Rotavirus strain diversity in Eastern and Southern African countries before and after vaccine introduction.

BACKGROUND: The African Rotavirus Surveillance Network has been detecting and documenting rotavirus genotypes in the African sub-continent since 1998 in anticipation of the rollout of rotavirus vaccination in routine Expanded Programme on Immunisation. This paper reports distribution of the rotavirus strains circulating in 15 Eastern and Southern African (ESA) countries from 2010-2015 as part of active World Health Organization (WHO) rotavirus surveillance, and investigates possibility of emergence of non-vaccine or unusual strains in six selected countries post-vaccine introduction. MATERIAL AND METHODS: Stool samples were collected from children <5 years of age presenting with acute gastroenteritis at sentinel hospitals pre- and post-rotavirus vaccine introduction. Samples were tested for group A rotavirus using an enzyme immunoassay by the national and sentinel laboratories. At the WHO Rotavirus Regional Reference Laboratory in South Africa, molecular characterisation was determined by PAGE (n = 4186), G and P genotyping (n = 6447) and DNA sequencing for both G and P types (n = 400). RESULTS: The six-year surveillance period demonstrated that 23.8% of the strains were G1P[8], followed by G2P[4] (11.8%), G9P[8] (10.4%), G12P[8] (4.9%), G2P[6] (4.2%) and G3P[6] (3.7%) in 15 ESA countries. There was no difference in circulating strains pre- and post-rotavirus vaccine introduction with yearly fluctuation of strains observed over time. Atypical rotavirus G and P combinations (such as G1P[4], G2P[8], G9P[4] and G12P[4]) that might have arisen through inter-genogroup or inter-genotypes reassortment were detected at low frequency (2%). Close genetic relationship of African strains were reflected on the phylogenetic analysis, strains segregated together to form an African cluster in the same lineages/sub-lineage or monophyletic branch. CONCLUSION: There has been considerable concern about strain replacement post-vaccine introduction, it was not clear at this early stage whether observed cyclical changes of rotavirus strains were due to vaccine pressure or this was just part of natural annual fluctuations in the six ESA countries, long-term surveillance is required.

A first report on the characterization of rotavirus strains in Sierra Leone.

In an effort to reduce the high mortalities associated with rotavirus infections, a number of African countries are considering introducing human rotavirus vaccines. The demonstrated safety and efficacy of the live-attenuate human rotavirus vaccines in several clinical trials worldwide has accelerated such initiatives. Although the percentage-mortality rates for Sierra Leone are top of the list for rotavirus-associated deaths in Africa, no study has reported the prevalent strains circulating within this country. In this study, stool specimens were collected from 128 Sierra Leonean children presenting with diarrhea in 2005. Almost 37.5% (48/128) were rotavirus positive by EIA, of which 89.6% (43/48) revealed a short electropherotype, and a further 6.98% (3/48) could not be assigned a PAGE pattern. Genotyping analysis revealed G2P[4] (30.23%), G2P[6] (13.95%), G8P[6] (11.63%), G2P[8] (4.65%), G8P[4] (4.65%), and G8P[8] (2%) strains. About 11% were only assigned VP7 genotypes (G2), while 20.9% had mixed G and P types. The frequent detection of G2 rotaviruses could be of concern considering data generated from some studies that suggests lower efficacy of Rotarix® vaccine against G2 rotaviruses. This underscores the need for extensive and continuous regional strain surveillance to support rotavirus vaccines introduction and guide future vaccine development efforts. Such information will be useful before considering administration of specific rotavirus vaccine candidates in countries like Sierra Leone where little is known about circulating rotavirus strains.

Characterization of human rotavirus recovered from children with acute diarrhea in Kinshasa, Democratic Republic Of Congo.

