Rotavirus group A genotype circulation patterns across Kenya before and after nationwide vaccine introduction, 2010–2018

Childhood diarrhea caused by rotavirus group A (RVA) infection remains a leading cause of morbidity and mortality in young children globally [1]. In 2016, RVA infections were estimated to be responsible for 1,537,000 hospitalization cases, 128,500 deaths globally and over 80% of these deaths occurred in developing countries [2]. In Kenya alone, it is estimated that rotavirus infection accounts for over 3000 deaths annually in children under 5 years of age [3].

Upon infection by the virus, immune response to RVA by the host is directed to the highly variable VP7 and VP4 genes found on two separate segments of the double-stranded RNA genome [4]. RVA G and P genotypes exist as multiple variants in nature, few of which have been found to infect humans [4]. Up to 36 G and 51 P genotypes have been detected globally in both humans and animals, with multiple G-P combinations [5]. Molecular studies have characterized circulating genotypes worldwide with predominance of genotypes G1P [8], G2P [4], G3P [8], G4P [8], G9P [8] and G12P [8] (in decreasing order of prevalence) [6, 7]. Although the distribution of these genotypes varies from region to region and from one season to another, genotype G1P [8] has remained the most dominant genotype globally [6]. In Africa, there is a high diversity of genotypes, most commonly G1P [8], G2P [4], G9P [8], G2P [6], G12P [8] and G3P [6], with G1P [8] and G2P [4] predominant [7, 8]. Significant RVA infections are also caused by strains of uncommon genotypes including G1P [4], G2P [8], G9P [4], G12P [4], G8P [6], G8P [8] and G12P [6] [7, 8]. Such uncommon strains also show a wide variation from one region to the other, and can spread globally to become common strains. For instance, genotypes G9P [8] and G12P [8] emerged and contributed to a larger proportion of global RVA infections [9]. An understanding of these genetic diversity after vaccine introduction is necessary for design and implementation of effective control programs.

In 2009, the World Health Organization (WHO) recommended the inclusion of RotaTeq® (Merck Vaccines, Whitehouse Station, New Jersey) or Rotarix® (GlaxoSmithKline Biologicals, Rixensart, Belgium) vaccines in the national immunization programs of countries that experience high diarrhea morbidity and mortality burden due to RVA disease [10]. The introduction and increased use of these vaccines have reduced up to 76% of rotavirus hospitalizations in children < 5 years [11] and averted up to 28,800 deaths globally, including 84% of the deaths in sub-Saharan Africa [2]. Furthermore, these vaccines showed overall good clinical protection against multiple homotypic and heterotypic RVA strains in humans [12]. Kenya incorporated the monovalent G1P [8] Rotarix® vaccine into the national immunization program in July 2014, administered in two oral doses offered at weeks 6 and 10 of age. The Rotavirus Immunization Programme Evaluation in Kenya (RIPEK) was established as a collaboration among existing rotavirus surveillance platforms across Kenya to monitor the impact of Rotarix vaccine introduction against rotavirus disease and circulating RVA genotypes. Substantial effectiveness of the vaccine in Kenya (vaccine coverage of 72% [13]) and the entire sub-Saharan region (where disease burden is high) has been recorded, and the decline in incidence of all-cause and rotavirus associated diarrhea admissions has been attributed to vaccine implementation [14‐18]. However, there are limited data on RVA diversity in post-vaccine introduction periods in this region. The current report describes the distribution and temporal patterns of RVA genotypes observed before and after Rotarix vaccine introduction in Kenya.

A total of 6562 stool samples were collected from health facilities in LMH, SCRH, TC and KCH. Of the 1312 (20.0%) RVA positive samples, individual G and P genotypes were identified in 928 (70.7%) and 904 (68.9%) samples, respectively, while 875 (66.7%) samples were successfully genotyped for both G and P genes [LMH (n = 38), SCRH (n = 161), TC (n = 84), and KCH (n = 592)]. Of these, 614 (70.2%) were observed in the pre-vaccine (January 2010 – June 2014) and 261 (29.8%) in the post-vaccine (July-2014 – December 2018) introduction periods, respectively.

Prior to vaccine introduction, G1P [8] was the predominant genotype. However, this genotype’s prevalence varied substantially from year to year (dominant in 2011, 2013, 2014 and 2015; less so in 2010 and 2012), emphasizing the limits of short-term surveillance and the potential for misrepresentation of patterns. During 2010 and 2012, a large proportion of children were infected with genotype G8P [4], which was not detected in any years following vaccination (except during 2017). Genotypes G1P [8], G2P [4] and G9P [8] have also been identified as dominant genotypes in a study involving six countries from Eastern and Southern Africa [7].

