-1999: emergence of G9 strains and P strains. Vaccine. 2003; 21:361–367
8. World Health Organization African Rotavirus Surveillance Network Manual. 2009; Geneva WHO
9. World Health Organization Manual of Rotavirus Detection and Characterization Methods. Immunization, Vaccination and Biologicals. 2009; Geneva WHO
10. MRC Diarrhoeal Pathogens Research Unit The 10th Rotavirus Surveillance Workshop Laboratory Manual. 2009; Ga-Rankuwa, Pretoria, SA University of Limpopo—Medunsa Campus
11. Gentsch JR, Glass RI, Woods P, et al. Identification of group A rotavirus Gene 4 types by polymerase chain reaction. J Clin Microbiol. 1992; 30:1365–1373
12. Gouvea V, Glass RI, Woods P, et al. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol. 1990; 28:276–282
13. Zuridah H, Kirkwood CD, Bishop RF, et al. Molecular characterization and epidemiology of rotavirus isolates obtained from children with diarrhoea in Malaysia. Med J Malaysia. 2009; 64:193–196
14. Potgieter N, de Beer MC, Taylor MB, et al. Prevalence and diversity of rotavirus strains in children with acute diarrhea from rural communities in the Limpopo Province, South Africa, from 1998 to 2000. J Infect Dis. 2010; 202:(suppl)S148–S155
15. Steele AD, Alexander JJ, Hay IT. Rotavirus-associated gastroenteritis in black infants in South Africa. J Clin Microbiol. 1986; 23:992–994
16. Nyangao J, Page N, Esona M, et al. Characterization of human rotavirus strains from children with diarrhea in Nairobi and Kisumu, Kenya, between 2000 and 2002. J Infect Dis. 2010; 202:(suppl)S187–S192
17. Page N, Pager C, Steele AD. Characterization of rotavirus strains detected in Windhoek, Namibia during 1998-1999. J Infect Dis. 2010; 202:(suppl)S162–S167
18. Steele AD, Page N, de Beer M, et al. Antigenic and molecular characterization of unusual rotavirus strains in Burkina Faso in 1999. J Infect Dis. 2010; 202:(suppl)S225–S230
19. Esona MD, Armah GE, Steele AD. Rotavirus VP4 and VP7 genotypes circulating in Cameroon: identification of unusual types. J Infect Dis. 2010; 202:(suppl)S205–S211