Between July and October of 2003, 2004, and 2005, outbreaks of acute gastroenteritis occurred among children <5 years of age in Kinshasa, Democratic Republic of the Congo. Stool specimens (67 in 2003, 108 in 2004, and 116 in 2005) were collected and screened for rotaviruses using either latex agglutination (Diarlex LAA; Orion Diagnostics) or enzyme immunoassay (IDEIA; DakoCytomation). The molecular characteristics of the rotavirus strains were then determined. Group A rotavirus was detected in 195 (76%) of 258 stool specimens. Polyacrylamide gel electrophoresis was used to observe the 11 rotavirus double-stranded RNA segments in 83% of the 195 rotavirus-positive specimens. Six rotavirus group A electropherotypic patterns were noted, predominantly within the short classic pattern (111 [69%]) and the long pattern (37 [23%]). Mixed patterns were noted in the 14 remaining specimens (9%). Of the 29 samples subjected to subgrouping VP6 enzyme immunoassay, subgroup I predominated. Some of the specimens collected in 2003 (n = 26), 2004 (n = 38), and 2005 (n = 52) were analyzed by reverse-transcription polymerase chain reaction, which showed that t G8P[6] and G8P[8] strains predominated in 2003, and G1P[6] strains with short electropherotypic patterns predominated in 2004 and 2005. The emergence in Kinshasa of G8 serotypes, unusually associated with the P[6] genotype, as well as uncommon G1 rotavirus strains showing a short RNA pattern, is significant in relation to the introduction of a rotavirus vaccine and underscores the need for continued rotavirus serotype surveillance in the Democratic Republic of the Congo.

Molecular characterization and genotyping of human rotavirus strains in Abidjan, Cote d'Ivoire.

In this study, we characterized human rotavirus strains recovered from infants and young children with acute diarrhea in Abidjan, Cote d'Ivoire, during 2000-2004. In total, 719 fecal specimens were collected from children aged 1-60 months with acute infantile gastroenteritis. Examination with a commercial enzyme-linked immunosorbent assay showed the presence of group A rotavirus antigen in 208 diarrheal specimens (28.9%). Polyacrylamide gel electrophoresis of the RNA extracted from rotavirus-positive stools yielded a variety of "long" and "short" RNA electropherotypes, which were used to help select strains for VP4 and VP7 genotyping. VP7 genotype G1 strains were circulating most commonly during the study period (53%), followed by G2 (22%) and G3 (5%) strains. Strains with multiple VP7 genotype reactivity were observed in 7.6% of specimens, and a similar number (8%) could not be typed at all. VP4 P[6] and P[8] strains circulated at similar levels (33%). Strains demonstrating multiple VP4 types were quite common (9%); however, 20% of the strains were untypeable by the methods used. Rotavirus strain diversity in Cote d'Ivoire was similar to that observed in other West African countries.

[Epidemiological and virological aspects Rotavirus diarrhoea in Abidjan, Côte d'Ivoire (1997-2000)]. / Aspects épidémiologiques et virologiques des diarrhées dues aux Rotavirus à Abidjan, Côte d'Ivoire (1997-2000).

Viruses, mainly rotaviruses are aetiological agents in more than 80% of the cases of acute diarrhoea in children. In order to determine the epidemiological characteristics and genotypes of human rotaviruses involved in gastroenteritis in diarrheic children aged from 0 to 5 years old in Abidjan, 642 specimens of stools were collected between 1997 and 2000 in the urban health centres and University Teaching Hospitals in Abidjan. The antigenic detection of rotaviruses carried out by ELISA test was followed by the antigenic (VP6 sub-groups) and molecular characterization: polyacrylamide gel electrophoresis and genetic typing. The general prevalence of Rotavirus diarrhoea was 27.9%. Among the children who were found positive, those whose age ranged from 0 to 11 months old accounted for 45.8% against 41.3% and 12.9% for those whose age ranged from 1 to 2 and 3 to 5 years old respectively proving thus the precocity of rotavirus infection. From an electrophoretypical and antigenic point of view 74.5% of 141 extracts of RNA had a "long" profile and belonged to the VP6 II sub-group against 24.8% of "short" profile belonging to sub-group I. The electrophoretypes with short profile were identified in majority in infants whose age ranged from 0 to 2 years old. Out of the P genotypes identified, the P [8] genotype (59.6%) was predominant followed by the P [6] genotype (26.2%), P [4] (2.8%) and one mosaic genotype P[6,8] which represented 11.4%. These results will need to be completed by the determination of VP7 genotypes in order to provide interesting information on rotaviruses before the introduction of anti-Rotavirus vaccines in the country.