Genotype prevalence varied year to year and from before to after vaccine introduction. Interestingly, while genotype G1P [8] showed no overall change in prevalence compared to the pre-vaccine period, it predominated only in the first year after vaccine introduction (2015), and declined in prevalence thereafter, in particular to be replaced by G2P [4] in 2016, and by G3P [8] in 2018. Genotypes G8P [4] and G9P [8], which were the second and third common genotypes in pre-vaccine period, significantly declined in the post-vaccine period. Predominance of G2P [4] and G3P [8] has been reported worldwide following universal Rotarix vaccine introduction. For instance, G2P [4] was observed in Australia [35] (in states using Rotarix) and Belgium [36] after vaccine introduction, while data from Ethiopia [37] and Madagascar [38] depicted an increase in prevalence of G3P [8] in post-vaccine introduction period. Studies on the G3P [8] have revealed the emergence and spread of strains of equine-like G3 genotype co-circulating with wild-type G3 strains [39‐41]. In here, 4.8% (2/41) were detected to be of equine-like type (data not shown). Further analysis on this genotype will illuminate of the genetic diversity and prevalence of the emerging equine-like in the post-vaccine introduction period. G2P [4] is one of the genotypes of concern since this fully heterotypic genotype has a different genomic constellation (DS-1-like) to the genotype in the monovalent Rotarix vaccine (Wa-like) [42]. There is no conclusive evidence associating the increase in prevalence of these genotypes to selective vaccine pressure created by implementation of Rotarix monovalent vaccine. Whilst the vaccine offers both homotypic and heterotypic protection [43, 44], the emergence of the fully heterotypic G2P [4] and partially heterotypic G3P [8] genotypes, and persistence in circulation of the homotypic G1P [8] genotype after vaccine introduction emphasizes the need for continued monitoring of impact of vaccine on genotypes.

We observed multiple uncommon G/P combinations including G1P [4], G1P [6], G2P [8],G4P [4], G8P [8],G8P [14], G10P [8], G12P [4] and G12P [6] at frequency of < 2%. Genotype P [14] has been described sporadically in humans and is believed to have originated from animal rotavirus and introduced into humans through interspecies transmission and/or reassortment events [45]. The circulation of these uncommon genotypes demonstrates the high diversity of RVA genotypes in Kenya and concurs with findings from other African countries [7]. However, genotype diversity was seen to decline in post-vaccine period (only 11 GP combinations, compared to 21 in pre-vaccine period), mirroring the experience of other countries, including Brazil and Zimbabwe, which also indicated a decline in genotype variation after vaccine introduction [46, 47]. This is in contrast to other countries such Australia which observed increase in genotypes after vaccine introduction [35]. Although diversity seems to decrease following vaccine introduction, it is unknown whether the observed trends will be sustained in the long-term, especially in African settings where pre-vaccine genotype diversity is high.

Phylogenetic analysis of genotype G1 revealed circulation of strains of lineages G1-I and G1-II while the P [8] strains formed three distinct clusters revealing circulation of strains of lineages P [8]–I, P [8]-III and P [8]-IV. The Kenyan strains were closely related to other global strains as they segregated together. G1-I and P [8]-III were the predominant lineages for both pre and post-vaccine strains. These lineages were distinct from the Rotarix vaccine lineages showing that the strains were distantly related to the vaccine strain. Similar findings were observed in data collected before and after vaccine introduction in Eastern and Southern Africa countries [7]. Although there were no lineage replacements after vaccine introduction, the genetic diversity between pre and post-vaccine strains, may suggest normal genetic fluctuations or an effect of the vaccine. Interestingly, a single case of vaccine-derived strain was observed in a sample isolated from a child in Kibera, who had received the first dose of the Rotarix vaccine. Phylogenetically, the post-vaccine strain clustered with the Rotarix vaccine, in G1-I and P [8]-I lineages, and showed sequence identities of 100% to the vaccine strain. These clusters also contained previously vaccine-derived strains isolated in Brazil, Korea, and Japan. Acute gastroenteritis, caused by vaccine shedding or horizontal transmissions of the vaccine strain have been reported at a higher rate in other countries [48‐50]. Further analysis will be necessary to determine whether the observed vaccine strain was as a result of vaccine shedding or horizontal transmission event.

This study provides substantial epidemiological information on changes in distribution and genetic diversity of RVA genotypes in Kenya. Some limitations of this study included fewer surveillance sites and unequal distribution of the number of samples among the sites, potentially underestimating the type of genotypes circulating in the post-vaccine period. However, it is important to consider that there was an overall decline in rotavirus circulation in the post-vaccine era, hence the fewer specimens and less diversity could be a reflection of less rotavirus circulation [14, 15]. Additionally, since this is an ecological study, the changes in distribution and diversity of genotypes in the post-vaccine era cannot directly be attributed to vaccine introduction. Furthermore, due to unsuccessful sequencing and/or contig assembly only two thirds of the total RVA positive samples were fully genotyped. Lastly these findings may not be generalized to the whole country because analysis was based on rotavirus cases observed in health facilities in three counties only.

In conclusion, we highlight the importance of monitoring the transition in the prevalence of genotypes for a better understanding of the performance of the currently available vaccines. The emergence of the fully heterotypic G2P [4] and partially heterotypic G3P [8] genotypes after vaccine introduction raises questions about the epidemiological dynamics following vaccine introduction. Previous analysis in Kenya showed that the vaccine had a significant impact on G1P [8] and non-significant G2P [4] (although with limited statistical power) [14], hence, continuous monitoring of the circulating genotypes in the post-vaccine era is necessary. Our findings also highlight existence of considerable variation and genetic diversity within and between Kenyan pre and post-vaccine strains. One strain identified in Kenya, was closely related to the Rotarix® vaccine strain, likely representing shedding or horizontal transmission of the vaccine strain. Additionally, continued surveillance of the genetic characteristics of circulating RVA strains is recommended to obtain a full view of the long-term effects of vaccine introduction. Since immunity to RVA involves immune responses conferred by genes other than the commonly studied P and G genes, vaccine effectiveness might be challenged by changes occurring on non-capsid genes. It is therefore recommended that full genome analysis of genotypes collected in different time or geographic regions be conducted to improve understanding of their evolutionary profile during the post-vaccine introduction period.