Group A rotavirus strains circulating in the eastern center of Tunisia during a ten-year period (1995-2004).

An epidemiological survey investigating rotavirus infections in children was undertaken in the Eastern Center of Tunisia between January 1995 and December 2004. A total of 982 faecal specimens collected from children less than 5 years in age were screened by enzyme-linked immunosorbent assay (ELISA) or latex agglutination assay for the presence of group A rotavirus antigen. Rotavirus-positive samples were used for G and P typing by multiplex semi-nested reverse transcription-PCR. Rotaviruses were detected in 22% (n = 220) of stools. Of these, 164 were typed for VP7: G genotypes found were G1 (59%), G2 (2%), G3 (9%), G4 (10%), G8 (1%), and G9 (1%). Sixteen specimens (9%) showed mixed G profiles. A total of 119 specimens were typed for VP4. P genotypes detected were P[8] (32%), P[6] (15%), and P[4] (13%). Mixed P profiles were also detected (6%). Although the distribution of the detected genotypes appeared to change annually, G1P[8] rotavirus strains always predominated during the 10-year period of study. This is the first report of rotaviruses in Tunisia with unconventional VP7 serotypes such as G8 and G9, highlighting the need for continual surveillance of emerging strains in Northern Africa. Indeed, the new commercial vaccines only contain the VP7 genes that dictate G1 or G1 to G4 specificities. These vaccines may protect less well against unusual strains circulating in countries planning to implement a rotavirus vaccine strategy.

Molecular characterisation of the rotavirus strains prevalent in Maua, Meru North, Kenya.

BACKGROUND: Rotavirus is the most common cause of severe infantile diarrhoea disease in infants and young children below five years worldwide. Rotavirus is associated with high cases of morbidity and mortality and it is estimated that up to 650,000 deaths in young children occur annually in the less developed countries and approximately 150,000-200,000 deaths occur in Africa alone. OBJECTIVE: To characterise the circulating rotavirus strains in Maua, Meru North district, Kenya. DESIGN: A prospective study to investigate and characterise rotavirus serotypes/genotypes and electropherotypes in infants and children with severe diarrhoea hospitalised and/or attending the outpatient department of Maua Methodist Hospital during the period April 2004 to September 2005. SETTING: Maua Methodist Hospital, Meru North, Kenya. SUBJECTS: Faecal samples were collected from 135 infants and children with acute diarrhoea and were screened first for the presence of human Group A rotavirus antigen using commercially available enzyme linked immunosorbent assay kit (ELISA). The positive samples were evaluated by sodium dodecyl polycrylamide gel electrophoresis (SDS-PAGE) to determine the viral RNA electropherotype profile. Rotavirus strains were also genotyped using reverse transcriptase polymerase chain reaction (RT-PCR) of the VP7 gene. RESULTS: Assay of these samples with commercial ELISA showed that 17.8% (24/135) were positive for group A rotavirus antigen. Twenty of these ELISA positive samples were also analysed by SDS-PAGE of which 75% (15/20) gave detectable electropherotype pattern with the long electropherotype being predominant 80.0% (12/15) followed by the short RNA profile 20.0% (2/ 15). Seventeen of the ELISA positive samples were genotyped for VP7 and the results showed that G9 was the most predominant genotype comprising 47.1% (8/17) followed by G8 29.4% (5/17), GI 17.4% (3/17) and the mixed genotype was G8/G9 5.9% (1/17). Most patients with rotavirus infection were of the age of 3 - 60 months, with 79% being less than 18 months old. CONCLUSION: The overall prevalence of rotavirus infection in young children with diarrhoea hospitalised and/or attending the out-patient department of Maua Methodist Hospital was 17.8% with the predominant serotype being G9. These results show that rotavirus plays an important role in severe viral diarrhoea in young children in Maua Meru North district, Kenya. Furthermore, this high G9 rotavirus prevalence in Kenya may require vaccine trials to be held in Kenya so as to determine the efficacy of new rotavirus vaccine candidates that do not include the G9 serotype.

Identification of viral agents causing diarrhea among children in the Eastern Center of Tunisia.

Viral diarrhea remains a major cause of childhood morbidity and mortality worldwide. In Tunisia, no comprehensive studies of all viral agents related to diarrhea in children have yet been conducted. The present study was performed to investigate the role of enteric viruses in acute diarrhea in the country. Six hundred thirty-eight stool samples were collected from children under 5 years of age seeking medical care for acute diarrhea between October 2003 and September 2005 in hospitals from the Eastern-Center Tunisia. All samples were tested for rotavirus, astrovirus, and adenovirus using commercial antigen enzyme immunoassays (EIAs). Positive samples for rotavirus and astrovirus were confirmed by an "in-house" reverse transcriptase-polymerase chain reaction (RT-PCR). Samples positive for adenovirus antigen were subjected to further EIA screening for species F enteric adenovirus types 40 and 41. At least one viral agent was found in 30% of the specimens. The frequency of rotavirus, astrovirus, and adenovirus was 20%, 7%, and 6%, respectively. Of the stool samples containing adenovirus, 57% (20/35) were found to be positive for species F adenovirus types 40/41. Dual infections were found in 9% (17/191) of the positive samples. Enteric viruses appear to play an important role in pediatric diarrhea in Tunisia. The introduction of affordable viral diagnosis in pediatric hospitals will improve patient care by reducing the unnecessary use of antibiotics.

Anticipating rotavirus vaccines: epidemiology and surveillance of rotavirus in South Africa.

Rotavirus infection is associated with acute infantile gastroenteritis in infants and young children globally. In South Africa, rotavirus infection has been shown to be associated with approximately one-quarter of all diarrhoeal admissions to hospital. Rotavirus infection predominantly occurs in infants less than 12 months of age (75%) and has a peak of shedding during the cooler, drier months of the year. A secondary peak during the spring has been observed. Multiple infections with rotavirus and at least one other microbial agent are common. The circulating VP7 serotypes and VP4 genotypes have been determined in various regions of South Africa and show a geographic specific distribution. A decade previously, P[8]G1 or G4 strains predominated, and P[4]G2 strains occurred in an epidemic pattern in one region. More recently, rotavirus strains with P[6] genotype have become common and novel VP7/VP4 genotype combinations are occurring across the country. G9 strains have been reported from Cape Town to Vendaland. The circulating rotavirus types observed in this study add to the knowledge of the natural history of rotavirus infection and provide the groundwork to consider future vaccine strategies.

Human group C rotaviruses identified in Kenya.

BACKGROUND: Rotavirus is associated with acute infantile gastro-enteritis in infants and young children worldwide. Rotavirus is associated with the high cases of morbidity and mortality and it is estimated that up to 650,000 deaths in young children occur annually in the less developed countries. Approximately a quarter of these deaths occur in African children yet minimal data exist on the circulating rotavirus strains in Africa. OBJECTIVE: To characterise the circulating rotavirus strains in Kenya. DESIGN: A prospective study to investigate rotavirus infection in infants and young children with acute diarrhoea in different areas of Kenya. Between September 2001 and February 2002, 119 faecal specimens were collected from children aged between 1 and 60 months with acute infantile gastro-enteritis. SETTING: Faecal samples were collected from clinics around Nairobi and its suburbs including Karen, Ngong, Ongata Rongai. SUBJECTS AND METHODS: Faecal samples were collected from 119 infants and young children with diarrhoea and were analysed by commercial ELISA and polyacrylamide gel electrophoresis (SDS-PAGE) to identify possible non-group A rotaviruses. Extraction of any potential rotavirus double-stranded RNA from faeces and characterisation by SDS-PAGE showed the presence of human group C rotaviruses. RESULTS: Examination of these samples with a commercial ELISA assay for the presence of group A rotavirus antigen showed that 13 specimens (11%) were positive. An analysis of all stool specimens was performed by polyacrylamide gel electrophoresis to identify possible non-group A rotaviruses. Extraction of any potential rotavirus double-stranded RNA from faeces and characterisation by SDS-PAGE showed the presence of human group C rotaviruses. CONCLUSION: This is the first report of group C rotaviruses in Kenya. Further studies are underway to continue the surveillance of rotavirus strains in Kenya; as this information will be useful in planning rotavirus vaccine trials in Africa.

Circulation of the novel G9 and G8 rotavirus strains in Nigeria in 1998/1999.

An epidemiological survey investigating the prevalence of rotavirus infection in infants and young children with acute diarrhoea was undertaken in Jos State, Nigeria, between January 1998 and April 1999. In total, 672 faecal specimens were collected from children aged between 1 and 60 months with acute infantile gastroenteritis. The 10-20% stool suspensions were examined by an ELISA for the presence of group A rotavirus antigen (Rotavirus IDEIA, Dako, UK). Only 116 specimens (17.3%) were positive for the group A rotavirus antigen detected by this ELISA. The rotavirus-positive specimens were analysed with monoclonal antibodies specific for rotavirus VP6 subgroup I and II, and for VP7 serotypes G1-G4, G8, and G9. Of the rotavirus strains that could be subgrouped, VP6 subgroup I and II strains circulated at similar levels. Amongst the strains that could be serotyped, VP7 G9 strains predominated occurring in 17 cases, with G3 (n = 10) and G1 (n = 9) strains occurring in lower numbers. Four G8 strains were detected and only one G2 and no G4 strains were identified. This report extends the description of the global distribution of G9 rotavirus strains.

Epidemiology of astrovirus infection in Zaria, Nigeria.

Astrovirus has been shown to be an important aetiological agent associated with gastroenteritis in children, although few studies have been conducted in Africa. In this study, stool specimens were obtained from 375 young children less than 5 years of age with acute gastroenteritis presenting at Ahmadu Bello University Hospital, and from a control group of 122 children without diarrhoeal illness. The specimens were examined for the presence of human astroviruses using a monoclonal antibody-based ELISA (Astrovirus IDEIATM, Dako, UK). Negative staining electron microscopy was performed on specimens to confirm the presence of astrovirus particles. Astrovirus was detected in 6.7 per cent (25/375) of the diarrhoeal stools compared to 5.7 per cent (7/122) of the control specimens. Astrovirus seemed to infect older children and more than half the children were between 1 and 4 years of age (15/25). Only four children were less than 6 months old. A winter peak of shedding was observed.

Viral diarrhoea in young children in two districts in Nigeria.

OBJECTIVES: To determine the prevalence of viral agents of diarrhoea in Ilorin and Lagos, two zones in Nigeria. DESIGN: A survey of young children who had clinically confirmed diarrhoea. SETTING: University of Ilorin Teaching Hospital in Ilorin, Kware State and the Massey Street Children's Hospital in Lagos State, Nigeria. SUBJECTS: 108 children under the age of five. RESULTS: The prevalence rates observed were 33.3% for rotavirus, 6.7% for adenovirus and 1.2% for astrovirus. The rotavirus strains were characterized further. PAGE determined the presence of seven patterns of RNA electropherotypes, with one short RN patterns and six long patterns. The G and P types of selected rotavirus positive samples were characterized by RT-PCR techniques. The VP7 G typing showed that GI was the most prevalent single strain found (8.3%), while G3 and G4 accounted for 6.7% and 1.7%. The rate of mixed G serotypes was 26.7%. The P[6] genotype was the most prevalent (52%) and the P[4] had a prevalence of 8%. The mixed P genotype accounted for 28% of the rotavirus strains. The high rate of mixed infection may have an implication on vaccine development. CONCLUSION: Rotavirus was the most prevalent virus in the study with astrovirus being the second most prevalent. There was only a single incidence of astrovirus.

Distribution of rotavirus VP7 serotypes and VP4 genotypes circulating in Sousse, Tunisia, from 1995 to 1999: emergence of natural human reassortants.

Rotavirus strains circulating in Sousse, Tunisia, between 1995 and 1999 were characterized antigenically by monoclonal antibodies to the VP6 subgroup and the VP7 serotype. The VP4 genotype was determined by reverse transcription-PCR, as were the strains with untyped VP7. Only 17% of 375 children were shedding rotavirus as determined by latex agglutination assay. Most rotavirus strains were G1P[8] (50%), followed by G4P[8] and G4P[6]. Reassortant G1P[4] strains emerged in Sousse during the 1998-1999 season.

Comparative studies of human rotavirus serotype G8 strains recovered in South Africa and the United Kingdom.

Epidemiological studies on the VP7 serotype prevalence of human rotaviruses in South Africa and the United Kingdom identified several strains which could not be serotyped as G1-G4 by monoclonal antibodies. Further analysis of these strains with a G8-specific monoclonal antibody and with probes for human rotaviruses confirmed them as G8 rotaviruses. These G8 strains exhibited a high degree of sequence identity when compared with each other and with other rotavirus G8 strains. Five South African strains were further characterized as VP6 subgroup I, but with a long RNA electropherotype, which is similar to the G8 strains previously isolated in Finland. In the UK strains, one was VP6 subgroup II with a long RNA electropherotype (similar to the Italian G8 strain). The other two were subgroup I with a short RNA electropherotype. None of these strains exhibited the super-short RNA electropherotype described in the prototype G8 strains recovered from Indonesia (69M).

Seroepidemiological study of genogroup I and II calicivirus infections in South and southern Africa.

Diarrhoea is associated with the daily death of between 180 and 200 children under the age of 5 years in South Africa. Until recently, many cases and outbreaks of diarrhoea were not associated with a known aetiologic agent. Previous studies using baculovirus-expressed Norwalk virus (NV) and Mexico virus (MxV) capsid antigens have shown that human calicivirus infection is common in South Africa. In this study, our surveillance was extended to different populations, as well as to four other southern African countries: Namibia, Angola, Zimbabwe, and Mozambique. More than 1,700 specimens, some involved in previous cohort studies of infectious diseases, were enrolled in the surveillance. The overall seroprevalence of antibody against NV was >90% for all cohorts except for Mozambican refugees that had 83. 8% sero-positivity. The MxV antibody prevalence was higher than NV, with >95% positivity for all cohorts, except for one in Namibia that had 81% exposure. This study is one of only a few reporting on the concurrent incidence of NV and MxV infections in a cohort study, and has determined that small round structured viruses are prevalent in the local populations of South and Southern Africa. These agents may account for a number of previously unknown or unidentified causes of diarrhoeal illness, in both adults and children, in southern Africa.

Rotavirus strains bearing the VP4P[14] genotype recovered from South African children with diarrhoea.

Human rotavirus VP4P[14] strains were previously recovered in South Africa [18]. The strains exhibited a long RNA pattern, VP6 subgroup II and VP7 serotype G1. Two of the VP8 genes were cloned and sequenced and demonstrated a high nucleotide homology with the prototype P[14] strains (PA169 and HAL1166).

Characterization of VP6 subgroup, VP7 and VP4 genotype of rotavirus strains in Lusaka, Zambia.

In a previous study, rotavirus infection was determined in young children at the University Teaching Hospital in Lusaka. In this study, selected rotavirus strains were characterized by monoclonal antibody assay to the VP6 subgroup antigen present and by hybridization analysis of the VP7 and VP4 genes carried by the virus. The majority of the strains were characterized as a VP6 subgroup II, VP7 serotype G1 strain with a long electropherotype and bearing the VP4 P8 genotype. A further four minor rotavirus strains with a long RNA electropherotype and subgroup II antigen were also observed to be circulating bearing G1 or G4 VP7 genes and the VP4 P8 genotype. Two electrophoretic strains with differing short RNA electropherotypes and subgroup I antigenicity were also present. These strains hybridized to the VP7 type G2 and VP4 P4 genotype probes.

Human Group C rotaviruses identified in Nigerian children.

A survey investigating the prevalence of rotavirus infection in infants and young children with acute diarrhoea was undertaken in Jos State, Nigeria during the first half of 1998. In total, 182 faecal specimens were collected from children aged between 1 and 60 months with acute infantile gastroenteritis. Examination with a commercial ELISA assay for the presence of group A rotavirus antigen revealed 16 specimens positive for this antigen. However, examination of the double stranded RNA of the viral genome by polyacrylamide gel electrophoresis showed the presence of three human group C rotaviruses. This is the first report of group C rotaviruses in Nigeria and West Africa.